Neurodegeneration Methods and Protocols

Introduction to Polymerase Chain Reaction Mark C. Miller and Laura Cunningham 1. Introduction The polymerase cham rea...

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Introduction

to Polymerase

Chain Reaction

Mark C. Miller and Laura Cunningham 1. Introduction The polymerase cham reaction (PCR) has become an indispensable tool of molecular biology (I-5). Smce its discovery in 1985 the PCR process has found its integration into all research areas mvolving the use of DNA and RNA. Using this technique, a small starting sample of DNA or RNA can be used to amplify a specific DNA or RNA target over a million-fold in as little as 2 h. This allows for the detection of as little as a single copy of a gene or part of a gene m cells, whether they be from blood, cultured cells, tissue biopsies, chromosomes, or any other biological system that contains DNA or RNA, mcludmg archival materials (formalin-fixed, paraffin-embedded). Thus chapter will drscuss the prmciples of PCR as it relates to the methodologies employed to successfully carry out the reaction on DNA targets. Protocols relating to DNA extraction, PCR conditions, amplification cycles, and post-PCR detection methods will be presented. RNA amplification (RNA PCR) will be discussed in another chapter. There is a wide variety of methodologies for the extraction of DNA for use in many molecular-biology applications. When preparing DNA for use m PCR, the DNA extracteddoes not have to be of the highest quality. A variety of kits are available on the market that allow for the relatively quick isolation of PCR-quality DNA. Two methodologies will be described. One mvolves a commercrally available krt for the quick isolation of DNA from cultured cells. The second procedure is for use with formalin-fixed paraffin-embedded brain tissue. Because of the destructive nature of formalin on DNA, special PCR consideratrons need to be taken mto account (i.e., length of PCR product). Because of the high lipid content of brain tissue sodium dodecyl sulfate (SDS) is often used in the extraction step. The reader is referred to the reference list for other methodologies mvolvmg paraffin-embedded bram tissues (6-8). From

Methods m Molecular Medrcme, vol 22 Neurodegeneratron Methods and Protocols Edtted by J Harry and H A T&on 0 Humana Press Inc , Totowa, NJ

1

2

Miller and Cunningham

1.1. Principles of PCR DNA replication m vivo requrres that the double-stranded DNA molecule be made smgle-stranded (accomplished through the use of specialized proteins) followed by the synthesis of a short RNA primer along a stretch of the smglestranded DNA. A DNA polymerase then binds to this primer-DNA complex and moves along the single-stranded DNA template polymerizing a new strand of DNA by mcorporatmg nucleotides that are complementary to the template (parent) strand of DNA. This highly integrated process allows for DNA to be replicated in a relatively short penod of time and requires specialized proteins, physiological conditions, the presence of nucleotides, and a vartety of enzymes used to catalyze the reaction. The PCR process, in a sense, mtmtcs this in vrvo DNA replication process. However, since it takes place in vitro, the PCR process requires modifications to the normal environment through the use of specialized enzymes and reaction components, which fosters its adaptation to a tube environment. Starting with double-stranded DNA (either genomtc or cloned), short single-stranded oligonucleotrde primers complementary to known sequenceson the template DNA, a thermostable DNA polymerase, as well as reagents needed to synthesize new DNA (namely deoxynucleottde trtphosphates), along with physrologrcal salts and buffers, the PCR reaction can carry out repeated amplifications of a target stretch of DNA until millions of copies of that target are synthesized (see Fig. 1). To compensate for a lack of a true physrologrcal state, the PCR process utilizes physical conditions (i.e., temperature) to accomplish the manipulations of the DNA that would, in an in vivo environment, be accomplished by protems. Specifically, the denaturing of the double-stranded DNA (converting to single strands) is accomplished in the PCR by raismg the temperature of the reaction to approx 95°C for a short period of time. The dtscovery of thermostable DNA polymerases (Tuq DNA polymerase) allows the PCR reactions to be carried out at high temperatures without the destructton of the polymerase after each hrgh-temperature step, which would occur rf a nonthermostable DNA polymerase were used. A typical PCR cycle involves three basic steps: 1 Denaturation: The PCR reagents are heated to approx 95°C for 15-30 s Thus causes the DNA template to denature (melt) into single strands. Cycling times may vary depending upon the thermal cycler model used. 2 Anneahng: The temperature is rapidly lowered to approx 55°C for 15-30 s. Thts allows the short, smgle-stranded DNA primers the opportunity to bind to then complementary regtons on the single-stranded DNA template. As wtll be dlscussed later, thus part of the reactton can lead to nonspecific annealing and extension of the primers to sues on the DNA other than their true complement, resulting m the generation of undesired, spurious products. Thus, selection of the annealing temperature 1scrltlcal to generating specific PCR products.

3

introduction to PCR double-stranded template DNA Denaturatlon (95 o C, 15-30

t

set )

3’,

smgle-stranded template DNA

s13 Annealmg (55o C, 15-30 set )

I

3’

6’ 5’

11111111 IIIIIIIIIIIIIll

l Ill

30 cycles

ll ll

5’

Pnmers bind to complementary regions on template DNA

Extension

(72oC, 30set ) I 3’

5”“““““““’ -c

Taq polymerase binds to 3’ ends of primers and Incorporates dNTPs as new DNA is synthesized in 5’4’ dlrection

New complementary strands are synthesized and results in doubling of starting target DNA

Fig. 1 Diagrammatic representation of the polymerase chain reaction. 3. Extension: The temperature is then rapidly raised to approx 72°C for 15-30 s. During this phase the AmpliTuq@ DNA Polymerase binds to the primer-DNA complexes and begins polymerizing a new strand of DNA by mcorporating deoxynucleottdes (dNTPs) that are complementary to the template strand. It should be noted, however, that this is the optimal extension temperature of AmplzTaq DNA Polymerase AmplzTaq DNA Polymerase has activity at lower temperatures, so care needsto be taken to avoid the opportunity for misprimed primers to be extended. These concerns will be addressedin Note 15

would involve approx 30 repeated cycles of the selected temperature and time parameters. In some cases, it is possible to do a twotemperature cycling step. A two-step PCR involves combining the anneal and extension steps in one. The anneal/extend cycle is run between 60 and 72°C for 45 s to 1 mm. However, rf the T,,, of the primers is below 60°C a three-step PCR cycle should be used. A typical PCR reaction


2. Materials 2.7. Reagents 2.1.1. Extraction Reagents 1 Puregene DNA Isolation from Cultured Cells Ku (Gentra Systems) to Include* cell lysis solution, RNase A solution, protein precipitation solutton (Mmneapohs, MN), 100% tsopropanol, 70% ethanol, and DNA hydration solution. 2 DNA from formalm-fixed, paraffin-embedded tissue. a. Phenol.chloroform.rsoamyl alcohol (25.24: 1) (AMRESCO [Solon, OH] cat. no. 0883 [Trts-buffered]). b. 95% ethanol (AMRESCO cat. no. E193). c. 1X TE, pH 7.4 (Dlgene [Beltsville, MD] cat. no. 3400-1039). d. 0.5 M EDTA (Digene cat. no 3400-1003) e. 20% SDS (AMRESCO cat. no E195) f. Protemase K (AMRESCO cat. no 0837) g. Chloroform (AMRESCO cat no. 0757)

2.7.2. PC/? Reagents 1 Autoclaved ultrafiltered water GoZdrM DNA Polymerase (250 U) (PE Applied Btosystems City, CA] cat. no N808-0241, (see Note 1, Table 1) 3 10X reaction buffer II, to include: a. 100 mM Tris-HCl, pH 8.3 (at 25°C). b. 500 mM KCl. c. 25 mM MgCl, solution. d. dNTPs (PE Applied Biosystems cat. no N808-0007). e. Primers (user provided, see Note 7) f. DNA template (user provided, see Note 22).

2 AmpliTaq

[Foster

2.1.3. Detection Reagents 1. Ethtdmm bromide solution (AMRESCO

2.2. Equipment 1. 2. 3 4 5. 6 7. 8

cat. no E406, see Note 23).

and Supplies

Microcentrifuge tubes High-speed microcentrifuge. Vortex. Heat block. Sterile scalpels. Sterile mortar and pestle UV spectrophotometer. Positive displacement pipe& 0 5-25 uL (PE Applied Biosystems

cat no N930-

6008) 9 Positive displacement ptpet, 25-250 PL (PE Applied Blosystems 6009)

cat. no N930-

5

Introduction to PCR

10. GeneAmp@ PCR System 9700 Thermal Cycler (PE Applied Blosystems cat. no. N805-000 1; see Note 24).

11. 12. 13. 14

Primer ExpressTM Software (PE Applied Biosystems cat. no. 402809). Pipet tips, 0.5-25 FL (PE Apphed Biosystems cat. no. N930-6010). Pipet tips, 25-250 FL (PE Applied Biosystems cat. no. N930-6011). MicroAmp@ Reaction Tubes with caps (PE Applied Biosystems

cat no.

N801-0540)

3. Methods 3.1. DNA Isolation 3 7.7. Cell Lysis

from Cultured

Cells

1. Add 0 5-l .O mllhon cells m approx 1 mL of balanced salt solution or culture medium to 1.5-mL tube (expected yield is 2-6 pg DNA). 2. Pellet cells by placing the tube in a centrifuge and spinning at 13,000-16,OOOg for 5 s. 3. Remove supernatant, leaving behind 5-10 pL of residual liquid 4 Vortex the tube vigorously to resuspend the cells m the residual supernatant. 5. Add 150 p.L cell lysts solution to the resuspended cells and plpet up and down approx 20 times to lyse the cells. If cell clumps are visible after mrxing, Incubate at 37’C until the solution is homogenous. Samples m the cell lysts solution are stable for at least 18 mo at room temperature.

3.1.2. RNase Treatment 1. Add 0 75 pL RNase A solution to the cell lysate. 2 Mix the sample by mvertmg the tube 25 times and incubate at 37°C for 15-60 mm.

3.7.3. Protein Precipitation 1. Cool sample to room temperature 2. Add 50 PL protein precipitation solution to the RNase A-treated cell lysate. 3 Vortex vigorously at high speed for 20 s to mix the protein precipitation solutton uniformly with the cell lysate. 4. Centrifuge at 13,000-16,000g for 3 mm. The precrpitated protems will form a tight pellet 5. If the protein pellet 1snot visible, repeat step 3 followed by incubation on ice for 5 min, then repeat step 4

3. I. 4. DNA Precipitation 1 Pour the supernatant containing the DNA (leaving behind the precipitated protein pellet) into a 1 5-mL microfuge tube containing 150 pL isopropanol (Zpropanol). 2. Mix the sample by gently inverting 50 ttmes. 3. Centrifuge at 13,000-16,000g for 1 mm; the DNA ~111be visible as a small white pellet on the bottom of the tube. 4. Pour off the supernatant and drain the tube on clean absorbent paper. Add 150 l.tL 70% ethanol and invert the tube several times to wash the DNA pellet

Table 1 Feature Chart for PCR Enzymes

Product name Organism Molecular weight Number of ammo acids Optimal extension temperature Extension rate m (72°C) Hot start Enzyme activation RT actrvity Half-life

Cycling half-life” Magnesium ion PCR optimum pH opttmum for PCR amphficatron pH optrmum for activatior+

(70)

AmpliTaq Gold DNA Polymerase

AmpllTaq DNA Polymerase

AmpllTaq DNA Polymerase, Stoffel fragment

rTth DNA Polymerase

Thermus aquaticus

Thermus aqua&us

Thermus aquatzcus

Thermus thermaphdus

94 kDa 832

94 kDa 832

61 kDa 544

94 kDa 834

Thermus thermophdus; Thermococcus lltoralls N/A N/A

72-80°C

72-80°C

72-80°C

72-80°C

72-80°C

24 kb/mm

2-4 kblmin

24 kb/mm

2-4 kb/mm

2-4 kblmm

Automated 10 mm at 95°C or 10 extra cycles Mimmalflow 97.5WlO mm 95W40 mm 92S”Ch130 min 160 cycles 15-4mM

Manual N/A

Manual N/A

Manual N/A

Manual N/A

Mmrmalllow 97 5WlO mm 95W40 mm 92 5”C/>130 mm 160 cycles 1-4mM

Mmtmal/low 97 5W20 min 95W80 mm

Yes, Mn2+-dependent 97 5”C/2 mm 95W20 mm

Yes, Mn2+-dependent 97.5W2 mm 95W20 mm

320 cycles 2-10 rrUI4

80 cycles 1.5-2.5 mi%Z

80 cycles 1.5-2 5 mM

7.0-7 5

7.0-7.5

7.0-7 5

7.0-7 5

7.0-7.5

8 3 at room temperature

N/A

N/A

N/A

N/A

rTth DNA Polymerase, XL

u

dNTP PCR optimum KC1 PCR optimum Primer PCR opumum Processivny Longest PCR product Extra A addition Y-3 exonuclease activity 3’-5’ exonuclease activity Contammatmg nucleases Supplied concentration Storage conditions

40-2OOpM

each

40-2OOpM 5omM

each

40-2OOpM each

40-2OOpM

10 mM 0 l-l.0 p.44

75-100 m&I 0.1-1.0 /.M

75-100 mM

each

40-2OOpM

5OlllM 0.1-1.0 /.tM

0 l-l.0

50-60 bases -5 kb

5CL60 bases -5 kb

5-10 bases N/A

3WO bases -5 kb

30-40 bases >40 kb

Yes Yes

Yes Yes

Yes (less) No

Yes Yes

No Yes

No

No

No

No

Yes

No

No

No

No

No

5 U/pL

5 U&L

5 U&L

5 U&L

5 U&L

-2OT

-20°C

-20°C

-2OT

-20°C

aAssumptions of 95”C, 15 s denaturanonlcycle bpH optrmum measured at 72°C

FM

0.4 pM

each

8

Miller and Cunningham 5 Centrifuge at 13,000-16,000g for 1 mm. Carefully pour off the ethanol. Because the pellet may be loose, you should pour off slowly and watch the pellet. 6 Invert and dram the tube on clean absorbent paper and allow to arr-dry for 15 min.

3.1.5. DNA Hydration 1 Add 25 ~.LLDNA hydration solution (25 pL will give a concentration of 100 pg/mL if the total yield is 2.5 kg DNA) 2. Allow DNA to rehydrate overmght at room temperature Alternatively, heat at 65°C for 1 h. Tap the tube periodically to aid in dispersmg the DNA. 3. Store DNA at 2-8°C

3.2. DNA Extraction from Forma/in-Fixed, Paraffin-Embedded Brain Tissue 3.2.1. Tissue Processing 1. Usmg a sterile scalpel, cut approx 100 mg from a paraffin block of brain tissue 2 Crush the tissue using a sterile mortar and pestle and mcubate with gentle agitation with 500 pL xylene for 1 h at room temperature. 3. Wash m 95% ethanol and then 70% ethanol for 5 mm each. 4. Centrifuge at 13,000g for 10 mm to pellet. 5 Dry pellets m a 50°C heat block. 6 Resuspend m 1X TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) 7. For tissue that is formalm-fixed but not paraffin-embedded, cut a 0 1-cm3 ptece from the tissue using a sterile scalpel and incubate in 10 mL of 1X TE overnight at 4°C.

3.2 2. Digestion/Extraction

and Purification

1 Digest sample in a 200 mL ahquot containmg a. 0.2 M Tris, pH 8.0. b 10 mM EDTA. c 1% SDS. d 4 mg/mL proteinase K. 2. Incubate for 16 h at 50°C 3. Add an equal volume of Tris-buffered, redistilled phenol.chloroform.isoamyl alcohol and vortex briefly. 4. Centrifuge at high speed (13,000g for 15 s). Make sure to separate the phases. Centrifuge longer if not completely separated 5. Pipet the aqueous upper phase to a fresh tube. Discard the orgamc lower phase and interface. When pipettmg the aqueous phase, care should be taken not to remove any of the organic phase with the aqueous phase. At this point you may “back-extract” the organic phase and mterfaces. These contain the proteins but some DNA may also be present. After removing the aqueous phase, add an equal volume of 1X TE to the organic and interface phases. Mix well and centrifuge at 13,000g for 15 s Transfer the aqueous phase to the previous aqueous phase

Introduction to PCR

6.

7. 8. 9. 10. 11 12 13 14. 15.

9

Discard the organic and interface phases in the appropriate organic waste disposal contamer Repeat steps l-3 until no protein is visible at the interface of the organic and aqueous phases, This may require extractions. Add the aqueous phases to the previously collected aqueous phases. Add an equal volume of chloroform and repeat steps 4 and 5 Adjust the volume of the combined aqueous phases to 300 pL using 1X TE. Add two volumes of me-cold 100% ethanol and mix by vortexmg. Incubate on ice for 15-30 mm. Centrifuge for 10 mm and carefully and completely remove the supernatant. Half-fill the tube with 70% ethanol and recentrifuge at 12,000g for 2 mm at 4°C in a microfuge Carefully remove all of the supernatant Store the open tube on the bench top until all of the hquid has evaporated. Add 1X TE to the desired volume Determine quantity of DNA via UV absorbance at 260 and 280 nm. See Note 20.

3.3. PCR Procedure 1. Prepare a master mix of PCR reagents (water, buffer, dNTPs, and DNA polymerase) for all of the samples (see Subheading 2.1.2.). The primers and template DNA are added separately. Using a master mix is recommended because it improves the plpettmg accuracy and mmtmlzes reagent losses on the pipet tips. To prepare a master mix, determine the final total volume of all of the reactions (i.e., 10 tubes x 50 @/tube = 500 pL total volume) Prepare a master mix tube with the appropriate volumes of water, buffer, dNTPs, and enzyme to accommodate the total volume of all of your reactions (i.e., 500 FL). Be sure to take into account the addition of primers and template DNA to your reaction tubes (see Table 2). If AmpliTuq Gold DNA Polymerase is the enzyme bemg used, all of the components (mcludmg primer and template DNA) can be added to the master mix. Gently vortex the master mix and dtspense 50 mL into each tube and place tubes in the thermal cycler. 2. Place tubes m the thermal cycler and perform cycling temperature and time

parameters(see Table 3). 3 Accurately

quantitate PCR components before and after PCR (see Note 25 and

Table 4). 4. 5 6. 7.

Optimize PCR reaction conditions (see Notes 26-29). If necessary, utilize Hot Start PCR protocol (see Notes 30-34) Low copy number templates may require further optimization (see Note 35). Crosscontaminatron prevention is critical during PCR amplification (see Note 36).

3.4. Defection of PCR Products At the completion of the PCR reaction, approx 5-10 PL of product is mixed with a gel loading buffer and run on an agarose gel (l-2%). DNA molecularweight markers are also loaded mto one of the wells as fragment size refer-

10


Table 2 PCR Reaction

Components

Components (50 pL rxn) Autoclaved ultrafiltered water [10X] Reaction buffer II 25 mM MgCI, solution dATP dCTP dGTP dTTP AmpliTaq Gold DNA polymerase User-provided primer #l User-provided primer #2 User-provided DNA template Total mix

Order

Volume

Final concentration

1

a

2 2 3 3 3 3 4 5 5 6

5.0 pL 2-8 ~JL

1X 1-4mMb

1 OpL 1OpL 10 pL 1.0 pL 05pL

200 pM 200 /AM 200 FM 200 PM 2.5 U

0.5-2.5 PL 0.5-2.5 ~.LL

0 2-10 FM

a

0 2-l .O /.LM cl pg/rxn”

50 mL

aAny combmation of water and template can be used as long as the total volume of the master mix, sample, and pnmers equals 50 pL bThe optimal magnesmm chloride concentration may vary, depending on the primer and template used and must be determined empmcally In most cases a final concentration of magnesium chloride m the range of 1 O-4 0 nul4 m the reaction mix will work well If using the 10X PCR buffer I, which already contains 15 mM MgC12, the concentration of the M&l, will be 1.5 mM. CPreferably >104 copies of template but concentration of Mg2+ is used and the dNTPs are at 800 PM, there would exist 0 7 mM (700 w of free Mg2+ available to react with the AmplzTaq DNA Polymerase. If one reduces the dNTP concentration (to increase fidelity) than a concomitant reduction m Mg2+ is also required. 29. Enzyme concentration: Recommended starting concentrations of AmpliTaq DNA Polymerase range from 1 25 to 2 5 U/50 PL reaction. Too little enzyme will result m insufficient amplification of target and too high a concentratron of enzyme can result in nonspecrfic amphfrcations, resulting m high background levels Ideal enzyme concentrations can be determined by adding enzyme to separate reactions over a range of 0.5-5.0 U/50 p-L (m 0 5-U increments) and assessing amphfrcation efficiency by agarose gel electrophoresis

4.8. Hot-Start

PCR-Minimizing

Nonspecific

Amplifications

30 One of the inherent shortcomings of the PCR process is the fact that unmtended PCR products are created. These nonspecific products are typically DNA fragments of varying molecular weights In addition, low-mol-wt primer-dimers may also form These occur when the primers bind to each other and are extended by AmplzTaq DNA Polymerase. Primers, being short, angle-stranded DNA molecules, can, and will, bmd to nonspecific sites on the template if given the

lntroductlon to PCR

19

opportunity to do so. These opportunities are created when primers, along with all of the PCR reagents, are present at temperatures below the optimum primerannealing temperature Each specific primer has a temperature at which it will melt or denature from the template. The melting temperature of the prrmer is referred to as the T, At temperatures below the T,, a primer will anneal (T,; annealmg temperature) to its complement on the template. There will be an optimum annealing temperature for each primer. Typically, the T, is set at approx 3°C below the calculated T, for the primers. At temperatures below the optimum T,, a primer can bmd to regions on the template that are not 100% homologous. The lower the temperature the less primer-template homology IS required in order for a primer to bind. Thus, at lower temperatures a primer can bind to nonspeciftc regions of the template AmpliTaq DNA Polymerase (and all thermostable DNA polymerases) have enzymatic activity at temperatures well below then- optimal temperature of activity of 72°C. If nonspecific primer annealing occurs and AmpliTaq DNA Polymerase is present m the system, extensions will occur from these sites and small. nonspecific products will be formed. This process is called pre-PCR mispriming. When thermal cyclmg commences, these nonspecific products can now serve as additional templates during each subsequent PCR cycle. Therefore, at the end of the PCR cyclmg, not only the desired fragment, but also these amplified nonspecific fragments will exist. These spurious products are referred to as background and can greatly interfere with the mterpretation of results via agarose gel electrophoresis. In addttton, there will be a reduction m the quantity of the desired fragment caused by competition for reagents. When amplifying a low-copy-number sequence, this can result m a negative reaction. Also, at these lower temperatures, the primers may bind to each other at the 3’ ends. If this occurs, these primer-dtmers can also be extended and result m the generation of low-mol-wt fragments of DNA. Thus, not only does temperature play a part m nonspecific amplification and primer-dimer formation, but also the design of the primers is critical for efficrent PCR. Figure 2 reflects examples of nonspecific amplification and primer-dimer formation. Lanes 2,4, and 6 reflect nonspecific fragments generated m the amplification of 2260-, 1940-, and 1620-bp fragments, respectively, of fibronectm. Lanes 3,5, and 7 reflect the same amplification in the presence of AmpliTaq Gold DNA Polymerase, an enzyme that virtually eliminates nonspecific amplification. To circumvent these anomalies, a variety of methods, Hot Start techniques, have been developed over the years. Each of these methods can help reduce the amount of nonspecific amphficatrons and primer-dimer formation, but none are 100% efficient. However, some hot-start protocols are not suited for high throughput applications and can increase the risk of crosscontamination and backcontamination between reactions Briefly, the following hot-start techniques have been used 31. Manual hot start: In these procedures, one typically adds all of the components of the PCR reaction to the tube except one or two critical components (1.e , AmpllTaq

20


DNA Polymerase and 10X buffer). The tubes are then placed in the thermal cycler and heated to approx 80°C. At this point, the cycler 1s stopped, the tubes are opened, and the rmssmg component(s) are added to each tube. The idea behmd this is to add the enzyme at a higher temperature, thus reducing the chances of pre-PCR mispriming from occurrmg When working with multiple tubes, this becomes quite cumbersome and also greatly increases the risk of crosscontammation among tubes. Each method, or variation of these methods, requires either extensive manipulations or the addition of some addItiona component(s), which mcreases the reaction cost. 32 AmpliWax PCR Gem 50. Developed by PE Applied Biosystems, this method involves the physical separation of PCR reactants m the tube usmg AmpliWax PCR Gem 50, a small wax bead. A subset of the reactants (typically, all components except AmplzZ’uq DNA Polymerase and buffer) are placed in the PCR tubes. The AmpliWax PCR Gem 50 1s placed on top of this “lower reaction mix.” The tubes are heated to 80°C melting the wax bead. The tubes are allowed to cool for several minutes, creating a wax barrier over the “lower reaction mix.” The PCR tubes are opened and the withheld components are then added on top of the wax barrier-“upper reaction rnlx.” The PCR thermal cycler parameters are then employed. When the tubes attam a high temperature, c8O”C, the wax melts and the “upper reaction” falls mto the “lower reaction” to combine all of the PCR reactants and the PCR commences Although more efficient than manual methods, this method 1s not suited for high throughput applications. This method can be cumbersome and mcreases the cost per reaction. 33. Tuq antibodIes. Developed by Clontech (Palo Alto, CA), this method mvolves the ad&ion of a Tuq antlbody to the PCR reaction In theory, the antibody will bind to the Tuq, rendermg it inactive. As the temperature of the reaction is raised during the PCR, the antibody will become inactive, thus allowing the enzyme to begin its polymerase activity. This method appears to work well with some apphcations, but the addItiona cost involved may make it prolnbltive for higher throughput apphcations. In addition, this method mvolves additional chemistries within the PCR reaction that may not be 100% efflclent. 34. AmpZiTuq Gold DNA Polymerase is a novel, highly efficient method that has been developed to elimmate nonspecific amplifications and primer-chmer formation and is becommg the method of choice for many people concerned with the efficiency and specificity of their PCR reactions It 1s a thermally activated enzyme that, prior to thermal actlvatlon, has no enzymatic activity. Thus, AmplzTuq Gold DNA Polymerase can be added along with all PCR reagents into a single tube. Because it has no activity before thermal activation, there 1s no prePCR misprimmg The enzyme is activated by programming the thermocycler to carry out a pre-PCR heat actlvatlon step (10 mm at 95°C). During this lo-mm activation step, the enzyme becomes active This method results m the virtual elimmation of both nonspecific ampllflcation and primer-dlmer formatlon Because there are no competing side reactions, the amount of desired product 1s greatly increased In addition, because there 1s no reagent competition within the

Introduction to PCR reaction, which can become hmttmg m traditional PCR reactrons, the number of cycles can be increased. This 1svery valuable when trymg to amplify a low-copynumber target. In very specific assays, such as quantitative PCR, sequencmg of PCR products, allelm-dtscrrmmation assays, and multiplex reactions, where the quality and purity of the PCR product is critical, AmpZiTuq Gold DNA Polymerase IS proving to be a highly versatile approach to obtaming high performance PCR In Fig. 2, lanes 3, 5, and 7 reflect the amplification of 2260-, 1940-, and 1620-bp fragments, respectively, of fibronectm m the presence of AmplzTaq Gold DNA Polymerase (note the absence of any nonspecific products).

4.9. AmpliTaq Gold DNA Polymerase and Low Copy Number Amplification 35 When working with low-copy-number DNA, there are special constderattons that need to be taken mto account m order to successfully amplify this low-copy target It IS important that the primers be desrgned carefully, to avoid nonspectfrc amphftcatton that may result m a primer bmdmg to other sites on the template Much nonspecific amplification can be eliminated through the use of AmpliTaq Gold DNA Polymerase Because there are few, if any, PCR side-reactions occurring, low-number ampliftcatton is more easily facilitated with this enzyme Traditionally, nested PCR was the method of choice for low-copy amphftcation. In this method, two separate PCR reactions are carried out. The first reaction involves a set of prtmers that amplify a larger target that contains the desired target. After this first amphftcatton, part of the product mtx is placed mto a second PCR mrx wrth another set of primers that flanks the desrred low copy target. Through two separate rounds of PCR, the low target amphcon is thus amphfted. Nested PCR was necessary because too many nonspecific products would normally be amplified and there would be little, d any, detectable desrred product. With AmpliTaq Gold DNA Polymerase, however, nonspecific amplification 1sno longer a concern Thus, low copy number amplification is unencumbered. Also, because there 1s no background, which traditionally limited the number of cycles possible, the number of cycles that can be run under AmpZiTaq Gold condtttons can be increased. This allows for the target numbers to be amplified to detectable levels much more efftctently than through tradttional means. AmpEiTaq Gold DNA Polymerase IS activated through an mtttal lo-mm mcubation/actrvation step at 95°C prror to commencement of the cyclmg parameters This step activates nearly all of the enzyme at one time Thus, when cyclmg begins many enzyme molecules are available for extension. However, some believe that the PCR reaction may be more efficient, particularly with low copy number, tf less active enzyme 1s available during the first few cycles. After the number of amplicons has been increased a higher level of enzyme activity is more favorable. When using AmpEzTuq Gold DNA Polymerase under low target conditions this “time-release” method of activation is possible. To perform “ttmerelease” PCR the reaction mix, wtth AmplzTaq Gold DNA Polymerase present, 1s not actrvated prior to PCR Instead, the enzyme IS allowed to activate durmg

22

Miller and Cunningham cyclmg. Each time the reaction reaches 95°C (denaturmg step) the enzyme is activated a bit more. Thus, as the reaction proceeds, increasing amounts of enzyme become active After 20-25 cycles, the enzyme would then be fully active. To compensate for this type of activation rt would then be necessary to increase the number of cycles by 20-25 cycles Using this method a 50-cycle protocol would be appropriate Some have performed a combmation of activation strategies: 5-mm inmal activation and Increased cycle number and have achteved excellent results. Whichever strategy is taken, AmpliTuq Gold DNA Polymerase has proven to be a superior enzyme when trymg to amplify low target numbers (16).

4.10. Contamination

Control

36 The PCR process generates mtlhons of copies of DNA As a result, contammanon of subsequent experiments can lead to false-positive results False positives can also result from crosscontammatton between samples and from Improper sample handling during PCR set-up. When settmg up a laboratory to perform PCR it is important to consider ways to mimmize cross-sample contammatton and PCR product carryover (I 7-20) a Physically separate the pre-PCR reagents and set up procedures from the PCR area and post-PCR analysis area b Separate pipets and other PCR devices be dedicated for both pre- and postPCR processes Items used in the post-PCR area should not be brought into the pre-PCR area, and vice versa. c. Pipets can be a convenient source of crosscontammation from previous experiments. To elimmate carry-over contammation use either postttve displacement pipets or filtered tips. d. Reagents should be stored as altquots to minimize reagent usage and samplmg. e. Change gloves frequently f Uncap tubes carefully to prevent aerosols. g. Mimmtze sample handling h. Add master mix prtor to addition of the DNA In addition to the physical and procedural precautions taken to mimmtze or prevent carryover between PCR reacttons, consider the use of an enzymatic system referred to as the UNG system. UNG, uracil-N-glycosylase, is an enzyme that cleaves DNA fragments containing uracrl. To utthze thts method, all PCR reactions are carried out m the presence of dUTP, instead of dTTP Therefore, all PCR products m a lab will contain uracil instead of thymidme. In preparing PCR reagents, UNG and dUTP are added to the master mix along with all of the other PCR components. Prior to PCR cycling, the thermal cycler IS set at 37-50°C for l-10 mm (dependmg on the level of UNG activity) During this mcubation period, UNG will cleave any DNA contammg uracil that may have been carried over from a previous reaction A lo-min mcubation at 95’C is then set to allow for mactivatton of UNG. Thus, durmg the subsequent PCR amphfication, all new

Introduction to PCR

23

1SubsequentPCR 1 reactions carried out in the presence I

Fig. 3. Diagrammatic

of dUTP and UNG

I

PCR products contammg dU

representation of the UNG system.

products generated will contam uractl. If AmpZzTuqGold DNA Polymerase is used, then this step also 1sused as the enzyme activation step (see Fig. 3). All PE Applied Biosystems PCR ktts are UNG compatible; thus, its inclusion in protocols is easrly facrhtated The UNG system 1s also used by some as a hot start method, since any pre-PCR products generated would be cleaved during the UNG mactivation step To ensure complete cleavage, tt is recommended that followmg the final cycle the samples are held at 72°C until they are removed from the Instrument. The reactions are than stored immediately at -2O”C, or quickly treated chemrcally with an equal volume of chloroform, to denature any remaining UNG acttvtty. This should prevent any degradation of dU-containing product by residual or reactivated UNG.

Acknowledgments The GeneAmp PCR Process is covered by U.S. patent nos. 4,683,202; 4,683,195; 4,800,159; and 4,965,188 owned by Hoffman-La Roche Inc. and other patents pending and issued patents in non-US countries owned by F. Hoffman-La Roche, Ltd., all of which are licensed for certain uses to The Perkin-Elmer Corporation. Tuq DNA polymerase and AmpZiTaq DNA Polymerase are covered by US patent nos. 4,889,818; 5,075,216; and 5,079,352 owned by Hoffman-La Roche, Inc. and other patents pending and issued patents in non-US countries owned by F. Hoffman-La Roche, Ltd., all of which

24


are licensed for certain uses to The Perkin-Elmer Corporation. PE Applied Blosystems and Primer Express are trademarks and Perkin-Elmer@ and MicroAmp@ are registered trademarks of The Perkin-Elmer Corporation AmpliTaq GolflM is a trademark and AmpliTaq@, AmphWax@, GeneAmp@, and UlTma@ are registered trademarks of Roche Molecular Systems, Inc. References 1. Higucht, R. (1989) Prtnciples

and Appltcatzons for DNA Ampltftcatton.

Stockton

Press,London, UK, pp. 3l-38 2 Inms, M. A , Gelfand, D H , Snmsky, J. J , and White, T. J., eds (1990) PCR Protocols. A Gutde to Methods and Applicattons Academic, San Diego, CA 3 Mulhs, K. S. and Faloona, F A (1987) Methods Enzymology 155,335-350. 4. Satkt, R. K , et al (1985) Enzymatic amplification of P-globm genomtc sequences and restriction sate analysis for diagnoses of sickle cell anenua Sctence 230, 1350-1354. 5. Fretfelder, D , ed. (1982) Phystcal Btochemtstry. Appltcations to Btochemtstry & Molecular Btology. Freeman, CA, pp 494-536 6. Kosel, S. and Graeber, M B. (1994) Use of neuropathologtcal tissue for molecular genetic studies parameters affecting DNA extraction and polymerase chain reaction. Acta Neuropathol. 88, 19-25. 7. Saldanha, J , et al. (1984) An improved method for preparing DNA from human bram J. Neurosct. Methods 11,275-279. 8. Savioz, J , et al. (1993) A method for the extraction of genormc DNA from human bram tissue fmed and stored in formalin for many years. Acta Neuropathol. 93,408-4 13. 9 Lawyer, F. C., et al. (1989) Isolatton, charactertzatron, and expression of Eschertchia coli of the DNA polymerase gene from Thermus aquattcus J Btol Chem

264,6427-6437. 10. PE Applied Biosystems PCR Catalog. PE Applied Btosystems, Foster City, CA. 11 Dieffenbach, C , et al. (1993) Setting up a PCR laboratory. PCR Methods Applzcations 3, S2-S7 12 GeneAmp@ PCR Reagent Ku Protocol. PE Apphed Btosystems, Foster Ctty, CA 13. Abramson, R. D. and Myers, T W. (1993) Nuclerc acid ampltficatton technologles Curr. Opm. Biotechnol. 4,41-47. 14. Rychhk, W , et al. (1990) Optimtzatton of the annealing temperature for DNA amphftcation in vitro Nucleic Aczds Res. 18,6409-6412. 15. Kebelman, C., et al (1998) Advantages of a new Taq DNA polymerase m multiplex PCR and time-release PCR Btotechntques 24, 154-l 58. 16 Mothk, J., et al (1998) Automated reportmg of RNA differential display patterns from pig granulosa cells. Biotechnzques 24, 148-153 17 Cone, R. W. and Fairfax, M R. (1993) Protocol of ultraviolet irradiation of surfaces to reduce PCR contammation PCR Methods Applzcations 3, S 15-S 17 18 Sambrook, J , et al. (1989) Molecular Clonmg, 2nd ed. Cold Sprmg Harbor Laboratory, Cold Spring Harbor, NY. 19. Dieffenbach, C., et al. (1993) General concepts of PCR design. PCR Methods Applicattons 3, S30-S37

Introduction to PCR

25

20. Zimmerman, K. and Mannhalter, J. (1998) Comparison sensitivity and specificity of nested PCR and single-stage PCR using a thermally activated DNA polymerase. Biotechniques

24,222-224

2 1. Salki, R. K., et al (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA Polymerase Science 239,487-49 1 22 Hartley, J A , et al. (1993) Handling reagents in the PCR laboratory. PCR Methods Applications 3, S 10-S 14

Methodological in Polymerase Alessandra

Issues Chain Reaction for RNA

D. Rasmussen

1. Introduction In order to understand the functionality of genes and the importance of their expression, it is important to be able to understand proteins and, therefore, amino acids. Many techniques allow one to look at proteins at the amino acid level; however, very few allow researchers to understand them at the nucleic acid level. It is obvious that m order to study proteins and amino acids at the nucleic acid level, one would need to look at RNA. Unfortunately, because of the nature of RNA (its single-stranded nature) it is a very labile substrate and difficult to study, especially if the RNA of interest is present in cells at a low abundance. 2. RNA Detection Methods Several methods have been developed and described for studying specific RNA molecules. These include in situ hybridization, Northern blots, dot blots, and nuclease protection assays. Of these, in situ hybridization (Table 1) is probably the most sensitive method and allows for the detection of lo-100 molecules in a single cell; the other techniques require at least 0.1-1.0 pg of the specific mRNA, which is equivalent to 105-lo6 molecules, m order to obtain results. Except for in situ hybridization, the lack of sensitivity with standard RNA detection techniques has traditionally been a drawback in investigating low abundant or rare transcripts. The application of the polymerase chain reaction (PCR) to the study of gene expression, therefore, represents an important technical innovation. From

Methods Edlted

m Molecular by

J Harry

Medrone, and

vol 22 Neurodegeneratron

H A Tilson

0 Humana

27

Press

Methods Inc , Totowa,

and Protocols NJ

Rasmussen

28 Table 1 RNA Detection

and Mapping

Method

Methods Sensitivity

Northern blot analysis Dot/slot blot analysrs Primer extension Nuclease S 1 analysis RNase protectron assay In sztu hybridrzatron RNA PCR RNA m mu PCR

105-lo6 molecules

lo-100 molecules l-10 molecules “

3. RNA PCR RNA PCR (or RT-PCR) has proven more sensitive and discrrmmating than Northern

blot analysis,

dot blot, nuclease protection

assay, and in sztu

hybrid-izatron (2). Because of its lability, RNA cannot serve as a direct template for PCR. The RNA must first be converted mto a complementary DNA (cDNA) using a reverse transcriptase (Fig. 1). This converted cDNA is then amenable to PCR. RNA PCR is a rapid and easy-to-handle technique and allows simultaneous analysrs of several transcripts from total RNA. It can also be used for relative and absolute quantification of mRNAs (34 and gene expression studies. This technique

is very powerful

in detecting

transcripts

that have a

low copy number or low rate of transcription and m detecting transcripts from a small number of cells or a small amount of tissue. It is also suitable for distinguishing between closely related transcripts independently of then abundance. Although the threshold of sensitivity of RNA PCR has been reported as being as low as 10 transcripts of a synthetic RNA (I), this threshold can vary considerably depending on a number of factors. The more important of these factors include: yield of RNA from extraction method, prevalence of RNA within cell, DNA contamination of RNA preparation, efficiency of reverse transcription, RNase H activity, and finally PCR amplification efficiency and reproducibility The total yield of RNA can vary from between 20 and 70%, making RNA preparation a critical issue. Various RNA extraction methods provide either

more or less pure nucleic acid. Guamdinium tsothyonate extractions provide among the highest purity RNAs, but this method is also the most time-consuming and cumbersome. Crude extractions (i.e., boiling methods, protease diges-

Polymerase Chain Reaction for RNA

29

mRNA

Synthesis cDNA

of first

with

reverse

PCR

components

Second wlth

Amplify

strand AmpllTaq

cDNA

strand

transcrlptase

added; cDNA DNA

synthesized Polymerare

cDNA

3’

5 5’

D

3’

3’

*

5’

5’

D

3’

Fig 1 RNA conversion

to cDNA

tion) are less time-consummg but provide less pure nucleic acid. The starting RNA material can also influence yield of RNA extracted. Different types of RNA are naturally found within the cell at different percentages and depending on the type of RNA isolated, more or less starting material may be needed (Table 2). Varying amounts of DNA can contaminate RNA preparations depending on the method used to isolate the RNA. If primers for PCR amplification are not designed properly (spanning an intron, for example), contaminating DNA will also be amplified and the RNA amplification will be indistinguishable from the DNA amplification. The efficiency of reverse transcription can also vary between 5 and 90% (Table 3). The enzyme chosen for reverse transcription may play a role in amplification efficiency depending on the specific RNA PCR application. Many reverse transcriptasesalso have RNase H activity associatedwith them This endonucleolytic activity cleaves RNA:cDNA hybrids and can lead to the degradation of template RNA. Finally, after the RNA has been reverse transcribed, the PCR may also add a degree of variability. RNA PCR allows one to overcome many of these limitations. The sensitivity of the PCR ampllftcations negates the need for a lot of starting mate-

30

Rasmussen Table 2 Types of RNAs and Their Natural Cellular RNAs 80-85% lo-15% l-5% , and add 20 pL (2 pg/pL) of strepavidm. 8. Repeat mcubation with strepavidm and extraction with phenol:chloroform* tsoamyl alcohol a total of four times. 9 After final phenol:chloroform:isoamyl alcohol extraction transfer aqueous layer to a new 1.5-mL tube and extract with 300 PL of chloroform, centrifuge for 5 mm, transfer aqueous phase to a new 1S-mL tube. 10. Add 25 pL of 3 M ammonmm acetate and 813 pL of ethanol Pellet cDNA by centrifugation at 15,000 rpm for 15 mm. 11. Wash the cDNA pellet with 70% ethanol, au dry, and resuspend m 11.5 yL of TE, pH 8.0. 12. Transfer the 11.5 pL of cDNA to a 0.5-mL Eppendorf tube, add 7.5 ltL (50 p.g) of biotmylated drover cDNA, 1 pL tRNA (1 kg) and heat at 100°C for 3 min. 13 Add 20 I.LL prewarmed 2X hybridization buffer and overlay with 1 drop of paraffm oil. Set short hybrtdizatton at 68’C for 2 h. 14. After hybrrdrzatron add 260 pL HEPES buffer (10 mM HEPES, 1 mM EDTA, pH 7.0) and heat at 55°C for 5 min. 15. Centrrfuge briefly. Transfer aqueous bottom layer to a new 1.5-mL Eppendorf tube. 16 Add 20 pL streptavrdm, incubate 20 mm, and phenol chloroform*rsoamyl alcohol extract as previously descrtbed. 17. Repeat a total of 4X exactly as described after long hybridization 18. After the last phenol:chloroform:isoamyl alcohol extraction transfer aqueous layer to a new tube, extract with 300 pL of chloroform, centrifuge for 5 mm, transfer aqueous phase to a new 1.5-mL tube 19 Precipitate with 25 pL of 3 M ammonium acetate and 8 13 pL of ethanol, centrifuge at 15,000 rpm for 15 mm. 20 Wash cDNA pellet with 70% ethanol, resuspend pellet m 5 pL of TE, pH 8 0

3.4. Preparation

of the Vector

1 Digest 10 mg of PCRII plasmid (Invnrogen) with &XI at 45°C for 3 h (see Note 6). 2. Add Not1 and incubate at 37’C overnight 3. The digest is run on 1% low-melting agarose gel and the 3.9-kb fragment 1s extracted, and purified with GeneClean (Bio 101) as described by the manufacturer 4. The fragment is dephosphorylated with 1 l.rL of calf intestinal phosphatase and 5 pL of 10X CIP buffer m a 50 pL reaction at 37°C for 15 mm 5. The enzyme 1s Inactivated by adding 1 rnM EDTA, and heating at 70°C for 10 min. 6 Load sample on 1% low-melting gel and purify the 3.9-kb fragment with GeneClean (Bra 101) as described by the manufacturer.

41

Subtractive Hybridization 3.5. Ligation

1. Ligate 5 FL of subtracted cDNA to 250 ng gel-purified PCRII (BstXIINotIICIP treated) with 1 yL of 10X ligation buffer and 1 PL T4 DNA ligase (400 U/10 pL) in a lo-pL volume. Incubate at 16°C overnight. 2. Precipitate ligation with 1 PL (1 pg) tRNA, 3 M ammomum acetate, and 100% ethanol. Centrifuge for 15 mm at 15,000 rpm. 3 Wash the pellet with 70% ethanol Resuspend the cDNA in 4 FL of water 4 Transform 2 FL ligated cDNA into 50 PL of Electromax DHlOB cells (Glbco-BRL) by electroporation (as specified by manufacturer). Culture transformed cells at 37°C for 1 h m 1 mL of LB media without antlblotics. 5. Dilute transformation 1’ 10 and plate 1, 10, and 100 yL on LB agar kanamycm plates (150 x 15 mm) by spreading technique. 6. Inoculate remaining transformation in a 1-L culture for freezing several 1 mL 30% glycerol culture stocks and generating a plasmid preparation

3.6. Evaluating

Subtracted

cDNA Library

1 Count colonies on overnight LB plates. 2. Multiply dllutlon factors to obtain actual number of independently derived clones. This IS the cDNA library size (see Note 7). 3. Three micrograms of the driver, tracer, and subtracted cDNA library plasmld preps should be digested with restriction enzymes that will liberate the inserts. 4. Run the digests on a 1% agarose gel with I-kb DNA markers, stain with ethidium bromide, and photograph (see Note 8).

3.7. Analysis

of Subtracted

cDNA Clones

1. Dilute 100 FL, of the 1 mL 30% glycerol culture stocks, mto 900 PL LB (1.10). Make nine 1: 10 serial dllutlons. 2. Plate 100 FL of dilutions 4,5,6,7, and 8 on LB kanamycm plates (150 x 15 mm). Incubate overnight at 37°C. 3. Choose dilution plate that has well-isolated colonies. Pick colonies for mmlpreps 4. Culture, and prepare mmlpreps by any standard technique (see Note 9). 5. Digest a sample of each plasmld prep with BstXI and Nut1 (or whichever enzyme releases the inserts in the vector used). Usually 1 pg in a 20 pL reaction is enough to visualize on an agarose gel. 6. Run restriction digest on 1.5% agarose gel, stain with ethldmm bromide, and photograph. 7 Sequence plasmld preps from clones that indicate the presence of inserts based on the gel of the digests (see Note 10).

4. Notes 1. If the starting cDNA libraries (1 e , driver and tracer) are m a vector that does not have BstXI and Not1 sites flanking the inserts, then use an appropriate rare cutting restrlctlon enzyme that will liberate the inserts. If possible, try to maintain

42

2. 3

4. 5.

6.

7

8.

Kelner and Maki cloning dlrectlonahty from five pnme to three prime for easier determination of the sequenced subtracted cDNA products onentatlon. If using different restnctlon enzymes than outlined m this procedure, then make certain to prepare the PCRII vector, which will be used for final hgatlon of the subtracted cDNA library, with compatible restriction enzymes to allow for ligation. The importance of the ScaI digestion 1s to destroy the driver vector, which will help to prevent its possible carryover and subsequent ligation into the final subtracted cDNA library. The solution after exposure to the lamp should have changed from an orange to a burnt-orange color. The addition of Tris, pH 9.5, is required to cause the excess photoprobe blotin to become soluble m the upper n-butanol phase Omitting this pH adJustment will cause all of the excess blotm to remam in the lower aqueous phase with the biotmylated cDNA. Do not carryover the thm upper 011layer. After the first centnfugatlon, there will be a white preclpltate at the interface of the organic and aqueous layer. This preclpltate contains the streptavidin-blotm complex With each subsequent extractlon, the amount of the precipitate ~111 dlmmlsh. Do not transfer material from the white preclpltate when transferring the upper aqueous phase to a new tube The upper aqueous phase contains the cDNA that did not hybridize to the photoblotmylated driver and therefore 1s enrlched for sequences that are unique to the tracer cDNA As previously mentloned m Note 1, this vector can be prepared using different restrlctlon sites to accommodate the inserts orlgmally digested from the tracer cDNA library Other vectors can be used. However, when choosing another vector, select one that has a different antlblotic resistance than the vector that the driver and tracer cDNA hbrarles contain. For example, if the driver and tracer vector are amplclllm resistant, then use a vector that 1s kanamycm reslstant for hgatlon of the subtracted cDNA library This eliminates the potentially high background of driver, and tracer-derived vector-containing clones. Also, it 1s important to select a vector in which the sites flanking the msert have convenient primer sites for sequencing (such as SP6, T7, T3, M13, and so on) In a subtracted cDNA library there should be far fewer clones than in the unsubtracted cDNA library. Where an unsubtracted cDNA library will have mlllions of clones, a subtracted cDNA library will have tens of thousands to a few hundred thousand clones, this 1s called the cDNA library size (no relation to the size of the inserts in the cDNA library) For example, if the plate that received 1 pL of the 1:lO dilution has 30 colomes, then the hbrary size will be 300,000 clones (30 colonies x 10 for the 1.10 dilution from the original transformation culture x 1000 for l/1000 of the dilution plated, or 1 pL of 1000 FL). The 10 mL and 100 mL plates m this example should have approx 300 and 3000 colonies, respectively Three dilutions are plated m order to obtam a plate that has a number of colonies which can be counted The profile of a successfully subtracted library 1s seen m Fig. 1. Both the driver and tracer cDNA library inserts appear as a smear ranging m size from 500 to

Subtractive Hybridiration

43

Fig. I. Evaluation of subtracted cDNA library. Plasmid preps from each cDNA library was digested with BstXI and Not1 to liberate the cDNA inserts. The restriction digest was loaded on a 1% agarose gel. Astrocyte cDNA lane 1, 12-h LPS activated astrocyte cDNA lane 2, astrocyte-subtracted 12-h LPS-activated astrocyte cDNA lane 3. Lanes 1 and 2 depict a complex mixture of cDNA inserts, and lane 3 demonstrates the enrichment of astrocyte-activation-specific cDNAs and an elimination of cDNAs common to the activated and unactivated astroyctes. 3000 base pairs. However, the subtracted cDNA library (which is the tracer cDNA library minus the genes in the driver cDNA library that were homologous) appears as a range of very discrete bands. In general, each band represents a gene that is unique to or upregulated in the tracer cDNA library as compared to the driver cDNA library. 9. There are many miniprep plasmid systems available. One such system is the Qiagen 96-well plasmid prep system (Chatsworth, CA). We have found that the 96-well format works well for larger scale sequencing, such as that performed on an automated sequencer. Any system that yields enough plasmid prep cDNA with quality that can be sequenced is acceptable. 10. The gel analysis of individual clones prior to sequencing provides information on the insert sizes and also eliminates the unnecessary sequencing of “empty” vectors which may be as high as 10% of the clones. Figure 2 represents a gel analysis of 96 clones from a subtracted cDNA library. Most lanes indicate the presence

44

Kelner

and Maki

Fig. 2. Analysis of subtracted cDNA clones. Plasmid minipreps from 96 individual clones of astrocyte subtracted 12-h LPS-activated astrocyte cDNA were digested with BstXI and Not1 were loaded on a 1.5% agarose gel. Molecular weight markers are in lanes M. of inserts, the vector is 3000 bp and appears in all lanes. The lanes that have only the 3000 bp band do not have inserts and are not sequenced.

Reference 1. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

4 The Use of Differential Display RT-PCR for Identifying Altered Gene Expression in lschemic Brain Injury Xinkang

Wang and Giora Z. Feuerstein

1. Introduction

Differential gene expression is essential for normal development, and a variety of pathophysiological conditions (including neurodegeneration, neurotrauma, and tschemtc injury) of the central nervous system.Vartous numbers of mRNAs are expressed m a given cell at any time point, and changes m relative mRNA levels may have important implications m the development of pathophysiologtcal processes. Therefore, elucidation of the differentially expressed genes is critical for understanding of the molecular mechanisms involved in normal and pathological states, as well as provrdmg new insights on molecular targets for pharmacological manipulation and drug development. Focal brain ischemia represents a pathophysiologic condition that modulates gene expression and functions in the brain. A number of genes have been identified for their altered expression after focal stroke, which include immediate-early-response genes (peak induction at l-3 h), such as c-fos and ztf268 (I), Intermediate-response genes (peak induction at 12 h), such as TNF-a and IL-lp (2,3), and delayed-response genes (induced after 2 d), such as TGF-P and IL-l receptor II (4,5). However, the overall molecular mechanisms of rschemic bram injury are not well understood. One way to better understand the molecular mechamsms associated with focal ischemic injury is to identify and characterize genes with altered expression specifically m ischemia. The polymerase chain reaction (PCR)-based mRNA differential display (6) is an mcreasmgly popular technique for isolating genes of interest m a variety of m vitro and in vivo systems (for review, see ref. 7) compared to the convenFrom

Methods m Molecular Medmne, vol 22 Neurodegensration Methods and Protocols Edited by J Harry and H A Tllson 0 Humana Press Inc , Totowa, NJ

45

46

Wang and Feuerstein

tional techmques such as differential screening and subtractive hybridization. The method of mRNA differential display consists of two basic steps: reverse transcription (RT) using a set of 3’-anchored primers, T,,M (M = G, A, or C), and PCR amplification of cDNA fragments using arbitrary primers (upstream) and anchored downstream primers. In this chapter, we describe the use of mRNA differential display for discovery of ischemia-induced gene expression. This technology can also be exploited for diverse manipulations of tissues and cells, and therefore, must be viewed asa highly versatile and adaptable technology. 2. Materials 2.7. RNA isolation 1 Lysis buffer (solution D)* 4 A4 guamdmium thiocyanate, 25 mJ4 sodium citrate, pH 7.0,0.5% sarcosyl. This solution can be stored at room temperature for up to a year. 0.1 A4 P-mercaptoethanol is added before use (which can be stored at room temperature for a few months) 2. Water-saturated phenol (nucleic acid grade). Stored at 4°C 3. Chloroform, rsoamyl alcohol, and isopropanol Stored at room temperature. 4. 70% ethanol. Store at -20°C. 5. 1.67 M sodium acetate, pH 4.0 Stored at room temperature

2.2. Differential

Display RT-PCR

1. RNA samples. Stored at -7O’C. 2. RNAmap kit (GenHunter, Nashville, TN). Stored at -20°C 3. Human placental ribonuclease mhrbitor (Boehrmger Mannhelm, Indranapolts, IN). Stored at -2O’C. 4. 10X PCR buffer. 100 mM Tris-HCl, pH 8.4, 500 mM KCl, 15 rniV MgCl*, and 0 01% gelatin. Stored at -20°C. 5. AmpZiTuq DNA polymerase (5 U/L, Perkin-Elmer, Norwalk, CT). Stored at -20°C. 6 [33P]-dATP (1000-3000 Ci/mmol, Amersham, Arlmgton Heights, IL). 7. 6% polyacrylamide/8 M urea sequencing gel.

2.3. Preparation of Differential 1. 6% polyacrylamtde gel.

Display PCR Products

2. DNA extraction solution (for acrylamide gel)* 500 mM NH,Ac, Stored at room temperature

2.4. Northern

1 mJ4 EDTA,

Analysis

1 RNA samples 2. GeneScreen Plus membrane (DuPont-New England Nuclear, Boston, MA) 3. Northern hybrtdizatton buffer 5X SSPE(750 mM MaCl, 50 m&Z NaH,PO+ pH 7.6, 5 mM EDTA), 50% deionized formamide (stored at -7O”C), 5X Denhardt’s solution (50X stock solution stored at 4”C), 2% SDS, 100 pg/mL polyA and 200 pg/mL boiled salmon sperm DNA. Freshly make the solutton prior to use

Differential Display

47

4. Random-priming DNA-labeling kit (Boehringer Mannhelm) 5. [32P]-dATP (3000 Cl/mmol, Amersham). 6 Washmg solution. 2X SSPE, 2% SDS Stored at room temperature.

2.5. DNA Sequencing

Analysis

1 Double-strand-DNA cycle sequencing system (Gibco-BRL, Gaithersburg, MD). Stored at -20°C. 2. [32P]-dATP (5000 Cl/mmol, Amersham). 3. PNK: T4 polynucleotlde kinase at stock concentration of 10 U: 1. Stored at -20°C. 10X PNK Buffer 700 mM Tris-HCl, pH 7.6, 100 mM MgC12, 50 mM dithlothreltol (DDT). Stored at -2O’C. 4. Blo-Spm 6 column (Bio-Rad, Hercules, CA). 5. 6% polyacrylamide/8 M urea sequencing gel. 6 pCRI1 vector (Invltrogen, San Diego, CA).

2.6. Computer

Database Search

1 Computer network to access genomlc databases, e g., GenBank.

3. Methods 3.1. RNA Preparation 1. Generate focal brain lschemla in rat by permanent occlusion of the middle cerebral artery (MCAO) as described previously (8). Dissect ipsilateral (lschemic) cortex and contralateral (nonischemic) cortex samples and freeze immediately m liquid nitrogen and then store at -80°C. 2. Homogenize the tissue sample m the lysls buffer (3.6 mL per cortical sample) m a 14 mL Falcon tube (Becton Dickinson, Rutherford, NJ) with a polytron at a medium speed setting for approx 10 s, and place on ice. 3 Add 448 yL of sodium acetate, pH 4.0,3.7 mL of water-saturated phenol, 732 FL of chloroform/lsoamyl alcohol (49.1) sequentially. Vortex the solution and place on Ice for 10 mm. 4 Centrifuge the samples at 10,OOOgfor 20 min at 4°C. Carefully remove the aqueous phase and transfer mto a fresh tube 5. Add equal volume of isopropanol and store at -20°C overnight. 6 Precipitate RNA by centrlfugatlon at 10,OOOg for 20 mm at 4”C, and then redissolve the RNA pellet m 0 5 mL lysis buffer, transfer into an Eppendorf tube, mix with equal volume of isopropanol, and place at 20°C for 2 h to overnight. 7. Preclpltate RNA by centrlfugatlon at 4°C for 10 mm in a mlcrocentnfuge, wash with 75% ethanol (centrifuge for 2 min), dry m a speed vacuum briefly, and then dissolve m 100 PL RNase-free water (see Note 1). 8. Measure the RNA concentration and store at -70°C.

3.2. Differential

Display RT-PCR (see Note 2)

1. Perform reverse transcription reactions in a volume of 20 PL, containing 0.2 yg total cellular RNA (e g., isolated from ischemic or nonischemlc cortical samples),

48

Wang and Feuerstein

2 FL of 2 PM HTA (H = AAGC, entended based for the HzndIII site) primers, 4 FL of 5X RT buffer (125 mM Tns-HCl, pH 8 3, 188 r&4 KCl, 7 5 mJ4 MgGl and 25 mM DTT), 1.6 pL of 250 yM dNTP, 0 5 FL RNase inhibitor, and 1 yL Maloney murme leukemia virus (MMLV) reverse transcnptase. Heat RNA at 65°C for 5 mm and place on ice prior to the addltlon of enzyme and RNase inhibitor. Incubate the RT reaction mixture at 37°C for 60 mm, and then heat at 95°C for 5 min and place on ice for PCR, or store at -2O’C for later use 2. Prepare PCR reactlon mixture containing 2 PL of 2 @! 5’ arbitrary primer, H-AP75 (5’-AAGCTTTTATTCG-3’) and 3’ HTA primer, 0.25 FL [PI-a-dATP, 2 pL 10X PCR buffer, 1.6 I.LL dNTP (25 mM), 2 pL RT products, and 0 2 mL AmpliTaq DNA polymerase (5 U/mL). Perform PCR for 40 cycles as follows: 94°C 30 s for denaturmg, 40°C 2 mm for annealing, 72°C 30 s for extension, followed by one cycle for extension at 70°C for 10 mm 3. Resolve the PCR products through an 8 M urea, 6% polyacrylamlde DNA sequencing gel and analyze the gene expression by autoradlography (see Fig. 1)

3.3. Preparation

of Differential

Display PCR Products

1 Localize the band of interest, cut out the band with a razor, release the DNA by boiling m 100 PL TE (10 n-&! Tns-HCl, pH 8.0, 1 mM EDTA) for 10 mm, and precipitate with 80% ethanol plus 5 mL glycogen (10 mg/mL) for 30 mm (to overnight) at -70°C. 2. Microcentrifuge for 10 min at 4°C to pellet DNA, wash the pellet with 80% Icecold ethanol by mlcrocentrlfuge at 4°C for 1-2 min Dry the pellet with speed vacuum and dissolve in 10 FL water. 3 Reamphfy the DNA using 4 pL resuspended sample, 4 FL of the same prtmers as for the primary amplification, and 3 2 PL dNTP (250 cLM>m a final volume of 40 FL All other condltlons are essentially the same for the initial amplification. 4. Resolve DNA bands on a 6% polyacrylamlde gel (see Note 3), stain the gel with ethidmm bromide. 5. Cut out the DNA band under long UV wavelength, mince the gel slice mto small pieces, and extract the DNA in 3 ~01500 pJ4 NHAc, 1 mM EDTA overnight at 37°C 6. Mlcrocentrifuge the samples for 3 mm at room temperature, collect the supernatant (repeat several times to remove the gel debris if necessary), extract the supernatant once with phenol/chloroform, and preclpltate with ethanol Resuspend in lo-20 pL TE and store at -20°C. The DNA 1snow ready for either direct sequencmg or probe labeling.

3.4. Northern

Analysis (see Note 4)

1. Resolve RNA samples (10 pg per lane) m a formaldehyde-agarose slab gel and transfer to a Gene Screen Plus membrane (DuPont). 2 Prehybridlze the membrane for 4 h, add 106 cpm/mL probe for overnight hybndlzation at 42°C 3 Wash the membrane m 2X SSPE at room temperature for 15 mm, followed by 2X SSPE, and 2% SDS at 65°C for 30 mm 2-3 times depending of signal mtenslty

49

Differential Display 3’ primer: 5’ primer:

IH-AP73

H-T, ,A ‘-1 H-AP76 H-AP75

1234

1234

1234

Fig. 1. Differential display analysis of upregulated gene expression after focal brain ischemia. mRNA differential display was carried out as described in detail in the text. A portion of differential display gel is shown. The 3’ primer used in this study contains the sequence of 5’-AAGCTTTTTTTTTTTA-3’, and the 5’ primers are H-AP73 (5’AAGCTTAGTTATC-3’), H-AP75 (S-AAGCTTTTATTCG-3’), and H-AP76 (5’AAGCTTGTTATAG-3’). The samples loaded in each lane are as follows: lane 1, 2 h ischemic; lane 2, 2 h nonischemic; lane 3,12 h ischemic; lane 4, 12 h nonischemic. Note the band (HSP-70) indicated with an arrow head showing a marked induction in the ischemic cortex was further analyzed. 4. Expose the membrane on X-ray film at -70°C with a Cronex Lightning-Plus intensifying screen (Fisher, Pittsburgh, PA) (see Fig. 2).

3.5. Subcloning

and DNA Sequencing

Analysis (see Note 5)

1. Subclone the PCR-amplified DNA fragment into a pCRI1 vector (Invitrogen) according to the manufacturer’s specification.

Wang and Feuerstein

50 +

*I

* m

-

;

ii

12

Ii

HSP-70

Fig. 2. Northern analysis of HSP-70 mRNA induction in rat cortex following MCAO. Total cellular RNA (20 pg/lane) isolated from rat ischemic and nonischemic cortical samples was resolved by electrophoresis, transferred to a nylon membrane, and hybridized to the differential display band (HSP-70 in Fig. 1) and ribosomal protein L32 (rpL32; loading control) cDNA probes sequentially. The loading order is the same as shown in Fig. 1. 2. Perform DNA sequencing analysis with either T7 or Sp6 primer using a dsDNA cycle-sequencing kit (Gibco-BRL) according to the manufacturer’s specification. 3. Resolve the sequencing reaction mixtures in an 8 M urea, 8% polyacrylamide sequencing gel, and subject to autoradiography.

3.6. Computer

Database Search

Apply Genetice Computer display gene products.

Group (GCG) program to analyze the differential

1. Use SeqEd to edit the sequence (enter at least 50 nucleotides of unambiguous sequence). 2. Use FastA for the search of nucleotide sequence identity (see Note 6) against computer databases, e.g., GenBank.

Differential Dtsplay

51

4. Notes 1. In many cases, we found that this RNA preparation is suitable for the mRNA differential display experiment if one handles every step with cautron. The RNA samples may be further treated with DNase I (Gibco-BRL) in the presence of human placental ribonuclease Inhibitor (Boehringer Mannhelm) for 30 min at 37°C under the conditions according to manufacturer’s specifications followed by phenol/chloroform extractron and ethanol precipitation. 2. In the mrtral protocol (6), short primers (lo-mers arbitrary primers and T12MN anchored primers, M = G, A, or C; N = G, A, T, or C) are used for differential display. Several modifications have been made using elongated primers by addmg extra nucleotides at the S-end of the primers. These extended bases usually include a restriction-enzyme site to facilitate subcloning if needed. The elongated primers can be directly used for differential display (9,IO) or used during the reamplification followmg original differential display method (II). In addition, the elongated primers are able to directly prime the sequencing reaction without subclomng of the differential display PCR products. 3 If DNA fragments are smaller than 500 bases, acrylamide gel electrophoresis is recommended to resolve and prepare the reamphfied products for its advantage to resolve small nucleotides. However, if longer fragments are generated, agarose gel (1.5-2%) electrophoresis is recommended and corresponding methods for DNA isolation should be applied. 4 Northern analysis is a cntrcal step that not only confirms the differential gene expression (see Fig. 2), but may also indicate the purity of DNA since heterogeneous hybrrdizatlon patterns usually indicate the presence of multiple genes. Similarly, other methods such as RNase protection assay, and nuclear run-on analysrs may also be used 5. The reamplified differential display bands could be directly sequenced if an elongated primer is used for either the initial amplification or reamphfication and there are no heterogeneous DNA species present Otherwise, the sequencing analysis could be carried out after subclomng. 6. Because the anchored PCR primers of differential display are located next to the poly(A) tail, the maJority of differential display products correspond to 3’ untranslated region (UTR). Therefore, a computer database search on the nucleotide (not ammo acid) level is recommended.

References 1 Wang, X. K., Yue, T L., Young, P R., Barone, F. C., and Feuerstein, G. Z. (1995) Expression of mterleukin-6, c-fos, and zif268 mRNAs m rat ischemrc cortex. J Cereb Blood Flow Metab 15,166171.

2 Lm, T , Clark, R. K , McDonnel, P. C., Young, P. R., White, R. R., Barone, F. C., and Feuerstem, G Z. (1994) Tumor necrosis factor a expressron in rschemrc neurons Stroke 25, 1481-1488 3 Wang, Z K., Yue, T. L , Barone, F. R , White, R. F., and Feuerstem, G. Z. (1994) Concomitant cortical expression of TNFa and IL-lb mRNAs follows early

52

4.

5

6 7.

Wang and Feuerstein response gene expression in transient focal rschemia. A401 Chem. Neuropathol. 23,103-l 14. Wang, X. K., Yue, T. L , Whrte, R. F., Barone, F. C , and Feuerstein, G. Z (1995) Transforming growth factor-p1 exhibrts delayed gene expression followmg focal cerebral ischemia. Brain Res. Bull. 36,607-609. Wang, X. K., Barone, F. C., Aryar, N V., and Feuerstem, G. Z. (1997) Interleukm1 receptor and receptor antagomst gene expression after focal stroke m rats. Stroke 28,155-162 Lrang, P. and Pardee, A. B (1992) Differential display of eukaryotrc messenger RNA by means of the polymerase cham reaction Sczence 257,967-97 1. Lrang, P. and Pardee, A B. (1995) Recent advances m differential drsplay Curr. Opm. Immunol. 7,274-280.

8 Barone, F. C., Price, W. J , White, R F., Wrllette, R N., and Feuerstem, G Z. (1992) Genetrc hypertension and increased susceptlbdlty to cerebral ischemia. Neurosci.

Btobehav. Rev 16,219-233.

9. Drachenko, L. B., Ledesma, J , Chenchtk, A. A, and Stebert, P. D. (1996) Combmmg the technique of RNA fingerprmtmg and differential display to obtam dlfferentrally expressed mRNA. Blochem. Bzophys. Res Comm. 219,824-828. 10. Zhao, S., 001, S. L , and Pardee, A B (1995) New primer strategy improves precision of differential display. BzoTechnzques l&842-850 11 Wang, X K. and Feuerstem, G Z. (1995) Dtrect sequencmg of DNA isolated from mRNA drfferenttal display. BzoTechniques l&448-452.

5 Analysis of Gene Expression by Multiprobe RNase Protection Anna K. Stalder, Axe1 Pagenstecher, and lain L. Campbell

Assay

Carrie L. Kincaid,

1. Introduction RNase protection assay (RPA) is becoming an increasingly popular method for the detection and quantitatron of RNA levels m cells and tissues (I-3). Hybridization is conducted in solution using an excess of a labeled antisense single-stranded RNA asprobe. Thus, hybridization of the probe with target RNA results in the formation of stable, double-stranded RNA-RNA hybrids. After hybridization, the excessprobe is removed by digestion with single-strand specific RNase, leaving behind only those probe molecules that were “protected” from digestion by virtue of having formed a duplex with their complementary mRNA target. These protected hybrids are denatured and separated from remainmg labeled probe using standard sequencing polyacrylamrde gel electrophoresrs. The separatedprotected probe can then be visualized using routine autoradiography. In comparison with other RNA detection methods, such as Northern blot analysis or RT-PCR, the RPA has a number of advantages. These include: 1. High sensitivity and speclflclty. 2. 3. 4. 5. 6. 7

Small sample requirement. Tolerant of RNA degradation. Easy quantltatlon Rapid and simultaneous analysis of multiple target transcripts. High throughput analysis. ConstructIon and use of “designer” probe sets.

As indicated above, with the RPA technique it is possible to develop “designer” RPA probe sets that can be used to analyze the expression of multiple genes within specrfic families (e.g., the cytokmes) or that are involved in common From

Methods

m Molecular

Medrone,

vol

Edlted by J Harry and H A Tkon

22

Neurodegeneratlon

0 Humana

53

Methods

Press Inc , Totowa,

and

NJ

Protocols

54

Stalder et al.

cellular processes(e.g., the host response). As described below, such multiprobe RPA sets can be conveniently constructed using routine molecular biological techniques. Further reflecting the increasing popularity of this technique, selected multiprobe RPA sets are becoming available commercially, e.g., Pharmmgen (Pharmingen, San Diego, CA) offers RPA probe sets that permit the detection of RNA transcripts for many different gene families, e.g., cytokines and chemokmes or gene families that are associatedwith specific cellular processes,e.g., apoptosis. The protocols discussedhere will cover the construction of multlprobe sets and the application of these probe setsfor RPA. 2. Construction of Probe Sets 2.1, Materials We show here the commercial suppliers we routinely purchase reagents from. However, these are not exclusive and these reagents can also be purchased from many other reputable suppliers. 1 MgCl,, ATP, TIT’, CTP, GTP, rATP, UTP, rC!TP, rGTP, and SP6 RNA polymerase, RNasin, AMV (avlan myeloblastosis virus) reverse transcnptase, 5X transcription buffer. 2. 10X T4 hgase buffer, T4 polynucleotlde kmase, T4 Llgase, EcoRI, HindIII, 10X restnctlon enzyme buffer, RQ-1 DNase, and pGEM-4, (Promega, Madison, WI). PCR buffer (10X), Taq polymerase, 1-kb ladder, and DTT (dithlothreitol) (GlbcoBRL, Galthersburg, MD) 3. Oligo d(T) primer (12-l&mer), Tns-saturated phenol/chloroform, competent bacteria (DHSa), (Invltrogen, Carlsbad, CA) 4 SeaKem ME-agarose and SeaPlaque GTG low-melt agarose (FMC Bloproducts, Rockland, ME) 5 6X DNA loading buffer 0 208% bromophenol blue, 0 208% xylene cyan01 FF, and 25% glycerol m water. Mix and ahquot Store at -20°C. 6 1X TE: 10 mM Tris, 1 mM EDTA.

2.2. Methods The initial step mvolves synthesis of the probe template, which can be done from RNA usmg reverse transcriptase-polymerase chain reaction (RT-PCR) or if available, directly from the cloned cDNA using PCR (see Notes 1 and 2). 2.2.1. RT-PCR 1 Combme the followmg Final Mm, (25 mu) PCR buffer (10X) Sterile Hz0

4 2 6

5mM 1x

Multiprobe RNase Protect/on Assay ATP, TTP, CTP, GTPs (each 10 m&I) RNasm (40 U/FL) AMV RT (10 U/pL) Ok0 d(T) (1 cLgW-) Total

55 2 1 2 1 18

1 r&f 2 U&L 1 U&L 50 ng/pL

2 Add 0.5 pg poly (A)+ or 1 0 kg total RNA. 3. Carefully mrx by gentle vortexmg. The reaction is performed m a standard thermocycler machme using the followmg condtttons 42°C 20 min; 99”C, 5 min; and 5°C. 5 min.

2.2.2. PCR Reaction 1 Combme the followmg:

MgCl, (25 mM> PCR buffer (10X) Sterile HZ0 Taq polymerase (5 U/pL) Total 2 Then add. 5’ primer (2 nM) 3’ primer (2 mI4) RT-PCR reaction product (from above) or cDNA template (0 5 n&L)

8 8 65.5 0.5 78

2mM 1X

1 1 20

20 pM 20 pM

20

0.1 ng/pL

0.025 U&L

3. Carefully mrx by vortexmg. The reaction is performed in the thermocycler machine using the followmg linked program: program 1: 94”C, 4 min; program 2: 94’C, 1 mm, 55’C, 1 mm, 72’C, 1 mm for 30 cycles; program 3. 72°C 5 min, and program 4: 4°C hold

2.2.3. Cloning the PCR Product tn pGEM-4 1. See Note 3. 2 After PCR, to confirm fragment size and concentration, perform dtagnosttc agarose gel electrophoresrs using 5 pL of PCR reaction mix in a standard 2-3% ME agarose gel containing ethrdmm bromide. Then do Tris-saturated phenol/chloroform extraction and ethanol precipitation on the remaining PCR mixture and resuspend dried pellet in 25 FL TE, pH 8.0. 3. To facilitate efficient restrtctron-enzyme digestion of the PCR DNA fragments, an end-hgation reaction 1s performed. Set up the following reaction: Final PCR mtx T4 ligase buffer (10X)

10.0 2.0

1x

56

Stalder et al. T4 polynucleotide

kmase (10 U/pL)

10 70

H2O

0.5 U/p.L

4. Carefully vortex mtx and incubate for 60 min at 37°C then add: T4 ligase (3 U&L) 10 015U/pL 5. Incubate for 60 mm at 37”C, then remove 2 pL for gel analysts (see item 9), the remaining reaction mixture is heat macttvated for 20 min at 72°C then the restriction enzyme digestion reaction is performed by adding to the end-ligation reaction mix

(End-ligation mix) Restriction enzyme buffer (10X) EcoRI (12 U/FL) Hind111 (10 U/pL) H2O

FL

Fur al

(19) 6 3 3 29

1X 0 6 U/uL 0.5 U/pL

6. In parallel with this reaction, set up a restriction enzyme digestion of the pGEM4 (see Note 4) transcription vector as follows

pGEM-4 plasmid (1 pg/pL) 10X restriction enzyme buffer EcoRI HzndIII H2O

PL 2 6 3 3 46

Final 33 ng/pL 1X 0 6 U/pL 0.5 U&L

7 Incubate both reactions for 2 h at 37”C, then add to each* 25 pL 3 M sodmm acetate, pH 5 2, 200 pL TE, and 700 pL 100% ethanol. 8. Freeze m dry ice for 20 mm then microcentrifuge for 20 mm at 16,000g. After drying, resuspend the pellet in 15 pL of TE and add 3 pL of 6X DNA loading buffer. 9. Prepare a 0.8% low-melt agarose gel containing ethtdmm bromide. Load the samples, the 2-pL ahquot removed from the end-ligation reaction and the 1-kb ladder, and run the samples approximately one-thud the way down the gel (see Note 4). Vtsuahze the gel under UV light, photograph, then carefully cut out the EcoRIIHindIII band using a clean razor blade. Trim all the excess gel from around the band and transfer fragment to a labeled 1 5-mL microfuge tube 10 For ligation of the fragments m pGEM-4, melt the gel fragments for 15 mm at 72°C and set up the followmg hgation reactions. a. 2 pL pGEM-4 gel, 5 pL EcoRIIHlndIII cut PCR gel fragment, hgase buffer, 1 pL T4 hgase, and 19 pL H20

3 pL 10X

b. 2 pL pGEM-4 gel, 3 pL 10X bgase buffer, 1 pL T4 hgase, and 24 uL H20. c 2 yL pGEM-4 gel, 3 pL 10X hgase buffer, and 25 p.L H20 11, Vortex mix and incubate overnight at room temperature.

Multiprobe RNase Protection Assay

57

12. Heat for 10 mm at 72°C to melt gel, vortex mtx, and remove 5 l.tL for transformatton of 50 l.tL of competent bacteria using standard protocols. Incubate at 37°C overnight. This should give the followmg result: (b) (cl (4 Number of colonies +++ +I13 Pick SIX colonies from plate (a) and do DNA minipreps using standard methods to confirm msertion of the PCR fragment using EcoRIIHindIII digestion of mimprep DNA. Retain an ahquot of each bacterial suspension and store at 4°C pending DNA maxrprep After confirmation, select a positive DNA clone containing the EcoRIIHindIII PCR fragment, and do a DNA maxiprep on the remaining ahquot of bacteria from which the positive DNA clone was derived The plasmid DNA is now ready for further mampulation

2.2.4. Linearization of Cloned pGEM-4 Constructs 1 For lmearization of the cloned pGEM-4 plasmids, set up two restriction enzyme reactions as follows

Cloned pGEM plasmid (12 p,g) Restriction enzyme buffer (10X) EcoRI or Hind111 H20

M-

Final

12 10 6 to

0.12 lJg/l.tL 1X 0 7 U&L 100

2 Incubate for a mmimum of 2 h at 37°C Remove 1 ltL reaction mtx for gel analysis (see step 8) to confirm lmearization. 3. To the remaining digest, add 20 ltL sodium acetate (3 M), 80 l.rL TE, mix, Trissaturated phenol/chloroform extract, and re-extract the aqueous phase with 180 l.rL of chloroform 4. Transfer the aqueous phase to a sterile tube and add 3 vol (600 FL) of 100% ethanol, mix, and freeze m dry me for 20 mm. 5. Thaw, centrifuge at 16,OOOg for 20 mm, wash pellet in 100 PL of 80% ethanol, recentrifuge, and remove all ethanol 6. Dry pellet and solubihze m 20 yL of sterile, RNase-free TE. 7. Determine concentration of hnearized plasmid by spectrophotometry at 260 nm using a 1:50 dilution of plasmid in sterile H,O. The concentration should be 0 3-0.6 pg/yL. 8. To confirm linearization, run a 0 8% diagnostic ME agarose gel electrophoresis using the 1+L aliquot removed after the enzyme digestion

2.2.5. Preparation of RPA Probe Set Dilute the purified EcoRI linearized DNA (prepared above) to give a workmg solution of 150 ng/pL. To prepare a 150 ng/pL solution apply the formula: Dil = (Imearized DNA j.tg/pL x 1000)/150

(1)

58

Staider et al.

The individual linearized plasmlds can then be combined to generate the probe set. Each 1s added to RNase-free TE such that the final dilution for each gives a concentration of 15 nglyL (see Note 5). After adding each of the components, the probe set IS vortex mixed and can be stored almost indefinitely at -70°C in a sealed microfuge tube. 2.2.6. Synthesis and Purification of Sense Target RNA 1. Set up the followmg transcription reaction

Transcrlption buffer (5X) DTT (100 mM) RNasm (40 U&L) rATP, rCTP, rGTP, UTP (each 2.5 mil4) Hind111 linearized DNA template (0.5 pg/kL) SP6 RNA polymerase (20 U/FL) H2O

W 20 10 10 20 10 2.0 37

Final 1x 1omM 0.4 U&L 0.5 mM 0 05 U/FL 0 4 U/FL

2. 3. 4. 5.

Incubate at 37°C for 90 mm. Add RQ 1 DNase (2 U/pL) 3.0 FL, to a final concentration of 0.06 U/pL Incubate at 37°C for 30 mm. To purify the sense RNA, add 100 pL Tris-saturated phenol/chloroform, extract and after centrifugation (5 min; 16,OOOg) carefully remove aqueous phase. Re-extract aqueous phase with an equal volume of chloroform and centrifuge as above Remove aqueous phase into a new tube. 6. Add* 20 FL ammonium acetate and 600 yL 100% ethanol. 7. Freeze in dry ice for 20 mm, centrifuge 20 mm at 16,OOOg, wash pellet m 80% ethanol (150 pL), centrifuge 10 mm at 16,OOOg, and remove ethanol. Dry pellet and add RNase-free TE (50 pL>. Determine concentration of RNA by UV spectroscopy at 260 nm

2.2.7. Preparation of Sense Target Set The final concentration of each sense target RNA in the set is 2 pg/pL. The dilution to achieve this should be m the order of 1:250,000 to >500,000. Prepare stock solutions of the sense RNA transcripts to give a concentration

of 40 pg/pL. Then combine each correspondmg sense RNA to give a final concentration

of 2 pg/pL

for each. Store in small (5-wL) aliquots

m -70°C

(see Notes 6 and 7). 2.3. Notes 1. The essential steps m designing and constructmg a RPA multiprobe set are deciding on the genes of interest to which probes will be generated, obtaining the cDNA sequence for the genes to be analyzed, determining the sequence length of

Multiprobe RNase Protection Assay

59

the probes to be generated, and synthesrzmg and clonmg of the probe fragments mto a suitable transcrtption-competent plasmrd. Since the protected hybrids m the RPA are routinely separated m 5-6% polyacrylamide sequencing gels, the recommended lengths of the probes should be approx 80-350 bp. Moreover, for multiprobe sets, the mdividual probes should differ in length by 20-50 bp to ensure adequate separation For quantitation it is necessary to include one probe against a “housekeeping” gene. For this purpose we routinely use a probe to the ribosomal protein gene RPL-32 (4). Since such housekeeping genes are usually expressed at high levels, It is advisable to make the length of their probes the shortest in the set. Other than the housekeepmg probe, m decidmg on the length of mdtvidual probes m the multrprobe set, it 1s helpful to have some mformation concerning the relative abundance of each RNA target For more abundantly expressed targets, the probe should be designed to be located toward the bottom (shorter length) of the probe set, whereas the inverse holds for rare targets In the case of abundant targets, there are often smaller breakdown products that are generated that can interfere with or obscure other protected fragments for shorter length probes in the set. An example of this point is illustrated in the case of GFAP m Fig. 1. The probes should be directed against unique sequences m the mRNA for the gene of interest and this should be established by comparing the sequences of gene families and other related molecules This can be done by using sequence alignment programs available on the Internet and from other sources. We routmely use BLAST (http,// www.ncbi.nlm.mh.gov/cgi-bin/BLAST/nph-blast), which is an efficient sequence analyzing program that is relatively easy to use. The specificity of the RPA is absolute and requires 100% sequence Identity between the probe and the target RNA Problems can therefore arise if the target gene is part of homologous gene family (e g , IFN-a or MHC class I). In this situation, varying degrees of sequence homology between the probe and these additional targets give rise, m addition to the authentic protected fragment, to the generation of multiple smaller RNase protected fragments In such cases, rt is advisable to run only a limited probe set contammg the probe for the gene of interest and the housekeeping probe. A final point that needs to be considered when designing the mdividual probes is to ensure that the selected target sequence does not contain sequence recognition sites for restriction enzymes used m the cloning of the synthesized probe fragments (see Note 3). 2. The probe fragments are conveniently synthesized using PCR. After deciding on the target sequence and length of the probes, upstream and downstream ohgonucleotide primers have to be designed that commence with the 5’ end of the target region of choice To facilitate subsequent clomng m the plasmid vector (see Note 3) the upstream and downstream primers should also include a 5’ sequence extension mcorporatmg a umque restriction-enzyme recognition site that will allow dnectronal cloning into the precut transcription vector. In the case of our own studies we routinely use EcoRI and HzndIII for this purpose, respectively. Probe cDNA synthesis can be accomplished by direct PCR using a cDNA clone from the gene of

Staider et al.

60

ICAM-

iNOS

MAC-l

EB22/5

GFAP

Fig. 1. Multiprobe RNaseprotectionassayfor the analysisof host-response geneexpressionin the spinalcord from mice with experimentalautoimmuneencephalomyelitis(EAE). A typical exampleof the resultsthat can be obtainedusing a designermultiprobe RPA set (A). This RPA permitsthe simultaneousdetectionof the six host responsegenesICAM- 1 (intercellular-adhesionmolecule-I), iNOS (inducible nitric oxide synthase),A20 (TNFinducedimmediate-earlygene), MAC-l (macrophage/microglialcell-activation marker), EB22/5 (an acute-phasereactant gene), and GFAP (glial fibrilliary acidic protein-an astrocyteactivation marker). In addition, a probe to the housekeepinggeneRPL-32 (L32) was also included and permitted quantitation of the levels of the host-responsegenes expressedat different stagesof EAE. (B) Note for the designof this multiprobe RPA set,

Multiprobe RNase Protect/on Assay

3.

4.

5. 6.

7

67

interest or if this is not available RT-PCR synthesis can be used to synthesize cDNA from cellular or tissue total or poly(A)+ RNA that IS known to express the gene of interest. For a more detailed discusston of PCR see Chapter 1 To reduce variation in transcriptional efficiency, it is advisable to use the same plasmid vector for the clonmg of each of the individual probes in the set We routinely use the pGEM-4 plasmid that has a multiple cloning site containing EcoRI and Hind111 restriction enzyme recognition sites and allows for T7 and SP6 RNA polymerase driven transcription. Other plasmtd vectors can also be used as long as they contain flanking RNA polymerase promoters to both sues of probe fragment msertlon. If the ligation reaction worked, a ladder of progressively fainter DNA fragments should be apparent when the gel IS visualized under UV. Conversely, there should only be a single band after the EcoRIIHindIII digestion. Once the probes of desired length have been cloned, they should be sequenced and their identity confirmed again using the BLAST program. For most cases the final concentration of the probe for the labelmg reaction should be 15 ng/pL. As an example to make a probe set of final volume 20 pL then 2 pL of each diluted lmearized plasmtd (150 ng/uL) would be added Clearly only 10 individual probes could be combmed in thus set. For probe sets containing more than 10 components the linearized plasmids above should be diluted to 300 ng/ pL thus giving a 1:20 dilutton. The diluted-sense RNA target sets are not stable to repeated freeze thawing and degrade progressively with each cycle. It is recommended that these be stored m small aliquots at -70°C and are used for no more than 3-5 cycles of freeze thawing or until the appearance of the sense bands in the autoradiographs begins to deteriorate.

3. The RNase Protection 3.1. Materials

Assay

1. See Note 8 2. RNase buffer (500 mL): 1 A4 Tris-HCl (pH 7.5): 0.5 mL, NaCl (5 M). 3 mL, EDTA (0.5 M, pH 8.0) 0.5 mL. Make up to 500 mL with dH*O. Immediately prior to use add to the RNase buffer: RNase A (100 mg/mL) 5 yL RNase Tl (125 U/mL) 1 I.LL 3. RNase-free tRNA: RNase-free yeast tRNA (Boehringer-Mannheim, Indianapolis, IN) is diluted in sterile, RNase-free TE at a concentration of 10 mg/mL in small (1-mL)

the probes for the constitutively and abundantly expressed GFAP and L32 RNAs were made the smallest m order to reduce interference from breakdown products (A; arrows). Immediately above the GFAP band m the symptomatic lanes, breakdown products are also highlighted that origmate from the highly upregulated EB22/5 RNA transcript

Staider et al

62

4 5.

6

7

8.

9 10

11. 12. 13.

ahquots. Store at -20°C. Yeast tRNA obtamed from other sourcescan be made RNase free by repeatedly phenol/chlorophorm extracting. 10% sodium dodecyl sulfate (SDS) m sterile H20. Protemase K stock, Proteinase K (Boehringer Mannherm, Indianapolis, IN) IS resuspended to a concentratron of 10 mg/mL m stertle H,O 1-mL ahquots are stored frozen at -20°C and are very stable Protemase K working solution: Make up fresh, for 25 samples: dH,O: 200 uL, SDS (10%): 150 pL, protemase K (10 mg/mL). 60 yL, yeast tRNA (10 mg/mL). 40 FL. Hybridization buffer (HB) 5X stock solutton: 200 mM PIPES (drsodmm salt of piperazine-1\!N’-bis(2-ethanesulfonic acid)) (pH 6 4), 2 M NaCl, and 5 mM EDTA. Working solution: 4 parts formamtde, 1 part 5X stock. Ahquot and store at 4°C. Kept m the dark it is stable for approx 6 mo RPA loading buffer: 80% formamtde, 10 mM EDTA (drsodmm salt of ethylendrammetetraacetate), pH 8.0, 1 mg/mL xylene cyan01 FF, 1 mg/mL bromophenol blue. Mix and store m 1-mL ahquots at -20°C 10X TBE. 1.3 M Trts, pH 8.0,450 mM boric acid, 25 m&Z EDTA. Dilute to 1X TBE usmg sterile water. 5% acrylamrde mix (1 L). 420.4 g urea (7 M), 125 mL 40% acrylamide bis (19: 1) solutron (125 mL for 5% gel stock). Dissolve urea m 500 mL of Hz0 and 50 mL 10X TBE on a heat plate. Add acrylarmde and adJUStvolume to 1 L. Filter (0 45-pm) and store in dark. Stable at 4°C for 2-3 mo. 10% APS (ammomum persulfate): Prepare fresh m stertle Hz0 Rain X (Unelko Corporation, Scottsdale, AZ) can be obtained from general automobile supply stores. However, any other stlicomzmg product may be used Sequencing gel apparatus. Standard sequencmg gel apparatus with 0.5-mm spacers can be used as long as the length (recommended mmimum 30 cm from the bottom of the well) is adequate for good separation of the protected fragments It is necessary to use square-bottomed wells rather than standard nucleotide sequencing combs

3.2. Methods

3.2.1. Probe Synthesis (see Notes 8-10) 1 Add to a 1 5-mL sterile nncrofuge tube 7.3 pL 10 mM UTP, 1.1. I.LL rGTP, rCTP, rATP (2.5 mMeach), and 12 0 l.tL [32P]UTP (3000 Ct/mmol; 10 mCt/mL) 2 Dry in a Speed-Vat for 15 mm or until totally dry. 3 Add to the above m order: Final CLL Stertle H20 Transcription buffer (5X) DTT (100 n-&Q EcoRI linearized template mixture

5.0 2.0 1.0 1.O

1x 1omM

Mu/t/probe RNase Protectron Assay

63

4. Vortex and centrifuge brlefly (10 s). 5. Add to the above in order

RNasin (40 U/mL) T7 RNA polymerase (I 5 U/FL)

NJ-

Final

0.5 0.5

2 U/yL 0.75 U/pL

6 Vortex and centrifuge briefly (10 s) Incubate at 37°C for 1 h 7 Heat mixture to 95°C for 5 min and place on ice. 8. Add. Final W TE Transcription buffer (5X) DNase I (2 U&L)

15 4 1

1X

0.06 U/pL

9 Vortex, centrifuge briefly (10 s), and incubate at 37’C for a minimum of 30 nun 10 Add to the above m order 29 PL 20 mM EDTA, pH 8.0, and 70 pL Tns-saturated phenol/chloroform. 11 Vortex for 30 s and centrifuge (16,000g) for 5 mm. 12. Transfer the aqueous phase (top) to a new tube and add 60 FL ammonium acetate (4 M), 600 PL 100% ethanol, and 5 PL tRNA (10 mg/mL). 13 Vortex and freeze m dry ice for 20 mm. Centrifuge (16,000g) at 4°C for 30 mm. 14. Wash the pellet with 100 PL 80% ethanol, centrifuge (16,000g) at 4°C for 10 mm 15 Remove all visible ethanol Dry wlthout heat. Add 10 FL hybridlzatlon buffer and solubihze pellet Count probe (see Note 3)

3.2.2. Hybridization 1 Add in order to a 1 5-mL sterile tube 8 PL hybrldizatlon buffer, 2 PL RNA sample (0.25-5 pg), and 2 PL diluted labeled probe (see Note 12) 2. Vortex, centrifuge briefly, add one drop of mineral ml and recentnfuge. Transfer the tubes to a heat block set at 95°C and immediately set the temperature to 56°C. Incubate overnight at 56°C

3.2.3. RNase Treatment Remove the tubes from the heating block and equilibrate to room temperature. Add 100 PL of RNase buffer containing

RNase A and RNase Tl.

Mix

gently by pipetting. Centrifuge briefly and incubate for 45 mm at 32°C (see Note 5).

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3.2.4. Protemase K Treatment After incubation, carefully transfer the reaction mix (lower phase) to a new tube containing 18 pL proteinase K working solution (see Note 6). Mix by pipetting. Incubate for 30 min at 37°C. 3.2.5. Purification and Precipitation 1 Followmg protemase K digestion, add 140 PL Trts-saturated phenol/chloroform to each tube. Vortex mix and centrtfuge (16,000g) for 5 mm (see Note 7) 2 Transfer the aqueous phase to a new tube and add: 60 yL ammomum acetate (4 M), 600 l.t.L 100% ethanol, 10 yL tRNA (10 mg/mL). 3 Vortex rmx then freeze m dry ice for 20 mm. Centrifuge (16,000g) at 4°C for 20 mm. 4. Wash pellet m 80% ethanol (150 uL) and recentrifuge for 10 mm. 5. Dry pellet and then add 4 uL loadmg buffer to each sample Include a sample with 6000 cpm of the probe and negative control (see Note 8), vortex mix, and centrifuge briefly. 6. Incubate samples for 3 mm at 95°C and then store on ice pendmg electrophoresis

3.2.6. Gel Electrophoresis 1. For gel electrophorests, carefully load the samples mto the wells and run until the bromophenol blue dye reaches about two-thirds the way down the gel (see Notes 9-13) 2. Carefully disassemble the gel plates leaving the gel on the nontreated plate 3. Place a presized piece of Whatman filter paper on top of the gel and remove the gel. 4. Cover with plastic wrap and dry m a gel-drying apparatus 5. Place dry gel on X-ray film and expose at -80°C to visualize and quantitate the separated radtoacttve protected fragments.

3.3, Notes 1. Whereas an RPA is not a particularly difficult assay to perform, certain standard precauttons that are applicable for all RNA work should be taken. One recommendation is to separate all reagents used on the first and second days of the assay. Furthermore, all plasticware, e.g., pipet tips, Eppendorf tubes, and so on) should be autoclaved and powder-free latex gloves should always be worn. We do not routinely treat solutions with DEPC. 2. Initially, both sensitivity and specifictty of the RPA probes should be tested by tttration against synthetic sense and target tissue RNA. This step 1s necessary to determme the sensittvlty and specificity of the probe set as well as to determme that the probe 1sm excess of the target over a range of RNA concentrattons. The potential for mtstdentification of the protected band increases with the number of probes m a given RPA Therefore, the correct identity of each protected band seen with the target RNA sample should be established by comparison with the correspondmg protected sense RNA band as well as by incubating each probe mdividually with target RNA 3. Smce the labeled probes are single-stranded RNA they are very susceptible to chemical and physical degradation and deteriorate m quality and specific activity progressively. The probes should therefore be made fresh and used immediately

Multlprobe RNase Protection Assay

4.

5.

6. 7.

8

9.

10.

(or within 24 h). The amount of probe to add to the hybridization reaction is calculated on the basis of 500 cpm/pLWUTP residue m the probe set. The number of UTPs per probe present m the RPA set therefore should be determined (for example a probe with 100 UTP residues would be diluted to 5 x lo4 CPM/pL). Prior to the hybridization, count the freshly made probe and dilute accordmgly For target RNA either poly(A)+ RNA or total RNA can be used. However, poly(A)” RNA offers greater sensitivity for detection of more rare target RNA and gives a higher signal-to-background ratio. For poly(A)+ RNA isolation, the protocol of Badley (5) is effective, whereas for total RNA isolation, commercially available reagents, such as TRIZOL (Gibco-BRL, Gaithersburg, MD) work well. It is convenient to utthze the time during the incubations to prepare for the following steps: a. Two sets of marked tubes, one set for the proteinase K digestion and the other for the transfer of the aqueous phase from the phenol/chloroform extraction and precipitation. b. The polyacrylamide gel. When taking the aqueous solutton from under the oil it is important to completely avoid the mineral oil since it may interfere with the subsequent purification steps The phenol/chloroform extraction step destroys the RNAse and efficiently purtfies the protected fragments. It 1s therefore very important to vortex for at least 20 s to obtain an emulsion of the phenol/chloroform with the aqueous solutron. Make absolutely sure to avoid the often turbid protein-containing interphase when removing the clarified aqueous phase. As a control for both the DNase and RNase digestion steps, a negative control sample should always be included in the assay This sample is simply probe (diluted to the same concentration used for the target RNA samples) alone incubated in the hybridization buffer and subsequently processed in parallel with the target RNA containing samples. Following autoradiography, the negative control lane should always appear completely clear. If, however, bands appear, in our experience, it 1s most likely that these are caused by incomplete digestion of the lmeanzed probe DNA template by the DNase 1 resulting in the formation of DNA/RNA hybrids. Thus, these protected RNA probe hybrids will run with the same apparent molecular size as the probe alone. In this event, discard the DNase and use a new batch. Additional bands in the negative-control lane likely indicate inefficient RNase digestion. In these circumstances, check RNase concentrations and mcubatton conditrons and adjust if necessary. The polyacrylamide gels used are standard sequencing gels of OS-mm thickness. It is necessary to coat one of the glass plates with an agent that prevents sticking of the gel For this purpose we use ‘ram X’ solution. After thoroughly cleaning the glass plates with 70% ethanol and drying, the ram X IS applied over the enttre surface of the glass In addition, to prevent sticking of the gel to the comb tt is also necessary to coat the comb wtth rain X. The wtdth of the gel and the number of wells should be chosen m a way that at least the outermost wells on either side are not loaded with sample, since these

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sometimes run distorted. It is also advisable to always load all wells that are not used with loading buffer to decrease distortion durmg running. 11. The gels (approx 20 cm width) are initially prerun for 30-45 min at 45-50 W and after sample loadmg run at the same power. For larger (approx 30 cm width) gels prerun and run at 55-60 W. 12. To Increase the comparability between different RPAs, we advtse that each gel electrophorests be run the same distance. To opttmally separate probes 300-87 bp length, we run the bromophenol blue dye band a distance of 30 cm from the bottom of the well. 13 Sometimes double or even more bands will appear for a specific probe m the RPA. Dependmg on their size, these may reflect inefficient RNase processmg or be caused by a sequence mismatch between the probe and the target RNA In the case of mefficient RNase processmg, the bands will be close together at the correct protected fragment size and include an additional band with the same size as the sensetarget RNA. This problem can usually be corrected by adJustmg the conditions for the RNase digestion step. Where sequence mismatch occurs, the protected bands will usually run at an mappropriately small size Carefully check the sequence of each synthesized probe to ensure it is identical to the target sequence. If there is no difference, the extra bands may indicate that the reported target sequence contains an error or that there are homologous target RNAs. It is advisable to resynthesize the probe to a different region m the target sequence If multiple bands still occur, it may be advisable to remove this particular probe entirely from the RPA set

Acknowledgments The studies presented were supported by USPHS grants MH 50426 and MH 47680. A. K. S. is a postdoctoral fellow of the National Multiple Sclerosis Society (NMSS). A. P. is a postdoctoral fellow of the Deutsche Forschungsgemeinschaft (DFG). Thts is manuscript number 10592-NP from the Scripps Research Institute.

References 1. Hobbs, M. V., Weigle, W. 0 , Noonan, D. J , Torbett, B E , McEvilly, R. J , Koch, R. J., Cardenas, G. J , and Ernst, D. N (1993) Patterns of cytokine expression by CD4+T cells from young and old mice J. Immunol 150,3602-3614. 2. Stalder, A K. and Campbell, I L. (1994) Simultaneous analysis of multiple cytokme receptor mRNAs by RNase protectton assay m LPS induced endotoxemia. Lymphokine Cytokme Res. 13, 107-l 12. 3 Chiang, C -S , Stalder, A K., Samimi, A., and Campbell, I L. (1994) Reactive gliosis as a consequence of IL-6 expression in the brain. Studies m transgemc mice Dev Neuroscl 16,212-221. 4. Dudov, K. P., and Perry, R. P. (1984) The gene family encodmg the mouse ribosomal protein L32 contams a umquely expressed mtron-contammg gene and an unmutated processed gene. Cell 37,457-468. 5. Badley, J E., Bishop, G. A., St. John, T., and Frelinger, J. A. (1988) A sample, rapid method of purification of poly A+ RNA Bzotechniques 6, 114-l 16

6 Molecular Probes for PNS Neurotoxicity, Degeneration, and Regeneration Arrel D. Toews 1. Introduction The functronal umt of the peripheral nervous system (PNS) is the neuron, with its long axon enveloped either by Schwann cells (unmyelinated axons) or by the multilamellar myelin sheath formed and maintained by these cells (myelmated axons) (Fig. 1A). Neuronal cell bodies may be located within the CNS (e.g., motor neurons m the ventral horn of the spinal cord) or within the PNS itself (e.g., sensory neurons m the dorsal root ganglia), but m either case, bundles of long axons course through various peripheral nerves to their target tissues (Fig. 1B). Normal operation of the PNS depends on the integrity of both neurons and glial (Schwann) cells. Additionally, however, function is critltally dependent on intricate interactions between these two cell types at molecular, structural, and functional levels. As an example, the intimate structural relationship between large axons and the myelin sheath that surrounds them is an absolute requirement for the rapid and efficient “saltatory conduction” of nervous impulses (1,2). Damage or insult to either the neuronal or glral component of this functional unit affects the other component, and most often has deleterious effects on normal nervous system function. In some cases,damage may be so severe that it is readily apparent as gross functional or morphological alterations, but in many casesthe initial insult, although eventually leading to serious pathological consequences, may be subtle and difficult to detect. In recent years, it has become possible to utilize molecular biological approaches as rapid and sensrtive probes for neurotoxic insults. Because of the highly specialized functions of various cell types within the nervous system (and the specialized metabolic machinery and structural elements required to carry them out), there are a number of protems that are either From

Methods Edlted

III Molecular by J Harry

Medmne, vol 22 Neurodegenerabon and H A Tiison 0 Humana Press

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Methods and Protocols Inc , Totowa, NJ

B

SENSORY

NEURON

(UNMYEUWJEDMON)

SENSORY

TOR

NEURON

(MYELINATED

NEURON

(MYELINATEDAXON)

AXON)

Fig. 1. (A) The neuron-Schwann cell functional umt of the peripheral nervous system. The functional integrity of the PNS depends on both the neuron and its ensheathmg Schwann cells (which may or may not produce a myehn sheath), as well as on vital interactions between these two components. The axon IS actually several orders of magnitude longer (in relation to Its cell body) than IS shown in the diagram, and each axon branches mto numerous nerve endings. (B) Generalized diagram of the penphera1 nervous system. The peripheral nervous system contams both myelmated and unmyelmated axons. Unmyelinated axons are also enveloped by Schwann cells (not shown in the figure). Neuronal cell bodies from which PNS axons arlse are located m either the dorsal root ganglia (sensory neurons) or in the ventral horn of the spinal cord (motor neurons). Sympathetic gangha with their associated neurons, also part of the peripheral nervous system, are not shown. The sciatic nerve is an easily accessible, major component of the peripheral nervous system. specific for a given cell type or are prominently expressed in them. Steadystate levels of mRNA for these proteins is often useful as a sensitive marker for

their function. In addition, toxic or metabolic insults that target a given cell type or function may be expected to result m somewhat specific alterations m mRNA expression for proteins involved m those functions. Conversely, mRNA expression for some protems, normally very low, is markedly upregulated following insult or injury. Thus, measurements of steady-state mRNA levels for selected proteins can serve as molecular probes or markers for altered nervous-

Molecular Probes for PNS Insults Table 1 cDNA Probes

for PNS Insults

Probe Schwann cells and myelm PO MBP MAG Neurons and axons GAP-43 Neurofilament proteins NF-L NF-M NF-H Neurotransmitter enzymes Choline acetyltransferase Acetylcholmesterase Dopamme P-hydroxylase Axon-Myelm interface NGF-R

Macrophages Lysozyme Housekeeping genes Ribosomal RNA Cyclophllm Glyceraldehyde-3 phosphate dehydrogenase

69

and injuriesa Remarks

Ref. #

MaJor structural protem of PNS myelin Other prominent PNS myelin proteins Involved in mitration of myelination

51 52 53

Growth-associated protein-43 kDa Major structural components of axons

58 59 60 Low-affinity nerve growth factor receptor (sensitive to alterations m either Schwann cell-myelin unit or neuron-axon unit)

61

Marker for active, phagocytlc macrophages

24

Involved in protein synthesis Involved m protein folding Glycolytlc enzyme

48 62 63

“References cited give cDNA sequences for rat genes and/or other mformatlon relevant to use of suitable cDNA probes.

system function. Such probes have also proven to be useful in elucidating the underlying nature and mechanism of action of some insults, and they are also of value in monitoring regeneration and recovery of function. A selection of specific probes potentially useful in studies of PNS neurotoxicity and damage are briefly discussed below, but the list is by no means comprehensive and it 1s mtended largely as a guide (see also Table 1). The same general approach is also applicable to the central nervous system (CNS), and some relevant information on selected probes is also briefly presented. Of particular interest m terms of a rapid, sensitive screen for a variety of insults to the PNS is the low-affinity nerve-growth-factor receptor (NGF-R). Because con-

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Toews

trol of its mRNA expression appears related to proper axon-Schwann cell interactions, it has proven to be a sensitive indicator of damage to either component of the neuron-Schwann cell functional unit (see Subheading 1.3. for details). 1.1. Schwann Ceils and Myelin Schwann cells are the predominant cells within the PNS, and synthesis and maintenance of myelin is the most prominent metabolic process within these cells. The protein composition of PNS myelm is relatively simple, with only a few proteins accounting for the bulk of myelm protem (2,3). Expression of mRNA for these proteins is very high during development when large amounts of myelin are being formed, and continues at lower but easily measurable levels throughout life, because of maintenance of existing myelm and, particularly in the rat, to continued myelination as the animal continues to grow. In rat sciatic nerve, approx 10% of total mRNA encodes myelm proteins during the early (first 2-3 wk of life) postnatal period marked by rapid myelin accumulation, and this value declmes to approx 2% of total mRNA in adult sciatic nerve (4). The PNS myelin-specific protein P,,is the most prominent protem in PNS myelin, accounting for about half of total myelin protein (and approx 8% of total sciatic nerve mRNA; 4), with myelm basic protein (MBP), PMP-22 (peripheral myelm protein, molecular weight of approx 22 kDa), and MAG (myelin-associated glycoprotein) accounting for most of the rest. Steady-state mRNA levels for all of the above myelin proteins decrease markedly followmg toxic insults that result m demyelmation (5-8), making them well-suited as markers for insults to myelin and/or the myelm-producmg Schwann cells. Note that for CNS myelm, the myelin proteolipid protem (PLP) is most prominent, but MBP, MAG, and the enzyme cychc nucleotide phosphodiesterase (CNP) are also present. Although mRNA for PLP and CNP is also expressed in the PNS, these are not good markers for altered myelm metabolism and function. Because myelin is a relatively lipid-rich membrane, assay of mRNA levels for enzymes involved in synthesis of lipids enriched in myelin are also useful as mdicators of altered metabolism. These enzymes include HMG-CoA reductase, the rate-limiting enzyme m biosynthesis of cholesterol (most promment myelin lipid), and ceramide galactosyltransferase (CGT), the rate-limiting enzyme in synthesisof the relattvely myelm-specific lipid, cerebroside. Both are also downregulated during demyelmating insults, m parallel with downregulation of myelin protein gene expression (7-9) 7.2. Neurons and Axons Axons comprise a large portion of the volume of peripheral nerves. As noted prevtously, small unmyelinated axons are enveloped by Schwann cells,

Molecular Probes for PNS Insults

71

whereas larger axons are ensheathed by multilamellar myelin produced and maintained by Schwann cells. The neuronal cell bodies from which these axons origmate are located either m the ventral horn of the spinal cord (motor neurons) or within the dorsal root ganglia (DRG), which parallel the spinal cord (sensory neurons). Proteins are synthesized in the neuronal perikarya, with those destined for axons or nerve endings moving down the axons by axonal transport processes. Insult or injury to axons may result in altered mRNA expression for axonal or other neuronal proteins in the neuronal perikarya. Because of their prominence in axons, cytoskeletal elements are good candidates for markers of axonal injury. Of particular relevance are the three neurofilament proteins, as well as classes II and IV of the P-tubulms. Also of relevance is GAP-43 (growth-associated protein of 43-kDa molecular weight). mRNA expression for both GAP-43 and P-tubulin is low m mature DRG neurons, but both are rapidly upregulated (within 1 d) following axonal injury, and mRNA expression remains elevated during regeneration (10). This pattern contrasts with that seen for neurofilament protein mRNA, which is normally high but is downregulated followmg axonal injury (II). However, downregulation of neurofilament mRNA expression occurs somewhat more slowly than upregulation for GAP-43 and tubulin, and thus it may not be as desirable as an early indicator of axonal insult. As an indication of the specificity of this response, the above markers are not altered in DRG by toxic insults that result m demyelmation but spare axons and neurons. For example, in telluriuminduced neuropathy, there is a rapid synchronous demyelmation with loss of about one-fourth of the total myelinated internodes, but there is little or no associated axonal degeneration. Although myelin gene expression is markedly downregulated (5), there are no alterations in mRNA expression for either neurofilaments or tubulin in the DRG (12). Interestingly, there is transient mRNA expression for NF-L and NF-M (low and medium molecular weight neurofilament proteins) and for class 2 P-tubulin in Schwann cells of the sciatic nerve during both primary and secondary demyelination (13). Alterations m gene expression for enzymes involved m neurotransmitter metabolism at nerve endings may also be useful as indicators of insult or injury to axons and/ or nerve endings. These enzymes are synthesized in the nerve cell bodies and must be delivered to nerve endings by rapid axonal transport. Although they have not been utilized specifically as markers for neuronal insults, such enzymes as dopamine j3-hydroxylase, choline acetyltransferase, and acetylcholmesterase are potentially useful candidates (see Table 1). 1.3. Axon-Myelin

Interactions

As noted above, there are close structural and functional interactions between axons and the myelm/Schwann cells that ensheathe them, and insults

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Toews

to either component of this functional unit might be expected to alter these important relationships. Molecules involved m these mteractions, or otherwise sensitive to alterations m them, might thus be useful as indicators for damage to either component. Recent studies suggest that mRNA levels for the low-affinity nerve-growth factor receptor may be especially useful in this context. NGF-R and its mRNA are expressed in precursor Schwann cells during early development, but as Schwann cells differentiate mto the myelmating phenotype, NGF-R is downregulated. The signal for downregulation involves axonal contact by the Schwann cell (1416), and NGF-R message levels in mature sciatic nerve are very low. However, a number of pathological situations involving insult to Schwann cells and/or their myelm, or to the underlying axon, result m marked upregulation of NGF-R mRNA. Such situations include drastic physical mjuries, such as those mduced by nerve crush or transection (14-17), in which both axons and myelin degenerate. However, upregulation also occurs m vanous models of primary segmental demyehnation, m which underlying axons are not damaged. These include tellurium-induced demyelination (6,181, lysolecithin-induced demyelination (19), and immune-mediated demyelination (19,20). Even early demyelination restricted to paranodal regions is sufficient for upregulation, and upregulation is restricted to those Schwann cells showing some demyelination (18); upregulation is related to loss of contact between axons and Schwann cells (20). Importantly, upregulation of NGF-R mRNA expression also occurs following exposure to compounds known to damage axons (e.g., acrylamide, isoniazid, carbon disulfide), and this upregulation occurs with exposure regimens that do not produce detectable morphological alterations (12,21). Based on the above studies, it appears that increased mRNA expression for NGF-R is an early and sensitive indicator of toxic insult or other damage to either the Schwann cell and/or its myelm, or to the underlying axon. As such, it should be useful as a rapid and sensitive screen for neurotoxic insults to the PNS. Such measurements should be even more informative when coupled with measurement of mRNA for myelin-specific components, such as POprotein. The latter should indicate if the insult primarily damages myelin or its supporting Schwann cell (downregulation of myelm gene expression) or is a consequence of mitral damage to neurons and/or then- axons (no early alterations in myelin gene expression). 1.4. Macrophages Following mjury to the PNS, circulating monocytes/macrophages invade the damaged tissue where they play vital roles during both degeneration and any subsequent regeneration. Such roles include phagocytosis of debris, salvage of cholesterol and other lipids for reutilization durmg regeneration, and modulation of Schwann cell prohferation and differentiation. Proliferation of

Molecular Probes for PIUS Insults

73

resident macrophages may also contribute to the increased number of these cells seen followmg mlury. The presence of macrophages m damaged PNS is typically monitored by immunocytochemical visualization of macrophages (the monoclonal antibody, EDl, is commonly used in rats; see ref. 22). However, immunocytochemical techniques are laborious and difficult to quantitate, because of both weakly staining cells and the distorted shapes of tissue macrophages. Expression of the macrophage-specific protein, lysozyme, correlates well with phagocytic activity (23), and lysozyme immunoreactivity colocalizes with ED1 staining during maximal PNS demyelination in several different experimental models (24). Expression of mRNA for lysozyme is thus a good marker for active phagocytic macrophages (24, and its assayin damaged nerve tissue is more quantitative and sensitive than the more tedious immunocytochemical approaches. 1.5. “Housekeeping Genes” “Housekeeping genes” are genes whose level of mRNA expression is presumed to remain relatively constant, at least under the variables or conditions under study (the term “housekeeping gene” is used in its operational sense herein, and not in the strict sense defined by properties such as absence of TATA boxes, presence of Spl binding sites near transcription start sites, and so on). Housekeepmg genes are potentially useful as benchmarks against which changes m expression for other genes can be compared. However, there are no perfect housekeeping genes and care and caution must be used when selecting and using them (for a detailed discussion of potential problems, see ref. 25). It is, in fact, not difficult to find examples of altered levels of all the so-called housekeepmg genes discussed below, but despite this, they can be useful in documenting relative specificity of alterations in expression of other genes. Commonly utilized housekeeping genes are generally constitutively expressed at relatively high levels in most cells, and their levels are considered to be generally proportional to the total amount of mRNA extracted. Cyclophilin (prolyl cis-truns rotamase), an enzyme involved in protein modifications, is commonly used, as is glyceraldehyde-3-phosphate dehydrogenase (an enzyme in the glycolytic pathway with a central metabolic role). Actin, an essential and prominent cytoskeletal protein, is also commonly used, but it may not be particularly suitable for the nervous system since many types of nervous system inluries involve alterations in cytoskeletal elements (reactive gliosis is the most obvious example). Use of ribosomal RNA (rRNA) is dually useful; in addition to its presumed relatively constant level of expression, it also constitutes a large proportion of the total cellular RNA. Its level thus provides a relative index of the recovery of total RNA from tissues, as well as an indication of variabihty introduced during sample handling and analysis. It may be worth-

Toews while to keep in mind that the protein products of the housekeepmg genes noted above are involved m basic metabolic and biosynthetic processes, and these processesmay themselves sometimes be operating at increased levels, particularly during the regenerative period that often follows PNS injury or msult. 1.6. Potential “Generic” or “Nonspecific” Markers for Toxic Insults An ideal nonspecific or generic marker for PNS insults might consist of a protein whose mRNA expression is normally very low but is rapidly and extensively upregulated by a wide variety of insults or mjuries, regardless of their specific site or mode of action. Conversely, such markers should also not be sensitive to hormonal, metabolic, or functional influences that are not directly related to toxicity. As might be expected, no such ideal markers are yet known. Several general classes of molecules are briefly noted below in this context, largely as candidates for further thought and study. These include stress-related genes, such as the heat-shock family of proteins, immediateearly-response genes, such as C-$X and c-jun, and various cytokines. With respect to usefulness as biomarkers for nervous system damage, all of these have been studied most extensively in brain, but some relevant studies regarding the PNS have also been published. Heat-shock proteins serve as molecular chaperones, assistmg m the assembly, folding, and translocation of other proteins. They apparently serve a role m protecting cells against Injury and other types of stress (see ref. 26 for a recent review), and increased gene expression for selected members of this family has been suggested as a useful marker for cellular injury in brain (27,28). Perhaps they might also be useful as markers for PNS insults or mjuries as well. The immediate-early-response genes c-fos and c-fun are among the first set of genes activated by cellular injury or ischemia in brain, but unfortunately, they are also activated in many other nonpathological situations, including various forms of stimulation. In fact, c-fos gene expression has been proposed as a marker for neuronal activity (29). With regard to PNS insults, upregulation of c-fos mRNA has been noted m both sciatic nerve (30,31) and m DRG (30) following a nerve crush injury. c-&n mRNA levels are also upregulated m DRG and ventral horn motor neurons followmg nerve crush or transection (32). A number of cytokines, including the early-response cytokmes mterleukm- 1 (IL-l p) and tumor necrosis factor-a (TNF-a), as well as IL-6, IL- 10, leukemia inhibitory factor (LIF), and granulocyte-macrophage-colony stimulating factor (GM-CSF) are upregulated m response to a variety of injuries or insults to the nervous system. As with the immediate-early-response genes noted above, virtually all studies examining mRNA levels of cytokines in damaged PNS have involved severe physical mjury models (nerve crushes or transections; e.g., see refs. 33-36) m which there is massive axonal degeneration and demy-


75

elmation, but upregulation may also be present followmg toxic or other insults. mRNA levels for cytokmes are generally very low, and RT-PCR methodology is usually necessary to detect any changes present (e.g., see ref. 37), although both Northern blot (38) and nuclease protection assays (39) have also been successfully utilized in models of Wallerian degeneration. Glial fibrillary acidic protein (GFAP), expressed in brain as part of the astroghal response to injury, has been proposed as a useful nonspecific marker for CNS insults (see refs. 40,41 for detailed discussion). Although there are no astrocytes in the PNS, Schwann cells do express GFAP under certain conditions, including the period of Schwann cell dedifferentiation that follows demyelinating insults (e.g., ref. 18). However, this upregulation is not as sensitive an indicator of PNS damage as is NGF-R rnRNA upregulation. 1.7. Utility of Approach Measurement of levels of specific mRNA species is useful as a screen for potentially neurotoxic compounds, and also provides valuable information regarding the sites and mechanisms of action of such toxicants. Isolation of mRNA from sciatic nerves (and DRG or spinal cord, if desired) followed by hybridization with selected cDNA probes provides quantitative measures of toxic insults rapidly, at a level of sensitivity not otherwise detectable or requirmg very tedious and time-consummg morphological examinations. In particular, NGF-R mRNA levels provide an early and sensitive general screen for many insults or injuries to the PNS. Additional probes can be included to determine if the toxicant’s primary effect involves the Schwann cell/myelin component or neurons and their axons. Additional probes are also useful to further delineate the specific sites and/or mechanisms of action of various toxicants, and to provide information regarding compensatory and regenerative responses in the damaged PNS. 1.8. General Methodological Approaches Although specific protocols are presented for the various required methodologies, they are but one of a large number of acceptable methods. The general overall approach to the use of selected mRNA levels as indicators of PNS damage mvolves isolation of RNA from nerves (and/or DRG or ventral spinal cord, if appropriate) of animals under study, followed by Northern blot analysis of steady-state mRNA levels for selected proteins of interest (Fig. 2). In our laboratory, we routinely isolate total RNA fractions by dispersion in the denaturing agent, guamdme thiocyanate, followed by centnfugation through cesium chloride, with subsequent ethanol precipitation and purification of RNA (42), but alternative methods are available (see Note 1). RNA is then separated according to molecular size on denaturing agarose gels containing formaldehyde (43),

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Toxicant exposure

v

(or other insult)

Remove sciatic newes (and DRG EL/or spinal cord) v Extract RNA - Guanidlnium lsothiocyanate dispersion - C&l gradient - EtOH

pmclpttation

v

Separate RNA on agarose gels; Transfer to nylon membrane

Hybrfdlze with JzPcDNA probes (e.g., NGF-R, PO, GAP43) v Autoradiography for visual image: quantitate radioactivity for mRNA levels v Strip probes from filter; hybridize with 32P-rRNA probe

22 3z 2E lzl

v Normalize mRNA levels to constant rRNA/lane

Fig 2. General approach for use of cDNA probes to detect and examme neurotoxic insults. to nylon membranes, and hybridized with 32P-labeled cDNA probes, synthesized using polymerase chain reaction (PCR) technology (44,45; see also Note 2). Filters are then washed to remove unbound probe and exposed to X-ray film to obtam a visual pattern of labeling. Relative levels of mRNA transferred

Molecular Probes for PNS Insults are then obtained directly from the filter by quantification of radioactivity, using an electronic autoradiography system (see Note 3). To control for vanability in sample handling, values obtained are normalized to the amount of 28s ribosomal RNA present m each lane, assayed with 32P-end-labeled oligonucleotides specific for rRNA (see Note 4). An alternative approach for measurement of mRNA levels involves quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) methodology. This approach is particularly useful if message levels are too low to allow reliable quantification by Northern blot analysis.

2. Materials 2.1, Labora tory Supplies 1. 2. 3. 4. 5 6 7.

1.5-mL mtcrofuge tubes. 50-mL conical centrifuge tubes Polyallomer tubes for ultracentrtfugatton. Zeta-Probe nylon blotting membranes (Bio-Rad, Hercules, CA) X-ray film (BMS 1 or XARS, Kodak, Rochester, NY). X-ray film cassette with intensifying screen. X-ray film developmg system

2.2. Equipment 1. Electronic autoradrography imaging system (Packard Instant-Imager, Downers Grove, IL) or laser densitometer. 2. Oven. 3 Vortex. 4. Water bath. 5. Speed-Vat concentrator (Jouan, Winchester, VA). 6. UV spectrophotometer 7 Polytron homogenizer (Brmkmann, Westbury, NY) 8. Low-speed centrifuge. 9. Ultracentrrfuge (Beckman, Fullerton, CA). 10. Swinging bucket ultracentrrfuge rotor (Beckman, SW50). 11. Horrzontal agarose gel electrophorests apparatus. 12. Power supply-constant voltage. 13. Handheld short-wavelength ultravrolet light source (Hoefer, San Franctsco, CA; TE-80). 14 Vacuum-transfer apparatus with vacuum pump or capillary transfer system 15 PCR-thermal cycler (Errcomp, San Drego, CA).

2.3. Reagents

(see Note 5)

1 Guamdme thiocyanate solutton (50 mL): 25 g guamdine thiocyanate, 0.25 g sodmm lauryl sarcosme (Sarkosyl), 1.25 mL 1 M sodium citrate buffer, pH 7.0,

78

2. 3. 4.

5.

Toews 0.35 mL P-mercaptoethanol, 0.165 mL 30% antlfoam A, double-dIstIlled water to give 50 mL. This solution may require brief heating for complete dissolution Filter sterlhze to remove particulates, then adjust pH to 7.0 with 1 N NaOH if necessary. Make fresh for same-day use Electrophoresls running buffer 10 mL 1 M sodmm phosphate buffer, pH 7.2, 82 mL formaldehyde, 908 mL double-distilled water Prepare just prior to use RNA denaturmg buffer (2 mL) 1.5 mL 100% delomzed formamide, 0.44 mL formaldehyde, 30 FL 1 M sodium phosphate buffer, pH 7 2,3 FL 0 5 M EDTA 5X RNA tracking dye (10 mL): 2 5 mL glycerol, 1.25 mL 1% bromophenol blue dye, 1.25 mL 1% xylene cyan01 dye, 10 PL 0.5 M EDTA, pH 8.0, 5 mL doubledistilled water MIX the above and add 0 1% DepC Incubate at 37°C for at least 30 min, then autoclave 15-20 mm rRNA hybrldlzatlon buffer (50 mL)* 12 5 mL filtered 20X SSC, 1 mL sheared salmon sperm DNA (5 mg/mL), 1 mL 1 M phosphate buffer, pH 6 7,0.5 mL 10% SDS, 0 25 g powdered milk, 5 mL 100% deionized formamide Add Dep-C water to give 50 mL. Make fresh prior to use. Boll approx 30 mL for 10 min, cool for 10 mm, and use to prehybrldize filter. Reserve remainder for rRNA hybrldlzatlon

3. Methods 3.1. Tissue Dispersion

and RNA Isolation

1 Dissect tissue as quickly as possible and freeze immediately m an RNase-free nucrofuge tube cooled on dry ice If samples are to be stored, place in ultralow freezer (-80°C) as soon as possible Because RNases work very fast, it 1s imperative that tissues be quickly frozen and that they remain deep-frozen until RNA IS isolated 2. Drop frozen tissue into 4.0 mL of guanidine thiocyanate solution in a 50 mL plastic conical centrifuge tube and disperse using a Polytron tissue homogenizer at full speed for 1 mm (see Note 6). 3 Spm the tissue dispersion in a low-speed centrifuge at approx 5000g for 5 mm to pellet any particulate matter 4 Carefully layer the supernatant from step 3 onto a 1 0-mL cushion of. 5 7 M RNase-free cesmm chloride, buffered with 25 m&J sodium acetate, pH 5.0, m an RNase-free polyallomer centrifuge tube Centrifuge at approx 100,OOOg for 18 h We routinely use the Beckman SW50 rotor, at 36,000 rpm overmght. (See Note 7 for a semlmicro modlficatlon ) 5. Following centnfugatlon, carefully remove the guanidme thlocyanate solution, the Interface, and a portion of the cesmm chloride cushion, using a sterile plastic transfer pipet Then carefully cut the tube below where the interface was (to ehmlnate possible contammatlon with RNases present m the dispersion medium) and remove the remammg ceslum chloride solution, taking care not to disrupt the clear and difficult-to-see RNA pellet 6. Add 50 pL of DepC water to the centrifuge tube. The RNA pellet 1s difficult to disperse and it 1sbest to transfer the partially dispersed pellet to a 1 5-mL RNase-free microfuge tube at this point. Rinse the centrifuge tube with an additional 50 PL of DepC water and transfer to the same mlcrofuge tube. Complete suspension of

Molecular Probes for PNS Insults the RNA pellet is aided by vortexing and brief heating to 55°C m a water bath, but this should be kept to a mmtmum to ensure RNA mtegrity. Once the RNA is m solutton, freeze the sample on dry me until ready to move to step 7 (to minimaze RNA degradation by RNases, the time the RNA solution is kept as a hquid should be mmimized as much as possible). 7. Add 0.1 vol of 2 M potassmm acetate, pH 5.0, mix, then add 2 5 vol absolute ethanol and mix. Store at -20°C for at least 3 h (overnight is acceptable) 8. Centrifuge for 20 mm m a microfuge at 4°C. Dry pellets in a Speed-Vat until almost dry, then suspend in 30 yL DepC water. 9. Drlute a small ahquot of the sample (usually 2 I.LL drluted I*lOO), and determme the concentration of RNA by measurmg absorbance at 260 nm Calculate the RNA concentration using the followmg formula: Itg RNA/mL

= ABSz6c x dilution factor x 40

Also measure absorbance at 280 nm. The 260/280 absorbance ratio gives an mdication of RNA purity and should be approx 1.8. Ratios below approx 1.5 generally indicate the presence of contammating protein. Store RNA samples at -80°C until used

1. Prepare a 0 8% denaturing agarose gel contaming 8.2% formaldehyde Dtssolve 0.56 g agarose m 63 mL double-distilled water m a 250 mL Erlenmeyer flask After weighing the flask, heat for approx 2 min m a microwave oven to dissolve the agarose Usmg the origmal weight, add water to replace that lost during boiling. Then add 0.7 mL 1 h4 sodmm phosphate buffer, pH 7.2, and 5.74 mL formaldehyde. Mix, pour mto a horizontal bed electrophoresis tray, and allow to polymerize. (We routinely use 10.5 x 14 cm gels in an IBI model MPH electrophorests apparatus, but other sizes and manufacturers’ products work equally well.) Prior to pouring the gel, rinse the gel tray, comb, and electrophoresis apparatus with an RNase mhibitor rinse (e g., RNase-ZAP by Ambion) and then with double-dtstrlled water to minimize the possibility of RNA degradation. After polymerization, place the gel tray m the apparatus, add gel-runnmg buffer to cover the gel, and carefully remove the sample well comb. 2 To an RNase-free rmcrofuge tube, add an appropriate volume of RNA denaturing buffer (e.g., if the RNA sample volume will be 10 pL, add 20 ~.LLof denaturing buffer). 3. Add RNA sample to the tube, mix, and incubate at 68°C m a water bath for 10 min to denature the RNA A constant volume (sample + denaturing buffer) should be used for each sample. We routinely dilute RNA samples to a constant concentration, usually 1.O or 2.0 pg/pL. Alternatively, DepC water may be added to each tube to give a constant final volume. 4. Allow tubes to cool to room temperature, then add an appropriate amount of 5X RNA trackmg dye 5 Load samples in the wells of the gel and run for 240-300 V-h This can conveniently be done overnight (e.g., 15 V for 16 h), but shorter times at higher volt-

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Toews

ages (up to 50 V) work equally well. Because the buffer and gel contam formaldehyde, gels should be run in a fume hood. 6. Followmg electrophoresls, remove the gel from the tray and examine the RNA pattern by placing the gel on a TLC plate contaimng a fluorescent mdlcator (e.g., SGG-254) that has been covered with transparent plastic wrap and lllummate with a hand-held short-wavelength ultraviolet light. The rRNA bands absorb ultravlolet light and create shadows on the TLC plate. Record the posmons of the rRNA bands, as well as those of the two marker dyes Cut off the upper left corner of the gel to maintain correct orientation durmg transfer 7 Transfer the RNA from the gel to a nylon filter, usmg 20X SSC. We routmely use a Hoefer TE-80 vacuum transfer apparatus with a Red-Evac vacuum pump and transfer at approx 5 kPa for approx 4 h. Capillary transfer usmg paper towels (see ref. 46, Sectlons 7.46-7.50) works Just as well and can convemently be done overnight if desired. 8. Followmg transfer, mark the upper left corner of the filter, as well as the posltlons of the sample wells. Illuminate with short-wavelength ultraviolet light and mark posltlons of the rRNA bands. Bake at 80°C for 2 h to lmmobillze the RNA, and store the nylon filter m a sandwich of filter paper mslde a zip-lock type plastic bag until used

3.3. cDNA Probe Synthesis

and Hybridization

Northern hybridization techniques are standard in most molecular biology labs, and a detailed protocol is therefore not presented. Reliable basic methods are detailed and discussed in Sambrook et al. (46) and other similar manuals. cDNA probe synthesis can be easily accomplished using polymerase chain reaction technology directly on the intact circular cDNA-containing plasmid (see Notes 2,8, and 9).

3.4. mRNA Quantification After filters have been rinsed to remove unbound probe, they may be exposed to X-ray film to obtain a visual pattern of mRNA levels, often useful for publication. Relative mRNA levels can then be quantitated by a number of approaches, the most reliable of which is use of an electronic autoradiography imaging system such as the Packard Instant-Imager to directly determme levels of radioactivity present m each band on the filter (see Note 3).

3.5. rRNA Hybridization Because of uncertainties about the absolute RNA concentrations determined by absorbance at 260 nm and because of experimental variability introduced during sample loading on the gel and transfer of RNA from the gel to the nylon filter, a measure of total RNA in each sample lane is useful for normahzatlon purposes. After mRNA levels of interest have been determined and bound


81

probe has been stripped from the filters, they can be hybridized with cDNA probes specific for rRNA (see Note 4). Because rRNA accounts for more than 95% of total RNA, measurement of rRNA levels provide a reliable index of the relative amounts of total RNA present in each sample lane, We routinely use 32P-end-labeled oligonucleotides (ref. 47, pp. 122,123) specific for bases 2673-2692 and/or 3361-3383 of the published sequence of rat rRNA (48). Conditions for prehybridization and hybridization are slightly different from those for longer cDNA probes (see Subheading 2.3. for rRNA hybridization solution composition). Filters are prehybridized with this solution for l-2 h at 30°C. End-labeling is accomplished m a microfuge tube containing: 1 5 FL 10X polynucleottde kinase buffer 2. 2-10 ng ohgonucleotide to be labeled

3. 50 uC132P-ATP. 4 1 yL polynucleotlde kmase. 5 DepC water to give a final volume of 50 mL.

by incubating in a waterbath at 37°C for 60 min. Labeled probe is then added to lo-15 mL of hybridization solution, which is placed m a boiling water bath for 10 min and then cooled to room temperature. The prehybridization solutton IS then replaced with this labeled hybridization solution and the filter hybridized overnight at 30°C. Because of the short probe length, different conditions are also necessary to remove unbound probe from the filters without also removing that bound to rRNA. Filters should be washed twice m 2X SSCYO.1% SDS for 30 mm each at 40°C followed by a final wash in 0.1X SSUO. 1% SDS for 10 min at 45°C. If additional hybridizations are desired, bound probe can be stripped from the filter by washing in 0.01X SSC/O.l% SDS for 45 min at 75°C. If care is taken to keep the filters moist, a single filter can routinely be stripped and reprobed with three to four different cDNA probes as well as with the rRNA probe. 4. Notes 1. Alternative methods for isolation of RNA from PNS (sciatic nerve) include various commerctal kits based on the method of Chomczynski and Sacchi (49) (e.g., RNAzol B from Ambion [Austin, TX]; Trlzol from Glbco-BRL [Garthersburg, MD] claimed to be suttable for simultaneous isolation of RNA, DNA, and protein fractions) These methods mvolve dispersion in an acid guamdme thtocyanate-phenol-chloroform reagent, followed by partittonmg and alcohol extracttons. Although not as tedious and arduous as the method described m Subheading 3.1., we have not found these alternative methods, when applied to sciatic nerve, to be sufficiently reproductble and reliable in terms of RNA yields and quality to satisfy our needs They are, however, m wide use by a number of mvesttgators

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2. Alternative methods for cDNA-probe synthesis mclude the more labor-intensive random pnmmg method (50) and various commercially available nonradioactive labeling approaches. 3 “Quantitative” laser densitometry of autoradiographs is an alternative method of determining relative mRNA levels on a given blot, but this approach reqmres multiple exposures at carefully selected exposure times and is more subject to variability than is direct determination of radioactivity on the filters 4 Because virtually all (>95%) cellular RNA is present as rRNA, its quantification provides a reliable mdication of the total RNA present m each sample lane on the gel. This is important because considerable variability may be introduced at a number of steps, including measurement of RNA concentrations, any subsequent dilutions, gel loading, transfer from gel to nylon filter, and so on. We utilize hybridization with 32P-end-labeled oligonucleotides specific for rRNA, but staming the filter with methylene blue can also be used as a considerably less quantitative measure of total RNA 5. Because RNA is very susceptible to degradation by RNases, extreme care must be taken to keep various solutions, glassware, and equipment that comes into contact with isolated RNA RNase-free. A discussion of required procedures IS beyond the scope of thts chapter, but adequate information is available m various molecular biology manuals (e g , see ref. 46). In addition, these manuals contain instructions for preparation of most commonly used solutions (e g., SSC, DepC water) and other materials, and their preparation 1s also not detailed here. 6. This volume IS suitable for tissue amounts rangmg from a single rat sciatic nerve to up to five pairs of sciatic nerves Complete disruption of sciatic nerve is difficult because of the large amount of connective tissue it contams, so rather drastic dispersion with a Polytron (Brmkman Instruments, Westbury, NY) or similar device is necessary. Adult nerves are even more difficult to disperse than those of young animals because of continued collagen deposition. Some RNA can be obtained with less drastic tissue dispersion, but the yield is much lower and may not be representative of the total RNA present m the tissue 7. This procedure can be scaled down to a senumicro level suitable for rsolatron of RNA from the distal stump of a single transected rat sciatic nerve. As an example, 1.7 mL of dispersed tissue suspension can be loaded onto a OS-mL CsCl cushion m a 2.2-mL ultracentrifuge tube and spun using a TLS-55 swinging bucket rotor m a Beckman TL-100 table-top ultracentrifuge. Smaller tubes can also be accommodated m larger rotors with available plastic adapters 8. PCR methodology is a widely established methodology standard in most molecular biology labs, and kits contammg thermostable DNA polymerase, buffer, and Mg*+ solutions are available from numerous suppliers, so a detailed protocol is not presented. In addition, exact conditions, such as Mg*+ concentrations, appropriate times and temperatures for denaturation, primer annealing, and DNA-synthesis steps during thermal cyclmg, number of cycles, and so on, may vary for different plasmids, and optimal conditions may need to be empirically


83

determined. Double-stranded PCR labeling (44) is usually satisfactory for labeled cDNA probe synthesis, but if message levels are very low, improved sensitivity can be gamed using single-stranded cDNA synthesis by PCR (see ref. 45 for detatled methodology and dtscusslon of advantages). The orientation of the cDNA in the plasmid must be known for single-stranded PCR probe synthesis, and the plasmtd is best linearized with an appropriate restriction nuclease to minimize the amount of extraneous plasmid DNA also synthesized, although careful selection of appropriately short cDNA synthesis times during thermal cycling can mmtmize this potential problem 9 The application of PCR technology to labeled probe synthesis can be further stmpllfted by use of common primer(s) We routinely use as primers 25mer oligonucleottdes specific for the P-galactostdase gene and lymg Just outside the multiple cloning site of pUC19 and related plasmids (e.g., pBluescript) This allows PCR synthesis of any cDNA cloned into pUC19-derived plasmids with a single set of primers. Although portions of the multiple cloning site are also synthestzed along wrth the desired cDNA, this does not significantly affect bmdmg of the probes to RNA during hybrtdizatlon. Because these common primers are unlikely to bmd to mammalian RNA, they have the further advantage of allowing the PCR synthesis reaction mixture to be used dtrectly m Northern hybrtdtzattons without puriftcation of the labeled probe

References 1, Rttchie, J M. (1984) Physiologtcal basis of conduction m myelmated nerve fibers, in Myelm (Morell, P., ed ), Plenum, New York, pp 117-145. 2. Morell, P., Quarles, R H , and Norton, W T. (1994) Myelm formation, structure, and biochermstry, m Baszc Neurochemistry, 5th ed. (Siegel, G J., Agranoff, B. W., Albers, R W , and Molmoff, P B., eds.), Raven, New York, pp. 117-143. 3. Norton, W T. and Cammer, W (1984) Isolation and characterization of myelin, in Myelm (Morell, P , ed.), Plenum, New York, pp. 147-195. 4. Stahl, N , Harry, J , and Popko, B (1990) Quantitative analysis of myelm protein gene expressron durmg development in the rat sciatic nerve. Mol. Brain Res. 8,209-212 5. Toews, A D., Lee, S. Y., Popko, B., and Morell, P (1990) Tellurmm-induced neuropathy a model for reversible reductions m myelin protein gene expression. J. Neurosci. Res. 26,501-507. 6. Toews, A D., Eckermann, C. E , Lee, S. Y., and Morel1 P (1991) Primary demyelmation induced by exposure to tellurium alters mRNA levels for nerve growth factor receptor, SCIP, 2’,3’-cychc nucleottde 3’-phosphodtesterase, and myelin proteohpid protein m rat sciatic nerve. Mol Brain Res. 11, 321-325. 7. Toews, A. D., Hostettler, J , Barrett, C , and Morell, P (1997) Alterations m gene expression associated with primary demyeiinatlon and remyelinatlon m the peripheral nervous system Neurochem. Res. 22, 1271-1280 8. Toews, A. D., Roe, E B., Goodrum, J F , Bouldin, T W., Weaver, J., Gomes, N D., and Morell, P. (1997) Tellurmm causes dose-dependent coordinate downregulation of myelm gene expression Mol Bram Res. 49, 113-I 19

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9. Toews, A. D , Goodrum, J. F., Lee, S. Y., Eckermann, E., and Morell, P. (1991) Tellurium-induced alterations in HMG-CoA reductase gene expression and enzyme activity: differential effects m sclatlc nerve and liver suggest tlssue-specific regulation of cholesterol synthesis. J. Neurochem. 57, 1902-l 906. 10. Hoffman, P. N. (1989) Expression of GAP-43, a rapidly transported growth-associated protein, and class II beta tubulm, a slowly transported cytoskeletal protein, are coordinated m regenerating neurons. J Neurosci. 9,893-897 11. Hoffman, P. N. and Cleveland, D. W. (1988) Neurofllament and tubulm expresslon recapitulates the developmental program during axonal regeneration mductlon of a specific beta tubulin lsotype. Proc. Natl. Acad. Scl USA 85,4530-4533 12. Roberson, M. D., Toews, A. D., Bouldm, T , Weaver, J , Gomes, N , and Morell, P. (1995) NGF-R mRNA expresslon m sclatlc nerve: a sensitive indicator of early stages of axonopathy Mol. Brain Res. 28,23 l-238 13 Roberson, M. D , Toews, A. D , Goodrum, J F., and Morell, P. (1992) Neurofilament and tubuhn expression m Schwann cells. J. Neuroscl. Res. 33, 156-162. 14. Tanmchi, M , Clark, H. B , and Johnson, E M., Jr. (1986) Induction of nerve growth factor receptor in Schwann cells after axotomy. Proc Natl. Acad. SCL USA 83,4094-4098.

15. Taniuchi, M , Clark, H. B., Schweitzer, J. B., and Johnson, E. M., Jr. (1988) Expression of nerve growth factor receptors by Schwann cells of axotomlzed peripheral nerves ultrastructural location, suppression by axonal contact, and bmdmg properties. J Neuroscr. 8,664-68 1. 16. Lemke, G. and Chao, M. (1988) Axons regulate Schwann cell expression of the maJor myelm and NGF receptor genes. Development 102,499-504. 17. Heumann, R., Lmdholm, D., Bandtlow, C., Meyer, M , Raedeke, M. J., Mlsko, T P , Shooter, E., and Thoenen, H. (1987) Differential regulation of mRNA encoding nerve growth factor and its receptor in rat sciatic nerve durmg development, degeneration, and regeneration’ role of macrophages. Proc. Natl Acad. Scl. USA 84,8735-8739

18. Toews, A. D., Griffiths, I R., Kynakldes, E., Goodrum, J F., Eckermann, C E., Morell, P., and Thomson, C E. (1992) Primary demyelinatlon induced by exposure to tellurmm alters Schwann-cell gene expression a model for intracellular targeting of NGF-receptor. J. Neurosci. 12,3676-3687 19 Stoll, G , Ll, C. Y , Trapp, B. D , and Griffin, J. W. (1993) Expresslon of NGFreceptors during immune-mediated and lysolecithin-induced demyelmation of the peripheral nervous system. J Neurocytol. 22, 1022-1029 20 Conti, G., Baron, P L., Scarpun, E., Vedeler, C., Rostaml, A., Pleasure, D., and Scarlato, G (1995) Low-affinity nerve growth factor receptor expression m SCIatlc nerve durmg P2-peptide induced experimental allergic neuritis Neuroscz Lett 199, 135-138. 21 Toews, A D., Harry, G J., Lowrey, K B., Morgan, D. L , and Sills, R. C. (1998) Carbon dlsulflde neurotoxlclty m rats. IV Increased mRNA expression of lowaffinity nerve growth factor receptor’ a sensitive and early mdlcator of PNS damage. Neurotoxicology 19, 109-l 16.


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22. DiJkstra, C. D., Dopp, E. A., Jolmg, P , and Krall, G. (1985) The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations m the rat recognized by monoclonal antibodies ED 1, ED2, and ED3. Immunology 54,589-599. 23 Keshav, S , Chung, L. P , Milan,

G., and Gordon, S. (1991) Lysozyme is an inducible marker of macrophage activation in murine tissues as demonstrated by in situ hybridization. 1. Exp Med. 174, 1049-1058. 24 Venezie, R. D., Toews, A D , and Morell, P. (1995) Macrophage recruitment in different models of nerve mJury: lysozyme as a marker for actrve phagocytosrs. .I. Neuroscl. Res. 40,99-107.

25 Spanakrs, E. (1993) Problems related to the interpretation of autoradiographic data on gene expression using common constitutive transcripts as controls. Nuclezc Acids Rex 21,3809-3819 26. Jmdal, S (1996) Heat shock proteins: applications in health and disease. Trends Bzotech. 14, 17-20. 27. Gonzalez, M. F., Shuarsht, K., Hisanage, K., Sagar, S. M., Mandabach, M., and Sharp, F R. (1989) Heat shock proteins as markers of neural injury. Mol. Bruin Res. 6,93-100.

28. Massa, S. M., Swanson, R. A., and Sharp, F. R. (1996) The stress gene response m brain. Cerebrovascular Brain Metab. Rev. 8,95-158. 29. Sagar, S. M , Sharp, F. R , and Curran, T. (1988) Expressron of c-fos protein m brain: metabohc mapping at the cellular level. Science 240, 1328-133 1. 30. Plantmga, L. C., Verhaagen, J., Wong, S. L., Edwards, P. M., Bar, P. R., and Grspen, W H (1994) The neurotrophrc peptide Org 2766 does not influence the expression of the immediate early gene c-fos following sciatic nerve crush in the rat. Znt. J Dev Neuroscz. 12, 117-125 31. Lm, H M., Yang, L. H , and Yang, Y J. (1995) Schwann cell properties: 3. c-fos expression, bFGF production, phagocytosis, and proliferation during Wallerian degeneration. J. Neuropathol Exp. Neurol. 54,487-496. 32. Jenkins, R and Hunt, S P (199 1) Long-term increase in the levels of c-jun mRNA and Jun protein-like immunoreactivrty m motor and sensory neurons followmg axon damage. Neuroscl. Lett. 129,107-l 10. 33. Bolm, L. M., Verity, A. N., Srlver, J. E., Shooter, E. M., and Abrams, J. S. (1995) Interleukin-6 production by Schwann cells and induction in sciatic nerve inJury J. Neurochem. 64,850-858.

34. Sun, Y. and Zigmond, R. E. (1996) Leukemia I inhibitory factor induced m the sciatic nerve after axotomy is involved in the induction of galanm m sensory neurons. Eur. J. Neuroscz. 8,2213-2220. 35 Wagner, R. and Myers, R. R. (1996) Schwann cells produce tumor necrosis factor alpha. expression in injured and non-inJured nerves. Neurosczence 73, 625-629 36. Jander, S., Pohl, J., Grllen, C., and Stoll, G. (1996) Differential

expression of mterleukin-10 in Wallerran degeneration and immune-mediated inflammation of the rat peripheral nervous system. J. Neuroscz. Res. 43,254-259.

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37. Bourde, O., Kiefer, R., Toyka, K. V , and Hartung, H. P (1996) Quantification of mterleukm-6 mRNA m Wallerian degeneration by competitive reverse transcription polymerase chain reaction .I Neuroimmunol 69, 135-140 38. Curtis, R , Scherer, S. S , Somogyi, R , Adryan, K. M., Ip, N Y., Zhu, Y., Lindsay, R. M., and DiStefano, P S. (1994) Retrograde axonal transport of LIF is increased by peripheral nerve injury* correlation with increased LIF expression m distal nerve. Neuron 12, 191-204. 39. Banner, L. R and Patterson, P. H (1994) MaJor changesm the expression of the mRNAs for cholinergtc differentiation factor/leukemia mhtbitory factor and its receptor after inJury to adult peripheral nerves and ganglia. Proc. Natl. Acad. Scz USA 91,7109-7113 40 O’Callaghan, J. P. (1988) Neurotypic and ghotyptc proteins asbiochemical markers of neurotoxicity. Neurotoxzcol. Teratol 10,445-452. 41 Billmgsley, M. L. and O’Callaghan, J. P. (1992) Molecular neurotoxtcology m Principles of Neurotoxzcology (Chang, L W., ed ), Marcel Dekker, New York, pp. 55 1-562. 42. Chirgwin, J M , Przybyla, A E , MacDonald, R. J , and Rutter, W. J. (1979) Isolation of biologically active rrbonucleic acid from sourcesenriched m ribonuclease.Biochemzstry l&5294-5299 43. Pauley, R. J., Parks, W. P., and Popko, B. J (1984) Expression and demethylation of germinally-transmitted BALB/c mousemammary tumor virus DNA in Abelson MuLV B-lymphoid cell lmes. Vzrus Res. 1, 381-400. 44. Jansen, R. and Ledley, F. D. (1989) Production of high specific activity DNA probes usmg the polymerase chain reaction Gene. Anal Techn. 6,79-83. 45. Bednarczuk, T. A., Wiggins, R C , and Konat, G. W. (1991) Generation of high efficiency, single-stranded DNA hybridization probes by PCR BioTechnzques 10,478. 46. Sambrook, J., Fritsch, E. F., and Mamatis, T (1989) Molecular Clonzng, 2nd ed , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 47 Mamatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Clonzng, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 122,123. 48 Hadjiolov, A. A., Georgiev, 0. I, Nosikov, V V., and Yavachev, L. P (1984) Primary and secondary structure of rat 28 S nbosomal RNA. Nuclezc Aczds Res. 12,3677-3693. 49 Chomczynski, P and Sacchi, N. (1987) Single-step method of RNA isolation by acid guamdmmmthiocyanate-phenol-chloroform extraction Anal Biochem. 162, 156-159. 50. Feinberg,A. P andVogelstem,B (1983) A techniquefor radiolabelhngDNA restnctlon endonucleasefragments to high specific activity Anal Bzochem 132, 6-13 5 1 Len&e, G. and Axel, R. (1985) Isolatron and sequenceof a cDNA encoding the maJor structural protem of peripheral myelm. Cell 40,501-508. 52 Roach, A., Boylan, K., Horvath, S., Prusiner, S. B., and Hood, L. E. (1983) Characterization of cloned cDNA representing rat myelin basic protein. absence of expression m brain of shiverer mutant mice. Cell 31,799-806.


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53. Lai, C , Brow, M. A , Nave, K.-A., Noronha, A. B , Quarles, R. H., Bloom, F. E , Mrlner, R. J., and Sutcliffe, J. G. (1987) Two forms of lB236/myelin-associated glycoprotem, a cell adhesion molecule for postnatal neural development, are produced by alternative sphcmg Proc Natl. Acad. Sci. USA 84,4331-4341. 54 Karns, L R., Ng, S -C., Freeman, J A., and Ftshman, M. C. (1987) Cloning of complementary DNA for GAP-43, a neuronal growth-related protem Sccence 236, 597-600. 55 Lewts, S. A and Cowan, N J (1985) Genetics, evolutton, and expression of the 68,000-mol. wt neurofilament protein: isolation of a cloned cDNA probe. J Cell Bzol. 100,843-850 56 Myers, M. W., Lazzarml, R A , Lee, V M.-Y., Schlaepfer, W W., and Nelson, D L (1987) The human mid-size neurofilament subunit a repeated protein sequence and the relationship of its gene to the intermediate filament gene family EMBO J. 6,1617-1626. 57. Shneidman, P. S , Carden, M J., Lees, J. F., and Lazzarmi, R. A (1988) The structure of the largest murme neurofilament protein (NF-H) as revealed by cDNA and genomic sequences. Mol. Brain Res. 4,2 17-23 1. 58 Brtce, A , Berrard, S , Raynaud, B., Ansieau, S., Coppola, T., Weber, M. J , and Mallet, J. (1989) Complete sequence of a cDNA encoding an active rat cholme acetyltransferase. a tool to investigate the plastictty of cholmergic phenotype expresston J. Neuroscl Res. 23,266-273. 59. Legay, C , Bon, S., Vernier, P., Coussen, F., and Massouli, E. (1993) Cloning and expression of a rat acetylcholmesterase subunit: generation of multiple molecular forms and complementarny with a Torpedo collagemc subunit. J. Neurochem. 60, 337-346. 60 McMahon, A., Geertman, R , and Sabban, E. L. (1990) Rat dopamine P-hydroxylase. molecular cloning and characterization of the cDNA and regulation of the mRNA by reserpme J. Neurosci. Res. 25,395-404. 61. Raedeke, M. J., Misko, T P , Hsu, C , Herzenberg, L. A., and Shooter, E M. (1987) Gene transfer and molecular cloning of the rat nerve growth factor receptor. Nature 325,593-597 62. Danielson, P. E., Forss-Petter, S , Brow, M A., Calavetta, L., Douglass, J , Milner, R. J., and Sutchffe, J G. (1988) plB 15. a cDNA clone of the rat mRNA encoding cyclophtlin. DNA 7,261-267 63 Tso, J. Y , Sun, X. H., Kao, T. H., Reece, K S., and Wu, R (1985) Isolation and characterization of rat and human glyeraldehyde-3-phosphate dehydrogenase cDNAs: Genomic complexity and molecular evolution of the gene. Nucleic Aczds Res. 13,2485-2502.

7 Mobility-Shift DNA-Binding Using Gel Electrophoresis

Assay

Yang Xiao and Keith Pennypacker 1. Introduction Transcription factors are induced in neurons to alter gene expression to adapt to brain injury caused by exposure to neurotoxins, neurodegeneration disease, or mechamcal damage. These DNA-binding proteins bind to specific recognition sequences m the promoter region of genes to regulate transcription. Genes related to both regeneration and degeneration are regulated by transcription factors. Therefore, the fate of a neuron is determined by which transcription factors are expressed within a particular cell. To identify the transcription factors that are induced after brain mJury, electrophorests mobility-shift assay(EMSA) exploits the specific binding between these DNA-binding proteins and their DNA-recognition sequence. A radrolabeled oligomer contammg a DNA-binding site is incubated with protein extracts. Transcription factors recogmzmg the site will form a DNA-protein complex whose electrophoretic mobility IS retarded compared to the unbound oligomers. EMSA is a simple, rapid, and sensitive method for the detection of sequence-specific DNA-binding proteins in crude whole cell or nuclear extract. This assay also permits the quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants (I-3), and binding specificity of DNA-binding proteins. Specific transcription factors within the Protein-DNA-binding complex can be identtfied by using anttbodtes produced against a transcription factor of mterest.The antibody can either recognize the protein-DNA complex causing further retardation of the mobility of the complex during electrophoresis or interfere with DNA binding, m whtch case no band is formed. Increased retardation of From

Methods Edited

m Molecular by J Harry

Medmne, vol 22 Neurodegeneratron and H A T~lson 0 Humana Press

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Xiao and Pennypacker

electrophoresis mobility by the formation of antibody-protein-DNA complex 1s termed a supershift. EMSA has been combined with Western blot analysis to Identify components of gel-shift assays, particularly when antibodies do not perform well to supershift the complex. This technique is termed “shift-Western blotting” (4). It is based on the idea that nitrocellulose and anton-exchange membranes dtfferentially bmd proteins and DNA. When the protein-DNA complexes are separated on polyacrylamide gels, the complexes can be transferred to nitrocellulose and anion-exchange membranes separately. The proteins, bound to nitrocellulose, can be identified by rmmunoblottmg, whereas the DNA, which migrates through the nitrocellulose to bind to the anion-exchange membrane, is detected by autoradiography.

2. Materials 2.1. Equipment 1 Radioacttvity-countmg system: Multipurpose scmtillation counter. a. Beckman LS 6500 or equivalent (Fullerton, CA). b. Scintillation cocktail: Ecolume 1 gal, ICN cat no. 882470 (Costa Mesa, CA). c. Scmtillation tubes and caps from Beckman 2. Imaging densitometer* Bio-Rad model GS-670 (Richmond, CA). 3. Apparatus chamber for the experiment. electrophoresis unit from Hoefer (San Francisco, CA) SE 600 series with matched glass plates and spacers Any electrophoresis apparatus will be fine. 4. Spectrophotometer with reagent for Bradford protein assay. Bio-Rad protein assay dye reagent 450-mL cat. no 500-0006. Bovine serum albumin, stock of 10 mg/mL (to make standard). 5. Microcentrifuge tubes. 6. High-speed microcentrifuge. 7. Gel dryer: Bio-Rad model 583 or equivalent 8. Dounce homogenizer. VWR, cat. no. 62400-595. 9 Vortex or mixer: any kind. 10. Nutator: Clay Adams Model 421105.

2.2. Reagents 1. Aprotmm (protease inhibitor)* ICN cat. no. 190382. Dissolve 10 mg m 2 mL H,O to make stock solution. Use 5 pL/mL m extraction solution. 2. Dithiothreitol (reducing agent)’ Gibco-BRL (Gaithersburg, MD) cat no. 1550% 013 Make stock solution 1 M, keep at -20°C. 3. Leupeptm (protease inhibitor): ICN cat. no. 151553. Dissolve 5 mg m 2.5 mL Hz0 to make stock solution. Use 2 pL/mL m extraction solutton. 4. Pepstatm A (protease inhibitor) Boehrmger Mannhemr (Mannhelm, Germany) cat no 253-286. Dissolve 2 mg in 3 mL Hz0 to make stock solution Use 0.7 pL/mL m extraction solution

Mobility Shift DA/A-Binding Assay

91

5. Phenylmethylsulfonyl fluoride (PMSF; protease Inhibitor): Sigma (St. Louis, MO) cat. no. P7626. Dissolve 34.8 mg m 2 mL anhydrous lsopropanol (Sigma cat. no 405-7) as a 0.1 M stock The stock solution 1s stable for 9 mo. (Note: Place on rocker to dissolve). 6 Radioactive isotope (for labeling oligomers): NEN Research (Boston, MA) products (Y-~~P) Adenosme 5’ trlphosphate cat. no. BLU-002~. 7. Noniomc detergent P-40 (NP-40): Sigma cat. no. N6507. 8 N,N,N’,N’-Tetramethylethylenedlamlmme (TEMED). Boehrmger Mannhelm cat. no. loo-139 or Sigma cat. no T-8133. 9 Acrylamlde/bls solution 37.5: 1 (2.6% C) solution: Blo-Rad cat no. 161-0158, or make It by using acrylamlde (Boehringer Mannheim cat. no 100-13) and blsacrylamide (Boehrmger Mannhelm cat. no. 100-140). 10. Ammomum persulfate: Boehrmger Mannhelm cat no. 100-735. 11. Salmon sperm DNA (ssDNA): Sigma cat. no. D7656. Stock is 10 mg/mL. Dilute to 1 mg/mL with H20, somcate three times for 5 s each and then incubate at 65°C for 10 min before ahquotmg and storing at -20°C. 12. Loading dye. bromphenol blue Sigma cat no. B 3269.

2.3. Buffers 1 Reaction buffer 10X (IOX TBE buffer) for sample and probe incubation. 24.22 g Tns-HCl, pH 7 8, 74.56 g KCl, 10.17 g MgC12, 3.74 g EDTA, 7.713 g dlthiothreltol, 0.5 g bovine serum albumin, and add water to 1 L. 2. Tris-borate EDTA, buffer (10X stock solution): 108 g Tris base, 55 g boric acid, 40 mL 0 5 M EDTA pH 8.0, add Hz0 to 1 L. 3 1X TBE solution* 89 mM Trls base, 89 mM boric acid, 2 mM EDTA. 4 Running buffer: 0.25-O 5X TBE buffer. 5. Buffer for nuclear cell extract. a. 250 n&l sucrose, 15 mM NaCl, 5 mM EDTA, pH 8.0,l mM EGTA, 0.15 mM spermme, 0 5 mM spermidine, 1 mM dlthiothreltol (DDT) b. 10 mM HEPES, pH 7 9, 1 5 mM MgCI,, 10 mM KCI. c. 0.5 A4 HEPES, pH 7.9, 0.75 mM M&l,, 0.5 mM EDTA pH 8.0, 0.5 M KCl, 12.5% glycerol, 0.1% NP-40. d. 10 mM Tns-HCl pH 7 9, 1 mM EDTA pH 8 0, 5 mA4 MgCl,, 10 mM KCl, 20% glycerol, and 0 1% NP-40, 1 mM DTT 6. Whole cell buffer: 20 mM HEPES, pH 7.9, 400 mM NaCI, 0.5 n&f EDTA, pH 8.0,O. 1 m&f EGTA, 1 mM MgCl,, 20% glycerol, and 1% NP-40.

2.4. Supplies 1. FuJifilm autoradlography film (8 x 10 m 100 sheets)ID no. 393339 or HyperfilmMP (Amersham cat. no RPN 1677, 25 sheets,cat. no. RPN.1678, 75 sheets). 2. G-50 column. STE select-D G-50,5 Prime-3 Prime (Boulder, CO) cat. no. 5301955312. 3. Whatman 3MM or equivalent filter paper. 4. Microcentrifuge tubes

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5 Dialysis tubing (presoaked m water before use)* SpectraIPor membrane tubmg molecular weight cut-off: 12,000-14,000, diameter* 6.4 mm, width; 10 mm (cat. no. 132 676)

3. Methods 3.1. Crude Nuclear Extraction Preparation A crude nuclear extract 1s prepared using this method. Other methods to isolate a purer nuclear sample reqwe more preparation time. All tissues should be frozen and buffers precooled

(see Notes 1 and 2).

3.1. I. Frozen Tissue 1 Add 0.5 PL of aprotmm, leupeptm, and phenylmethylsulfonyl fluoride (PMSF) from stock solution to 500 PL buffer 1 (Isotonic solution) for one sample (4 1 buffer to tissue weight). 2 Add 500 pL buffer 1 with protease mhlbltors directly to approx 100 mg of frozen tissue from -80°C (see Note 1). 3 Homogenize for 10 strokes, avolding bubbles with a loose pestle m the dounce homogemzer (VWR, cat no. 62400-595) on ice 4. Place homogenate mto Eppendorf tubes and centrifuge at 2000g m mlcrocentrifuge for lo-15 mm m the cold room (4°C). 5. Remove and discard the supernatant 6 Add 0 5 FL from stock of aprotmm, leupeptm, and PMSF to 500 PL buffer #2 (hypotomc solution), mix gently. 7. Add 500 pL of buffer 2 with protemase inhibitors to the pellet. MIX gently with a plpet and homogenize for 10 strokes with the dounce homogenizer tight pestle. 8. Let the tubes sit on ice for 10 min 9. Centrifuge at 5000g for 10 mm m a microcentrlfuge in the cold room. 10 Remove the supernatant (cytosohc protein) and set aslde if interested m cytosohc fraction 11. Add 500 FL of buffer 3 solution (hypertonic) with 0.5 PL of aprotmm, leupeptm, and PMSF from stock to each Eppendorf tube. 12 Shake on Nutator for 30 mm in the cold room 13 Centrifuge m the cold room at 16,OOOg m a mlcrocentrifuge for 30 mm (If pellet IS not well formed, repeat) 14. Making dlalysls buffer by adding to buffer 4. DTT l.lO,OOO, leupeptin 1: 10,000, aprotmin l:lO,OOO, pepstatm A 1 10,000, and PMSF l.lO,OOO (eqmvalent to 100 FL/L) from stock solution. 15 Plpet supernatant into dialysis tubing that IS presoaked m water Clamp the tubmg and dialyze m dialysis buffer overnight in the cold room to remove extra salt Put a stir bar into the beaker and stir gently overnight 16. Take dlalysls tubing and clamps out from dialysis buffer Remove one clamp and squeeze content mto a Eppendorf tube. Spm all samples at 16,000g in a mlcrocentrifuge for 15-30 mm to remove any excess debris. Plpet supernatant (nuclear extract) mto new Eppendorf tubes.

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Mobility Shift DNA-Bindmg Assay

17. Protein concentration IS assayed by Bradford method with Bio-Rad protein assay reagent on spectrophotometer to determine the volume of sample to be loaded m each well m electrophoresls mobility shift assay. 18. Aliquot the rest of samples mto tubes and rapidly freeze by using liquid nitrogen or dry ice with methanol Store the extracts at -8O’C (-20°C will decrease DNA bmding actlvlty). Avoid more than one cycle of freezmg/thawing. Frozen extracts should be thawed on ice.

3.1.2. Cell Culture 1. 2 3. 4 5. 6. 7. 8.

Wash the cell with cold PBS twice to remove media. Scrape the cells m PBS and plpet It to a centrifuge tube. Spm down the cells and discard the PBS Add 500 yL of buffer 2 (containing 0 5 PL of each aprotinin, leupeptm, and PMSF from stock solution), mix gently Homogemze 10 strokes with a tight pestle in dounce homogenizer. Plpet the samples m Eppendorf tube and place them on ice for 10 mm Centrifuge at 5OOOg in a microcentrifuge for 10 min at 4°C. Continue the following steps as in nuclear extract preparation for the tissue (from steps lo-18 from Subheading 3.1.1.).

3.1.3. Whole-Cell Extract 1. Add 0 5 PL of each protease inhibitors aprotmin, leupeptm, and PMSF from stock to 500 PL of whole cell buffer. 2. Add 400-500 FL of whole-cell buffer mto the frozen tissue directly (4.1 buffer to tissue weight; see Note 2) 3. Homogenize with 10 strokes m the dounce homogenizer on ice. 4. Rotate sample m Eppendorf tubes on a Nutator at 4°C for 20 mm. 5. Centrifuge Eppendorf tubes at 13,800g in a microcentrifuge for 15-30 min at 4°C. 6. Plpet the supernatant mto dialysis tube. Clamp the tube and dialyze overnight m buffer 4 with stock solution of DTT and protease inhibitors (leupeptm, aprotinin, pepstatin, and PMSF) 1: 10,000 Stir gently with a stir bar in the container m the cold room (see Note 3).

3.2. Labeling

DNA Probe with Desired Binding

Site (6)

1. Y~~P-ATP (adenosme 5’ tnphosphate) should be ordered fresh and stored in -2O’C freezer. Before use, put the container of isotope in a 37°C hot water bath for 1 h 2 Remove one Sephadex (G-50) spm column (5 Prime-3 Prime) for each sample. Mix resm by inverting column. Allow column to drain by removing top and bottom covers for 5 mm. Centrifuge the column for 1lOOg for 4 min Keep the column 3. End labeling of ollgomers with 32P: in an Eppendorf tube, add 39 FL ddH,O, 2 PL ollgomer 1 75 pmol/pL, 5 PL 10X T4 kinase buffer, 30 ~CI (-3 PL of 10 mCi/mL) f2P-ATP, and 1 FL (10 U) T6kmase (USB) for a total volume of

94


50 FL. Add H20, ohgomers, and buffer first, followed by isotope and then kmase. Incubate for 37°C for 30 mm or longer. 4 Place G-50 column m a collection tube, apply the 50-p.L sample directly to the center of the gel bed, and centrifuge at 1lOOg for 4 min. 5. The labeled nucleic acid will be recovered m the collection tube m approx 50 pL Because >99% of the mcorporated dNTPs will be retained m the column gel, the used column should be discarded m an appropriate radroactive waste container. 6 Four microliters of scmtillation cocktail is added in one scmtillation tube Then pipet 1 pL of labeled probe to the scinttllatton tube and count m Beckman scmtillation counter. The count should be higher then 100,000/mm/scmtillatton tube.

3.3. Mobility Shift DNA-Binding 3.3. I. Polyacrylamide Gel

Assay Using Gel Electrophoresis

1. Make stock solution 45 mL for 5% gel (45 mL is enough for two 16cm-long gels.). 7.5 mL 30% polyacrylamide, 2 25 mL 10X TBE, 35 25 mL ddHaO 2 Wash the glass plates and treat the mside of one plate with Rain-X. (This will aid m removing the gel from the plate, see Note 4) Assemble washed 16-cm-long glass plates and 0.75-mm spacers for casting the gel. Put assemblies m base and secure with black cam. 3. Add 120 uL of 30% ammonmm persulfate and 45 pL TEMED to 45 mL stocking solution, mix gently. 4. Pour the gel mixture between the plates and insert a comb. For optimal results, use a comb with teeth that are 2 7-mm wide. 5. Allow the gel to completely polymerize for approx 30 mm. Remove the comb and rinse wells thoroughly with distilled water. Mark the wells with a marker before they are submerged m the running buffer (see Note 5) 6. Remove the fasteners and disconnect the glass plates from the base. Place the glass plates into the top of the holder of gel running apparatus. Secure with the fasteners. Place the top of runnmg apparatus with glass plates into the top part of electrophoresis apparatus. Then fill the lower reservoir with 0 5X TBE electrophoresis buffer until it covers a little over the button of the glass plates Fill the upper reservoir of the tank with 0.5X TBE electrophoresis buffer. With a needle syringe or a Pasteur pipet, remove air bubbles trapped m the wells of the gel and flush out the wells. 7. Prerun the gel at 100 V for approx 90 mm to equilibrate. At 100 V the gel should mltially draw a current of approx 22 mA, and should decrease to approx 18 mA after 90 mm

3.3.2. DNA-Binding Assay 1. In a microcentrifuge tube, combme the following* a Protein. x pL (volume is calculated from protein concentration). We recommend 20-30 pg of crude protem extract b. Buffer 4. 15 - x l.r.L (Dependent on volume of protein, contain glycerol ). c. 10X reaction buffer 2 yL.

Mobility Shift DNA-Binding Assay

2.

3. 4.

5 6

7.

8. 9. 10. 11.

95

d. Sheared salmon sperm DNA 2 pL (for nonspecific hlockmg). e. Radioactive probe (32P). 1 pL (100,000 counts/p1 as determined from scmtillation counter or 0.5 ng of probe). This will result m 20 pL total volume. Let stand for 15 mm at room temperature Add 2 pL of loading dye (bromphenol blue) m the well to track the sample durmg electrophoresis. Centrifuge if necessary to pull down all the liquid before loading the sample mto the wells (see Note 5). Pipet all of the sample into the loading well with a long loadmg tip. Electrophoresis at approx 20-30 mA until the dye approaches the bottom of the gel. Normally it takes l-2 h. Bromphenol blue (the dye) migrates at approximately the same position as a 70-bp DNA probe. If electrophoresis 1sperformed at room temperature, the glass plates should be allowed to become only slightly warm. Decrease the voltage if the plates become any hotter. Heat may break down complex. For probes c70 bp, do not run the bromphenol blue to the bottom of the gel. To run the gel faster, place the apparatus in a cold room. Higher voltages may then be used without overheating the glass plates. Colder temperature can cause a contraction of the gel, mcreasmg its sievmg properties. As a result, protein-DNA complexes may appear as sharper bands. Remove the glass plates from the electrophorests apparatus and carefully remove the side spacers Using a spatula, slowly pry the glass plates apart, allowing au to enter between the gel and the glass plate The gel should remain attached to only one of the plates. Prying the plates apart too quickly may tear the gel or cause it to stick to both plates If this occurs or If the gel has become distorted, squirt a stream of water underneath it This will reduce the stickiness of the gel. Be careful not to let the gel slide off the plate or tear apart Lay the glass plate (with the gel attached) on the bench with the gel facing up. Place a sheet of Whatman filter paper cut to size on top of the gel (use it to hft the gel off the plate). Support both sides with your hands and carefully flip the sandwich over so that the Whatman paper is on the bottom and the glass plate is on the top. Carefully lift up one end of the glass plate and peel the Whatman paper with the gel attached to tt from the plate. Cover the gel with plastic wrap and dry under vacuum for 10-20 min with a Bto-Rad gel dryer (model 583). The dried gel is placed in a cassette with X-ray film for antoradtograph Visualize the protein-DNA complex after overnight exposure. Adjust the time to get proper exposure and analyze the data by using Bio-Rad Imaging Densitometer. Another way to quantify the radioactivity is to apply a Molecular Dynamics imager analyzer (Sunnyvale, CA) to gel directly.

3.4. Shift- Western

Blotting

(4)

1. After running the EMSA in TBE buffer, electroblottmg of gel shifts is performed. Any suitable electrophoretic transfer apparatus umt can be used, such as

96

2. 3.

4

5.

Xiao and Pennypacker Multiphor II, NovaBlot from Pharmacra (Uppsala, Sweden). Current (mA) 1s fixed at 0.8x the gel surface area (cm2) for 1.5 h Transfer for standard protem-DNA analysis is done at room temperature in 48 mM Tris, 39 mM glycme, 20% methanol, pH 8.5. For transfers, membranes are stacked. The first membrane below the gel is nitrocellulose (BA85, Schleicher & Schuell) followed by a second anion-exchange filter like DEAE membrane (Schleicher & Schuell, Keene, NH) or DE 81 paper (Whatman). Nylon (HyBond, Amersham, Arlington Heights, IL) and polyvmylidene difluoride (PVDF) membrane (Immobilon P, Mrlhpore, Bedford, MA) can also be used. All filters are soaked m water, whereas PVDF membrane 1s soaked m methanol, all filter papers (Whatman 3MM) and towels are equihbrated m the appropriate transfer buffer The different membranes/filters should be separated by a Whatman 3MM paper during the transfer. Radiolabeled components are detected by autoradiography, DE 81 papers are dried for lo-20 min with a Bra-Rad gel dryer (model 583) before autoradiography on a X-ray film. Analysis the data on film by using Bio-Rad Imaging Densitometer. Radioactivity on gels can also be quantified by Molecular Dynamics Image Analyzer using Image Quant software. Proteins are identtfted by tmmunoblottmg on nylon or mtrocellulose membrane, as with a typical Western blot procedure The protems are visualized after mcubation with specific antibody using the ECL Western blottmg detectmg reagents (Amersham cat no. RPN2209) or other detecting reagents.

4. Notes 1. To maintain the DNA-binding activity, tissues are frozen on dry ice immediately after the dissection and stored at -80°C. 2. To keep a better DNA-binding activity, it is recommended that all centrifugation and procedures are performed at 4°C. Buffers and equipment are precooled. Samples should always be placed on ice. All solutions can be stored at 4°C for 1 mo. 3. Protease mhibttors are used during the procedure of whole-cell or nuclear-cell extraction. Dnhiothreitol (DTT) and phenylmethylsulfonyl fluoride (PMSF) must be added to buffers immedtately before use DTT 1sa reducmg agent that is absolutely necessary for DNA binding of transcription factor such as Activator Protein-l 4. To facilitate removal of the gel after electrophoresis, srhcomzed glass plates can be used. Wash the inside of one glass plate with X-rain, Just like treating a wmdshield, so the gel will not stick to it. This is very helpful to separate the plates without tearing the gel apart 5. It may be difficult to see the wells for loading samples Before the glass plates are submerged mto the running buffer, mark the well with a marker. The use of long pipet tips with flat ends also helps to load the sample.

References 1. Riggs, A. D., Suzuki, H , and Bourgeois, S. (1970) Lac repressor-operator mteraction I. Equilibrium studies. J. Mol. Biol. 48,67-83.

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Shift DNA-Bindmg

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2. Frted, M. and Crothers, D. M. (1981) Equilibrium and kinetics of Zac repressoroperator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9, 6505-6525. 3 Ghodosh, L. A., Carthew, R. W., and Sharp, P A. (1986) A single polypeptlde

possesses the binding and activmes of the adenovirus maJor late transcription factor Mel Cell Biol. 6,4123-4133 4. Demczuk, S., Harbers, M., and Vennstrom, B. (1993) Identification and analysis of all components of a gel retardation assay by combmatton with tmmunoblotting. Proc. Natl. Acad. SCL USA 90,2574-2578.

5 Kmgston, R. E , ed (1990) m Current Protocols in Molecular Btology, vol 12, Wiley, New York, pp 12.0.3-12.2.7. 6 Sambrook, J., Fritsch, E. F., and Mamatts, T. (1989) Molecular Cloning A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. E 37-E 38

8

Transfection of Mammalian Cells In Vitro Use in Analysis of Neuronal Damage Melvin L. Billingsley

and Theresa A. Thompson

Introduction Transfection of specific elements of DNA into cultured mammalian cells allows for the analysis of a range of functional and toxicologic mechanisms. At the heart of this technique is the ability to promote the uptake of DNA into actively growing cells, and to detect and analyze the expression of the gene(s) encoded by the DNA (1). The two basic types of transfection analyses are transient transfections, in which the DNA is expressed during the few days postapplication, and stable transfections, in which cells expressing the gene of interest are actively selected via comtroduction of a marker for positive selections (2). Choosing which technique is appropriate for a given experiment is determined by the temporal aspects of the question, the types of assaysperformed, and whether the gene of interest is expressed constitutively or via an inducible promoter. Illustrations for both methods will be provided. The key problem that must be overcome is the barrier posed by the cell membrane toward the uptake of DNA. Initial experiments used naked DNA, which carries a highly negative charge and a relatively large size; these physical factors strongly prevent uptake into cells. Nonetheless, small numbers of cells took up the DNA, and in some cases, incorporated the target DNA into the genome. Since this time, numerous efforts have been directed at increasing the efficiency of transfection. First-step improvements used calcium phosphate coprecipitation of DNA (hydroxyapatite) prior to application to cells; microparticulate material containing DNA was taken up, possibly via pmocytosis, resulting in increased uptake efficiencies (3). It 1simportant to note that little is actually known about the exact mechanisms of DNA uptake, nuclear transport, 1.

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pMSGISV40 small t

Fig. 1. Representative vectors used for mammalian cell transfections. (A) Vector used for the conditional expression of the SV40 small-T antigen. The MMTV-LTR promoter/enhancer responds to glucocorticoids, leading to conditional expression of the SV40 small-T antigen. The plasmid also harbors the ampicillin-resistance gene (Amp) for selective growth in bacteria and the neomycin (Neo)-resistance gene for selection in mammalian cells via G418 resistance. This vector would be used for stable transfections in which the gene product, small-T antigen, is under conditional expression since constitutive expression would inhibit cell growth. (B) Vector that could be used for the stable transfection of a gene product (stannin) under the regulation of the strong constitutive CMV promoter. This plasmid contains genes for ampicillin resistance (Amp) and for neomycin resistance (Neo). The plasmid also has a bovine growth hormone polyadenylation and termination signal (PA) after the gene of interest. This vector would be used for stable transfections in which the gene of interest was continually expressed. (C) Vector that could be used for analysis of transient expression of a gene product. This vector contains the gene of interest (stannin) under the control of a CMV promoter. There is an ampicillin resistance gene (Amp) that allows selection in bacteria. There is no selectable element for mammalian cells.

and integration into DNA. Current approaches rely on incorporation of DNA into cationic liposomes (Lipofectin; Life Technologies, Bethesda, MD) to enhance transfection efficiencies (4). Important parameters must be optimized for each cell type in culture, and include the ratio of DNA to liposomal suspension, cell density, and time of exposure to the DNA-liposomal complex. A major variable is the construction of an appropriate vector for transfection. Three examples of vectors used for transfection are shown in Fig. 1. The first vector represents a construct that could be used for transient transfections. The strong constitutive cytomegalovirus promoter (CMV) is used to express the gene of interest at high levels. The ampicillin gene is used for expansion and selection of the plasmid in bacteria. There is no marker for stable selection of this vector in mammalian cells (5). The second vector is designed for use in stable transfections. The gene of interest is under the control of the strong CMV promoter, but the DNA also contains a neomycin (NEO) resistance gene, which allows for positive selection in G418 antibiotic media. Only cells that

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have incorporated the transfected DNA will survive in this media. Finally, the third vector represents a model construct m which the gene of interest IS under control of a glucocorticold inducible MMTV promoter. This vector would be used for analysis of stable transfectants 2. Materials 1 Mammalian cells, such as PC-12 cells, COS cells, or NIH 3T3 fibroblasts, m exponential growth. 2 Appropriate culture media such as RPM1 1640 (Glbco-BRL, Gaithersburg, MD), Opt+MEM (Glbco-BRL), or Dulbecco’s minimum essential medium (DMEM); serum-free; also DMEM contaming lo-20% fetal calf serum (complete medium) 3. Llpofectm reagent (Glbco-BRL) stored at 4°C. Do not freeze Lipofectm consists of a preformulated stock (1:l) of N-[1-(2,3-dioleyloxy)propyl]-n,n,ntnmethylammonmm chloride (DOTMA) and dioleoyl phosphatidylethanolamme m sterile HzO. 4. Either six-well culture plates or 35-mm2 flasks (Corning, Corning, NY). 5. Sterile polystyrene tubes (15- and 50-mL, Falcon, Los Angeles, CA) The use of polystyrene is highly recommended 6. Cell culture hood and 37°C Incubator with 5% CO,. 7. Reagents for detecting gene expression (antibody; substrates, and so on) 8 G418 antibiotic for stable transformants (Gibco-BRL). 9. The pRC/CMV/neo and pcDNA3 mammalian expression vectors can be purchased from Invurogen (San Diego, CA) 10 Qlagen (Chatsworth, CA) Mmlprep plasmrd isolation kits.

3. Methods 1 Cell lines are maintained m a humidified, 37°C incubator with 5% C02. Cells are plated in 75-mm2 flasks or m multiwell plastic dishes NIH-3T3 (murine fibroblast) cells are grown m Dulbecco’s modified Eagle’s medium with 10% heatinactivated newborn bovine serum (NBS), 1 mM glutamme, 10 mM HEPES; pH 7.4, and pemcillm-streptomycin (1 mg/mL). PC-12 (rat pheochromocytoma) cells are grown m RPM1 1640, 10% FBS, 5% heat-inactivated horse serum (HS). To mduce differentiation, PC12 cells were incubated with 50 nM nerve growth factor (NGF) in RPM11640 with 5% HS. 2. Approprrate mammalian expression vectors are utilized to optimize transfection efficiency Vectors are chosen that contam the CMV enhancer and promoter (a strong, constrtutive promoter); a polyadenylation site; the ampicillm resistance gene, allowmg for selection of bacteria harboring the recombmant plasmid; and, for stable transformations, the neomycin gene conferring resistance to G418, allowing for antibiotic selection-of eukaryotic cells harboring the recombmant plasmid (Fig. 1, see Note 1). 3. The vectors are used to transform the HBlOl strain of Escherlchlu cob. Transformants are plated on ampicillin plates and grown overnight Colonies are picked and screened for insertion of the cDNA of interest using restriction diges-

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6. 7 8. 9

10.

11.

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Billingsley and Thompson tion or Southern blotting. Plasmtd DNA can be isolated by either cesmm chloride density-gradient centrifugatton or by Qiagen munprep ktts. The Qiagen kit is highly recommended, since residual cesmm from DNA purification can interfere wtth mammalian cell vtabihty (see Note 2). Mammalian cells are replated in six-well plates at a density of 2-5 x lo5 cells/ well Cells are then grown m complete media overnight to reach 50-60% confluence. This 1s a good starting point, however, certain cells may respond to transfection better at higher or lower densities. Prepare the DNA-liposome complex. Ftrst dilute plasmid DNA (0.5-2 pg DNA/ mL) into 1 mL serum-free media (DMEM or Opti-MEM). Vortex for 1 s, then add hpofectin (range of 5-10 pL/mL). Incubate for 10 min at room temperature to allow DNA to complex with the cationic liposomes (see Note 3). Aspirate serum-containing media from 50% confluent cells; wash once with 1 mL of serum-free media Add the 1 mL of DNA-hposome complex directly to the cells, mix gently to distribute the complexes across cells, incubate for 3-6 h at 37°C m 5% CO,. To each well, add 3 mL of complete medium (containing serum) and incubate for a total of 48-72 h at 37°C m 5% CO,. At this point, the protocol would shift dependmg on whether transient or stable transfections are being performed, For transient transfections, cells would be harvested via centrifugation, washed, and cell extracts assayed for gene activity or expression Expression assays could consist of either enzyme assays, Northern blots, or Western blots of extracts usmg appropriate anttbodies For stable transfection analysts, after step 8 (48 h after addition of the DNAhposome complexes), the media 1s removed, and Incubated m 2-3 mL of complete media (contammg serum) which also contains 400 mg/mL of G418 antibiotic to select for positive clones. G418 wtll kill mammalian cells that do not express the neomycin resistance gene. After 14 d, individual colonies are Isolated and placed m plasttc multiwell dishes. When these cells reach 80% confluence, they should be transferred to 75-mm2 flasks (subcloning and expansion). G418-resistant clones can now be analyzed for gene expression. If the gene used is under control of a strong constttutive promoter (CMV), then expression should not be condttional and can be analyzed using blotting or enzyme assays If the gene is under the control of an inducible promoter, the approprtate inducer molecule (dexamethasone, 1 m for the MMTV promoter) can be added and expresston examined 12-24 h post induction (see Note 4) As a positive control, cells can be transfected as descrtbed above wtth a commercially available reporter gene construct, such as the pSVneo-LacZ construct In this case, positive transformants will express B-galactosidase, which can be easily detected usmg either htstochemical or spectrophotometric methods (see Note 2) The experimental design will dictate whether the assay of gene expression 1s performed on a cellular basis (histochemistry; immunocytochemistry) or m cell extracts (radiometric, spectrophotometric or blot-derived assays; see Note 5).

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4. Notes 1. The most drffrcult aspect of transfectron is to find the optimal conditions that promote full expression m a specific cell type. It may be necessary to vary the density, amount of DNA-liposome ratio, and the time of cellular exposure to the hposomes in order to fmd conditions that are best 2. Use of parallel posmve controls using commercially available reporter genes allows one to isolate problems that may stem from vector constructron vs transfection protocols 3. The hposomal method can also be used to enhance delivery of antisense oligonucleotrdes mto cells. In this case, significantly more antisense oligonucleotide (lo-100 pg) is allowed to complex with the liposomes. The other procedures are as described above, except that the end-point for assay is a reduction m expression of the gene product targeted by the antisense construct. 4 Care must be taken m the design of transient transfectron experiments m which only a percentage (2-20%) of all cells express the gene of interest. Homogemzalion of cell extracts “normalizes” this difference. However, use of stable transfections allows selection of a cell population which expresses the gene of interest in most cells 5 Other caveats include loss of gene expression over time, because of insertion at fragile sues, or because high-level expression of certain genes causes cellular compensatory mechanisms that either suppress or remove the gene of interest. However, these techniques provide a powerful approach for analysis of genes that affect the responses of cells to toxrcants. Transfections can be used to study genes that both sensrtrze and protect cells from toxicant actrons.

References 1 Krueger, M. (1990) Gene Transfer and Expression. Freeman, New York 2. Borrelh, E , Heyman, R , Hsi, M., and Evans, R. M (1988) Targeting of an inducible toxic phenotype m animal cells Proc. Natl. Acad Sci. USA 85,7572-7576. 3. Chen, C. and Okayama, H. (1987) High efficiency transformatron of mammalian cells by plasmld DNA. Mel Cell Biol. 7,2747-2754. 4. Feigner, R., Gadek, P L., Helm, T. R., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Rmglod, G. M., and Danielson, M. (1987) Lipofectm: a hrghlyefficient lipid-mediated DNA/transfection procedure. Proc. Natl. Acad. SCL USA 84,7413-7417. 5. Sussman, D. J. and Mrlman, G. (1984) Short-term, high efficiency expression of transfected DNA Mol. Cell. Biol. 4, 1641,1642.

9 Detection of Apoptotic Cells in the Nervous System Terri Kagan and Zahra Zakeri 1. Introduction Over the course of an organism’s life, cells divide, grow, differentiate, and die. For many years cell death has been recognized as significant in normal neuronal development. More recently, interest has grown in the mechanisms that regulate both cell death and cell survival in neurons during homeostasis and aging Understanding these mechanisms depends largely on the ability to identify dead or dying cells. Several markers have been identified and developed for the detection of cell death m various tissues. In this chapter, we will present an overview of different approaches used to identify cell death in the nervous system while focusing on a few specific protocols. 1.1. Neuronal Cell Death The importance of cell death in neurons is best exemplified during embryonic differentiation, Here, an initial overabundance of neurons is produced and then “pruned” allowing the final number of neurons to match the size of the target tissue. In the superior cervical ganglion alone, naturally occurrmg cell death results in a loss of up to 50% of the sympathetic neurons (1). The molecules critical to the determination of cell survival or death are target-derived neurotrophic factors, such as nerve growth factor (NGF). NGF is synthesized and released in minute quantities by the dendritic processes on neuronal targets. The message induces a complex set of morphological, biochemical, and functional alterations in the differentiating cell. Exogenously administered NGF can prevent naturally occurrmg neuronal-death during development (2,3). Continual accessto NGF is absolutely necessary for the survival of the developing neuron. It is required to a lesser extent for the maintenance of the mature differentiated neuron (45). Once development is complete, neuronal cell loss From

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is not normally a major factor in either orgamsmal homeostasis or aging, however it is a critical aspect of neurodegeneration caused by trauma or disease. All cells have discrete morphologies that reflect both their origin and physiology activity. These distinctions further influence the production of specific cell-death markers, allowing us to categorize cell death. Cells may die as a result of injury or pathogen infection through a process called necrosis. Necrosis does not require the input of energy or the synthesis of either protems or nucleic acids. Necrotic cells die in large groups as a consequence of compromised cellular ionic homeostasis. They typically swell resultmg in the rupture of cellular membranes and the leakage of cellular contents mto the extracellular space, which leads to a significant inflammatory response (6,7). In contrast to the blatant lytic death characteristic of necrosis, cells may also die via a subtle, physiological death, called programmed cell death (PCD). PCD was first described in a developmental context, yet it appears increasingly to be an essential tool used by many cells when subjected to stress. PCD 1s both genetically regulated and evolutionarily conserved (8,9). It is a tightly regulated process that often requires energy Input and either the synthesis of macromolecules or de ltovo gene transcriptton. Cells may die individually within areas of healthy cells or in synchronous masses(d-10). Cells that die by PCD can also be categorized by distinct morphology and biochemical differences. Apoptotic (type I) cells exhibit DNA fragmentation before any other morphological changes are evident. The DNA fragments range m size m multiples of 180-bp length and are visible by agarose gel electrophoresis as a characteristic “DNA ladder.” There is no conspicuous formation of cytoplasmic vacuoles or vesicles. DNA fragmentation is followed by loss of the nuclear envelope, shrinkage of the cytoplasm, blebbing of the plasma membrane, and coalescence of both organelles and nuclear chromatin. As apoptosis continues, the cell breaks up and forms numerous small apoptotic bodies that contain largely intact organelles. These bodies are rapidly engulfed and degraded by phagocytic scavenger cells (J&11). Both cultured mature sympathetic neurons deprived of NGF and embryonic neurons “pruned” during dtfferentiation die via apoptosis (12-14). Lysosomal (type II) cell death is more frequently observed in secretory cells, generally possessmg relatively large cytoplasms (16-18). These cells are characterized by the early formation of cytoplasmic vacuoles and promment lysosomal activity. In lysosomal cell death, DNA fragmentation appears to be a late event and is evident only in the final stages of death prior to autophagrc degradation (19,20). Despite fundamental distinctions between these varrous forms of cell death, there are also areas of overlap. Both necrosis and apoptosis have been shown to result from either traumatic injury or hypoxia (15,21-23) and apoptotic DNA fragmentation has been observed in cells characterized by prominent lysoso-

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mal actrvity (8,15,18,24). Both apoptosrsand necrosishave been implicated in some forms of excitotoxm- and neurotoxm-induced cell death (25,26), and in disorders as various as neuronal loss caused by ethanol poisoning (fetal alcohol syndrome), senile dementia characterrsticof Alzhermer’s disease (SDAT), cerebral rschemia, and stroke (27-29). Nevertheless, the question of whether PCD always underlies the neuronal loss observed in neurodegenerative diseasesis still unanswered. There are clearly generalized markers and features that can be used to distinguish between the different mechanisms of cell death. The markers described m this chapter have been established by studying cell death m both isolated cells and intact tissue. The identification of dying cells m an in vivo system may prove difficult when a few cells die amidst many hundreds of live cells. In addition, the kinetrcs of cell death in VIVOmay vary such that although a general cell-death signal IS initiated, the time required for the signal to be received and acted on may be very different for individual cells within a population. For these reasons,advances in understandmg the actual processesof cell death on a cellular level have been made by studying the death processin isolated neuronal explants. Culture systems are sources of stable, homogenous cells that can be grown in large number, and can be manipulated both experimentally and genetically to study the effects of various signals in cell death. Despite these advantages, culture systems are limited by the absence of both extracellular matrix and cell-cell interactions and therefore may not manifest the same cellular markers of PCD as those produced m vivo. Thus, a common approach used to evaluate neuronal cell death is to first identify possible mechanisms and markers in cultured cells followed by an examination of postmortem brain tissues for the presence or absence of srmrlar events. Keeping these facts in mind, m this chapter we survey different approaches used to examine programmed cell death in neuronal cells. We will further detail several protocols used to evaluate cell death by examining nuclear and nucleic acid alterations. 1.2. Approaches to Determine 1.2.1. Cellular Morphology

Cell Death

The classical method used to identify dead and dying cultured cells IS a careful examination of cellular morphology using both light and electron microscopes. Early apoptotic cells can be identified by a marginated coalesced chromatin, dilated endoplasmic rettculum, and swollen mitochondria. At later stages, the cytoplasm appears amorphic with few intact mitochondria, and the membranes are blebbed (I&30,31). 1.2.2. Alterations in Membrane Function to Assess Viability Dying cells, whether necrotic or apoptotrc, exhibit alterations in membrane function. A determination of membrane integrity can be made only on fresh,

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unfixed material. The loss of membrane integrity m dead and dying cells allows the preferential uptake of dyes like trypan blue. Ahquots of cells may be briefly stained with a 0.4% solution of trypan blue m PBS (32). We have also found that slightly longer periods of mcubation may be used to stain populations of cultured cells in situ on microscope slides or coverslips (32). Several commercial sources produce convenient and highly efficient kits that assessmembrane viability in cultured cells. One commonly used kit is the Live/Dead ViabihtyKytotoxicity kit from Molecular Probes (Eugene, OR) (33-35). This assay utilizes calcem AM and ethidmm homodimer-1 to differentiate between live and dead cell populations. Calcem AM is a fluorogenic esterase substrate that is hydrolyzed to a green fluorescent product (calcein) m living cells, whereas ethidium homodimer-1 is a high-affinity red fluorescent nucleic acid stain that is able to pass through only the compromised membranes of dead cells. Cells that fluoresce green possess both esterase activity and an intact membrane, whereas cells that fluoresce red are dead. It should be noted that although this kit is sensitive and efficient, it cannot differentiate between necrotic cell death and the various forms of PCD, and it is not useful for in vivo situations. Changes on the cell surface during the early stages of apoptosis can also be monitored. These changes include the translocation of phosphatidylserme (PS) from the interior side of the plasma membrane to the outer leaflet. Efficient and convenient kits from several commercial sources are now available using a Ca2+-dependent phospholipid binding protein known as Annexin V as the basis for identification of the apoptotic phenotype (i.e., Boehringer Mannheim, Indianapolis, IN). The PS binds Annexin V with high affinity during early stages of cell death. Since necrotic cells also lose membrane integrity and expose PS, cells that die via PCD must be differentiated from necrotic cells by the simultaneous application of a DNA stain like propidmm iodide. Apoptotic cells ~111have distinctly demarcated DNA positive bodies, whereas necrotic cells will have lysed. 1.2.3. Measurements of Organelle Function to Assess Viability Biochemical assaysuse alterations m cellular function during PCD as markers of cell death to allow the identification of specific events during this process. Many assay kits are commercially available to test various organelle functions that are known to change durmg programmed cell death. 3-(4,5dimethylthiazol-2-yl)-2,5diphenyltetrazolium (MTT; 5 mg/mL; Sigma, St. Louis, MO) is a water-soluble tetrazolmm salt that yields a yellowish solution when prepared in media lacking phenol red. Dissolved MTT is converted to an insoluble purple formazan when the tetrazolium ring is cleaved by active mitochondrial dehydrogenases in living cells (36). The formazan precipitate can be


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solubilized and quantified spectrophotometrically yielding a measure of absorbency as a function of concentration of converted dye. MTT activity can also be examined in situ in tissuesthat can be labeled in culture, like insect glands (17,37). Lysosomes are another organelle whose altered activity during PCD has been used as a marker of cell death. Lysosomal acid phosphatase activity has been shown to increase during certain forms of cell death and can be measured spectrophotometrically with a simple widely available biochemical assaybased on the hydrolysis of p-nitrophenol phosphate (Sigma cat. no. 181-A). The intensity of color produced is proportional to phosphatase activity and can be used to identify dying cells. Lysosomal activity can also be measured qualitatively and quantitatively by in situ examination under a microscope (17,18). This method can be used for both cultured cells as well as tissue sections. Vital dyes that stain the acidic compartments of dead and dying cells in living tissue, like nile blue sulfate and acridine orange, can also be used to identify areas of cell death by identifying phagocytosed apoptotic fragments (29). 1.24. Nuclear and DNA Fragmentation to Assess Apoptosis Nuclear changes are the most widely used markers of programmed cell death. We therefore will concentrate on presenting in more detail some of the ways in which one can use these alterations for the identification of cell death. These methods have been used m our laboratory to detect programmed cell death in neuronal and nonneuronal tissues. It is important to keep in mind that depending on tissue type and method of fixation, individual methods may not yield satisfactory results. We suggest for definitive results that a variety of complementary methods be used. 1. Detection of fragmented nuclei: During apoptosis the nucleus is broken into

nuclear fragments called apoptottc bodies. One assayused to visualize the fragmentedchromosomespresentm theseapoptoticbodies is the DNA fluorochrome Hoechst 33258, bls benziamide(Sigma) which actsby intercalating between the bases in DNA fragments. Specific methodology for Hoechst staining of fragmented nuclei can be found in Subheading 3.1. (38,39). 2 Detection of fragmented DNA: The fragmentatron of a dymg cell’s nucleus IS typically accompanied by the cleavage of Its DNA into oligonucleosome length fragments that are multiples of 180 bp. These regular-sized fragments are a hallmark of apoptosrs and can be rdentrfred by agarose gel electrophoresis (40). The Subheading 2. below ~111 descrrbe the specific assay conditions used by our laboratory for high-efficiency DNA extraction and radioactive end-labeling (41). Since agarose gel electrophoresis does not provide mformation about indrvrdual cells in a given population or in intact tissues, fragmented DNA may also be examined in situ The process of DNA degradation by endonucleases yields both double- and smgle-stranded DNA breaks, known as nicks. Efficient and convenient kits now available from several commercial sources can detect nicks

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Kagan and Zakeri by enzymatlcally labeling the free, newly generated 3’ OH ends with modified nucleotides by DNA polymerases (nick translation) or with a terminal deoxynucleotidyl transferase (TdT) mediated dyTP nick end labeling (TUNEL) techmque (31,42,43). Methodology for the labeling of apoptotrc nuclei in situ either by fluorescent or nonfluorescent methods (Apop-Tag, Oncor, Garthersburg, MD) can be found in the following section. Other commercially available krts are equally sensitive in detecting apoptotlc DNA fragmentation and these assays may all be performed either on tissue (whether paraffin embedded, frozen fixed, or nonfixed) or on fresh or fixed cells.

2. Materials 2.1. Nuclear Fragmentation in Apoptotic Bodies 2.1.1. Hoechst Bis Benzimide Staining (See Notes 2-4) 1. 2 3. 4

1X PBS: 50 m&I sodium phosphate, pH 7.4, 200 mM NaCl Keep me-cold 3% Paraformaldehyde in 1X PBS. Keep me-cold. Hoechst 33258 stock solution: 16 pg/mL in 1X PBS, pH 7 0. Store protected from light at 4°C This solution IS stable for approx 1 mo

2.1.2. Agarose Gel Electrophoresis

and Analysis of Low-Mob Wt DNA

1. Lysts buffer: 0.2% Trrton X-100, 10 n-r&fTns-HCl, 10 r&4 EDTA, pH 7.5. 2 RNase A. Drssolve 100 mg (Sigma) m 10 mL of 15 rmI4 NaCl, 10 mM TrrsHCl, pH 7 5. Boll at 100°C for 15 mm. Cool slowly to room temperature. Aliquot and store at -20°C for 2-4 mo. 3 Phenol:chloroform:tsoamyl alcohol (24.24: 1) Protect from light. Chlorofonnlsoamyl alcohol (24.1) 4. 5 MNaCl. 5. TE buffer: 10 nuI4 Tris-HCl, 1 mM EDTA, pH 8 0 6 10X loading buffer (0.25% bromphenol blue, 0.25% xylene cyanol, 25% Ficoll [type 400 in dHZO]). 7. 1X TAE running buffer: 0.04 M Tris-acetate, 0.002 M EDTA. 8. 2 g Agarose in 100 mL 1X TAE. 9. Ethrdrum bromide (Sigma).

2.2. Agarose Gel Electrophoresis and Analysis of Low-MobWt 2.2. I Low-MO/- Wt DNA Extraction

DNA

1. Lysis buffer. 0.2% Trrton X-100, 10 m&I Trrs-HCl, 10 nuI4 EDTA, pH 7.5. 2. RNase A. Dissolve 100 mg (Sigma) m 10 mL of 15 mM NaCl, 10 m&f Trts-HCl, pH 7.5. Boil at 100°C for 15 mm Cool slowly to room temperature. Aliquot and store at -20°C for 2-4 mo. 3. Phenol:chloroform:rsoamyl alcohol (24.24: 1) Protect from light 4 Chloroform lsoamyl alcohol (24: 1) 5 5MNaCl.

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6. TE Buffer 10 mM Tns-HCl, 1 mM EDTA, pH 8.0. 7 10X loading buffer* 0.25% bromphenol blue, 0.25% xylene cyanol, 25% Flcoll (type 400) in dH20.

2.2.2. Agarose Gel Electrophoresis 1. 1X TAE runmng buffer. 0 04 M Tns-acetate, 0.002 M EDTA. 2. 2 g Agarose m 100 mL 1X TAE 3. Ethichum bromide (Sigma).

2.3. Radioactive 1 2. 3 4 5. 6. 7. 8. 9. 10.

End Labeling

of Low-Mob Wt DNA

Low-mol-wt DNA. 5X dTd buffer (Boehrmger Mannhelm). Cobalt chloride (Boehrmger Mannhelm). 32P ddATP (Amersham, Arlington Heights, IL, 10 mCl/mL). Terminal transferase (Boehrmger Mannheim). 500 mM EDTA, pH 8.0. Phenol:chloroform.isoamyl alcohol (24:24:1). Protect from light. 3MNaAc. Glycogen (10 mg/mL). TE buffer.

2.4. TUNEL Labeling: In Situ DNA Fragmentation 2.4. I. Specimen Preparation 1. 2. 3. 4 5. 6 7. 8 9.

Detection

4% Paraformaldehyde. 1X PBS: 50 mM sodium phosphate, pH 7.4, 200 mM NaCl Ethanol*acetic acid 2 1 PAP pen (Fisher, Pittsburgh, PA) Xylene. Cryostat. Slide oven. Microscope slides. Humidified chamber (see Note 1).

2.4.2. Nonfluorescent Detection of In Situ DNA Fragmentation (Apop-Tag Peroxidase) 1. 1X PBS 50 mM sodium phosphate, pH 7.4, 200 mM NaCl. 2 Hydrogen peroxide solution: 2% H20, in 1X PBS. It is very important that this solution be freshly made. 3 Equillbratlon buffer (Oncor cat. no S7100-1) 4. TdT stock enzyme (Oncor cat no. S7100-3) 5. Reaction buffer contaming dlgoxlgemn- 1 l-dUTP (Oncor cat. no S7100-2) 6 35X stop/wash buffer (Oncor cat no S7 100-4) 7 Antldlgoxlgenm peroxldase (Oncor cat no S7100-5).

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8. DAB (diammobenzrdme) solution: This is prepared by combmmg 0.015 g DAB, 400 mL DMSO, 50 mL 1X PBS and spinnmg for 1.5 h Immediately prior to use, the DAB IS filtered and 10 PL Hz02 added 9. Methyl green: 0.5% methyl green in 0.1 M NaAc, pH 4.0. 10 Toluene.

2.43. Fluorescent Labeling of In Situ DNA Fragmentation 2.4.3 1. DIRECT ASSAY (APOP-TAG-DIRECT) 1. 2. 3. 4.

1X PBS. 50 mM sodium phosphate, pH 7.4, 200 mM NaCl. Equilibration buffer (Oncor cat no. S7160-1). TdT stock enzyme (Oncor cat no S7160-3). Reaction buffer containing fluorescem nucleotide (Oncor cat no S7160-2), 35X stop/wash buffer (Oncor cat no S7160-4) 5. Propidium iodide/antifade (Oncor cat no. S1370-6).

2.4.3 2. INDIRECT ASSAY (APOP-TAG PLUS) 1. 2 3 4 5. 6 7 8. 9. 10.

1X PBS: 50 mM sodium phosphate, pH 7 4,200 mM NaCl 1X PBST: 1X PBS + 0 1% Tween 20. Equilibration buffer (Oncor cat. no S711 l-l) TdT stock enzyme (Oncor cat no S7111-3) Reaction buffer contammg digoxigenm- 11-dUTP (Oncor cat no. S7 11 l-2) 35X stop/wash buffer (Oncor cat. no. S7 11 l-4) Blocking solution (Oncor cat. no. S7 11 l-5) Antidigoxigenm-fluorescem (Oncor cat. no. S7111-6). 0.1% Triton X-100 m PBS (May be stored at 4°C for 1 mo). Glycerol (90%)

3. Method 3.1. Nuclear Fragmentation

in Apoptotic

Bodies

1. Collect cells from culture flask and centrifuge for 10 min at approx 1OOOg. Remove supernatant and wash pellet m ice-cold 1X PBS. 2 Centrifuge cells for 10 mm, remove 1X PBS, and resuspend pellet m 200 l.rL fresh, ice-cold 3% paraformaldehyde. 3. Incubate on me for 6 mm. 4. Microfuge for 3 min at 4°C. Remove paraformaldehyde supernatant. Resuspend pellet m 40-50 PL Hoechst solution (see Note 2). 5. Incubate completely covered at room temperature for 25 mm. 6 Microfuge at room temperature for 5 mm at 5OOOg 7. Gently remove the Hoechst solution, leaving the pellet intact. 8. Add 40-100 PL 1X PBS and resuspend the pellet Cell suspensions may be stored at 4°C in a light-protected box for up to 2 wk 9 Apply 20 PL of cell suspension to microscope slide, apply a coverslip, and immediately examme by fluorescence microscopy (see Notes 3-8)

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3.2. Agarose Gel Electrophoresis and Analysis of Low-Mob Wt DNA 1. Wash cells (at least 3 x 106) m 1X PBS. 2. Lyse cells in 500 pL lysis buffer and transfer samples to Eppendorf tubes (see

Note 9). 3. Incubate on ice 15 mm 4. Centrifuge at 4°C at 12,OOOg for 20 mm. 5. Transfer the supernatant that contains the low-mol-wt DNA to a new Eppendorf tube and add RNase A to a final concentration of 100 pg/mL. 6 Incubate m a water bath at 37°C for 1 h. 7. Add 500 pL phenol chloroform.isoamyl alcohol (24 24: 1) to each sample 8 Vortex to mix the two phases. Centrifuge at room temperature for 3 mm. Remove the top layer that contains the low-mol-wt DNA and place m a new Eppendorf tube. 9. Repeat steps 7 and 8. 10. Add 500 ltL of chloroform*tsoamyl alcohol (24: 1). 11. Repeat step 8 12 Precipitate the DNA overnight at -20°C by adding 25-30 yL of 5 M NaCl (final concentration 300 mM) and 2 5 vol 100% ethanol. 13. Centrifuge at 12,000g for 30 mm at 4°C. 14 Gently pour off the NaCl solution checking to ensure the DNA pellet remams mtact. 15. Gently add 1 mL 75% ethanol, pouring away from the side where the pellet is located so you do not dislodge the pellet. 16 Centrifuge at 12,000g for 10 min at 4°C 17 Pour off ethanol and invert Eppendorf on paper towels m a hood for at least 15 mm or until completely dry 18. Resuspend the DNA m 18 pL of 1X TE by fhckmg the tube a few ttmes Mtcrofuge for 5 s to get all the liquid to the bottom of the tube. (For DNA end labelmg, resuspend m 20 pL TE buffer) 19 Incubate in water bath at 45°C for 15 min. 20. Put DNA samples on ice and add 2 l.tL 10X loadmg buffer. 21. Melt 30 mL 2% agarose Add 1.5 pL ethidmm bromide (0 5 l.rLWmL of agarose gel), swirl, and pour into gel apparatus. Place gel comb to form wells (see Note 10). 22. After gel hardens (20-30 mm) remove gel comb and add enough 1X TAE running buffer to Just cover the entire gel. Make sure wells are located at the anode end of gel apparatus 23 Gently ahquot mdtvidual DNA samples to wells, using one well per sample Run DNA at approx 80 V for about 1 h or until the dye 1s two-thuds of the way across the gel (see Notes 11-15).

3.3. Radioactive

End Labeling

of Low-Mel-Wt

DNA

1. Make reaction mixture For each sample of DNA (equivalent to 20 pL) add 10 pL 5X dTd buffer, 5 p,L CoCl, 0.5 pL 32P ddATP, 1 pL terminal transferase, and 13 5 pL dH20, which will yield a total volume of 50 yL/sample (see Note 16).

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2. Incubate reaction mixture at 37°C for 1 h. 3. Stop the reaction by adding 5 PL EDTA to each reaction tube and heating at 70°C for 10 mm. 4. Extract once with 600 I.IL phenol:chloroform*lsoamyl alcohol, taking care to remove the upper layer to a clean tube. (The phenol may be back extracted to ensure that all extracted DNA 1srecovered.) 5 Precipitate the DNA overnight at -20°C with 1: 10 vol sodmm acetate, 1 FL glycogen, and 2 5 vol absolute ethanol. 6. Mlcrofuge at 12,000g for 30 mm at 4°C and wash the DNA pellet m 75% ethanol 7. Dry DNA pellet and resuspend m 30 pL TE buffer. 8 Use 1.5pL of DNA to run a 2% agarose gel. 9 Dry agarose gel at 60°C for 4 h and expose gel to film overnight (see Note 18)

3.4. TUNEL Labeling Tissue on slides may be fixed or unfixed, frozen or paraffin embedded, and cells in culture may be fixed as suspensions or grown directly on slides. 3.4.1. Specimen Preparation For unfixed tissue cryosections or for cells cultured on slides, slides should be processed as follows: 1. 2. 3. 4. 5

Fix with 4% paraformaldehyde (m PBS) for 6-10 mm. Wash in 1X PBS twice for 5 mm/rinse Post fix in ethanol: acetic acid 2.1 for 5 mm at -20°C. Dram but do not allow to dry Wash slides m 1X PBS twice for 5 mm/rmse at room temperature Circle the area of tissue sectlon or cells with PAP pen (Fisher).

For paraffin-embedded slides, slides must be deparaffmed as follows: 1, 2. 3. 4. 5. 6.

Prewarm xylene at 60°C for at least 30 mm. Incubate shdes m prewarmed xylene twice for 10 mm each time at room temperature. Wash slides m 100% ethanol twuze for 5 mm/rmse Wash slides m 50% ethanol twice for 5 mm/rinse. Wash slides m 1X PBS once for 5 mm. Circle the area of tissue sectlon or cells with PAP pen (Fisher).

For cell suspensions: 1. 2. 3. 4.

Fix cells with 4% paraformaldehyde (in PBS) for 6-10 min Aliquot 50-100 yL of cells onto a microscope shde and allow to dry. Wash slide twice in 1X PBS for 5 mm/rinse. Circle the area of tissue section or cells with PAP pen (Fisher).

3.4.2. Nonfluorescence Detection of In Situ DNA Fragmentation (Apop-Tag Peroxidase) 1. Rinse sections twice in 1X PBS for 5 mm/rinse. 2. Quench endogenous peroxldase with hydrogen peroxide solution for 20 mm at room temperature (see Note 20).

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3 Rinse secttons twice m 1X PBS for 5 mm/rinse. Gently tap off excess hquid and blot around sectron. 4. Apply equilibration buffer (13 pL/cm2), cover with plastic coverslip and mcubate for 10 min (up to 30 min) in a humidity chamber at room temperature 5 Prepare working strength TdT enzyme. Calculate how much enzyme is needed to completely cover specimens using 10 pL/cm2. Mix one drop TdT stock enzyme with two drops reaction buffer to yield 108 FL of solution. Vortex mixture well. If desired, a greater quantity of solutton can be prepared but solution should be used immediately and may be stored on ice no more than 6 h. 6 Dram slides of equrhbratton buffer by gently tapping off. Blot plasttc coverslips dry to reuse. Apply workmg strength TdT enzyme (10 pL/cm2), cover again with plastic coverslips and incubate for at least 60 min in humidity chamber at 37°C (see Note 21). 7. Prepare working strength stop/wash buffer by mixing 1 mL 35X stop/wash buffer with 34 mL dH20 This solutton may be stored at 4°C for up to 1 yr. 8. Prewarm stop/wash buffer to 37°C for 30 min. Place slides in prewarmed stop/ wash buffer and incubate for 30 mm at 37°C Agitate slides by dipping in and out of buffer once every 10 mm. Blot coverslips dry to reuse. 9. Rinse three times in 1X PBS for 5 mm/rmse. Tap off slides. 10. Apply antidtgoxygenm peroxidase to section (13 pL/cm2), recover with plastic coverslips, and incubate m a humidity chamber for 30 min at room temperature. 11 Rmse four times m 1X PBS for 5 min/rmse. 12 Introduce slides to DAB solutton m a cophn Jar at room temperature and stain for 20 mm 13 Wash three times with tap water for 1 min/wash 14. Wash with dHzO for 5 mm. 15. Counterstain with methyl green for 5 mm. 16. Rinse slides with dH20 twice agitating each slide 10 times up and down per rinse. 17. Rinse slides with dH,O for 30 s without agitation. 18. Rinse slides m butanol twice agitating each slide 10 times up and down per rinse. 19 Rinse slides in fresh butanol for 30 s without agitation. 20 Dehydrate slides in toluene three times for 2 mm/rinse. 21. Do not let slides dry. Immediately mount using perrnount (Fisher) and glass covershp.

3.4.3. Fluorescence Labeling of In Situ DNA Fragmentation 3.4.3.1.

(see Notes 20 and 27)

APOP-TAG-DIRECT (SEE NOTES 22 AND 23)

1. Calculate how much enzyme is needed to completely cover specimens using 10 pL/cm2. Prepare working strength TdT enzyme by mtxmg one drop TdT stock enzyme and two drops reaction buffer to make 108 pL (see Note 24). Vortex mixture well. If desired, a greater quantity of solution can be prepared but solution should be used immediately and may be stored on ice no more than 6 h. 2 Prepare working strength stop/wash buffer by mixing 1 mL 35X stop/wash buffer with 34 mL dHzO. This solutron may be stored at 4°C for up to 1 yr.

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Kagan and Zakeri Rinse shdes twice m 1X PBS for 5 mm/rinse Gently tap off excess hquld and carefully blot around sections. Apply eqmhbratlon buffer (13 pL/cm2) directly to specimen, cover with a plastic coverslip, and incubate for 5 mm in a humidity chamber. Tap off eqmhbratlon buffer. Rinse and dry plastic covershps. Blot around sections and apply working strength TdT enzyme (25 yL/cm2) (see Note 25). Cover sections again with plastic coverslips and incubate for at least 60 mm m humidity chamber at 37°C. Prewarm stop/wash buffer to 37°C for 30 mm before next step Remove plastic coverslips. Place slides in CoplinJar containing prewarmed stop/ wash buffer and incubate for 30 mm at 37’C Agitate slides by dlppmg m and out of buffer once every 10 mm (see Note 26). Rinse and dry covershps. Tap off liquid Rinse three times m IX PBS for 3 mm/rmse Apply Oncor propidmm lodide/antlfade Mount under glass coverslip. If storage 1s required, apply clear nail pohsh to the edges of the coverslip. Store at -20°C m the dark.

3.4.3.2

APOP-TAG PLUS

1. Calculate how much enzyme 1s needed to completely cover specimens using 10 yL/cm2. Prepare working strength TdT enzyme by mixing one drop TdT stock enzyme and two drops reaction buffer to make 108 pL. Vortex mixture well If desired, a greater quantity of solution can be prepared but solution should be used immediately and may be stored on ice no more than 6 h. 2 Prepare working strength antldlgoxlgenin fluorescem by mixing 56 pL blocking solution with 49 FL antidlgoxlgemn-fluorescem. Vortex well Since this solution 1s light and temperature sensitive, it must be covered with aluminum foil and can be stored on ice no more than 3 h. 3. Prepare working strength stop/wash buffer by mixing 1 mL 35X stop/wash buffer with 34 mL dH20. This solution may be stored at 4°C for up to 1 yr 4 Rinse slides twice m 1X PBS for 5 min/rmse Gently tap off excess liquid and carefully blot around sections Take care that slides do not dry out during the following steps 5 Apply equlhbratlon buffer (13 yL/cm*) directly to specimen, cover with plastic cover slip and incubate for 5 min m a humidity chamber Tap off eqmhbratlon buffer Rinse and dry plastic coverslips 6. Blot around sections and apply working strength TdT enzyme (25 pL/cm2) Cover sections again with plastic covershps and incubate for at least 60 mm m humidity chamber at 37°C Prewarm stop/wash buffer to 37°C for 30 mm before next step. 7. Remove plastic cover slips. Place slides m CophnJar containing working strength stop/wash buffer Agitate slides by dipping m and out of buffer and incubate for 10 min at room temperature. Rinse and dry covershps. 8. Tap off liquid and gently blot around sections Rinse three times m 1X PBS for 3 min/rmse. 9 Apply 30 PL antldigoxlgenm-fluorescem to sections on slides Cover with plastic coverslips and incubate m humidified chamber for 30-45 mm at room temperature.


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10. Remove coverslips and wash three times m 1X PBS/Triton X for 5 mm per wash 11. Mount with glycerol and cover slip. Store protected from light at 4°C.

4. Notes 1 A humidified chamber IS required for the incubation steps in these assays To construct this chamber, any clear plastic tray wrth lid may be lined wrth paper towels that have been soaked m water Slides are placed transversely across two ptpets that are arranged on top of the paper towels. 2 Since bzs benzrmide fluoresces under UV radiation, nuclei with condensed chromatin or nuclei that are fragmented into smaller dense bodies will appear bright and are considered apoptotic Nuclei with uncondensed and evenly dispersed chromatm will appear less bright and are considered alive or nonapoptotic 3 Hoechst stammg is quick and inexpensive and gives few false positive tdentificattons. However, tt can only be used to identify cells m late stages of cell death in which fragmented nuclei are present 4, Hoechst staining is most useful for tdentifymg cell death m cells mamtamed m ttssue culture. However this protocol may be modified to identrfy cultured cells in sztu on slides Slides should be briefly washed with ice cold PBS and fixed with 3% paraformaldehyde The Hoechst solution is then pipetted directly onto the slides and left for 25 min Followmg incubation the slides are again washed. A cover slip 1s applied and clear nail polish is used to seal the edges and prevent excess evaporation This method might be tried with thm sections of tissues, however smce bzs benztmtde does not usually penetrate well mto layers of tissuestt is not likely to be a very good method for quantitative m vtvo ttssue section studtes. 5 Hoechst staining may not be uniform. Sometimespopulations of cells may appear to contam both bright and dim cells. This usually indicates that assaycondmons were not optrmum Reagents may not have been sufficiently cold, or may not have been adequately mixed into cell suspension. 6 Stammg may be too intense. Because the degree of staining is relative to the length of mcubation in Hoechst stock solution, it may be necessary to vary the length of mcubatton time 7 It is important to examme completed shdesas quickly aspossible.Since Hoechststainedcells are light sensitive any excessexposure, even to standardindoor hghtmg, can causethe cells to photobleach, obscuring identificatton. 8. When doing in sztuHoechst staining, the reverse side of the cultured shdesshould be carefully wiped to remove all traces of solutions (i.e., media, PBS, formaldehyde, or bzsbenzimide) otherwise the solutions will dry and crystallize. This may interfere wtth viewmg the specimens. 9. Fresh tissuemay be analyzed by this method after it is lysed overnight at 37°C m PBS contammg sodium dodecyl sulfate (SDS) and proteinase K (I 0 mM Tns, pH 7.8, 5 mM EDTA, 0 5% SDS and 0 6 pg/pL protemase K). Frozen ttssue cannot be usedfor this assay, asthe freezing processwill causesomeadditional degree of DNA fragmentation.

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10. In this method, the fragmented DNA 1s first isolated and then stained by ethidmm bromide. Smce this compound is highly carcmogemc, extreme care should be taken to prevent contact with skin or eyes. There are also several very efficient, less hazardous nucleic acid stains commercially available (for example SYBR Green I and II and SYBR Gold from Molecular Probes) These probes should also be used as directed by the manufacturer. 11. A profile of DNA isolated from tissues undergoing PCD should show signs of characteristic DNA “laddermg” when run on a 2% agarose gel This umformly spaced “ladder” represents the fragments of DNA that are roughly 180 bp m lengths. A mmimum of 3 x lo6 cells must be used to ensure sufficient fragmented DNA, depending on the tissue type, circumstances of cell death and kinetics of cell death in mdivrdual cells withm a population, the number of cells needed may vary and a larger population of cells may be required. 12 A large band at the top of the gel may be evident after electrophoresis. This band represents large, semifragmented pieces of DNA and indicates mcomplete apoptotic fragmentation m the sample material. 13. In some instances, electrophoresis of total cellular DNA is effective and ~111 result in a good fragmentation ladder. Nevertheless we prefer to extract only the low-mol-wt DNA for electrophoresis. Smce cells whether m tissue or m culture are not homogenous m the kmetics of their death, separating the low mass DNA from the total DNA can greatly increase the sensitivity of this assay. 14 Occasionally after a gel is run, there is no evidence of DNA. The DNA may have been lost during the extraction process, or it may have been extracted but lost before electrophoresis as a result of DNA leaking out of the wells m the agarose gel Such leakage can occur if too much DNA is loaded mto a lane or if there is a high ratto of high-mol-wt DNA in the extract. To ensure that extracted DNA enters the gel and is not lost, the gel apparatus may be filled halfway with buffer and run on low power (5-10 V) while the DNA is being added to the wells. The electromc current will pull the DNA into the agarose and after the DNA has entered the gel, the apparatus may be completely filled with buffer and run at normal voltage If the ethidmm bromide is forgotten when the gel is prepared, the DNA will not be visible by UV illumination However, the entire gel may be stained with a solution of ethidmm bromide (1 10,000 m 1X TAE) for 1 h on a shaker, followed by destammg m 1X TAE if necessary at the completion of the run, to visualize the fragmented DNA. 15. When the DNA of a cell is completely fragmented, the DNA laddermg effect appears as a smear. Thts may represent either a necrotic event mvolvmg complete lysis of the cell and its contents, or DNase actrvity and contamination in the extractlon process. 16. The sensitivity of the assay may be further increased by labeling the free 3’-ends of the fragmented DNA with either radioactive (15,37,39) or nonradioactive probes. Although we have not described their use, less hazardous nonradioactive probes may be used to end label the fragmented DNA before electrophoresls.


17.

18.

19

20

21.

22

These probes are currently available from several commercial dtstributors and should be used as directed by the manufacturers. The methods descrtbed here label all free OH- groups regardless of their derivation. Although the presence of these groups 1s usually indicative of DNA fragmentation during programmed cell death, it may also denote active DNA synthesis. Consequently tt is possible that cells labeled by the TUNEL method are undergoing DNA synthesis and are not actually dying. Although this is an uncommon situatton, one ttssue m which this false-positive label IS prevalent IS the labial (salivary) gland of the tobacco hornworm (3744). In this tissue polyplord cells exhibit extensive DNA synthesis, which results m a strong positive signal. In cells that are not polyplord there are fewer chromosomes and thus fewer free OH- groups are detected It 1s very important to have both positive and negative control slides for each assay. Negative controls should be run as duplicates of each section. For this purpose, sham staining can be performed by substituting distilled water for the TdT enzyme m the stammg procedure. For positive controls, cell death should be induced by using a known cell killer. Alternatively, positive control slides may be created by adding varying concentrations of DNase directly to sections, which results m DNA fragmentation similar to that caused by apoptotlc cell death. The labeling targets of this assay are the free 3’-OH DNA ends generated during DNA fragmentation and typically localized to the apoptotrc bodies. Residues of digoxtgemn nucleottde (dtgoxtgemn- 1 I -dUTP) are catalyttcally added to these DNA ends by TdT The antidrgoxrgemn antibody fragment subsequently carries a conjugated peroxrdase enzyme to the reaction site where tt generates an intense signal from chromogemc substrates Although the sensitivity of this assay 1s lower than that of the fluorescence assays presented next, the signal IS permanent and more specific In addition, this assay creates less background noise and does not require the use of a flourescence microscope Occastonally, the stammg characteristic of this assay is masked by excess background staining This may result from endogenous peroxidase activity mterfermg in the assay reaction. Such endogenous activity is quenched with the freshly made HzOz solution described m the assay. An addrtronal precaution taken to avoid excess background is the use of frequent rinses. It is important to be aware that the timing of wash conditions may vary for different tissue types. Preliminary studies on your material should evaluate the exact timing of each step Some tissues or cells may be more or less sensitive to TdT labeling If a light postttve staining 1s found on nonapoptotrc cells in addition to a strong posittve staining on apoptottc cells, the TdT enzyme may need to be further drluted or the length of mcubatron time may need to be shortened The indirect fluorescent method utilizes similar steps for identifying DNA fragmentation as described above for the nonfluorescence peroxtdase assay In the first step, the free 3’-OH- ends of the fragmented DNA are extended with restdues of dtgoxigenm nucleottde (dtgoxrgenin-1 l-dUTP). In the second step, rmmunohrstochemrcal staining of the extended DNA with antrdigoxrgenm anti-

120

23.

24

25.

26.

Kagan and Zakeri body carries a fluorophore (fluorescem) to the reaction site. The labelmg targets of the direct assay are the free 3’-OH- DNA ends generated durmg DNA fragmentation that are typically localized to the apoptotic bodies Residues of fluorescem nucleotide are directly added to these DNA ends by the catalytic activity of TdT. Regardless of whether the assay is direct or indirect, when excited by light of 494 nm, fluorescem generates an intense signal at 523 nm This signal may be evaluated by either fluorescence or confocal microscopy Using a direct fluorescence assay ensures a good rate of cell recovery because there are few mampulations. However, it is not as sensitive an assay as the indtrect method Indirect fluorescent labeling results m a more sensmve staining of apoptotic DNA fragmentation. However it also produces both higher background and higher signalto-noise ratios. False positive results are more commonly produced by this method when compared to either nonfluorescent or direct fluorescent methods Some tissues or cells may be more sensitive to TdT labeling and specimen variables, such as tissue type or length of fixation, can affect the length of mcubation time required Therefore if a light posmve stammg is found on nonapoptotic cells m addition to a strong positive staining on apoptotic cells, the TdT enzyme may need to be further diluted or the length of mcubation may need to be shortened Uneven or spotty stammg may result when too httle reagent is used, or when reagents are not apphed evenly Care should be taken that reagents entirely cover the surface of the specimen. One precaution taken to avoid excess background is frequent rmsmg. It is important to be aware that the timing of wash conditions may vary depending on speclmen vartables. In addition, although this assay does not use peroxidase to label DNA fragmentation, we have found that quenching endogenous peroxidase activity with a solution of 0.3% H20, m methanol before Subheading 3.4.3.1., step 3, or Subheading 3.4.3.2., step 4, followed by several washes in 1X PBS may lower the background stammg

Acknowledgments We thank Richard Lockshin, Malki Blelcher, Claudette Davis, and Chrrstopher Paoloni for critical reading of this manuscript. We also thank R. Halaby, N. Karasavvas, H. Smgh-Ahuja, T. Latham, and Y Zhu for their roles in developing the different methods described m thts work. This work was supported by the National Institute of Agmg Grant K04-AGO031 (to Z. Z.) and the Alzheimer’s Allied-Signal Foundation Pilot Research Grant (to Z. Z.).

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36 Mossman, T. (1983) Rapid colonmetric assay for cellular growth and survrval. apphcatton to proliferatton and cytotoxtcity assays.J. Immunol. Methods 6555-63. 37 Jochova, J , Zakeri, Z., and Lockshin, R. A. (1997) Early collapse of the cytoskeleton in the programmed cell death of the Drosophila salivary gland Cell Death Dzff.5, 140-149. 38. Oberhammer, F. A, Pavelka, M., Sharma, S., Tiefenbacher, R., Purchto, A F., Bursch, W., and Schulter-Hermann, R (1992) Induction of apoptosis m cultured hepatocytes and in regressing hver by transforming growth factor pl Proc. Natl. Acad. SCL USA. 89,5408-5412.

39 Karasavvas, N., Erukulla, R. K., Btttman, R., Lockshm, R., Hockenbery, D , and Zakert, Z (1996) Bcl-2 suppresses ceramide induced cell killing. Cell Death Dzff. 3,149-151. 40. Sambrook, J , Fritsch, E F , and Mamatis, T. (1989) Molecular Cloning A Luborutory Manual, 2nd ed. Cold Sprmg Harbor Laboratory, Cold Sprmg Harbor, NY. 41 Karasavvas, N., Erukulla, R K., Bittman, R., Lockshin, R , and Zakeri, Z. (1996) Stereospectftc induction of apoptosts m U937 cells by N-octanoyl-sphmgosme stereoisomers and N-octyl-sphmgosme. Eur. J, Biochem 236,729-737 42 Wijsman, J H., Jonker, R R , KetJzer, R., Van de velde, C J., Cornehsse, C J , and van Dierendonck, J H. (1993) A new method to detect apoptosrs m paraffin sections’ m situ end labeling of fragmented DNA. J Hzstochem. Cytochem. 41,7-12. 43. Gorczyca, W , Gong, J., and Darzynkiewrcz, Z. (1993) Detection of DNA strand breaks m individual apoptotic cells by the in sztu terminal deoxynucleotldyl transferase and nick translation assays. Cancer Res. 53(8), 1945-1951. 44. Jochova, J , Quaglmo, D , Zakeri, Z , Woo, K., Sikorska, M , and Weaver, V (1998) Protein synthesis, DNA degradation and morphological changes during programmed cell death m labial glands of Manduca sexta. Devel. Genet. 21, 249-257.

Translocation Assays of Protein Kinase C Activation Ginger G. Wescott, Christina

M. Manring, and David M. Terrian

1. Introduction The protein kmase C (PKC) family members include at least 11 different rsoforms that, based on their different requirements for actrvatton, have been divided into three subfamthes, the Ca*+-dependent (cPKCa, PI, &, and r), the Ca2+-independent (nPKCG,&,q,B, and CL),and phorbol ester-insensitive (aPKC< and r, the human counterpart of mouse PKCh) subfamilies. Much research on this growing family of protein kinases has concentrated on the possibility that these enzymes may have assumed distinct responsibilities for the control of complex and diverse cellular functions. The current working hypothesis is that the differential sensttivlty of PKC isoforms to endogenous agonists and their differential targeting to discrete subcellular domains may dictate what substrates are phosphorylated by a given isoform. For this reason, an important initial goal in the analysis of the connection between PKC activation and a cellular response, such as neurodegeneration, is to identify the endogenous PKC isoforms that become “membrane-associated” in response to an approprtate sttmulus. Translocatlon assays have classically been used to screen the individual isoforms of PKC for the production of active PKC-membrane complexes. The principal criteria used to distinguish between inactive and active forms of PKC are that, m the later case, the solubility of the enzyme IS reduced when intact cells are treated with an appropriate agonist (e.g., phorbol esters) and that the associatron of a given PKC isoform with the particulate fraction resists extraction with Ca*+-chelators, but not nonionic detergents. The method described below is an extension of that previously described by Kazametz et al. (I), m which cellular homogenates are initially separated mto cytosohc and particulate fractions. The partrculate fraction is then extracted using the nonFrom

Methods

m Molecular

Medune,

vol

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Edlted by J Harry and H A Tllson

Neurodegeneration

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ionic detergent Trrton X-100, to separate the detergent-soluble (membrane) and msoluble (“cytoskeletal”) fractions Many rsoforms of PKC have been reported to become associated with cytoskeletal protems upon activation (2) and, using thus approach, we have recently shown that PKCE IS an actin-bmdmg protein (3).

2. Materials

2.1. Subcellular Fractionation 2.1.1. Equipment and Supplies 1. 2 3 4. 5

Sonicator. Refrrgerated ultracentrrfuge with mrcrocentrrfuge rotor. Hot plate. Spectrophotometer. Mmrocentrrfuge tubes wrth screw-on caps

2. f 2. Reagents 1. Homogemzatron buffer. Trrzma base (20 mM, pH 7.4), EDTA (10 n&f), EGTA (2 rmI4), P-glycerophosphate (100 m&I), phenylmethylsulfonyl fluoride (PMSF, 0.05 mg/mL). Store at 4°C PMSF is toxic and has a extremely short half-life m this buffer (9.5% of the cells phenotyplcally express GFAP (9,101, enabled the whole pantheon of experimental techniques of neuroscience and cell brology to be brought to bear on the study of astrocytic cultures. Over the last two decades numerous critical concepts regarding astrocyte speciahzation, functional heterogenerty, and the complexity of astrocyte-neuron mteractlons have emerged from these studies (4,11-24). Primary astrocyte cultures have been found, not surprismgly, to also be a potent tool for predicting and studymg the mechanisms of central nervous system (CNS) toxicity. Although a viable tool both m elucidating neurotoxlc phenomena and in aiding reciprocally in the search for astrocytic function, how fatthfully these cultures mnnic the state of affairs in VIVOremains an unresolved issue, since in culture they are devoid of integrative functions with other CNS cells (neurons, oligodendrocytes, microglia, Just to name a few). Although admittedly still imperfect, coculture methods have provided additional information about integrated responses. For example, studies on glutamate-induced neuronal toxicity in astrocyte-poor vs astrocyte-rich cocultures underscore the importance of astrocytes m both the normal physrology of glutamate, as well as in their ability to markedly attenuate glutamate-induced neuronal toxicity (15). Therefore, conditions that alter astrocyte function (hypoxia, hypoglycemia, metabolic poisoning, and so on) might be expected to manifest themselves in glutamate-mediated neuronal mJury and death, and suggest that the sensmvrty of neurons to glutamate cytotoxicity IS dependent on astrocytic integrity. Thus, cocultures are of greater heuristic value in probing cell-cell interactions, both in health and disease.

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The first section of thus review will describe the methodology associated with neonatal rat cortical astrocyte cultures The method described for astrocytic isolation and culturing is largely based on that described by Frangakis and Kimelberg (IO). This is followed by description of a cortical mixed neuronal-glial culturing method. 2. Materials 2.1. Asfrocyte Cultures 2.1.1. SupplIes 1. Stertle filter, Nalgene filter flask with 0.2~pm pore size m a membrane (Nalgene [Rochester, NY], cat no. 158-0020). 2. Cellulose acetate syringe filter with 0.2~ym pore size (Corning cat no. 21052-25). 3 Conical 15-mL polystyrene centrifuge tubes (Corning, cat no. 4. loo-mm culture dishes (Cornmg, cat no. 25020-100). 5. Six-well plates (Corning, cat. no. 25810-6). 6 60-mm dishes, Falcon easy-grip tissue-culture dish (Beckton mouth, UK], cat. no. 3004). 7. 18 x 18-mm cover shps (Cornmg, cat. no. 2875-18).

cellulose acetate [Corning,

NY],

253 11-15).

Drckmson

[Ply-

2.7.2. Reagents 1. 2 3. 4. 5. 6. 7. 8. 9. 10

Protease (Sigma [St. Louts, MO], cat. no. P-3417). Complete MEM (Grbco-BRL [Grand Island, NY], cat. no. 61100-061). Jokhk, Jokltk-modified MEM (Grbco-BRL, cat. no 22300-024). Penictllm-streptomycin (Gibco-BRL, cat. no 15140-015) Deoxyrtbonuclease I from bovine pancreas type IV (Sigma, cat no. D-5025). Trypan blue (Gibco Chemical [St. Lotus, MO], cat. no 630-5250AG). Heat-inactivated horse serum (Gibco Chemtcal, cat. no. 26050-021). Phosphate-buffered salt (PBS) (Sigma, cat. no P-4417). Primary antibody for GFAP (Dako [Carpinterta, CA], cat. no. 20334). Secondary antibody for GFAP staining (Biosource [Camarillo, CA], cat no. AL10408). 11. Joklik medium: With a stir bar, add 800 mL sterile Milh-Q water to a clean beaker Empty contents of one Gibco premtxed powdered medmm packet and rinse the packet thoroughly for any residual powder. Mix solutions and allow the powder to dissolve (it should dissolve immediately). Add 2 0 g NaHCO,/L medium. Gently stir until dissolved (1 min) AdJust the pH to 0.2-0.3 pH units below the desired pH (7.4) with 1 N HCl or 1 N NaOH. Add penicillin and streptomycm (10,000 U/mL and 10,000 p.g/mL, respectively), since the Jokhk mix does not contam antibiottcs. Add Milli-Q water to 1 L and stertltze through a 0.2+m filter (Nalgene) using a penstalttc pump The solution may be stored at 4°C for up to 2 mo m sterrle 500~mL bottles, Add pemcillm and streptomycin (10,000 U/mL and 10,000 pg/mL, respectively), since the Jokhk mix does not contam anttbtotics.

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12. DNase preparatton: Reconstitute DNase (deoxyrtbonuclease I from bovine pancreas Type IV) powder with Milli-Q water. Mix gently The final concentration should be 4 mg DNase powder/ml water. Mix gently, and brmg up to the final volume. Filter through a 0 2-pm pore cellulose acetate syringe filter (Corning) Before filtering the DNase, rinse the filter itself with sterile Mtlh-Q water. It eliminates any detergents on the filter. Store at -20°C m ahquots of 1 mL The solution may be stored for up to 3 mo. 13 Dispase preparation: Dtspase (Protease, Sigma) works optimally at 2.5-3.0 U of activity per milhhter of Jokhk It is the optimal protease concentration m terms of yteld, vtabihty, and conservation of the enzyme (10). The solutton is stable and It may be stored at 37°C for several months

2.2. Neuronal Cultures 2.2.1, SupplIes and Equipment 1 Plastic Petri dishes (60-mm) (Corning, cat no. 25010-60) 2. Plastic Petri dishes (35mm) (Corning, cat. no. 25000-35). 3. Sterile filter, Nalgene filter flask with 0.45~pm pore size (cellulose acetate membrane) (Nalgene, cat. no. 155-045). 4. Conical 15-mL polystyrene centrifuge tubes (Fisherbrand [Atlanta, GA], cat. no 05-539-2). 5. Lab-Tek tissue culture slides, 8 chamber, (Nunc [Naperville, FL], cat. no. 177402) 6 Dissecting microscope (or hghted 4X magnifying lamp). 7 Miniblade scalpel knife (Roboz [Rockville, MD], cat no. RS 6270). 8 Pasteur pipets (borosihcate-coated, fire-polished; see Note 1) 9. Centrtfuge (Sorvall [Newton, CT], GLC-2) 10 Sterile pipet tips. 11. Adjustable pipets 20, 100, 200, and 1000~pL 12 Hemocytometer (Ftsherbrand, cat. no. 0267110) 13 Inverted-phase contrast microscope

2.2.2, Reagents 1. Trypan blue (Gibco-BRL, cat. no. 630-5250AG) 2. DMEM (Dulbecco’s modified Eagle’s medium), 4.5% glucose, no glutamme (Gibco-BRL, cat. no. 1000687). 3. F12 nutrient supplement (Gibco-BRL, cat. no 11765-054). 4. Fetal bovine serum (Gtbco-BRL, cat. no 26050-088). 5 EBSS (Earle’s balanced salt solution) (Gibco-BRL, cat. no 14015-069). 6. OlMPBS 7. Vectastain Elite Kit (Vector [Burlmgame, CA], cat. no. Pk6102), mouse IgG. 8. Paraformaldehyde fixative (4%) in PBS. 9. Microtubule-associated protein-2 (MAP-2) antibody (1: 10,000); personal grft, but available from various commercial sources 10. Diammobenzidme (DAB) (Sigma, cat. no. D-5905)

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11 Hydrogen peroxide (30%) (Sigma, cat. no. H-1009). 12 Alcohols (50, 70, 95, and 100%) for dehydrating and xylene for defattmg. 13 Permount (Sigma) for cover slippmg (Sigma, cat. no. SP15-100).

3. Methods 3.7. Astrocyte Cultures 3. I. I. Tissue Collection and Cell Preparation 1. All procedures are carried out m a laminar-flow culture hood. Remove the cerebral hemispheres of l-d-old newborn rats (Sprague-Dawley), employing a sterile technique. Remove the cerebellum, midbrain, and brain stem and discard Remove the meninges expedltlously, yet carefully and completely (as best as possible) with the aid of a dissecting microscope. Place the cortices m ice-cold serum-free Jokhk medium (m a 50-mL conical tube) until the desired number of brams is collected Then pour the Joklik mechum off and chop the cortices with sterile scissors For the next 10 mm, stir the cortices at low speed (approx 60 rpm) m 10 mL Jokhk medium plus protease (Dispase [Sigma]; 2.5-3 0 U/mL Joklik), at 37°C This leads to dlssociatlon of the cells (see Note 1) 2. Aspirate 10 mL of the solution containing the dissociated cells from the beaker and place in a pair (5 mL m each) of sterile conical centrifuge tubes (Corning, 15-mL polystyrene centrifuge tubes, cat. no. 2531 l-15), each containing 5 mL of mmlmum essential media (MEM) Add 10 mL of Dlspase in Joklik and three drops of deoxyrlbonuclease II (Sigma cat. no. D-5025, 4 mg/mL) with a sterile Pasteur pipet to the tissue remaining m the beaker containing the cortlces Deoxyribonuclease II 1sadded only once (after the first extraction) and 1snot repeated thereafter (1.e , add only 10 mL of Dispase in Jokhk). Each such step (removal of 10 mL of dissociated cells and placing 5 mL each in a pair of conical tubes containing 5 mL MEM each) is referred to as an extraction. Repeat this procedure 5-S times until only residual, fibrous-looking tissue remains m the beaker. 3. Process the individual extractions independently. Each of the extractions 1s initially left undisturbed until any undlssoclated tissue has settled to the bottom of the tube (10 mm). Remove this undlssociated tissue by careful aspiration with a Pasteur pipet and discard rt (or place back into the ongoing extraction) Spin each cell suspension at 400g for 10 min m a bench centrifuge. Pour off the supernatant and resuspend the cell pellet in 3-4 mL of fresh, complete MEM and allow to set for 5 mm Again, during this stage, any cell clumps and/or undissoclated tissue that settle out during the resuspension are discarded. 4. Next, pool the resuspended cells in the mdlvldual conical centrifuge tubes, and dilute a small ahquot (0.5 mL) of the resuspended cells 1:l with trypan blue (20% [v/v] of 0.4% staining solution). The number of viable and nonviable cells 1s determined with a hemocytometer. 5. Then adJust the cell density to 1 x lo4 cells/ml and plate at a density of l-2 x lo4 cells/cm2 m plastic tissue-culture dishes, multiwell plates, or covershps (see Subheading 2.1.1, for types of plates and covershps that are used m our lab).

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Aschner and Bennett Grow the cells m 5% C02/95% an humidified atmosphere at 37’C, and change the media the next day, and thereafter twice weekly

3.2.2. Immunocytochemical Staining for Glial Fibrillary Acidic Protein (GFA P) 1. At 3-4 wk m culture, a time in which the cells reach confluence, stain the monolayers growing on coverslips for GFAP. The procedure is carried out with each coverslip mdividually m a six-well plate Prior to fixation, each coverslip is washed three times with PBS/Ca2+/Mg2+ (8.1 mM Na,HPO,-7H,O, 138 mM NaCl, 1.2 mM KH2P04, 2 7 mM KCl, 0.5 n-u!4 MgCl,-6H,O, 0.9 mM CaCl, anhydrous) at 37°C. 2 Then immerse the cells for 5 mm m chilled methanol at -20°C and then rinse three times m ice-cold PBS contammg 140 mM NaCl, 3.5 mM NaH2P04, 12 mM Na2HP04 at pH 7.25. 3 Block the cells with 80 PL of 10% normal horse serum m PBS for 20 mm in a humidified chamber. 4. Wash the cells three times with PBS and incubate for approx for 30 mm in 1 mL of a 1.100 dilution of polyclonal rabbit anticow GFAP (1”) in PBS. All antibody solutions are made with 10% horse serum m PBS at room temperature. 5. Wash the cells again three times in me-cold (4°C) PBS 6. Apply 1 mL of goat antirabbit IgG (2” antibody) conjugated with fluorescem and diluted 1:20 m PBS for 30 mm. 7. Rinse the coverslips three times in PBS at room temperature, mount on slides with Vectashield (Vector, cat no. H- lOOO), and store at 4°C protected from light 8. Establish controls by staining m the absence of the primary antibody, and adhermg to the remainder protocol. View the cells through a microscope at 2040x magnification (see Note 3).

3.2. Mixed Neuron-Astrocyte Ceil Culture 3.2.1. Tissue Collection and Cell Preparation 1. Obtain timed-pregnant rats The rats used m this procedure were 14-15 d pregnant, although they can be up to 16 d gestation and still utilize this dispersion protocol. Sacrifice the pregnant mother and obtain the rats by cesarean section. Typically, a pregnant rat will have 8-14 fetuses Remove the fetuses from the uterus and place in cold, sterile EBSS. In a sterile environment (lammar flow or horizontal flow hood), dissect the cortices on 60-mm sterile Petri dishes and then place m filtered DMEM m 35mm Petri dishes. Transfer the cortices to a 15-mL conical centrifuge tube and rinse twice with sterile DMEM (2 mL each). Then triturate the cortices with a large-bore pipet (approx 8-10 triturations). After settling for approx 30 s (there should be a small amount of fibrous-looking material), transfer the supernatant to another 15-mL tube. Then, add 2 mL of DMEM to the undispersed tissue left in the tube and repeat the process with a pipet having a slightly smaller bore Repeat this procedure another time and each time add 2 mL to the undispersed tissue remammg in the tube Cap the second tube (hold-


139

ing the supernatant) and centrifuge at a very low speed (400g) for 5 min (see Note 2). 2 After centrifugatron, there should be a very loose pellet m the bottom of the centrifuge tube Remove the supernatant and add 2-8 mL of DMEM to the tube (this varies depending on the number of fetuses that were obtained). We typically add approx 6 mL to a tube that contains cells derived from 40 cortices. Then gently triturate the tissue with a sterile large-bore pipet to resuspend the loose pellet Next, measure the density of the cell suspension (i.e., number of cells per volume). We generally place 90 uL of DMEM m an Eppendorf and add 10 pL of cell suspension (1: 10). This is diluted 1: 1 with trypan blue. Place a small amount of this on a hemocytometer under an inverted microscope and count the number of viable, round cells per 16-grid square (trypan blue IS excluded from viable cells). If 200 cells are counted, your suspension contains 400,000 cells per 10 pL of suspension. For plating onto the astrocytic cultures, the number of cells added will depend on the size of the dish as well as the desired final density. For example, using 60-mm dishes, one would typically add 3-5 million cells (or 75-150 PL of the above-described suspension). After plating, place the cultures in the incubator. 3 Add this mixture of neuronal-gha cells to pure astroglia cultures (>95% astrogha) that have been maintained for at least 3 wk. The mixed neuronal-ghal cells will rapidly adhere to the astrocytic layer of cells and one can readily ascertain that the density is dramatically increased. These cultures, because they are so dense, can only be maintained for 7-10 d because of the dimmution m neuronal integrity after this time point. Therefore, experimental analyses should ensue 4-5 d after addition of the neuronal cells and no later than 10 d after addition

3.2.2. lmmunocytochemical for Microtubule-Associated

Staining Protein (MAP)-2

1 Prior to fixation, wash each coverslip three times with PBS/Ca*+/Mg*+ at 37’C. Fix the cells for 30 mm at 4°C in 2% paraformaldehyde in ice-cold (4’C) 10 mM HEPES-buffered, 1 mM CaC12 solution, pH 7.4. Then immerse the cells for 5 mm m chilled methanol at -20°C and then rinse three times m me-cold PBS, pH 7 25. Then block the cells with 80 pL of 10% normal horse serum in PBS for 20 mm m a humidified chamber Subsequently, wash the cells three times with PBS, and incubate with MAP-2 antibody (1: 10,000) added to the slide dishes for approx 16 h and then rinse with PBS (2X). 2. Follow the Vectastain protocol utilizing a mouse reagent kit since the primary antibody is a mouse monoclonal antibody (16). The chromogen, diaminobenzidine (DAB), is made up from a 10 mg pellet of DAB obtained from Sigma that is placed into 30 mL of PBS. Make a solution of hydrogen peroxide from a 30% concentrate by dilutmg 1 mL of 30% hydrogen peroxide into 100 mL of dH,O From this diluted solution of hydrogen peroxide, add 150 FL to the DAB solution. Then titurate this working solution for several minutes to ensure that the DAB pellet is thoroughly broken up and dissolved. This DAB solution should not be made up more than 30 mm ahead of time

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3 After the Vectastain procedure IS completed, cover the tissue with approx 100 pL of the DAB solution and immediately observe with the inverted microscope Stammg is allowed to proceed for approx 2 mm, and the DAB solution 1s removed. Rinse the tissues twrce with PBS and then coverslip. Discard the DAB solution by adding bleach to the mixture (which turns the entire solution black) and then pourmg down the drain with the water runnmg to ensure proper dilution of the neutralized solution A typical neuron-astrocyte coculture stained for MAP-2 and GFAP, respectively, is shown m Fig. 1.

4. Notes 1 Always start with clean, sterile tissue-culture ware This includes beakers, graduated cylinders, stir bars, sctssors, and any other surgical equipment Use only sterile, double-distilled Milh-Q, or some other highly pure water. All tissue culture ware should be washed prior to the procedure with Cytoclean (Islolab, Akron, OH) After washing and drymg all the tissue-culture ware should be autoclaved. If cells are grown on covershps, the latter should also be autoclaved. 2. For neonatal dissections, once the CNS IS exposed, with curved forceps, pinch off the olfactory bulbs. Note: Pull cortices up and out shghtly on top, to avoid retammg the olfactory bulbs Gently slip the curve of the forceps under each cortex and pull up while moving the forceps from one side to the other (generally beginning where the cortices Jam and move to the outer edges) This will unfold the cortices and separate them from other structures Do not separate the cortical hemispheres Gently pull both cortices (both hemispheres) up and away from the remamder of the brain and place on sterile gauze under a dissectmg mrcroscope. With curved forceps, gently unfold the corttces. If there is a large section of CNS tissue on the outer edge(s) that is not cortical, remove it. With watchmaker forceps, gently tease away the menmges. This must be done quickly, but gently and thoroughly. If too many menmges remain, they will lead to fibroblast contaminants in the cultures. A good rule of thumb is to spend approx 10 mm per brain removing the menmges. With curved forceps, gently loosen tissue from the gauze and flip the cortices This step is qmte tricky. Flip the edge closest to you upward and slightly back. However, do not squash the tissue onto the gauze When the hippocampal crescents are visible, remove the hippocampus with curved forceps Tease away any remaining memnges with watchmaker forceps Roll the cortices together gently with curved forceps and lift Place m 10 mL Joklik-modified MEM (in a sterile lOO-mL beaker) on ice until all the brain dtssections have been completed 3. This procedure 1s to isolate astrocytes from any other CNS region. However, as cellular yield will not be as high as from the cortlces, you will have to increase the number of dtssections. To mcrease cellular yield you may also increase the number of extractions 4. Bactertal neutral protease (Dispase) extends the dtssocratron time of the cortices, but it appears to be milder, allowmg for more controllable and reproducible dissoctation vs other techniques, such as trypsnuzation, where the drssociation

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Fig. 1. Neuron-astrocyte coculture system: Freshly isolated neurons were plated on 3-wk-old cultured astrocytes, The cocultures were maintained for 1 wk prior to fixation and staining. The cells were fixed with 4% paraformaldehyde for 30 min. (A) Fluorescence staining of astrocytes for GFAP. (B) Neuronal networks (dark perikarya and fibers), corresponding to the neuron-specific staining for MAP-2 visualized with DAB. Images represent the same fields at 500x. For further details, refer to the text. period is very short. Both Dispase (IO) and trypsin dissociation (6,17,18) result in comparable cell yield, but the Dispase-dissociated cells appear to have higher viability (10). Cell-growth rate and cell homogeneity appear also to be very reproducible among the two methods (IO). Mechanical dissociation (cell filtration through nylon sieves) may also be used to isolate astrocytes (6). However, it is more coarse and stressful to the cells, reducing both the cellular yield and cell viability.

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5. The average yield & SD m cells/brain and cell viabihty for cultures prepared according to the above method is 5 + 1.4 x lo6 cells and 78.3% f 2 0, respectively (10) 6. Adherence of Isolated astrocytes to glass cover shps is markedly increased by pretreatment of the glass with poly+lysine (Srgma, cat. no. P-1274). Start by preparing a borate buffer. Add 1.24 g boric acid (Gibco-BRL, cat. no 15583-016) and 1.9 g borax (Sigma, cat. no B-9876) to 400 mL of Mrlli-Q water, and pH to 8.4 Add 25 mL of borate buffer to 25 mg of poly-L-lysine. Store as stock m a refrigerator (10X stock of poly-L-lysine) Dilute 1: 10 with borate buffer for workmg concentration and sterile filter Place the sterile cover slips mdividually m six-well plates and add poly-L-lysine (approx 1 mL per well) Place the dishes overnight at 4°C. Next, rmse the plates three times with sterile dHzO. 7. The cultured cells are sparse during the first few days of the culturing procedure. As they divide, they form clusters of spread and flattened cells. They can, however, assume many morphological characteristics, dependent on the environment m which they are cultured Growth m serum-free medium (hormone supplemented), treatment with fibroblast growth factor, treatment with agents that stimulate mtracellular CAMP levels, as well as direct contact with neurons (cocultures), Just to name a few, lead to morphological changes where the cells transform from a spread, flat morphology into a stellate shape (19). The cultures also contam occasional neurons, macrophages, and microglial cells (these may be identtfied by specific staining techniques). The addition of dibutyryl cyclic adenosme monophosphate (dBcAMP), final concentration 0.25 mM can be employed to arrest cellular division. It should be noted, however, that this treatment causes profound changes m cell morphology as well as biochemical parameters. The treatment may also be used to remove cellular impurities, such as the macrophages (20). If the aim of your studies 1s to evaluate the effect of extraneous variables on cell morphology it is imperative that control and treated cells grow m the same medium (1.e , same serum concentration, dBcAMP, and so on) You may also need to consider that upon treatment changes in astrocytic morphology may not be directly correlated with the extraneous variable, but rather with changes m the composmon of the medium, such as mcreased or decreased concentrations of endogenous molecules, factors secreted by astrocytes, or any other cellular impurities within the culture 8. Because of the complexity that IS associated with the isolation and culturing procedure, it is apparent that occasionally thmgs ~111go awry. As a rule of thumb, if the cells appear granular in their morphology, or do not attam confluency within 3-4 wk postisolation, it is wise to discard them. Despite our meticulous and cautrous approach to the astrocytrc rsolation and culturmg, we have had to deal with “extraneous” contammants. Most recently, we were confronted with a mycoplasm contamination If you suspect contamination (bactertal, viral, or of any other source), it is recommended that a media sample be sent to a certified microbiology lab for analysis. Accurate characterizatron of the contaminant is likely to save you hardship m the long run, and will greatly facilitate the speedy re-establishment of your cultured cells.


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9. A note of cautton! It seems premature to correlate astrocytes m culture to astrocytes zn situ. At this pomt, it remains unclear if cultured astrocytes correspond to their type 1 or type 2 astrogha m vtvo It is likely that both of these types corresponds to cultured astrocytes, and that the type 2 astrocytes reflect the plasticity of astrocytes m vitro, but do not have a counterpart in VIVO during normal development (18) 10. The best means for provldmg staining of the cocultures is to utilize a specialized dish for the rmmunocytochemrcal studies. We utilize a tissue-culture slide (made by Lab-Tek, available from Nunc) that is adapted with slide-well chambers varying from 2 to 16 total chambers. We typically use the eight-chamber slide wells. This allows one to utilize several different antibodies on one slide dish, which makes comparisons more efficient After the fixation and staining is complete, the upper chamber unit is removed as well as the gasket, and the slides are then dehydrated and cleared like other slide preparations (i.e , 50, 70, 95, and 100% ethanol, followed by xylene rinses) and cover slipped. 11 Pasteur pipets, cotton-plugs added, are obtained from Fisher (cat no 13-678-20B) and fire polished by placing them in an open flame (bunsen burner) for a few seconds. This should just be enough time for the end of the pipet to begin to close off. This is done for 30-50 prpets, producing ptpets with varying size openings. Each of these is then coated with Sigmacote and dried (we use an automated pipeter to attach the pipet to and draw up Slgmacote into each pipet). We then place these ptpets m steel camsters (one each for small-, medium-, and largesized bores) designed for autoclavmg pipets, seal them with autoclave tape, and autoclave. These are then placed in the hood and opened only under sterile conditions. Pipets should be placed m the canisters such that the tips are m the bottom and you touch only the other end when choosing one. Gloves should always be worn when handlmg them smce it IS imperative that they be kept sterile.

Acknowledgments This review was partially supported by grants from PHS, ES 0733 1 (M. A.), DA 05073 and DA 10647 (B. A B.), and the U. S. Envrronmental

Protection

Agency (EPA) R-819210 (M. A.). References 1. Vuchow, R. (1846) Ueber das granulierte Ansehsn der Wandungen der Gehirnventnkel. Allg. 2. Psychiatric 3,242-250 2. Somjen, G. G (1988) Nervenkltt: Glia 1,2-9.

notes on the history of the concept of neurogha

3 Lugaro, E (1907) Sulle Funzioni Della Nevrogha. Riv. D. Par. Nerv. Ment. 12, 222-233. 4 Kimelberg,

H. K. and Aschner, M. (1994) Astrocytes and their functions, past and present, m National Institute on Alcohol Abuse and Alcohohsm Research Monograph 27, Alcohol and Gllal Cells NIH Publication No. 94-3742, Bethesda, MD, pp. l-40

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5. Kuffler, S. W., Ntcholls, G., and Orkand, K. (1966) Physrologrcal properties of ghal cells m the CNS system of amphtbia. J. Neurophysiol. 29,768-787. 6. Booher, J. and Sensenbrenner, M (1972) Growth and culttvatron of dtssoctated neurons m glial cells from embryonic chick, rat and human brain in flask cultures Neurobtology 2,97-105. 7 Eng, L. F , Vanderhaeghen, J. J , Brgnami, A., and Gerstl, B. (1971) An acidic protein isolated from fibrous astrocytes Brutn Res. 28, 351-354 8. Brgnami, A. and Dahl, D. (1976) The astroglial response to stabbing: rmmunofluorescence studtes with antibodies to astrocyte-specific protein (GFA) m mammalian and submammahan vertebrates Neuropathol. Appl. Neurobzol 2,99-l 10. 9. McCarthy, K. D and De Vellis, J. (1980) Preparation of separate astroglial and ohgodendroghal cell cultures from rat cerebral tissue. J. Cell Bzol. 85,890-902. 10. Frangakis, M V and Krmelberg, H. K (1984) Drssoclation of neonatal rat brain by dlspase for preparation of pnmary astrocyte cultures. Neurochem. Res. 9,16891698 11. Ktmelberg, H K. and Norenberg, M. D. (1989) Astrocytes Scz. Amer. 260, 66-76 12. Abbott, N J , ed (1991) Gltal-Neuronal Interactzons Annals of the New York Academy of Science, New York, pp. 663. 13. Murphy, S , ed (1993) Astrocytes. Pharmacology and Functton. Academic, New York 14 Aschner, M and Kimelberg, H K., eds (1996) The Role of Glta in Neurotoxtctty CRC, Boca Raton, FL. 15 Rosenberg, P. A. and Arzenman, E. (1989) Hundred-fold increase m neuronal vulnerabthty to glutamate toxtctty m astrocyte-poor cultures of rat cerebral cortex. Neuroses. Lett. 103, 162-168. 16. Vallee, R B., Bloom, G S., and Luca, F. C. (1986) Differential structure and drstributton of the high molecular weight bram mtcrotubule-associated proteins, MAP-l and MAP-2. Ann. NYAcad. Set 466, 134-144 17. Kimelberg, H. K., Narumi, S., and Bourke, R S. (1978) Enzymatic and morphological properties of primary rat brain astrocyte cultures, and enzyme development zn vivo Brain Res. 153, 55-77 18. Manthorpe, M , Adler, R., and Varon, S. (1979) Development, reactivity and GFA immunofluorescence of astroglia-containing monolayer cultures from rat cerebrum J. Neurocytol 8,605-62 1. 19. Levlson, S. W. and Goldman, J. E (1993) Astrocyte ortgms, m Astrocytes Pharmacology and Functton (Murphy, S., ed ), Academic, New York, pp. l-22 20 Hertz, L., Juurlmk, B. H J., Szuchet, S , and Walz, W (1985) Cell and tissue culture, in Neuromethods 1: General Neurochemtcal Techntques (Boulton, A A and Baker, G B , eds ), Humana, Chfton, NJ, pp. 117-167.

12 Viability Assays for Cells In Vitro The Ethiclium/Calcein Assay and the lmmunofluorescence

Combination Assay

Theresa A. Thompson 1. Introduction In neurotoxicology, studies have become more sophistrcated and focus more on changes evident at the cellular and molecular level rather than on wholeanimal morbidity. For this and other reasons, in vitro model systemsare being mcorporated into neurotoxicrty screens and mechanistic studies. The use of in vitro systems allows for the isolation and culturing of cells specifically targeted by neurotoxrcants and for the determination of cell viability following treatment wrth a neurotoxrcant. A reliable m vitro cell viability assay IS a critrcal component of any drug or neurotoxrcant study (I). The two viability assays described in this chapter include the ethidium/ calcem viability assay and the ethidium/calcein viabrlrty assay combined with double-label immunofluorescence. For the ethidium/calcein viability assay, cells are incubated with ethidmm homodimer and calcein-AM. Ethidium homodimer enters the nuclei of cells with damaged membranes and undergoes a 40-fold enhancement of red fluorescence upon binding nucleic acids (2). Calcem-AM is hydrolyzed by mtracellular esterases within live cells to yield the green fluorescent product, calcein (3). Calcem and ethidium homodrmer are excited in the range of 485-500 nm; fluorescence is visualized using a fluorescein filter set (4). Live cells generate an intense, uniform green fluorescence, whereas the nuclei of dead cells fluoresce red. A log dose-response curve IS generated from the cell-vrabrllty data, and the toxrc concentration that kills 50% of the cells (TC,,,) 1s determined by linear extrapolation (5). This assay can be used with cell lines and with primary cells. The advantages of this assay From

Methods III Molecular Medrcme, vol 22 Neurodegeneratron Methods and Protocols Edited by J Harry and H A Tkon 0 Humana Press Inc , Totowa, NJ

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include the srmultaneous visualization of live and dead cells and the rapid quantrtation of live and dead cells. Thus assay IS particularly useful for homogeneous cell populations. The ethidmm/calcem viability assay combined with double-label immunofluorescence IS effectively used with cells growing m a mixed population and 1s comprised of two separate procedures. One set of cells is incubated with ethidium homodimer and calcem-AM in order to ensure that cells remaining on the cover slip are alive, and a parallel set of cells is incubated with cell typespecific primary antibodies and fluorophore-tagged or chromogenic secondary antibodies. The surviving cells are identified and quantitated according to cell type. A log dose-response curve can be generated using the numbers of survrvmg cells, and the toxic concentratron that kills 50% of the cells (TC,,) can be determined by linear extrapolation (5). There are three major differences between this combination assay and the one-step ethidium/calcein viability assaydescribed above. First, with the combinatron viability assay,the cells are not harvested before incubation with ethidium homodimer and calcein-AM; therefore, cell disruption is minimal. Secondly, only live cells are visualized m the combination assay. Thirdly, the cells are identified and quantrtated via rmmunofluorescence with the combmation viability assay. The advantages of the combinatron viability assay include: mimmal cell disruption, maintenance of good cell morphology, rdentrfrcation of cell phenotype in a mixed population, and persistence of cell rmmunofluorescence for several weeks postassay. The combinatron vrability assayis especrally useful in primary cultures derived from neonatal rat brain that are maintained as a mixed population of neuronal and glial cells. The combination viability assay is also useful when testing selective neurotoxrcants that may target one cell type m a mixed populatron. This assay is ideal when harvesting the cells is not desirable or feasible. Both of the viability assays described m this chapter are easily performed using readily available materials. Furthermore, both assaysyreld highly reproducible results and are superior to other commonly used viability assays, including trypan blue dye exclusion (4) and the LDH release assay. Necessary information concerning the toxicity of a particular drug or toxrcant can be generated from the cell quantrtatron data, including log-dose response curves and TC5a values. 2. Materials 2.1. EthidiumXalcein

Viabilify Assay

1 Ethldmm homodlmer and Calcem-AM. Components of the Llve/DeadTM Vlablllty/Cytotoxlclty Assay avallable from Molecular Probes (Eugene, OR) (4). Store stock solutions at -2O’C in the dark The diluted solution should be prepared fresh and kept in the dark for each assay, it cannot be stored

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2. Dulbecco’s phosphate-buffered saline (Dulbecco’s-PBS): 200 mg/L KCl, 200 mg/L KH,P04, 8000 mg/L NaCl, and 1150 mg/L Na2HP0, in dH*O. Prepare thts buffer using 500 mL dHzO, sterile filter mto a 500 mL bottle using a 0.2ym bottle top filter, and store at 4°C. Dulbecco’s-PBS 1s a modification of standard PBS and does not contain Ca2+, Mg2+, or a colored pH indicator. Do not use standard PBS because it will interfere with the assay. 3. 25-mm sterile glass cover slips. Soak in ethanol and pass through a flame or autoclave to sterilize these coverslips.

2.2. EthidiumKalcein Viability Assay and Double-Label lmmunofluorescence 1. Hanks’ balanced salt solution (HBSS) with Ca2+ and Mg*+: sterile, cell-culture grade buffer (10X) stock solutron available from Gibco-BRL (Gaithersburg, MD). Store at 4°C. 2. HBSS without Ca2+ and Mg*+: contams no calcium chloride, magnesium chloride, magnesium sulfate, or sodium bicarbonate; sterile, cell-culture grade buffer; (10X) stock solution available from Gibco-BRL. Store at 4°C 3 Sodturn pyruvate (100 mM solution)’ standard cell-culture grade buffer (100X) sterile stock solution available from Gtbco-BRL. Store at 4°C. Final concentration is 1 mM sodium pyruvate in HBSS 4. HEPES buffer: standard, sterile cell-culture grade buffer. Store at 4°C. Final concentration IS 10 mA4 HEPES in HBSS 5. NeurobasalTM medmm: (1 X) liquid medium specifically formulated for culturing primary neuronal cells m serum-free medmm; available from Gtbco-BRL Store m the dark at 4°C 6. B-27 supplement* serum replacement specifically formulated for culturing primary neuronal cells; (50X) sterile stock solution available from Gibco-BRL Store at -20°C. Must be added to Neurobasal medium prior to use. 7 L-Glutamic acid: sterile, cell-culture grade amino acid solution. Final concentration is 25 @I L-glutamtc acid Must be added to Neurobasal medium after initial plating of primary cells. 8. L-Glutamme: sterile, cell-culture grade ammo acid solutton. Store stock solution at -20°C. Final concentration is 0.5 m&I L-glutamine in Neurobasal medium. 9. Penicillin and streptomycin. standard, sterile, cell-culture grade anttbiottcs Store stock solutions at -20°C. Final concentrations are 2 U/mL pemcillm and 2 pg/mL streptomycin m Neurobasal medium. The use of these antibiotics is optional. 10. Ethidium homodimer and Calcem-AM: as above. 11. Dulbecco’s-PBS: as above 12. 12-mm sterile glass coverslips. sterilize as described above. 13 Cell-type-specific primary antibodies* These antibodies are used to recognize neurons and glia m a mixed population. The two primary antibodtes must be generated m different species. For example, we used rabbit antineuronal anttbody and mouse anttghal antibody. Antibody dilutions may be adjusted to achieve maximal epitope recognition

148

14 15 16. 17

18.

19

20. 21

Thompson a. Affinity-purified rabbit polyclonal antibody to the C-terminus of the tau protein: prepared according to the method of Garver et al. (6) Prepare fresh as required at a dilution of 1500 m PBS. A commerctally available neuronal antibody (generated in rabbits) can also be used; the appropriate dilution must be determined empirically This primary antibody recognizes neurons in the mixed population of primary cells b. Mouse monoclonal antibody to ghal frbrrllary acidic protein (GFAP): avarlable from Boehrmger Mannherm (Mannheim, Germany). Prepare fresh as required at a dilution of 1: 10 in PBS. This primary antibody recognizes gha m the mixed population of primary cells. Sterile phosphate-buffered saline (PBS). 3.7% formaldehyde: This solution should be new, rf possible. 0 1% Trrton in PBS* This solutton should be made fresh as required Normal goat serum’ available from Jackson Immunoresearch (West Grove, PA) Stock solution should be stored at -20°C. Prepare fresh as required at a dilution of I:20 m PBS Species-specific secondary antibodies: The two secondary antibodies recognize the two primary antibodies (see Subheading 2.2., step 13) and are tagged with two different fluorophores Antibody drlutrons may be adJusted to achieve maximal cell fluorescence wnh minimal background fluorescence. Neurons can exhibit a red fluorescence and gha can exhibit a green fluorescence, depending on fluorophore choice. Alternatively, chromogenic-linked secondary antibodies can be used a Cy3-conjugated goat antirabbit IgG (min human, murine, bovine, horse, serum proteins): available from Jackson Immunoresearch The lyophihzed antibody should be reconstituted and stored as a 50% glycerol stock solutron m the dark at 4°C. Prepare fresh as required at a dilution of 1500 in sterile PBS. The Cy3-conjugated goat antu-abbrt IgG recognizes the primary anti-tau antibody (or other rabbit-generated primary neuronal antibody). b. Fluorescein rsothiocyanate (FITC)-comugated goat antimouse IgG (min human, murine, bovine, horse serum proterns). available from Jackson Immunoresearch. The lyophilized antibody should be reconstituted and stored as a 50% glycerol stock solution m the dark at 4°C. Prepare fresh as required at a dilution of 1:lOO m sterile PBS. The FITC-conjugated goat antimouse IgG recognizes the primary anti-GFAP antibody Poly-o-Lysme hydrobromide (high molecular weight): used to coat plastic plates for culturing of primary cells; available from Collaborative Biomedical Products (Bedford, MA) Store solubrlized ahquots at -20°C for up to 3 mo The final coating concentration is 5 pg/cm2. Pro-Texx mounting medium. used for mounting coverslips to slides, available from Sclentrfrc Products (McGraw Park, IL) Final concentratrons for solutrons used in these protocols: a. HBSS(A) HBSS with Ca2+ and Mg2+ (10X stock diluted to IX), 1 m&Y sodmm pyruvate, 10 mA4 HEPES, sterile dHzO. b. HBSS(B): HBSS without Ca*+ and Mg*+ (10X stock diluted to 1X), 1 mM sodium pyruvate; 10 r&4 HEPES; sterile dH20.

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c. Neurobasal medmm (-Glu): Neurobasal medium (1X bottle), B-27 supplement (50X stock diluted to 1X), 0 5 mJ4 L-glutamine, 2 U/mL pemcilhn, and 2 pg/mL streptomycin. d Neurobasal medmm (+Glu): Neurobasal medium (1X bottle); B-27 supplement (50X stock diluted to 1X), 25 pM L-glutamic acid, 0.5 mM L-glutamme, 2 U/mL pemcllhn, and 2 pg/mL streptomycin.

3. Methods 3.7. EthidiumKalcein

Viability Assay

1 Plate the desired number of cells and grow to 50-75% confluence m six-well culture plates usmg normal growth condltlons (I.e., 37”C, 5% COZ, 95% air) For toxicity assays, add fresh medium containing the drug or toxin for the appropnate mcubatlon time. The six-well culture plates are useful for toxicity assays since each plate can hold five treatment groups and a negative control well (culture medium only). A vehicle control treatment well should also be included, especially if the vehicle itself may be toxic (e.g., DMSO, ethanol, and so on). 2 Remove spent medium containing the dead cells from each well and place m SIX 15mI. conical tubes Gently harvest the remainmg cells (e.g , trypsinize or detach mechanically) and add to the spent medium. Centrifuge the cell mixture for 3 mm at 200g and wash the cells once or twice with Dulbecco’s-PBS (Note 1). Resuspend the cell pellet m a small volume of Dulbecco’s-PBS (e.g., 100 pL to 1 mL; see Note 2). 3. Prepare the calcem-AM and ethldium homodimer solution m a 15-mL conical tube wrapped with aluminum foil. The final concentrations should be 2 @Z calcem-AM and 4 PM ethldmm homodlmer m Dulbecco’s-PBS. The final concentrations of calcem-AM and ethldmm homodimer may be adjusted if the desired fluorescence 1snot achieved. 4. Place a 25-mm glass cover slip into each well of a six-well plate. Place a 15 PL ahquot of the cell suspension and 15 pL-30 pL of the calcem-AM and ethidium homodlmer solution onto the glass cover slip (see Note 3). Incubate these cells for 30-45 mm m the dark at room temperature The ethidium homodlmer enters the nuclei of dead and dying cells, and the nuclei of these cells fluoresce red. Esterases within live cells hydrolyze calcem-AM; thus, live cells are green. 5 Immediately before counting, gently place a second 25-mm cover slip onto the cell suspension Observe and count live cells (green cells) and dead cells (cells with red nuclei) via a standard fluorescem filter set. Score multiple fields (4-6) per cover slip using an ocular grid (see Note 4). The cell counts are expressed as the fraction of dead cells (number of dead cells divided by the total number of cells [dead + alive]). A log dose-response curve 1s generated by plotting the fraction of dead cells on the y-axis and the log dose values on the x-axis (Fig. 1). Values are expressed as the mean + the standard error of the mean (SEM) of the fraction of dead cells compared with the total number of cells The toxic concentration that kills 50% of the cells (TC,,) 1s determined by linear extrapolation. The T& value should be expressed as TC& _+1 standard deviation (SD) If the cell count data are to be analyzed statistically, use non-

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H-NEURONS C-NEURONS

TBT

(uM)

Fig. 1. Use of the ethidmm/calcem viability assay m cell lines. Viabihty of NIH-3T3 rat fibroblast, HTB- 14 human ghoma, and S2OY murine neuroblastoma cell lines was determined followmg a 48-h treatment with tributyltm (TBT) at doses ranging from 0.1 to 100 l.tM Cell viability data were obtained using the smgle-step ethidmm/calcem viabihty assay. Values are expressed as the mean f SEM of the fraction of dead cells compared with the total number of cells. parametric analyses (e.g., Kruskal being compared.

Walhs analysis)

since fractional

values are

3.2. Ethidium/Calcein Viability Assay and Double-Label lmmunofluorescence 1. The isolation and plating of neonatal rat hrppocampal and cortical neurons and glial cells described below is based on the method developed by Brewer et al. (7) Anesthetize neonatal rats (1 -d-old) with halothane and decapitate Place the head mto a sterile 35 mm culture dish on ice containing HBSS(A). To remove the brain, use scissors to cut the skm along the skull, use microscissors to cut open the skull, carefully remove the skull fragments, and use fine forceps to ease the bram from the skull. Immediately place the intact brain into another sterile 35 mm dish on me containing HBSS(A). With the aid of a dissectmg microscope, use microscissors to separate partially the brain hemispheres and push one hemisphere to the side. Use microscissors and fme forceps to remove the htppocampus by carefully cutting around the perimeter of the hippocampus and then lifting the hippocampus from the rest of the brain Remove the meninges from the hippocampus usmg fine forceps and place the hippocampus into a sterile 35mm dash on me containmg HBSS(A) Cut the cortex from the rest of the brain usmg microscissors, remove the menmges from the cortex and place the cortex into another sterile 35 mm dish on ice contammg HBSS(A).

In Vitro Viability Assays

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2. Transfer all hippocampi mto a sterile 15mL conical tube (pool 12 hrppocampi from 6 brains) and all cortices (pool 12 cortices from 6 brains) mto another sterile 15mL comcal tube containing 1 mL of HBSS(B) (Note 5). Place the conical tubes mto a beaker filled with ice and isolate individual cells by triturating lo-12 times with a sterile, silicomzed Pasteur pipet. Fire-polish the end of the Pasteur pipet to avoid shearing the cells. Add 3 mL of HBSS(A) to each 15-mL conical tube. After 3 mm, transfer the supernates to two 15-mL conical tubes and centrifuge for 1 min at 200g Resuspend the cell pellet m 1 mL of HBSS(A) per bruin (6 mL total volume) Plate the cells at a density of 250-400 cells/mm2 on sterile 12-mm glass cover slips m six-well plates coated with a 0.05 mg/mL solutton of poly-n-lysine (see Note 6) For this cell density, use the equivalent of two brains (i.e., 2 mL of cell suspension) per six-well plate. Use three, srx-well plates for plating the hippocampal neurons and another three six-well plates for plating the cortical neurons. (If SIX brains are mitially removed, this amount equals two brains per plate). 3 For the initial plating of the cells, ahquot Neurobasal medium (-Glu) mto each of the wells and add the appropriate amount of cell suspension. Neuronal and ghal cells are maintamed as a mixed population m this medium (see Note 7). Cells are maintained m a humidified, 37°C incubator with 5% CO2 and 95% air. On d 4 after plating, add fresh Neurobasal medium (+Glu) For drug or toxicity studies, add the drug or toxm directly to the Neurobasal medium (+Glu) and add to the cells for the appropriate incubation time 4. For the double-label immunofluorescence step, remove the 12-mm cover shps from the six-well plate using forceps and place one mto each well of a 12-well plate (cell-side up); use the remaining adhered cells m the six-well plate for the ethidmrn/calcein viability assay. For the ethidium/calcein viability assay, remove the spent medium and gently wash the remaining cells m each well twice with Dulbecco’s-PBS After the second wash, aspirate the Dulbecco’s-PBS; leave a very thm layer in the well. Add 50-150 pL of 2 pM calcem-AM and 4 pjt4 ethrdium homodimer m Dulbecco’s-PBS directly to the cells in the six-well plate. Incubate for 30-45 mm (m a dark, humidified chamber) at 37°C or at room temperature Immediately before counting, place a 25-mm cover slip mto each well. Observe live cells using a fluorescem filter set (see Note 8). 5. For the double-label immunofluorescence (see Note 9), use the 12-well plate with the 12-mm cover shps. Wash the cells twice with room temperature PBS. (When adding solutions to the cells on the cover slrps, place the pipet on the side of the well and allow the solution to run slowly into the well.) Fix the cells for 30 mm at room temperature with 3.7% formaldehyde (500 pL to 1 mL per well; see Note lo), aspirate the formaldehyde, and rinse the cover slips twice with room temperature PBS Permeabihze the cells with 0.1% Triton m PBS (500 pL to 1 mL per well) for 20 mm at room temperature Aspirate the Triton solution from around and underneath the cover slip. Leave a very thin layer of Triton on the cover slip itself The thm layer of Triton will allow the primary antibody solution to spread evenly over the coverslip.

Thompson 6. Dilute the two primary antibodies m PBS For this procedure, a 1 500 dilution of the anti-tau antibody and a 1.10 dtlutton of the anti-GFAP antibody were used Incubate the primary antibodies with the cells for 60 mm at room temperature m a humidified chamber (see Notes 11 and 12). After three 5-mm washes with PBS, incubate the cells with normal goat serum (1.20 dilution) for 15 mm at room temperature. Wash the cells three times wrth PBS and incubate for 30 mm at room temperature with Cy3-conjugated goat antirabbit IgG (1 500 dilution m PBS) and FITC-comugated goat antimouse IgG (1’100 dilution u-rPBS). Wash the cells twice wtth room temperature PBS and twice with sterile dH,O. During the final wash with dH20, remove the coverslips from the 12-well plate using forceps and place on a labeled paper towel (cell side up) to dry. Mount the cover slips (cell srde down) on glass shdes with Pro-Texx mounting medmm (see Note 13) Allow the mounted coverslips to dry overnight and store the slides at 4’C m the dark The fluorescence can be maintained for several weeks if the cells are not exposed to light. 7 Observe and count the neurons and gha at a set magnification m several fields (46) using an ocular grtd Neurons, when tagged with a Cy3-conjugated secondary antrbody, are observed using a rhodamme filter set and will fluoresce a bnlhant red Glia, when tagged with an FITC-conlugated secondary antibody, are observed using a fluorescein filter set and will fluoresce green Cell viabihty IS expressed as the number of surviving cells, for example, neuronal or ghal, counted per field. A log dose-response curve can be generated by plotting the number of hve cells on the y-axts and the log-dose values on the x-axis (Fig. 2) Values are expressed as the mean 4 SEM of the numbers of surviving cells As described above, the toxic concentration that kills 50% of the cells (TC,,) 1sdetermined by linear extrapolation and should be expressed as TCsa + 1 standard deviation (SD)

4. Notes 1. Wash the cells gently but thoroughly; ensure that no growth medmm remains. Components in serum from the growth medium will increase background fluorescence, and a hazy fluorescence will be seen. 2 The final resuspenston volume is important and must be determined empirrcally The cells will be difficult to count if the final cell suspension is too dense, conversely, there will not be enough cells to count If the final cell suspension is too dilute 3 The volumes of the cell suspension and the calcem-AM and ethidmm homodrmer solution can be adjusted so that the entire suspension drop stays on the cover slip If the volumes are too large, the cell suspenston ~111 run off the cover slip, and there will be no cells to count When placing the cell-suspensron drop onto the covershp, use the tip of the prpet to spread the cells around the covershp. 4. The fluorescence will remam at maximal levels for 1 h after the mmal 30X mm incubation period, therefore, count quickly. Be sure to count enough cells per field and ensure that the randomly chosen fields are representative of the entire cover shp Ethidmm homodrmer enters the nuclei of cells with damaged membranes, thus, the nuclei of dead cells fluoresce red Live cells generate an intense, uniform green fluo-

In Vitro Viability Assays

153

4

NIH-313 0

----o----

HTB-14 SZOY

oom 0

’

[TBT]

uM

lo

100

Frg. 2 Use of the ethtdmm/calcem vrabrhty assay combined with double-label immunofluorescence m primary neuronal cells Viability of primary neuronal cells was determined followmg a 48-h treatment with trrbutyltm (TBT) m doses ranging from 0.01 nM to 10 p&zf.Neurons were isolated from the hippocampi (H-neurons) and the cortices (C-neurons) of postnatal d 1 rats Cell-vrabrhty data were obtained using the ethrdium/calcem viability assay combined with double-label immunofluorescence. Values are expressed as the mean I!ZSEM of the numbers of hrppocampal and cortical neurons survrvmg after the 48-h TBT treatment. rescence on hydrolysis of calcem-AM to calcem. Cells that are green with a red nucleus are counted as dead. For counting purposes, generate a data sheet with three columns: Number of dead cells, number of live cells, and total number of cells (hve + dead). To calculate the fraction of dead cells, use the following formula. fraction of dead cells = number of dead cells / total number of cells

(1)

5. Placing the brains into the HBSS(B) without Ca2+ and Mg2+ 1snecessary in order to dissociate the cells For the remaining steps, HBSS(A) with Ca2+ and Mg2+ must be used to mamtam cell viability. All dissectron steps and cell-rsolatron steps must be performed usmg ice-cold solutions; keep all 35-mm dishes and conical tubes on me unttl the final plating of the cells The Neurobasal medium should be warmed to 37’C prior to adding rt to the cells For optimal primary neuronal viability, keep the time from the removal of the brain to the final plating of the cells to a minimum At the most, pool only 6-8 brains for one plating. 6 Cell-culture plates should be coated with poly-b-lysme on the same day that prrmary neuronal cells are to be plated The final coating concentration IS 5 pg/cm2. When coating cover slips for primary cell culture, place a 12-mm cover slip into each

Thompson

7.

8.

9.

10.

11.

12.

well of a six-well plate, add 1 mL of the 0 05 mg/mL poly-o-lysme solutton for 1 h, aspirate the solutton, and rinse with sterile dH,O. Add Neurobasal medium (-Glu) to each well and place in the incubator unttl the cells are to be plated. Use a stenle pipet to push the cover slips to the bottom of each well smce the cover slips tend to float, For optimal primary neuronal attachment and growth, do not store poly-o-lysmecoated plates and do not allow the six-well plates to dry after coating In a defmed medium of Neurobasal and B-27 supplement, primary neuronal cells grow very well, and the number of ghal cells is reduced to ~25% of the total cell population, without resorting to the use of anti-mttotlc drugs such as cytosme arabmoside. Cell viabthty IS maintained for 7-8 d using thts method. Drug studies must be planned wtthin this time period. For example, the drug or toxin can be added on d 4 and incubated wtth the cells for up to 72 h If a longer drug exposure ttme IS required, the drug must be added earlier so that the vtabdity assay 1s performed by d 7 after plating Compare the number of live cells m the control group to the number of live cells m the different treatment groups Note that wtth thts assay, only live cells remain on the covershp. Counting the cells at thts point is not particularly useful if the cells are growing in a mrxed population. Since cell type cannot be determined by the ethtdium/calcem viabrlity assay, cells can be double-labeled with cell-specific markers tn order to identify surviving cells. For example, neuronal markers (microtubule assocrated proteins) and gltal markers (GFAP) are useful m identifying neurons and gha, respecttvely, mamtamed m a mixed population of prtmary cultured cells. See ref. 5 for immunofluorescence photographs of prtmary cultured cells. Use of the appropriate fixative is critical in order to mamtam cell morphology and to achieve optrmal antigen recogmtion and fluorescence For this procedure, the best results were obtained by fixing the cells wtth 3 7% formaldehyde for 30 mm and by permeabihzing the cells with 0.1% Tnton for 20 mm. Very few cells hfted off the cover slip, and good cell morphology was mamtamed. Alternatively, cells can be fixed for 5 mm with 1 part acettc acid and 3 parts 95% EtOH, washed twice with PBS, and permeabrhzed with 0.1% Trrton for 2 min. If many cells are lifting off the cover slip during the fixation step, or If antigen recogmtton and fluorescence are poor, try a different fixative The appropriate antibody dilutions (primary and secondary) must be determmed empirically A small volume of the diluted antibody solutton (lo-20 pL per cover slip) can be used if a very thin layer of 0.1% Triton remains on the cover slip and if the antibody solution 1s placed carefully onto the cover shp so it does not run off the edge To construct mdivldual humidified chambers, cut a paper towel to the size of the 12-well plate, saturate the towel with dH,O, remove the excess dH20 from the towel, place the towel on top of the 12-well plate, and replace the lid Do not allow any dHz0 to drip mto the wells Include the appropriate controls when performing the double-label immunofluorescence. For a negative control for the primary antibody, incubate the cover slips with PBS only (no primary antibody) To ensure primary antibody specific-

In VItro Via bdity Assays

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ity, incubate the cover slips wtth a primary antibody that will not recognize an antigen m the observed cell type. 13. Do not mount wet cover shps. If wet cover slips are mounted, crystals will form over the entire cover slip, and no cells will be visible For ease in mounting the cover shps, use the wooden end of a cotton-tipped applicator to place a small dot of Pro-Texx mounting medium on the slide Place the cover slip (cell side down) on the shde and press down gently with the blunt end of forceps to remove au bubbles The mounting medmm should extend only to the edge of the cover slip; do not allow the mounting medium to cover the top of the cover slip.

References 1 Leeder, J S (1989) Fluorescence-based viabthty assay for studies of reactive drug intermediates Anal. Biochem 177,364-372 2 Gaugain, B. (1978) DNA bifunctlonal intercalators. II. Fluorescence properties and DNA binding mteractron of an ethtdium homodlmer and an acridine ethidmm heterodimer Biochemistry 17,5078-5088. 3 Moore, P L , MacCoubrey, I. C , and Haugland, R. P. (1990) A rapid, pH msensrtrve two color fluorescence viability (cytotoxicity) assay J. Cell Biol 111,58a 4 Haugland, R P. (1992) Set #25 fluorescent dyes for assessing vital cell functions, m Handbook of Fluorescent Probes and Research Chemicals. Molecular Probes, Eugene, OR, pp 172-175 5. Thompson, T A, Lewis, J. M., DeJneka, N S., Severs, W. B., Polavarapu, R , and Blllmgsley, M L (1996) Induction of apoptosis by organotin compounds m vitro: neuronal protection with antisense oligonucleottdes directed against stannin J. Pharm. Exp. Ther 276,1201-1215. 6 Garver, T D , Harris, K A, Lehman, R. A. W., Lee, V. M Y , Trojanowski, J Q , and Billmgsley, M. L (1994) Tau phosphorylatron m human, primate, and rat brain evidence that a pool of tau is highly phosphorylated in vivo and is rapidly dephosphorylated m vitro J. Neurochem. 63,2279-2287 7 Brewer, G J , Torricelh, J., Evege, E. K , and Price, P. J. (1994) Neurobasal medium/B27 supplement: a new serum-free medium combmatron for survival of neurons. Focus 16.6-9

13 Measurement of Nitric Oxide Synthase Activity Using the Citrulline Assay Thomas

Ft. Ward and William R. Mundy

1. Introduction The free-radical gas nitric oxide (NO) recently has been identified as an important biological messenger molecule in both the central and peripheral nervous system. NO is generated by the enzyme NO synthase (NOS) by the oxidation of the ammo acid L-arginine. As a dissolved gas, NO 1s an unusual neurotransmltter. It is not packaged in synaptic vesicles and released by exocytosis upon membrane depolarization, but rather diffuses from its site of production to surroundmg neurons where it acts directly on specific intracellular targets. The activity of NO terminates when it chemically reacts with a target substrate. Although all of the targets of NO are not yet known, NO can bmd to the iron associated with heme groups or result in nitrosylation of proteins, leadmg to conformational changes. One of the best-described targets of NO m the central nervous system is the heme-containing protein guanylyl cyclase. NO is a relatively long-lived free radical and does not react readily with most cellular components. This allows it to diffuse to several surrounding neurons and integrate neuronal activity on a local scale. NO is involved m a number of physrological processes including morphogenesis and synaptic plasticity. However, under conditions in which NOS IS overstimulated, excessive formation of NO may mediate cell injury m a variety of disorders of the nervous system that result in neurodegeneration (1). Because the cell does not sequester and store NO, the key to regulating NO levels is the control of NO synthesis by NOS. Molecular-cloning experiments have identified three distinct lsoforms of NOS: neuronal (nNOS, type I), inducible (iNOS, type II), and endothelial (eNOS, type III). Both neuronal and endothelial NOS are constltutively expressed and are briefly activated by From

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H2N >-

Neutral

+

&N

NH2

N

0

>N

NOS

+

NO

NADPH &N 2

COOH

2 Ca2+/CaM

L-arginine

b

HrLN1

COOH

L-citrulline

Ftg 1. Biosynthesis of NO* NOS catalyzes the oxrdatlon of L-argmme to generate NO and L-citrulhne The reaction requires NADPH, O,, and Ca2+/calmodulin and IS enhanced by tetrahydrobtopterin increases m mtracellular calcium. They are located mainly (but not excluwely) in central and peripheral nerves and m vascular endothelial cells. In

contrast, inducible NOS is synthesized de lzovo within selected cell types (including macrophages and hepatocytes) after exposure to bacterial endotoxins or various cytokines. Immunohistochemmal localization of NOS in bram demonstrated that NOS is widely distributed and occurs in a heterogenous population of neurons, with no glial staining (2,3). Although only 2% of neurons contain NOS, these neurons are branched in such a manner that all CNS neurons are within a few microns of a NO source (4). NO is formed by the stoichiometric conversion of L-arginme to L-citrullme and NO in the presence of oxygen and NADPH through an oxidative-reductive pathway that requires 5 electrons (5). Neuronal NOS is activated by an increase m intracellular calcium that results m calcmm/calmodulm (CaM) complexes (Ca2+/CaM) that bmd to NOS dimers. The binding of Ca*+/CaM to the dimer results in a conformational change allowing the flow of electrons from the reductase segment of the enzyme (containing flavin mononucleottde and flavin adenine dmucleotide binding domains) to the catalytic site of the enzyme (containing a heme moiety). NADPH and tetrahydrobiopterin (BH4) are required as cofactors (Fig. 1). Because the reaction 1sstoichiometric, a sensitive and specific assay for the enzymatic formation of NO was developed by Brendt and Snyder (67) by monitoring the conversion of L-[3H]arginme to L-[3H]citrulline. In a standard assay, radiolabeled arginine is added to intact tissues or extracts. After incubation, the reaction is terminated by adding a stop buffer (pH 5.5) containing

EDTA

(which binds calcium).

Radrolabeled

crtrul-

line, which at a pH of 5.5 is neutral, 1s separated from the positrvely charged arginme using a column containing

a cation-exchange

resin. The use of radro-

labeled compounds makes the assay sensitive to the picomolar range. In the

Measurement of NWc Ox/de

159

nervous system, the direct conversion of argmine to citrulline is unique, so that the assayis a specific determination of NOS activity. Thus, the following procedures are optimized for the determination of neuronal NOS activity in preparations derived form CNS tissue. 2. Materials 2.1. r-Arginine L-[3H]arginine (New England Nuclear, Boston, MA; approx 35 Wmmol) is received as a solution of 1.OmCi/mL (1000 lKi/mL) in deionized water containmg 2% ethanol. It is stabile for up to 6 mo when stored at 4OC. 2.2. Dowex Columns 1. Analytical-grade cation-exchange resm (Bio-Rad, Richmond, CA; AG Dowex SOW-X8 resin) 2. Polystyrene chromatography column (Evergreen Scientific, Los Angeles, CA) 3. Preparatton of Dowex columns. Wash 100 g of Dowex twice with deionized water. Excess water is removed by allowing the Dowex to settle and decanting the liquid. Add 300 mL of 1 M NaOH, stir, and allow to sit overmght at 4°C. Decant the liquid and remove excess NaOH by washing the Dowex with detonized water until the pH is below 8.0. Add a 1 mL bed volume to each column. Before use, equilibrate each column by adding 5 mL of stop buffer (see

Subheading

2.5.).

4. The Dowex can be reJuvenated by elutmg the L-[3H]arginine from the columns with 1 M NaOH. Remove the Dowex from the columns and wash as described in step 3.

2.3. Cell Cultures 1. Incubation buffer: 154 mM NaCI, 5.6 mM KCl, 3.6 mM Na&O,, 10.0 mM HEPES, 1.3 mM CaCl,, 5.6 mM o-glucose, pH 7.35. Store at 4°C. 2. Rinse buffer: 154 mM NaCl, 5.6 mM KCl, 3.6 mM Na,C03, 10.0 mM HEPES, 1 3 mM pH 7.35. Store at 4°C. 3. Stop solution: 0 3 M HClO,. 4. Neutralizing solution 1.5 M K&O,. 5. NaOH solution* 1 M NaOH

2.4. Tissue Slices 1 Krebs-Henseleit Buffer (KHB). 118 mM NaCl, 4.7 mM KCl, 2 mM CaC12, 1.2 mM KH2P04, 25 mM NaHC03, 11 mM o-glucose, pH 7.4. Store at 4°C. 2. HEPES buffer: 20 mM HEPES, pH 7.0. 3 Argmme-EDTA solution. 5 mM L-arginme, 4 mM EDTA. 4. Dtethyl ether 5 TCA* 1 M m detomzed water

Ward and Mundy 2.5. Tissue Homogenates 1 Homogenization buffer: 20 mM HEPES, 0 5 m&I EGTA, 1 nnV2 DTT, 0 32 M sucrose, pH 7.4. Store at 4°C 2. Assay buffer 20 mM HEPES, 0 5 n&f EGTA, 1 mM DTT, 0.32 M sucrose, 0 5 m&I CaCI, (1 pM free Ca2+), 200 pM NADPH, 1 pM L-argmme, 1 pCr/mL r.-[3H]argmme, pH 7 4. Store at 4°C. 3. Stop buffer: 20 mM HEPES, 2 nuI4 EDTA, pH 5 5. Store at 4°C

3. Methods 3.1. Cell Cultures 1 This method has been used on primary neuronal cultures (mcludmg cerebellar granule cells and cortical cells) grown on 12-well plates 2 Wash cells two times and incubate for 10 mm at 37°C with 1 mL of mcubatron buffer 3. Preload cells with 1 pCi/mL L-[3H]argmine m 0 9 mL of fresh mcubatron buffer for 5 mm at 37°C 4. Stimulate cells by adding 0.1 mL of agonist (or test solutton) made up in incubatron buffer Swirl gently to mix and incubate an additional 5 mm. A O-mm blank should be stopped at the time the cells are stimulated. 5. The reaction IS termmated by rapid aspnatron of the medium, two washes with 1 mL of me-cold rinse buffer, and the addition of 1 mL stop solutton 6 The stopped reaction mixture IS then neutralized with 0 18 mL of neutrahzmg solution. The addmon of the neutralizmg solution results m bubbling and a prectpttate will form. The pH should be checked to be certain that the solutron IS neutral or basic. 7. Allow the precipitate to settle and take a 200~pL aliquot of the neutralized extract for determmatron of radroactrvrty by lrquld scmtrllatron spectroscopy. Thus sample represents the total uptake of L-[3H]arginme and is the sum of total L-[3H]argmine remaining and L-[3H]citrullme generated 8. Take a separate 400~PL aliquot of the neutralized solutron and add to the Dowex columns (whtch are suspended over 20-mL scmtrllatron vrals) for the separation of L-[3H]citrullme from L-C3H]arginine. Wash the column twice with 1 mL deionized water and collect the flow-through m the 20 mL scmtrllatron vials. Determine radioactrvrty by hqutd-scmtrllatron spectroscopy L-[3H]cttrulline formation IS expressed as a percent converston of the total L-[3H]arginme taken up into cells.

3.2, Tissue Slices 1. Slice preparation. Bram mmlprrsms are prepared from the regton of interest using a McIlwam tissue chopper. The brain region IS sliced at 0 4-mm mtervals in both the sagrttal and coronal planes. The slices are rinsed once with KHB then incubated for 1 h at 37°C while being continuously gassed wrth 95% 0,/5% CO*. 2. After 1 h, transfer the slices to a 15mL polypropylene centrrfuge tube and allow them to settle

Measurement

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3 Transfer 20-PL ahquots of gravity packed slices to 12 x 75-mm plastic test tubes contannng 250 PL of KHB and 3 ~CI [3H]argmine and incubate under 95%/5% Oz/C02 for 15 min Agonists may be added at this time. A O-time blank should be stopped at the time the agomst are added. 4 Stop the reaction by adding 0 75-mL of me-cold argmlne-EDTA solution. Centrifuge tubes at 10,OOOgfor 1 mm to pack the slices. Discard the supernatant and add 1 mL of 1 M TCA to denature and precrpitate the protein. Sonicate and transfer to 1.5-mL mmrocentrtfuge tubes 5. Centrifuge at 10,OOOg for 15 min to pellet the protein. 6. Discard the supernatant and extract the TCA precipitate three times wrth 2 mL of water-saturated ether. 7. Remove a 0 5-mL ahquot of the aqueous phase, neutralize with 2.0 mL HEPES buffer, and apply to Dowex columns suspended over 20-mL scintillation vials Wash the column with 2 mL deionized water and collect the flow-through. Add scintillation cocktail and determine radroactivlty by llqurd scmtrllatron spectroscopy. 8 The precipitated protein can be dissolved m 0.6 mL of NaOH (0.5 M) and quantified by the method of Lowry et al. (8)

3.3. Tissue Homogenates 1 Thus assay IS optimized to determine the activity of constttutively expressed neuronal NOS. 2. Disrupt the tissue m 20 vol of me-cold homogenization buffer using a polytron or equivalent tissue homogenizer. 3 Centrifuge the homogenate at 20,OOOg for 15 mm at 4°C. Determine the amount of protein m the supernatant and use for the assay of NOS activity 4 The supernatant (contammg approx 150 kg protein) IS incubated m 1 mL assay buffer for 15 mm at 37°C (mcubatrons may be carried out for lo-60 mm at 22-37°C depending on the tissue used). 5. Nonspecific actrvrty IS determined by running a blank condmon using assay buffer that does not contain calcium or NADPH. 6. Stop the reaction with the addition of 2 mL me-cold stop buffer 7. Apply the entire sample to Dowex columns suspended over 20-mL scintillation vials. Wash the columns with 2 mL deionized water and collect the flow-through. Add scmttllatlon cocktail and determine radroactrvity using liquid scintillation spectroscopy.

4. Notes 1. NADPH IS recommended to be stored at -20°C. NADPH contammg buffers should be made fresh and stored at 4°C 2. NOS activity is relatrvely unstable, therefore brain dissection should be performed on ice. Homogenates should be kept at 4°C until mcubatron. Brain samples may be stored at -70°C but NOS activity will decrease with time even at this temperature

162 3 As with all enzymatic assays, condrtrons should be checked for linearrty with respect to time of mcubation and amount of protein 4. Thin-layer chromatography can be used to verify the product from the Dowex column. Ten-microhter ahquots of eluant are resolved on sihca gel 60 plates (20 x 20, alummum Alltech Associates, Los Altos, CA) with chloroform methanol:ammomum hydroxrde:water (1:4.2: 1) as a mobile phase This results m Rf values of 0 38 for arginme and 0.83 for citrulline

References 1. Varner, P D. and Beckman, J. S. (1995) Nitrrc oxide toxrcrty m neuronal mJury and degeneration, in Nitric Oxide m the Nervous System (Vmcent, S R., ed ), Academic, New York, pp. 191-206. 2. Bredt, D. S., Hwang, P M , Glatt, C. E., Lowenstein, C., Reed, R R., and Synder, S. H (1991) Cloned and expressed mtrrc oxide synthase structurally resembles cytochrome P-450 reductase. Nature 351,714-718. 3 Southam, E. and Garthwaite, J (1993) The nitric oxrde-cyclic GMP signallmg pathway m rat brain. Neuropharmacology 32, 1267-1277. 4 Bredt, D. S , Hwang, P M., and Synder, S H. (1990) Localization of nitric oxrde synthase mdicatmg a neural role for mtrrc oxide. Nature 347,768-770. 5. Marletta, M. A. (1993) Nitric oxide synthase structure and mechanism. J Blol Chem. 268, 12,231-12,234. 6 Bredt, D. S. and Snyder, S H. (1989) Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum. Proc N&Z. Acad. SCL USA 86, 9030-9033. 7 Bredt, D. S. and Snyder, S. H (1990) Isolatron of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc. Natl. Acad. Sci. USA 87,682-685. 8. Lowry, 0. H , Rosebrough, N. J., Farr, A. J , and Randall, R J. (195 1) Protein measurement with the folm-phenol reagent. J. Bzol. Chem. 193,265-275.

Measurement of lntraneuronal Free Calcium Using the Fluorescent Probe Technique Prasada Rao S. Kodavanti 1. Introduction The distribution of Ca2+within the cell is complex and involves binding to cell macromolecules and compartmentalization within the subcellular organelles (1). Normal physiological functions of the cell are regulated by changesin intracellular free Ca*+ ([Ca2+],), which ranges from 0.1 to 0.3 pJ4. This low concentration is regulated by energy-dependent transport systemslocated in plasma membrane, endoplasmic reticulum, and mitochondria (I). Cytosolic [Ca2+],within the cell are elevated either through Ca2+-influx or by Ca*+-release from mtracellular stores. Such increases in [Ca2+], have been reported to activate several intracellular Ca2+-dependentreactions mcludmg production of second messengers(2,3), spontaneousrelease of neurotransmitters (4), phosphorylanon of proteins (5), and actrvity of proteases(6). Cells needto mamtam a fine balance of these Ca2+homeostatic mechanisms in order to function normally, and any major disturbance in these systemswill activate destrucnve events leading to neuronal injury (7-10). Alterations in Ca2+homeostasishave been reported to be pathophysiologrcally important m aging and several neuronal disorders including Alzheimer’s disease and Huntington’s disease(11,12). Deficits in the movements of Ca2+acrossmembranes may lead to the formation and phosphorylatron of abnormal neurofilament proteins during Alzheimer’s disease (13,14). In organotypic hippocampal cultures, neuronal calcmm loading has been attributed to neurodegeneration caused by hypoxia/ischemia. Further, enhancement of neuronal calcium buffering attenuated or delayed the onset of anoxic neurodegeneratron (15). Kater and Mills (16) indicated that neuronal growth-cone motility and neurite outgrowth are inhibited if [Ca2+], falls below an optimal level, or rises significantly above it. From

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Also, recent work suggests that a linear correlation exists between total Ca2+ loading and neurodegeneration (17). Therefore, Ca2+ homeostasis is an important event to determine whether the neurons are functioning at the optimal level or not, either during neurodegeneration or toxic challenge. In this chapter, measurement of [Ca2+], m a single neuron as well as a population of neurons using a fluorescent-probe technique is described. In Chapter 15, calcium-buffering mechanisms, which exist within the neuron and play a greater role in mamtainmg normal calcium homeostasls, are described in detail. At the present time, the most widely used method for the measurement of neuronal [Ca2+], is to monitor the fluorescence of Ca2+ indicators such as Qum-2, Fura-2, Flue-3, or calcium green (18-21) These calcium indicators (e.g., Fura-2) are loaded into Intact neurons by mcubating with a membrane-permeant acetoxymethyl ester derivative (Fura- AM). Cytosolic esterases cleave the ester groups and leave the membrane-impermeant Fura- trapped m cytosol. This Fura- binds to Ca2+ present m cytosol and produces fluorescence (Fig. 1). The fluorescence can be measured qualitatively using an imaging system or quantitatively using a photomultiplier system.

2. Materials 1, Fura- AM (1 mM)* Dissolve 1 mg completely in 1 mL volatile solvent, such as acetone or chloroform, and then transfer 50-uL aliquots to 20 microfuge tubes. Evaporate all the volatile solvent and store in a desiccator at -20°C or below This sample can be reconstituted as needed usmg 50 yL dlmethylsulfoxide (DMSO) to obtain 1 mM concentration 2 Locke’s buffer (Mg2+-free): Dissolve 9.0 g sodium chloride (NaCl; 154 mM; formula weight [FW] 58.44), 0.418 g potassium chloride (KCl; 5 6 mA4, FW 74.56), 0.303 g sodium bicarbonate (NaHC03, 3 6 mM; FW 84.01), 0.339 g calcium chloride (CaCl,; 2.3 mM; FW 147.02), 1.009 g o-glucose (5.6 mM, FW 180.2), and 1 192 g N-[hydroxyethyllpiperazme-N’-[2-ethanesulfonic acid] (HEPES; 5 mM; FW 238.3) in distilled water, adJust the pH to 7.4 with 1 N NaOH, and make up the volume to 1 L. Store in refrigerator 3. Ionomycm (1 in&‘, FW 747.1): Dissolve 0.747 mg in 1 mL of DMSO A loo-fold dilution will give a final concentration of 10 FM. Store m freezer. 4. CaCl, (5 mM; FW 147.02). Dissolve 73.5 mg in lOO-mL distilled water. Store in refrigerator 5. Ethylene glycol-bu@-ammoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA, 5 mA4, FW 380.4): Dissolve 190 mg in 100 mL Locke’s buffer Store m refrigerator

3. Methods 3.1. Loading

Neuronal

Cells wifh Nuorescent Probe

1 Warm up Locke’s buffer, CaClz, and EGTA solutions to room temperature. 2 Observe the neurons, grown on 25-mm cover slips in Petri dish or six-well cul-

lntraneuronal

Free Ca*+ Measurement

765

Fv.wa-2 AM

Fig. 1. Mechanism of neuronal free calcium measurement using Fura-2. In this figure, the principle involved in the measurement of intraneuronal free calcium using fluorescent probe technique is shown. The prototypic fluoroprobe, Fura- is impermeable where as its acetoxymethyl ester form, Fura- AM is cell permeable. When the neuronal cells are incubated with Fura- AM, it can cross the cell membrane and intact neurons are loaded with Fura- AM. Within the cells, cytosolic esterases cleave the ester groups and leave the membrane-impermeant Fura- trapped in cytosol. This Fura- binds to Ca2+ present in cytosol and emits fluorescence at 505 nm when excited alternatively at 340 and 380 nm. ture plate, under microscope for dendritic network and to make sure that the neurons grew normally. 3. Wash the neurons once carefully with 2.0 mL of Mg*+-free Locke’s buffer at room temperature and then add 2 mL of Locke’s buffer containing 2 w furaAM (see Notes 1 and 2). 4. Incubate the cells with fura- AM for 40 min at room temperature in the dark (see Notes 3 and 4). 5. After dye loading, wash the cells with Locke’s buffer and incubate in the dark for an additional 20 min to allow for complete de-esterification (see Note 5).

3.2. Measurement

of [CS+]i in Neurons

1. Rinse the cells after loading and de-esterification and place them in a recording chamber. 2. Make sure that the neurons face the upper side of the chamber. 3. Mount this recording chamber containing neurons on to the stage of an inverted Nikon (Melville, NY) fluorescence microscope equipped with a 100X Fluor oil immersion objective. 4. Observe the cells under microscope and determine whether to focus on a single neuron or a group of lo-15 neurons.

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5. Measure the fluorescence m the selected neurons using a computer controlled PTI Deltascan system (Photon Technology, South Brunswick, NJ) 6 Excite the cells alternatively at 340 and 380 nm, and filter the emission signals at 505 nm with a narrow bandpass filter. 7 Equilibrate the cell system for few minutes before adding the test chemical to the chamber. 8 Monitor the fluorescence signal for the required time after adding the test chemical (see Notes 6 and 7). 9. Calculate neuronal [Ca*+],) usmg the followmg equation as described by Grynkiewicz et al. (19).

(1) where R 1sthe ratio of fluorescence intensity excited by 340 and 380 nm after correcting for background fluorescence. Determine Rm,,,, R,,, and F,,/F$ under identical conditions m sister cultures exposed for 10 mm to either Locke’s buffer containing 5 mM EGTA with Ca*+ omitted (R,,), or Locke’s buffer contaming 5 mM CaCl, and 10 pM tonomycm (R,,,). FJF, is the ratio of the excrtation fluorescence of 380 nm at zero and saturated Ca*+ levels, respectively. A K,, of 265 nM for furaand Ca*+ bmdmg may be used after correction for room temperature (22). 10. Determine the background fluorescence before each experiment in a single cell or groups of cells m Locke’s buffer (as per the experimentation), not exposed to fura- AM and subtract from all the values before calculatmg the absolute [Ca*+],) values.

4. Notes 1. Make sure that the neurons are healthy by observing them under a microscope before loading the cells. Subtract autofluorescence of the cells and reagents before calculatmg the absolute values. Always conduct a preliminary dry run with a known agonist like KC1 or ionomycin as positive controls. 2. Make sure that the neurons do not come off the cover slips during washing, dye loading, and/or addition of test chemical. Slow addition of solutions would help solve this problem. 3. One of the problems with ester-loaded Fura- is the accumulation of dye m mtracellular organelles, such as mitochondria, endoplasmic reticulum, nucleus, lysosomes, and so on (23). It has been reported that longer loading times enhance the appearance of dye within subcellular organelles and that a lower loading temperature favors uniform cytoplasmic distribution. Such a situation is controlled in this procedure. 4 There is a potential for photobleachmg and photodamage. The loss of fluorescence intensity by photobleachmg of fura- is accompanied by the appearance of Ca*+-insensitive fluorescence species (24). Careful attention should be paid by keeping cells m the dark. 5. Incomplete de-esterification is another major problem Fura- and other rattometrtc fluorescent dyes are available as cell-permeable acetoxymethyl (AM)

Intraneuronal

Free Ca2+Measurement

167

esters which are hydrolyzed by mtracellular esterases, trapping the Ca*+-sensitive free acid inside the cell. This technique makes dye loading simple; however, some cells may not completely convert the AM form of the dye to its free acid The partially de-esterified species are highly fluorescent but msensltive to Ca*+, and there are no spectral shifts when Ca*+ concentration is changed (25). In order to prevent this problem, cells may be incubated for a longer time for proper de-esterification. 6. The basal intracellular free calcmm levels m neurons vary from 40 to 150 nM (26,27) If any abnormal values are seen during experimentation, the above listed five notes may be verified. 7. Several pharmacological agents may be used to delineate the sites responsible for increases in [Ca2+], (1,28,29). These agents include: Verapamil and diltrazem (L-type Ca*+ channel blockers) Tetrodotoxm (Nat channel blocker) 3-(2-carboxypiperazm-4yl)-propyl1-phosphonic acid (CPP) and MK-801 (glutamate/NMDA receptor channel blockers). 6-cyano-7-mtro qumoxalme-2,3-dione (CNQX; AMPA channel blocker) 3’,4’-dichlorobenzamyl (Na+-Ca2+ exchange mhibitor). Thapsigargm and cyclopiazomc acid (ER Ca*+ pump inhibitor). Caffeine and ryanodme (openmg Ca*+ release channel). Preincubatmg these agents with neurons before chemical exposure will provide important mformatron about the sites responsible for altered Ca*+ homeostasis

Acknowledgments This chapter has been reviewed by the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. References 1. Farber, J. L. (1990) The role of Ca*+ in lethal cell injury. Chem. Res. Toxicol. 3, 503-508. 2. Patel, J., Kreth, R. A., Salama, A. I., and Moore, W. C. (1991) Role of calcium m regulation of phosphoinositide signalling pathway. J. MoZ Neuroscl. 3, 19-27. 3. Fischer, S. K., Heacock, A. M., and Agranoff, B. W. (1992) Inositol lipids and signal transduction m the nervous system. J, Neurochem. 58, 18-38. 4. Drapeau, P. and Blaustem, M. P. (1983) Initial release of [3H]dopamine from rat striatal synaptosomes. correlation with calcium entry J Neuroscz. 3,703-7 13 5. Browning, M. D., Huganir, R., and Greengard, P. (1985) Protem phosphorylation and neuronal function J Neurochem 45,1 l-23. 6. Zimmerman, U.-J P and Schlaepfer, W W. (1982) Characterization of a brain calcium-activated protease that degrades neurofilament proteins. Biochemistry 21, 3977-3983

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7. Nicotera, P., Bellomo, G , and Orrenms, S. (1992) Calcium-mediated mechanisms m chemically mduced cell death Ann. Rev. Pharm. Toxzcol. 32,449470 8 Verity, M. A. (1992) Ca*+-dependent processes as mediators of neurotoxtctty Neurotoxacology 13, 139-148 9. Fehpo, V., Mmana, M.-D., and Gnsoha, S. (1993) Inhtbttors of protem kmase C prevent the toxicity of glutamate m primary neuronal cultures. Brain Res 604, 192-196. 10. Kodavantt, P. R. S , Mundy, W. R., Tilson, H. A., and Harry, G. J. (1993) Effects of selected neuroacttve chemicals on calcmm transportmg systems m rat cerebellum and on survrval of cerebellar granule cells Fund. Appl. Toxacol. 21,308-3 16 11 Farber, J L (1982) Biology of disease: membrane mJury and calcium homeostaSIS m the pathogenests of coagulative necrosis. Lab Invest 47, 114-123 12. Ctcchettt, F and Parent, A (1996) Striatal mterneurons m Huntmgton’s disease selective increase m the density of calretmm-tmmunoreacttve medium-stzed neurons. Mov. Disord. 11,619-626. 13 Sternberger, N. H., Sternberger, L. A , and Ulrich, J (1985) Aberrant neurofilament phosphorylatton in Alzheimer disease. Proc Natl. Acad. Scz USA 82, 4274-4276. 14. Peterson, C., Ratan, R. R , Shelanski, M. L., and Goldman, J E. (1986) Cytosohc free calcium and cell spreading decreases m fibroblasts from aged and Alzheimer donors. Proc Nat1 Acad. Scz USA 83,7999-8001 15 Abdel-Hamtd, K M. and Tymianski, M. (1997) Mechanisms and effects of mtracellular calcium buffering on neuronal survival in organotyptc htppocampal cultures exposed to anoxia/aglycemia or to excttotoxms. J Neurosa. 17,3538-3553. 16 Kater, S. B. and Mills, L R. (1991) Regulation of growth cone behavior by calcium. J. Neurosci. 11,891-899. 17. Tymianski, M. (1996) Cytosohc calcium concentrations and cell death zn vitro Adv. Neural. 71,85-105.

18. Tsien, R. Y., Pozzan, T., and Rink, T J. (1982) Calcium homeostasts m intact lymphocytes* cytoplasmtc free calcium monitored with a new, mtracellularly trapped fluorescent indicator J. Cell Bzol. 94,325-334. 19. Grynkiewtcz, G., Poenie, M , and Tsien, R. Y (1985) A new generation of calcmm indicators with greatly improved fluorescence properties. J. Bzol. Chem 260, 3440-3450 20. Minta, A., Kao, J. P Y., and Tsten, R. Y. (1989) Fluorescent indicators for cytosohc calcium based on rhodamme and fluorescem chromophores. J. Biol. Chem 264,8171-8178 21. Hayashi, H. and Miyata, H. (1994) Fluorescence imaging of mtracellular Ca*+ J. Pharmacol

Toxic01 Methods 31, I-10

22 Shuttleworth, T J and Thompson, J. L. (1991) Effect of temperature on receptoractivated changes m [Ca*+], and their determmations usmg fluorescent probes. J Blol Chem. 266,1410-1414. 23. Blatter, L A. and Wter, W G. (1990) Intracellular dtffusion, bindmg, and compartmentalization of the fluorescent calcium indicators mdo- 1 and fura- Biophys J. 58,1491-1499

Intraneuronal

Free Ca*+ Measurement

169

24. Becker, P. L and Fay, F. S (1987) Photobleachmg of fura- and its effect on determination of calcmm concentrations Am. J. Physiol. 253, C6 13-C6 18 25 Scanlon, M., Wtlhams, D W., and Fay, F. S. (1987) A Ca2+-msensitive form of fur-a-2 associated with polymorphonuclear leukocytes. J. B~ol. Chem. 262,6308-63 12. 26. Shafer, T. J , Mundy, W. R , Trlson, H. A., and Kodavantr, P R. S (1996) Drsruption of inositol phosphate accumulation in cerebellar granule cells by polychlorrnated biphenyls. a consequence of altered Ca2+ homeostasis. Toxlcol Appl Pharmacol. 141,448-455 27 Dubmsky, J. M. and Rothman, S. M (1991) Intracellular calcium concentratrons during “chemical hypoxia” and excitotoxrc neuronal Injury. J Neuroscl 11, 2545-255 1. 28. Xu, Y.-J., Shao, Q , and Dhalla, N S. (1997) Fura- fluorescent techmque for the assessment of Ca2+ homeostasrs in cardiomyocytes Mol. Cell Biochem 172, 149-157. 29 MilJanich, G. P and Ramachandran, J (1995) Antagonists of neuronal calcium channels: structure, functron, and therapeutrc imphcatrons. Ann. Rev. Pharrnacol. Toxicol. 35,707-734

15 Measurement of Calcium Buffering by Intracellular Organelles in Brain Prasada Rao S. Kodavanti 1. Introduction Cells maintain low concentrations of intracellular free calcium ([Ca2+],,by the effective operation of Ca2+pumps located in plasma membrane as well as mtracellular organelles, such asmitochondria and endoplasmic reticulum (microsomes) (1,2). Under normal condmons, Ca2+enters the cell by diffusion down an electrochemical gradient through voltage-dependent or receptor-mediated Ca*+-sensitive channels (2). Calcium can also be released from intra-cellular stores such as endoplasmicreticulum and mitochondria.As cytosolicfret Ca2+mcmases,Ca2+-bindmg proteins, mitochondria, and microsomes initially sequester the Ca2+ from cytosol. However, if there is a sustainedinflux of Ca*+,low cytoplasmicCa*+ level is maintainedby active extrusronthrough plasmamembraneCa2+-ATPaseand by the Na+/Ca2+exchanger(1,2). Mitochondrra and microsomesdiffer in the mcyhanismsby which they sequestercytoplasmicCa*+. Microsomal Ca2+-sequestrationis an active processinvolving ATP hydrolysisby Ca2+-ATPase.On the other hand, mitochondrial Ca2+-sequestration is anelectrophoreticuniport processdriven by the potential differenceestabhshedacrossthe mitochondrral inner membraneby an ATP-energized proton pump (I). These calcium-buffering processeswithin the neuron are illustrated in Fig. 1. The efficient operation of calcium sequestrationand extrusion mechanisms within the cell is crucial for the mamtenanceof normal calcium homeostasis. Several chemical agents including drugs, pesticides, and environmental pollutants are known to alter Ca2+homeostasis(3). For some agents, a close association between altered Ca2+homeostasisand symptoms of neurotoxicity has been established (4). Several environmental chemicals, which are known neurotoxrcants including polychlorinated brphenyls (5,6), chlordecone (71, triorganotms (8), lead (9), and mercury (IO), have been reported to inhibit Ca2+-sequestration by intracellular organelles and/or Ca2+-ATPase involved in the Ca2+-extrusion From

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Fig. 1. Different calcium-buffering processes involved in the maintenance of normal cellular Ca2+ homeostasis. The inlracellular free Ca”+ ranges from 0.1-0.3 nM, in where as extracellular calcium is in millimolar concentrations. There is about lO,OOOfold concentration gradient across the plasma membrane. This gradient is maintained by the effective operation of calcium pumps located in mitochondria, endoplasmic reticulum, and plasma membrane. All of these processes are energy-dependent and require the hydrolysis of adenosine triphosphate (ATP). process. I have previously reported that neuroactive chemicals belonging to different categories inhibit Ca2+-transport systems in the cerebellum to different extents (II). The importance of Ca2+ buffering by intracellular organelles has also been recognized in the neurodegeneration process. It has been shown that neuronal Ca2+ loading, attributable to hypoxic/ischemic injury, is believed to trigger neurotoxicity. Enhancing neuronal Ca2+ buffering by pharmacological treatments has been shown to attenuate the hypoxic/ischemic-induced neurodegeneration (12). All these studies clearly indicate the importance of intracellular Ca2+ buffering mechanisms in toxic phenomenon as well as in neurodegeneration. This is in continuation with the previous chapter describing the measurement of intracellular calcium-buffering mechanisms in neurons.

2. Materials 2.1. /so/a tion of In trace/War Organelles (Mitochondria and Microsomes) 1. Homogenizing buffer: Dissolve 42.78 g sucrose (formula weight [FW] 342.3; 250 mM), 0.596 g N-[hydroxyethyllpiperazine-N’-[2-ethanesulfonic acid]

173

Bra/n Organelle Calcium Buffermg

(HEPES; mol wt 238.3, 5 mM), 0 373 g potassium chlortde (KCl; FW 74 6; 10 mM), and 0.102 g magnesium chloride (MgCl,; FW 203.3; 1 mM) m dlstilled water, adJust pH to 7 05 wtth 1 N NaOH, and make up the volume to 500 mL 2. 1 2 M Sucrose* Dtssolve 41 076 g sucrose (FW 342.3) m distilled water and make up the volume to 100 mL. 3 0.32 M Sucrose. Dtssolve 10.954 g sucrose (FW 342 3) in distilled water and make up the volume to 100 mL.

2.2. Measurement of %$+-Uptake

by Mitochondria

and Microsomes

1. 60 mM Htstrdine-rmrdazole buffer, pH 6 8: Dissolve 1 862 g histidme (mol wt 155.2) and 0 817 g imtdazole (mol wt 68.1) in distilled water, adJust the pH to 6.8, and make up the volume to 200 mL. 2. 1 5 M KCl. Drssolve 11.2 g (FW 74 56) m distilled water and make up the volume to 100 mL. 3. 15 mM Ethylene glycol-bts-[P-aminoethyl ether] N, N,N’, N’-tetra-acetic acid (EGTA): drssolve 0.57 g (mol wt 380.4) in 100 mL histrdme-rmrdazole buffer. 4 75 mM MgC12: Drssolve 1 525 g (FW 203 3) in 100 mL dtstrlled water 5 75 mMSodmm aztde (NaN,)* Dtssolve 0.488 g (FW 65.01) in 100 mL dtsttlled water. 6 75 mM Ammonmm oxalate: Drssolve 1.005 g (FW 134) m 100 mL drsttlled water. 7. 5 805 mM Calcmm chloride (CaCl& Dissolve 85.3 mg (FW 147.02) m 100 mL drstllled water. 8. 45CaC12(0.1 pCt/lO pL) Drlute the stock obtained from NEN to get 0 1 pCr/lO FL. 9. 100 mMTrts-buffer, pH 7 4. Dtssolve 12.01 g Trrzma-base (mol wt 120.1) m 900 mL distilled water, adJust the pH to 7.4 with HCl and make up the volume to 1 L 10. 10 mM Tars-buffer Dilute 100 mM Tns buffer 10 times m drsttlled water before use. 11. 75 mM Adenosme tnphosphate (ATP, 3H20): Dtssolve 450 mg (FW 551.2) m 10 mL dtstdled water Just before use

3. Methods 3.1. Isolation of Mitochondria and Microsomes The method described here for subcellular fractionation is similar to that of Dodd et al. (.Z3) with slight modlflcatlons (II). 1. Decaprtate the animal, dissect distmct brain regions on ice-chilled metal plate, and keep them on ice at 4°C. 2 The entire fracttonatron (homogemzatron and centrifugation) should be done m cold at 4°C 3 Homogenize the brain by hand m 9 vol of cold homogemzing buffer with a Teflon pestle and glass homogemzer. 4. After homogemzatton, centrifuge the homogenate at 1OOOg for 10 mm. Discard the pellet.

5. Take the supernatantand centrifuge at 9000g for 20 mm. 6 Save the supernatant for mrcrosome preparatron.

7 Suspend the pellet m 10 mL of 0.32 A4sucrose. Layer it on 1.2 M sucrose (8 mL). 8 Centrifuge at 150,OOOgfor 20 mm in Beckman Instruments (Schaumburg, IL) L565, rotor T170. 9 Collect the pellet contammg mltochondrla and suspend m homogemzmg buffer (l-2 mg protein/ml) 10 Take the supernatant after centrlfugatlon at 9OOOgand centrifuge at 105,OOOg for 60 mm. 11. Collect the pellet containing mlcrosomes and suspend in homogenizing buffer (l-2 mg protein/ml) 12. Determine protein content m these fractions using Folm-phenol reagent (14) or Coomassle brilliant blue dye (15) 13 Divide mlcrosomal and mltochondrlal fractions mto 1-mL allquots and use them fresh for 45Ca2+-uptake studies (see Note 1)

3.2, Measurement of 45C$+-Uptake by Mitochondria and Microsomes 1 The 45Ca2+-uptake by intracellular organelles such as mltochondna and microsomes can be determined by incubating the fractions with radioactive CaCl, as outlined by Moore et al. (16) 2. The reaction mixture of 1.5 mL should contam the followmg reagents: Reagent

Volume

Final cont.

Hlstldme-lmidazole Potassium chloride EGTA Magnesium chloride Ammonium oxalate Sodium azlde

0.75 mL 0.10 mL 0.10 mL OlOmL OlOmL O.lOmL

30 mM 100 mM lmh4 5mM 5mM 5 m&Z

(Add 0 I mL water instead of sodium azlde for mltochondrlal

Calcium chloride 45CaC12 (see Note 2) Protein (microsomal or mitochondrial suspension) ATP

OlOmL 0.01 mL 0.04 mL OlOmL

uptake, see Note 3)

5@4 0 1 kc1 60-80 pg 5mM

3. Add all the above reagents serially except ATP into the test tubes. 4 Conduct nonspecific bmdmg studies by keeping one set of tubes on ice wlthout the addition of ATP 5. For m vitro studies, determine a dose-response by adding different concentrations of test chemical and appropriate vehicle solutions (see Note 4). 6. Keep all the tubes m water bath maintained at 37°C for 5 mm (preincubatlon) 7. After 5 mm premcubatlon, start the 45Ca-uptake by adding ATP (Do not add to nonspecific tubes) to each tube at 10-s intervals. 8. Incubate for 20 mm and stop the 45Ca-uptake by keepmg the tube m cold on ice and adding 5 mL cold 10 mM Tris-buffer at 10-s intervals

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Buffering

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9 Fdter the contents through a 0.45~pm Mlllipore (Bedford, MA) nitrocellulose or mixed cellulose ester filters presoaked m 10 mM Trls buffer using Milhpore filtering device (sampling manifold) under vacuum. 10. Wash the filters twice with 5 mL of cold 10 mM Tris-buffer each time. 11 Remove the filters carefully and place them in vials containing 10 mL scintlllatlon fluid (e g., Ultlma GoldT”). Count them in liquid scintillation counter. Express as pmol/mg protein/min. 12. Normal actlvlty of brain mlcrosomal and mltochondrlal uptake in adult rats 1s approx 40 and 14 pmol/mg protem/mm, respectively. In cerebellum, the activities are 40-50 pmol/mg protein/mm for mlcrosomal 45Ca-uptake and 15-22 pmol/mg protein/min for mltochondrlal 45Ca-uptake (II) (see Note 1). 4. Notes 1. Freezing the subcellular frachons and usmg them at a later time will result in loss of 45Ca-uptake activity. There will be no substantial loss of actlvlty if the microsomes are frozen in liquid nitrogen and stored at -80°C for later use. However, there will be a significant loss of actlvlty if mitochondria are frozen and used at a later time 2 The half life of 45CaC12is 165 d Care must be taken to account for the loss of radioactivity when using 45CaC12in the assay 3 Care must be taken while adding sodium azide, since this would mactlvate mltochondrial electron transport and 45Ca-uptake. Do not add sodium azide to the tubes when measuring mitochondrial 45Ca-uptake. 4 Use known mhlbltors of 45Ca-uptake process as positive controls before testing new chemicals, e.g., chlorpromazme (inhibitor of microsomal and mitochondrlal 45Ca-uptake), Ruthenium red (mhlbltor of mltochondrlal 45Ca-uptake), thapsigargin, and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (inhibitors of microsomal 45Ca-uptake).

Acknowledgments This chapter has been reviewed by the National Health and Environmental Effects Research Laboratory, US Envxonmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial product constitute endorsement or recommendation for use. References 1 Carafoh, E. (1987) Intracellular

calcium homeostasis. Annu. Rev Biochem. 56,

395-433. 2 Miller, R J. (1991) The control of neuronal Ca2+-homeostasls. Prog. Neurobiol. 37,255-285 3 Komulamen, H and Bondy, S C (1988) Increased free intracellular Ca2+ by toxic agents: an index of potential neurotoxlcity. Trends Pharmacol. Sci. 9, 154-156

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4. Johnson, J. D., Melsenheimer, T L., and Isom, G E (1986) Cyanide-induced neurotoxlaty: role of neuronal calcmm. Toxzcol Appl. Pharmacol. 84,464-469 5 Kodavanti, P. R S., Shin, D., Tllson, H A., and Harry, G J. (1993) Comparative effects of two polychlorinated biphenyl congeners on calcmm homeostasis m rat cerebellar granule cells ToxicoE Appl. Pharmacol 123,97-106. 6 Kodavantl, P. R. S., Ward, T. R , McKmney, J D , and Tilson, H. A (1996) Inhlbltion of mlcrosomal and mltochondrlal Ca*+ sequestration in rat cerebellum by polychlormated blphenyl mixtures and congeners: structure-activity relationships. Arch Toxzcol. 70, 150-157. 7. Desaiah, D , Chetty, C. S., and Prasada Rao, K S. (1985) Chlordecone inhibltlon of calmodulm activated calcium ATPase m rat brain synaptosomes J. Toxicol. Envw Hlth. 16, 189-195. 8. Prasada Rao, K. S , Chetty, C. S., Trottman, C. H , Uzodmma, J E , and Desaiah, D (1985) Effect of trlcyclohexylhydroxytm on synaptosomal Ca*+-dependent ATP hydrolysis and rat bram subcellular calmodulm. Cell Bzochem. Funct. 3,267-272. 9 Pounds, J. G. (1984) Effect of lead mtoxlcatlon on calcium homeostasls and calcmm-mediated cell function. a review. NeuroToxzcology 5,295-332. 10. Binah, O., Mein, U., and Rahamlmoff, H. (1978) The effects of mercuric chloride and mersalyl on mechanisms regulating intracellular calctum and transmitter release Eur J. Pharmacol. 51,453-457. 11 Kodavanti, P. R. S., Mundy, W. R , Tllson, H. A., and Harry, G. J (1993) Effects of selected neuroactive chemicals on calcium transporting systems in rat cerebellum and on survival of cerebellar granule cells Fund. Appl. Toxicol. 21,308-3 16 12. Abdel-Hamld, K M. and Tymianskl, M (I 997) Mechanisms and effects of mtracellular calcium buffering on neuronal survival m organotypic hippocampal cultures exposed to anoxlafaglycemla or to excltotoxms J. Neurosci 17,3538-3553. 13. Dodd, P. R., Hardy, J. A., Oakley, A. E., Edwardson, J A , Perry, E K , and Delaunoy, J. P. (198 1) A rapld method for preparing synaptosomes: comparison with alternatlve procedures Brain Res 226, 107-l 18 14 Lowry, 0. H., Rosebrough, N. J , Farr, A L., and Randall, R. J (1951) Protem measurement with the folin-phenol reagent. J. Biol. Chem. 193,265-275. 15. Bradford, M. M. (1976) A rapid and sensitive method for the quantltation of mlcrogram quantities of protein utilizing the prmclple of protein-dye bmdmg Anal. Biochem. 72,248-254.

16 Moore, L., Chen, T., Knapp, H R., Jr., and Landon, E. L (1975) Energy dependent calcium sequestration actlvlty m rat liver microsomes. J Blol. Chem. 250, 4562-4568

Receptor-Mediated Release of lnositolphosphates in Brain Slices PushpaTandon 1. Introduction Target-cell response to a number of neurotransmitters, growth factors, hormones, and other stimuh are initiated by cell-surface receptor-mediated activation of phosphohpase C (PLC) and the rapid hydrolysis of phosphomositides (J-3). The activation of PLC by receptors for most neurotransmitters and growth factors occurs through a mechanism involvmg a guanine nucleotide regulatory protein or G protein. The PLC-catalyzed hydrolyses of phosphatidylinositol 4,Sbisphosphate (PI(4,5)P2) results in the formation of inositol1,4,5 trisphosphate (Ins( 1,4,5)P3) and diacylglycerol (DAG). Both Ins(1,4,5)P3 and DAG have second-messenger functions inside the cell. Ins( 1,4,5)P3 mobilizes mtracellular Ca2+by binding to specific mtracellular receptors that promote opening of calcium channels in vesicular storage sites associated with endoplasmic reticulum (4,5), whereas DAG binds to and activates protein kinase C (PKC), resultmg m the phosphorylation of a number of intracellular proteins (1,2). The termination of the second-messenger activities of Ins( 1,4,5)P3 can occur by two metabolic routes: phosphorylation by a 3-kinase and dephosphorylation by a 5-phosphatase. Ins(1,4,5)P3 can thus be metabolized by dephosphorylation into Ins( 1,4)P2, Ins(LF)P,and then into free inositol or phosphorylated first into Ins(1,3,4,5)P4 (which may also have second-messenger function) and other higher inositol phosphates (IPs) including IP5 and IP6 (phytic acid), and then dephosphorylated into lower IPs (Ins( 1,4,5)P3 and its metabolites, or Ins(1,3,4)P3 to Ins(I,4)P2 and Ins(3)P). The concentration of Ins(1,4,5)P3 m basal cells ranges between 0 1 and 0.2 pM and can go up from 1 to 25 pM on strmulatron with high concentrations of receptor agonist (3,6). From

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The last two decades have seen an explosion of interest in the study of receptor-mediated release of second messengers. The use of lithium to inhibit phosphatases preventing the rapid metabolism of inositoltnsphosphate (IP3) to mositolbisphosphate (IP2) and mositolmonophosphate (IPl) has proved to be a useful tool in the study of receptor-stimulated release of IP3 (7-9). Since Ins( 1,4,5)P3 is an intracellular second messenger in a number of neuronal cells, considerable effort has been invested in understanding the receptor-stimulated metabolism of IP(s) in the brain (610-12). Receptor-mediated release of inositolphosphates has been studied in brain regions under numerous conditions including development and aging (13-16), following lesions of various neuronal populations, and exposure to neurotoxicants (1,17-20) and pesticides (21) The following protocol gives a description of the commonly used procedure for the measurement of receptor-mediated release of IPs m brain slices 2. Materials 2.7. Supplies 1. 2. 3. 4. 5.

6

7 8.

9. 10.

McIlwain mechamcal ttssue chopper with a movable platform to make tissue slices. Paintbrushes to wet the blade and handle tissue. 5% O,-95% CO2 gas tank with multiple connectrons Shaking water bath for mcubatron at 37°C 6-mL polypropylene tubes with snap au--tight caps (Falcon, Los Angeles, CA, 2063 tubes work well) and 15-mL polystyrene capped tubes (Falcon 2095 or any 15mL graduated polystyrene tubes) Chromatography columns, total volume 10 mL Columns can be made from glass Pasteur pipets by inserting a glass wool plug at the bottom. The total volume of these columns is small, which can be problematrc. Poly-Prep (Bio-Rad, Rrchmond, CA) chromatography columns that have a filter disk near the tip work well and can be reused several times. A stand to hold multiple columns that can fit over a box of scintillation vials such that the eluate from each column can be collected mto separate vials Pipets: Apart from Pasteur prpets and a IO-mL glass pipet, it is also good to have at least two repeat ptpets, one to be used for stopping the reaction, and the other for water and solutrons to be used for elutron of IP(s). [3H] Myomositol (15 Wmmole, DuPont, Boston, MA; New England Nuclear, Boston, MA; or Amersham, Arlington Heights, IL) [3H] labeled mosrtolmonophosphate (IPl), mosrtolbrsphosphate (IP2), mosrtoltrisphosphate (IP3), mositoltetrakrsphosphates (IP4) (DuPont, New England Nuclear) are also needed as standards, if various IP(s) are to be eluted separately from the ion exchange column. If total IP(s) are to be eluted together, a mixture of [3H] labeled IP(s) can be used as standard. These standards are important to test the elution profile of various IP(s) from the ion-exchange column Slight varratron between batches of Dowex AGl-X8 (ion-exchange resin) can occur, makmg slight admstments in elution volume and/or salt gradient necessary to get

Receptor-Mediated

11 12. 13. 14 15.

lnositolphosphates

Release

179

mmlmum overlap and good resolution for separation and elutlon of each mositolphosphate species Receptor agonists and other compounds used for receptor-stimulated release of IP(s). Dowex AGl -X8,200-400 mesh, formate form (Blo-Rad, analytical grade anionexchange resin). Liquid scintillation counter Scintlllant for counting organic and high salt concentration aqueous solutions 12 and 20 mL scmtlllatlon vials

2.2. Solutions 1 Krebs Ringer bicarbonate buffer (KRB) For 500 mL buffer. 3 45 g NaCl (118 mM), 175 mg KC1 (4 7 mM), 80mg KH,P04 (1.18 mM), 145 mgMgS0, (7H,O) (1.18 mM), 1 04 g NaHC03 (24.8 mM), 0.9 g glucose (10 mM), and 55 mg CaCl, (2H20) (0 75 mM, add last after all other salts have been dissolved m dH,O) Dissolve m 450 mL of water, oxygenate, and equilibrate with 5% 02/95% CO2 gas mixture by bubblmg the gas into the buffer for several min, adJusting pH to 7 4 with 1 M HCl, and makmg up to 500 mL. The buffer should be made the day before the experiment (stored at 4°C) and pH set on the day of the experiment. It 1s not advisable to make and store the buffer for longer periods since it turns cloudy and forms a precipitate. Stock solutions of all salts can be made and stored separately at 4”C, the buffer mixed and diluted with dHzO to get final concentration of various salts, oxygenated, and the pH set just before use. 2 8 mM hthmm chloride m Krebs Rmger Buffer: For 500 mL of buffer weigh* 169.6 mg LlCl(8 mM), 3.21 g NaCl(l10 mM), 175 mg KC1 (4 7 mM), 80 mg KH,PO, (1.18 mM), 145 mg MgSO, (7H20) (1 18 mM), 1 04 g NaHCO, (24.8 mM), 0.9 g glucose (10 m&Z), and 55 mg CaCl, (0.75 mM; add last after all other salts have been dissolved m dH20) The receptor-agonist-stimulated release of IPs 1sperformed in the presence of LiCl. LiCl 1san uncompetltlve inhibitor of inosttol phosphatase (an enzymes that metabolizes IPs), allowmg for the accumulation and measurement of total IPs possible. Notice that the concentration of NaCl(ll0 IIUV) 1sdifferent than in the KRB (118 n&f) without LICI Keep oxygenated with O&O2 gas mixture The buffer can be made the day before the experiment, oxygenated, and the pH set just before use (see Note 1). 3 [3H] Myoinosltol: [3H] Myoinositol used at a concentration of 25 pCi per 100 mg brain tissue in 0.5 mL KRB at a concentration of 3.2 yM (usually obtained at a concentration of 1 mCi/mL, specific activity 15 Wmmole; DuPont, New England Nuclear, or Amersham) Take 50-pL of stock [3H] myomosltol solution and add to 950 pL of KRB for incorporation into slices from two hlppocamp1 from adult rat ([3H] myoinosltol obtained as stock solution is stable for several months at 4°C) All necessary precauttons for working with radioactivity should be taken to prevent exposure to radloactlvlty and contammatlon of work areas.

180

Tandon

4. Agonlsts and antagonists: Receptor agomsts and antagonist to be used m the experiment are always made fresh m KRB contammg LiCl. Some of the mosrtolphosphate-linked receptors and the common agonist used for receptorstimulation are: muscarmic cholmerglc (carbachol), noradrenergm (norepinephrme), serotonergic (5hydroxy tryptamme), metabotropic glutammergic (ibotemc acid, quisquahc acid). It is important to get water-soluble salts of the compounds to be used. The weights and concentrattons for some of the agonists and antagonist are listed below. Carbachol 1.369 mg/3 mL (2 5 x 1 n&f) Norepmephrme (HCl) 1.408 mg/3 mL (2.5 x 1 mM) 5-hydroxy tryptamme (serotonm) 1 595 mg/3 mL (2 5 x 1 mA4) Ibotemc acid 1 19 mg/3 mL (2 5 x 1 mM) Quisquahc acid 1.42 mg/3 mL (2.5 x 1 nuU) Atropine (sulfate) 5 08 mg/3 mL (2 5 x 1 mM) Plrenzepme (dihydrochloride) 318mg/3mL(2Sxtn-&f) AP-3 1 268 mg/3 mL (2 5 x 1 mM) 5. Stop solution chloroform* methanol. concentrated HCl (1.2 0 02) To 100 mL chloroform add 200 mL methanol and 2 mL concentrated HCl 6. Solutions for elution of IPs. Various IPs are separated by an ion-exchange column (Dowex AGl-X8) and eluted using a salt gradient a The glyceromositolphosphates (GIPs) are eluted by a solution of 5 m&4 sodmm tetraborate (Borax) containing 60 mM sodium formate (solution 1) For 500 mL of solutron, weigh 92 5 mg of sodium tetraborate and 2.04 g of sodium formate, dissolve in dHzO The solution can be made and stored at room temperature b. IPl to IP5-6 are eluted from the Dowex column by increasing concentrations of ammomum formate contaming formic acid A stock solution of ammonium formate and formic acid can be made and stored at room temperature. A. 5 M ammonium formate (157.65 g of ammomum formate m 500 mL of dH*O). B. 1 M formic acid (20 mL of 95% formic acid m 500 mL with dHzO) C. For 500 mL of salt solutions of different concentratrons to be used for the elution of various IP(s), add the following amounts of A (5 M ammomum formate solution) and B (1 M formic acid): IPl(s)* 20 mL A + 50 mL B + 430 mL Hz0 (final concentration’ 0.2 M ammonmm formate with 0 1 M formic acid, solution 2) IP2(s) 50 mL A + 50 mL B + 400 mL Hz0 (final concentration: 0 5 M ammomum formate with 0 1 M formic acid, solution 3) IP3(s). 80 mL A + 50 mL B + 370 mL Hz0 (final concentratron: 0.8 M ammonium formate with 0.1 M formic acid, solution 4) IP4(s)* 120 mL A + 50 mL B + 330 mL Hz0 (final concentration. 1.2 M ammonmm formate with 0 1 M formic acid, solutron 5) IP5-6(s): 200 mL A + 50 mL B + 250 mL Hz0 (final concentration. 2 M ammonium formate with 0 1 M formic acid; solution 6) All salt solutions can be made and stored at room temperature.

Receptor-MedIated

lnosltolphosphates

Release

181

7. Dowex activation: Empty the contents of a bottle (500 g) of Dowex AGl-X8, (8% crosslinked, 200-400 mesh, formate form, analytical-grade anion-exchange resin from Bio-Rad) mto a 2-L beaker. Add 1000 mL of 1 M NaOH, stir well, and let settle for lo-15 mm. Decant the top solution and the floating resin. Add 1 M NaOH again and repeat. Wash twice with dH20. Now repeat the process with 1 M formic acid. Wash once again with dH,O By this time there should be no resin floating on the top. Let the Dowex settle and add dH20 to 1.5 times the volume of Dowex (on top of the resin). The Dowex is now ready for use and should be stored at 4°C Dowex AGl-X8 can be reused after reactlvatlon, by repeatmg the activating procedure mentioned above (see Note 3). The elutlon profile for each batch of Dowex can be different. It is important to elute standards for each IP(s) usrng the salt concentration mentloned above for each IP before starting the experiment. (Also read Subheading 3.5.).

3. Methods Make sure that all materials are available and arranged as needed before starting the experiment Although some of the solutions have to be made fresh, it 1s advisable to collect all other things and arrange them such that they are easily assessable. Since the viability of the slices decreases significantly with time if not kept under optimal conditions and the receptor-stimulated release of IPs is time dependent (with changes occurring in seconds), the success of the experiment depends upon the preparation done before hand. The procedure consists of several steps containing multiple tubes. Prepare and number tubes for each step to avoid confusion during the experiment.

3.1. Slice Preparation 1. Remove the brain from the skull onto a piece of wet (with oxygenated KRB) filter paper Do not put on Ice. 2. Dissect the brain regions to be used with minimum amount of handling Try not to hold the tissue between forceps. The tissue should be dissected quickly with minimum handling since the viability of the tissue slices depends upon slice preparation. Use a sharp razor blade to cut the brain region out rather than a forceps or spatula and do not pinch any of the brain regions out (see Note 2). 3. Have the tissue chopper adjusted to cut 350~pm thick slices. Make sure that the blade 1ssharp, the speed and the force of the blade adjusted for cutting. (The force of the blade should be Just enough to cut the slices only not the filter paper) (see Note 3). 4 Gently lift the tissue by putting a spatula under it, weigh and position on the wet filter paper placed on the platform of the tissue chopper. Wet the blade and the tissue with KRB to prevent the slices from sticking to the blade Start the tissue chopper and section the tissue all the way through, stop and turn the platform 90” and cut again such that the tissue 1scut into 350 x 350~pm slices. 5. Using a paint brush lift the slices from the filter paper and put m a round-bottomed capped tube containing warm (37°C) KRB (15-20X volume of slices)

182

Tandon Using a 10 mL glass pipet, gently pull the solution into the plpet to separate &es. Repeat once if necessary Oxygenate the slices and maintain under constant 02/C02 atmosphere (see Notes 4 and 5).

3.2. Preincubaiion

and Washing of Slices

1. Start and set the timer for 30 mm. Put the tube containing the shces m the water bath at 37°C with gentle shakmg. From this point on mamtam all solutions made m KRB or KRB with LlCl oxygenated and at 37°C Adjust the pressure of the gas such that there 1s gentle agitation of the solutions or slices bemg oxygenated 2. Within the next 30 mm wash the slices three times as follows. lift the tube gently out of the water bath lettmg the slices settle for a few seconds The top solution will be cloudy and may contam fragmented shces and pieces of membrane at this point Aspirate carefully without dlsturbmg the slices m the bottom, replace with fresh KREJ fill with 02/C02, and place the tube back in the water bath. Repeat twice (see Note 6). 3 At the end of the preincubatlon period, take the tube out, let slices settle, and aspirate the buffer By now the buffer should be clear without any floating pieces of tissue. Separated slices should be seen wlthout any debns when the tube 1sshaken gently. If this 1s not the case, the slices can be washed once more with KRB.

3.3. Incorporation

of PH] Myoinositol

1. After the second wash during the premcubatlon period, dilute the [3H] myomosito1 m KRB as mentloned earlier (25 jMZ1/100 mg tissue m 0 5 mL buffer at a concentration of 3.2 FM) and keep at 37°C. 2 At the end of the premcubatlon period and after the last wash let the slices settle, aspirate, and add [3H] myomositol m KRB. Set the timer for 120 min and put the tube back m the shaking water bath. The slices should be mcubated under constant flow of 02/C02 Attach a glass Pasteur plpet to the tube connected to the O,/CO, gas tank, make a couple of holes m the cap and put the tip of the Pasteur plpet mto the tube through the hole m the cap. Be careful to adjust the flow of the gas to agitate slices gently (see Note 6) 3 At the end of the mcorporation period (120 mm) remove the tube from the water bath and aspirate the buffer into a separate container for radloactlve waste Be careful since this solution 1s highly radioactive. Wash the slices three times with 10X volume warm (37°C) KRB to remove umncorporated r3H] myomosltol. By the third wash, the slices should be separated and the wash buffer clear. Now add oxygenated KRB containing LlCl mto the tube and let the slices settle. The shces are now ready to be ahquoted mto tubes for the agonist stimulated release of IP(s) (see Note 7)

3.4. Stimulated

Release of Inositol

Phosphates

1 Durmg mcorporatlon of [3H] myomosltol into slices, set up tubes for the release of inositolphosphates by agonist stimulation. Small (6-mL) polypropylene tubes with air-tight caps that can be snapped on work best for this The experiment should be done in trlphcate It IS very important to include tubes for each bram

Receptor-Mediated

lnositolphosphates

Release

Table 1 Experimental Protocol of lnosltol Phosphates

for the Stimulated (IPs)

Release

Experimental condition

Basal group A

Stimulated group A

Basal group B

Stimulated group B

Tube # KRB + L&i Slices

1,2,3 100 pL 50 pL

45,6 100 p.L 50 pL

7J-U 100 l..tL 50 yL

10,11,12 100 jtL 50 pL

Premcubate under 02/C02 Agonist KRB+LrCl Incubate under O&O2 at Stop solutron (cold) Vortex, put on ice Chloroform dHzO

at 37°C for 5 mina 0 100 pL 100 pL 0 37°C (5-30 mm) 1 mL 1 mL

0 100 l.tL

100 pL 0

1 mL

1 mL

400 pL 400 l.tL

400 pL 400 pL

400 pL 400 pL

400 pL 400 pL

183

“The mcubatron period must be emprncally determined

2. 3 4.

5.

6.

7

8.

regron and each animal per treatment group without agonist (basal release) Number and arrange tubes m a stand. Make the agonists needed for the experiment m KRB + LiCl and keep warm Add the appropriate volume (Table 1) of oxygenated KRB + LrCl into each tube, cap, and keep in water bath till needed. Following incorporation of [3H] myoinosrtol into slices and washing slices to remove unmcorporated [3H] myoinositol, pipet 50 pL of gravity-packed slices (approx 600-700 pg protein) mto each tube. Approximately lo-12 tubes can be obtained from two hrppocampi weighing 200 mg total werght. Start the stop watch, fill each tube with the O&O, mixture, close the tube wrth the cap, and put mto the water bath with gentle shaking. Put each tube in the water bath 30 s apart and time each tube such that an exact premcubatron (5 mm) and mcubatron (5-30 mm) period can be achieved Add the appropriate volume of warm agonist solution or KRB (Table 1) at exactly 5 min of premcubatron time, fill the tube with 02/C02, cap, and put back m the water bath with gentle shaking. Always start with the basal release tubes to prevent accidental contamination of the tube with agonist Be very careful to maintain the order of tubes when starting and stopping the reaction. The final volume m the reaction tube is 250 pL at this point. Shut the gas tank off since it will not be needed any more Make sure the stop solution is ready for ptpettmg and kept on me. A container of ice with a test tube stand to hold the reaction tubes will also be needed At the end of incubation period take the tube out, add 1 mL cold stop solution, vortex, and put on ice. Stop the reaction m each tube m the order it was started m,

184

Tandon such that the reaction time m each tube 1s consistent to prevent wtthm-expertment variability Add 400 pL each of chloroform and dH,O, vortexmg after each addition to extract the water soluble IPs into the aqueous phase and the mosrtol lipids into the organic phase (see Notes 8 and 9). The total volume in the tube is now 2.05 mL. Keep the tubes at 4°C for 30-45 mm (see Note 10).

3.5. Separation

of Inositol

Phosphates

1. Centrtfuge at approx 1200g for 10 mm m a table-top centrifuge to separate the two phases. The orgamc phase (0 92 mL) is at the bottom, the tissue at the mterphase and aqueous phase (1 13 mL) at the top. Note the volume of the phases, these are important m the calculations for IP release later. 2 Count 50 l.tL of the organic phase (contammg phosphomosttol hprds) and the aqueous phase (contaming water soluble IPs) from each tube, in the liquid scmttllation counter set to count for trmum These values will be used to calculate total mcorporation (see Note 8). 3. Number and arrange 15-mL polystyrene tube correspondmg to the tubes m the stimulation reaction Ahquot 800 pL of the top aqueous phase (without dlsturbmg the interphase) and add to the correspondmg tube containing 5 mL of dH,O and vortex well Remember to take due precautions since the aqueous phase is radtoacttve If the organic phase has not been well separated resulting m chloroform being present m the aqueous phase, the solution gets cloudy and should clear upon vortexmg. (This, however, IS not desirable and can be avoided by increasing the centrifugation speed and the separation time at 4°C). 4 Get the activated Dowex from the refrigerator The resin settles at the bottom of the beaker and has to be stirred with a glass rod Put on a magnetm stirrer and stir slowly (see Notes 11 and 12). When an even slurry 1s formed, pipet 2 mL mto of the slurry mto each tube containing the diluted aqueous phase. 5. Arrange the small chromatography columns in a stand (after opening the column tip by removing the small plasttc tab if using Blo-Rad Poly Prep columns) that can fit a box of 20-mL scintillation vials Put the stand over a plastic tub or contamer and wash with dH,O to make sure that the filter dtscs are m place and not blocked. The columns are now ready for use. (This can be done while the reaction tubes are sittmg m ice before centrifugatton). Number and arrange the columns to match the tubes m the stimulation reaction 6 Vortex each tube contammg the diluted aqueous phase and the Dowex and qurckly pour the solution into the correspondmg column As the Dowex settles it should create a 0 8 mL bed volume m the column 7. Wash each tube by adding 5 mL of dH20, vortexmg, and pourmg mto the corresponding column Let the water run through the column and wash twice for a total of three washes with 15 mL of dH,O (5 mL x 3) The water wash elutes free mosltol from the Dowex Since all the eluate from the Dowex column is radioactive, it should be treated accordmgly. 8. Add 10 mL of solution 1 (sodium borate/sodium formate) to each column, trymg not to disturb the resin bed. Let the solution run through. Lift the stand with the

Receptor- Media ted lnosltolphospha tes Release Table 2 Separation

of lnositol

Phosphates

185

on AGI-X8

IP(s) eluted

Salt concentration

Elution volume

Free mositol GPIP( s) IPl(s) IP2(s) IP3(s) IP4(s) IP5-6(s) Total IP(s)

dH,O 5 m&f borax/60 m&Z sodium formate 0.2 M AF” I 0.1 M formic acid 0 5 M AF J 0.1 M formic acid 0.8 M AF J 0 1 M formic acid 1 2 M AF J 0 1 M formic acid 2 0 M AF J 0 1 M formic acid 2 0 M AF J 0 1 M formic acid

15 mL (5 mL x 3) 10 mL 8mL 8mL 8mL(4mLx2) 8mL(4mLx2) 8mL(4mLx2) 10 mL (5 mL x 2)

aAlJ ammonutmformate columns and place over 20-mL scintillation vials (or other tubes) to collect the eluate from each column (seeNote 13). Solutions 2-6 are usedto elute IP(s) from the column (Table 2) IPl(s) and IP3(s) are eluted from the column by a single 8mL fraction of solution 2 and solution 3. The elution of IP3 to 6 is achieved m two fractions by adding 4 mL x 2 (8 mL total) of solutions 4, 5, and 6 respectively. This procedure allows for better elution and separation of the higher IP(s) If all IP(s) are not to be separated, then total IP(s) can be eluted in one fraction as follows* Wash with 15 mL (5 mL x 3) of dH20 as mentioned earlier. Add 10 mL solution 1, and let run through. Elute total IP(s) by the addition of 5 mL x 2 (10 mL total) of solution 6 Both fractions can be collected and counted together The high concentration of salt elutes all IP(s) from the column together Count each fraction for tritium m a liquid-scmtillation counter. High salt concentrations used in the elution procedure often make the dilution of some fractions necessary before counting. The stimulation reaction tubes containmg the tissue (and the remammg organic and aqueousphase) can be dried down under nitrogen, and the tissue used for protein estimation if needed (seeNote 9). The followmg flowchart summarizesthis method. 1 2 3. 4. 5. 6. 7.

Slice preparation (350 x 350 pm). Wash with KRB and premcubate at 37°C (30 mm). Incorporate [“HI myomosttol(37”C, 90-120 min). Wash with KRB + LiCl. Stimulation of agonist-stimulated IP release(37°C 5-30 mm). Stop reaction and extract water soluble IPs Separate IPs over Dowex column.

3.6. Calculations The expertment results in

worksheet.

a Iarge series of values that are best handled m a

Tancfon

186 Total incorporation

of inosltol

(DPM orgamc phase

x

into slices in each tube = TI Cl) + (DPM aqueous phase x C2) = TI

Cl = 18.4 and C2 = 22.6, if DPM represent 50 PL of both phases from a total of 0.92 mL of organic and 1.13 mL of aqueous phase m the reaction tube. Release of IP(s) = R DPM IP(s) x C3 = R

(1)

C3 = 1.4125, if 0.8 mL of the aqueous phase used for elutlon of IPs from a total of 1.13 mL. The release of IP(s) obtained from the tubes without agomst (basal, BR) 1s basal release, whereas the release of IP(s) in the presence of agonist is stlmulated release (SR). The stimulated release (SR) of IP(s) 1s often reported as a fraction of total incorporation or as a fraction of basal release. SRJTI

(2)

or, (SR/TI)/(BR/TI)

(3)

When calculating release of various IPs, the DPM values obtained for each fraction are used in the equation. For IP3-6 eluted m two fractions, DPM values from both fractions are added to get the final DPM value to be used m the equation.

4. Notes 1 Oz/COz should be contmuously bubbled m the buffer to maintain equihbnum. Equilibrating the solution with O,/CO, alters the pH of the solution, making it important to oxygenate the buffer before adjusting pH to 7.4 and keeping the buffer under Oz/CO, atmosphere It is also advisable to add CaCI, last (specially if using stock solutions), it can precipitate and turn the buffer cloudy, in which case it should be made again. 2 Tissue taken from all treatment groups should always be run together within one experiment Keep the time for premcubatlon and agonist stimulation constant (within seconds) between all tubes. Tubes for basal release from each ammal (or group, if the tissue 1s pooled) should always be included and serves as an internal control. Brain regions that are small (striatum and hlppocampus) may have to be pooled from two or more animals to get enough tissue for the appropriate number of tubes needed for a complete study. 3. Make sure all tubes are numbered and labeled accordingly Prepare for the experiment a day m advance. Check the blade m the tissue chopper, the micrometer setting (350 pm), the speed, and the blade force before starting the procedure. Wet the filter paper, tissue, and the blade with oxygenated KRB to prevent sticking All these points are important m making viable shces Be careful when hft-

Receptor-Mediated

4.

5.

6

7

8.

9.

lnosltolphosphates

Release

187

mg the slices from the filter paper, if the filter paper has been sliced along with the tissue it ~111come up with the tissue and can be removed during washing and can be problematic. If the shces are sticky, they can be separated by using a lo-mL pipet. Be careful not to use a pipet with a narrow tip, this can break the slices rather than separate them A paintbrush can also be used for the purpose. High basal release with little or no receptor-mediated release of IPs mdicates loss of viability of tissue slices during the experiment. This can occur for several reasons, the most common being improper handling and slice preparation The importance of correct handling of the tissue cannot be stressed enough. As the tissue slices loose viabihty, myomositol is not taken up m the slices leading to low mcorporation, or if this happens after mcorporation has occurred to some degree, then it results m high basal release (>20% of total mcorporation) and little or no receptor-mediated release of IPs. Practice dissecting the tissue quickly and with little manipulation. Grippmg the tissue between forceps for lifting, a common procedure, tends to decrease viability of the slices significantly This can be avoided by lifting the tissue from below Time taken m slice preparation should be minimum, no more than lo-15 mm period between sacriftcmg the animal, taking the brain out, drssecting the tissue, making slices, and placing them in oxygenated KRB at 37°C. Keeping the tissue wet with oxygenated KRB maintained at 37°C during the whole procedure is also helpful. If the top solution durmg premcubatron and washing does not clear after repeated washes, the tissue has not been sliced correctly. Small fragments of tissue rather than clean slices will be seen floating around If this is the case a decision has to be made if the experiment should be continued further as it may not be successful. The time taken for mcorporation of myoinositol varies between various tissues and experimental conditions. Incorporation of myomositol m tissue slices increases with time until an equilibrium is reached with m the cells. However, viability of tissue shces can decrease significantly after a few hours even under optimal conditions. Thus, the experiment has to be performed such that the slices are viable during the whole procedure, the mcorporation of myoinositol is optimal, there is little basal release, and significant agonist-mediated release of IP(s) is observed. Pilot experiments varying time for mcorporation and agomst-stimulated release of IPs should be performed such that optimal conditions can be achieved for the specific brain region to be studied. For most brain regions, 90-120 min for incorporation of myoinositol and 5-30 mm for agonist-mediated release of IP(s) are sufficient. Shorter periods for both incorporation and stimulated release of IPs have also been used and can work well. Count ahquots from organic and aqueous phases for calculating total mcorporation to verify sufficient incorporation of the [3H] myomositol. In most brain regions, ~20% of the DPMs incorporated as [3H] myomositol are released as basal release within 30 mm at 37°C under experimental conditions. If this value is higher, the experimental protocol should be re-examined. The organic phase contammg labeled mositol lipid, can be separated, dried under mtrogen and used to separate phosphomositol phosphates by thin-layer chromatography.

188

Tandon

10 The whole procedure can take up to 10 h to complete and can be performed over 2 d After the agonist-stimulation reaction has been stopped, the aqueous phase can be separated after centrifugatton and stored overnight m au-tight tubes at 4°C without loss of IPs 11. The elution profile for various IP(s) can vary between batches of Dowex AGl-X8 To confirm that the volumes and salt concentration of various solutions used for elution is sufficient and provides a clean separation of each IP, standards for [3H] IPl(s), [3H] IP2(s), [3H] IP3(s), [3H] IP4(s) and [3H] IP5-6(s) are used Dilute a known amount (DPM) of each IP m 5 mL of dH20, add 2 mL of activated DowexAGl-X8 slurry, and pour into columns Wash with 15 mL of dH,O (5 mL x 3) and elute each IPs using solutions 2-6 as mentioned m Table 2 Collect eluate, and count If more than 10% of DPM from one IP fractton are eluted wtth higher ammonmm formate salt gradient, than a change in elutton volume or salt concentration gradient 1srequired Increasing the elution volume to 10 mL (instead of 8 mL) is often sufficient to elute all the specific IP from the column. If all of the specific IP is still not eluted, the salt concentratton can be increase by 0 05-O 15 M An overlap between various IPs will occur if the salt concentration is increased further. 12. It is Important to check the elution profile for each batch of Dowex after acttvatmg it. One bottle of Dowex can last for several runs (300-350 samples) After elutmg all IPs, the Dowex should not be radioactive. Even when elutmg total IPs by solution 6 (2 M ammonmm formate) it is important to make sure that all radioactivity has been eluted This can be checked after the Dowex has been activated and before the start of the experiment. Load a sample of standard mixture of [3H] IPs to the column, wash with 15 mL dHz0 (5 mL x 3) and elute with 10 mL (5 mL x 2) of solution 6. Move to separate vials, elute with further 2 mL solution 6 and collect m I-mL fractions. These fraction should have very few counts (DPMs). If this 1snot the case, increase elution volume until all radiolabel is eluted out of the Dowex When all IP(s) have been eluted, a sample of the Dowex should be taken and counted and should not contain any radioactivity Dowex AGl- X8 can be reused after reactivation. Reactivation of Dowex is done by washing the used Dowex several times with dH20 and then repeating the activation procedure. Note the slight change in color of the Dowex resin upon proper activation The activated Dowex can be stored at 4°C for up to 2 mo. It is advtsable, however to reactivate the unused Dowex after 30 d Dowex can be washed and activated in small batches 13 Each eluted fraction can also be collected m test tubes or 15-mL polystyrene tubes and small ahquots taken for scmttllation countmg. The higher IPs (IP3IP6) are eluted m two fraction resulting m doubling the number of scintillation veals to be counted This can be mmimized by collectmg both fractions tn one tube and then counting a small ahquot Make sure to adJust for changes m volume while calculatmg IP release as needed Before adding scmtillatton cocktail, make sure that the scmtillant IS capable of handling salt concentrations as high as 2.0 M, the eluant can be diluted with dH,O if this is a problem

Receptor-Mediated

lnositolphosphates

Release

189

References 1. Berridge, M. J. and Irvine, R F (1989) Inositol phosphates and cell signaling. Nature 341, 197-205 2 Fisher, S. K and Agranoff, B W. (1987) Receptor activation and mosttol hpid hydrolysis m neuronal tissues J. Neurochem. 48,999-1017 3 Osborne, N. N, Tobm, A B., and Ghazi, H. (1988) Role of inositoltrisphosphate as a second messenger m signal transduction processes. an assay Neurochem Res. 13, 177-191. 4. Irvme, R E. (1987) Inosrtol phosphates and calcium entry. Nature 328,386. 5. Steb, H., Irvine, R F , Berrrdge, M. J., and Sculz, I. (1983) Release of Ca2+ from a nonmitochondrial store m pancreatic cells by mositol 1,4,%trisphosphate. Nature 306,67-69

6. Nahorski, S. R , Kendall, D. A , and Batty, L. L. (1986) Receptors and phosphomosmde metabolism m the central nervous system. Biochem. Pharmacol. 35, 2447-2453. 7. Berridge, M. J , Downes, C P , and Hanley, M R. (1982) Lithium amplifies agomst-dependent phosphatidylmosttol response in bram and salivary glands Biochem J 206,587-595

8 Irvine, R. E. (1990) Methods m Inositzde Research. Raven, New York 9. Jenkmson, S., Patel, N., Nahorski, S R., and Challiss, R. A. (1993) Comparative effects of lrthium on the phosphomositrde cycle m rat cerebral cortex, hippocampus and striatum J. Neurochem. 61, 1082-1090. 10 Gonzales, R A. and Crews, F T (1984) Characterization of the cholmergic stimulation of phosphomosrtrde hydrolysis m rat brain sbces. J. NeuroscJ. 4,3 120-3 127. 11. Janowsky, A., Labarca, R., and Paul, S. M. (1984) Characterization of neurotransmuter receptor-mediated phosphomositide hydrolysis m the rat hippocampus. Life Scz. 35,1953-1961 12. Willars, G. B., Chalhss, R. A., and Nahorski, S. R. (1996) Acute regulation of the receptor-mediated phosphomositide signal transduction pathway. J. L&d Metab. Cell Sqnalmg 14, 157-168. 13 Balduim, W., Sheldon, D. M., and Costa, L. C. (1987) Developmental changes m muscarimc receptor-stimulated phosphomosmde metabolism m rat brain J. Pharm Expt. Ther. 241,421-427 14. Heacock, A. M , Fisher, S. K., and Agranoff, B. W. (1987) Enhanced coupling of neonatal muscarmic receptors m rat brain to phosphoinositide turnover. J Neurochem. 48,1904-1911. 15. Rooney, T A. and Nahorskt, S. R (1987) Postnatal ontogeny of agonist and depolarization-induced phosphomositide hydrolysis m the rat cerebral cortex. J. Pharmacol

Expt. Ther 243,333-341.

16 Tandon, P., Ah, S F , Nanry, K., and Tilson, H A (1991) Age-dependent changes in the receptor-stimulated phosphomosmde turnover in the rat hippocampus Pharmacol.

Bzochem Behavior. 38,861-867.

17 Reed, L. J. and de Belleroche, J. (1988) Increased polyphosphoinositide responsiveness in the cerebral cortex induced by cholinergic denervatron J. Neurochem. 50,1566-1571

190

Tandon

18. Tandon, P., Harry, G. J., and Ttlson, H. A (1989) Colchtcme-induced alterations m receptor mediated phosphoinositrde hydrolysis m the rat hippocampus Brain Res 417,308-3 13 19. Tandon, P , Ali, S. F., Bonner, M., and Ttlson, H. A, (1989) Characterrzatron of receptor-coupled phosphoinosrtrde hydrolyses in the rat htppocampus after intradentate colchicine J. Neurochem 53, 1117-l 125. 20. Tandon, P , Padrlla, S , Pope, C. N., Barone, S., Jr., and Tilson, H. A. (1994) Fenthion produces persistent decreases m muscarmtc receptor functton m the adult rat retma. Toxicol. Applied Pharmacol. 125, 271-280 21. Nicolettr, F , Wroblewski, J. T., Alho, H., Fadda, E., and Costa, E (1987) Lesions of putative glutammerglc pathways potentiate the increase of mositolphosphohptd hydolysrs elicited by exrcttatory ammo acids. Brazn Res. 436, 103-l 12

17 The Use of Enzyme-Linked lmmunosorbent to Quantitate Proteins in Biological Fluids

Assays

Dori Ft. Germolec 1. Introduction Immunoassays are techniques for measuring the concentration or activity of a substance using immunological reactions. Several types of immunoassay are commonly used, mcluding precipitation assays using antibody/antigen complexes, agglutination assaysusing coated erythrocytes or other particles, and radio-(RIA) and enzyme immunoassays. Multiple factors determine the best assay for a particular use, including sample concentration, technical difficulty, required precision, and availability of specialized equipment. The most sensitive assaysare the radio- and enzyme immunoassays, however safety and environmental issuesregarding the use of radioisotopes have limited the application of RIAs, and thus enzyme-linked immunosorbent assays (ELISA) tend to be the method of choice when sample concentration is a critical issue. The potential for robotic automation and the availability of inexpensive microplate dilutors, pipetors, and readers has allowed for the generation of highly reproducible results for large numbers of samples in an short time. ELISAs may be classified as either homogeneous or heterogeneous. Homogeneous rmmunoassays are based on detecting antibody-mediated changes in enzyme activity. Because the binding of the antibody to enzyme-labeled antigen generates the detectable signal, no physical separation of bound and free label is needed (1). A number of compounds give modified signals on binding immune complexes, including free radicals, fluorescent dyes, and several enzymes (2). Homogeneous ELISAs have been particularly useful in detecting low-mol-wt compounds, such as haptens and drugs, but are of limited value in the detection of most proteins. From

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Heterogeneous ELISAs are the method most commonly used for detection of specific antibodies, soluble mediators, or cell-surface antigens (3). In the most common designs, soluble protems are removed from a sample or standard solution by binding to a solid-phase component. In these assays,the enzyme is not directly involved m the antibody/antigen bindmg reaction, and 1sadded as a secondary detection step. Whereas heterogeneous ELISAs can also be performed as fluid-phase assays,i.e., without protein binding to a solid material, such as plastic or mtrocellulose, solid-phase assays are generally much more sensitive and can detect antigen or antibody well below their usual affinities (3). Variations of the heterogeneous assays differ m the number of steps and antibodies used to increase the sensittvity and which component of the reaction mixture (antigen or antibody) is mitially bound to the solid phase. Four basic variations of the heterogeneous ELISA allow the flexibility to quantitate proteins under a wide variety of concentrations and conditions (Fig. 1). Selection of the appropriate methodology is based on a number of considerations, including avarlabillty of standards and relevant antigens/antibodies, concentration of reactants m test samples, cost, reproducibility, purpose of the test (i.e., research or diagnostic) and desired sensitivity and specificity. Direct ELISAs are the simplest of all of the ELBA techniques (Fig. 1A). In this method, antigen or antibody is bound to the solid phase by passive adsorption and detected using enzyme-labeled antibody or antigen, respectively. Direct assaysare similar to RIA techniques, and are frequently used m the titration of monoclonal antibodies (MAbs) and for estimation of hormone concentrations, however, they are generally poor methods of detecting antibodies in multiple test antisera or culture supernatants. Indirect ELISAs are often used in clmical settings for the detection of specific antibodies from serum samples, for assessment of immune status to infectious agents and identification of allergen-specific antibodies and autoantibodies. We have used this method in our laboratory to detect human antibodies to tetanus toxoid produced in immunodeficrent mice reconstituted with human cells (4). This technique involves passive adsorption of antigen to a solid phase, addition of test samples or standardized antibodies directed against the antigen, and detection using an enzyme-labeled secondary antibody (Fig. 1B). Selection of the secondary antibody 1s critical for both the sensitivity and specificity of the assay, and care must be taken to identify species- and isotype-specific antibodies appropriate to each specific system. A number of competitive ELISA designs can also be put to use. Competitive assays can utilize either antigen or antibody bound to the solid phase and include an additional step in which the test sample is mixed with a known quantity of unlabeled antibody or antigen (Fig. 1C). These methods have the advantage of being easier to quantitate and offer increased specificity, as they

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Fig. 1. Types of ELISA. (A) Direct ELISA: In this example, antigen is attached to the solid phase, followed by incubation with enzyme-labeled antibody and then detection with the appropriate substrate. (B) Indirect ELISA: In this example, antigen is attached to the solid phase, followed by incubation with test samples containing nonlabeled primary antibodies. An enzyme-labeled antibody specific for the isotype and species of the primary antibody is used as the detecting antibody and quantitation after incubation with the appropriate substrate follows. (C) Competitive ELISA: In this example, a known quantity of antigen is attached to the solid phase. Sample containing an unknown quantity of antigen is added simultaneously along with the detecting antibody. Unbound antigen/antibody complexes are removed in the washing steps. The amount of antigen in the test sample is inversely proportional to intensity of the calorimetric reaction. (D) Sandwich ELISA (indirect): A primary antibody is attached to the solid phase, followed by incubation with sample containing the protein of interest. The antigen is “sandwiched” by a secondary antibody which may bind to similar or different antigenic determinants on the protein of interest, and may or may not be enzyme-labeled. In this example, the sensitivity of the assay is increased by the use of a tertiary enzyme-labeled antibody, followed by detection with the appropriate substrate. are less likely to be influenced by cross-reactive proteins in a heterogeneous sample (3). However, they are significantly more labor intensive and, as both antigen and antibody are present at limiting concentrations, the results are more likely to be influenced by errors in pipetting, diluting, and so on. A variation on the indirect ELISA is the capture or sandwich ELISA (Fig. 1D). In these assays, a capture antibody is bound to the solid phase, followed by incubation with the test samples and standards to allow antigen binding. This

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step is followed by bmding of an enzyme-labeled detectmg antibody, thus forming an “antibody sandwrch” around the antigen. If the antigen contams multiple similar antigenic sttes,the capture and detecting antibodies can be the same. However, it is much more common to use capture and detectmg antibodies to different antigenic epitopes. In general, low-mol-wt antigens are not appropriate for these types of assays, as limited antigenic sites are available (5). Use of a monoclonal capture reagent provides a high degree of specificity during the capture phase, although caution must be exercised, as many MAbs are not efficient capture reagents (3). When monoclonal capture antibodies are used in combination with polyclonal detecting antibodres, sensitivity is also maximized, as the polyclonal reagent will detect multiple epitopes on the antigen. Use of polyclonal reagents for both detection and capture can lead to decreased specificity, crossreactivity, and higher levels of background, although use of affinity purified antibodies and isotype fragments (F(ab)‘2) may help optimize the signal and cut down on background noise (6). Whereas the sandwich technique 1s perhaps the most sensitive method for accurately determining antigen concentratton, tt can be costly because it requires relatively large quantities of semipurified or purified antibodies for antigen capture. Sandwich ELISAs are the most frequently used ELISA techniques and the sample protocols included wtth this chapter are for sandwich ELISAs used to detect soluble proteins from serum or culture supernatants. The relative ease and convenience of the methods, then wide applicability to numerous biological parameters, the increasing availability of commercial reagents including enzyme-labeled antibody conjugates and chromogenic substrates, as well as advanced technologies, such as work stations or robotic instruments, that can perform dilutions, washes, incubations, and plate readings ensure that ELISA techniques will continue to be important tools in the research laboratory. 2. Materials 2.1. Indirect Sandwich ELISA 2.1.1. Reagents 1. Washing solutions.phosphate-buffered saline (PBS), PBS+ 0.05% Tween 20 2. Blockmg solutions’ PBS + 1% bovine serum albumin, appropriate culture medium + serum or serum substitute (complete medium: i.e., RPM1 1640 +

10% FCS). 3. 4. 5 6.

Standard: recombinant murine IL- 1p 5 l.tg/mL Capture antibody:monoclonal hamster antimouse Primary antibody: polyclonal rabbit antimouse Detecting antibody. brotinylated goat antirabbit stock solutron

stock solution. IL-lb 0 05 mg/mL stock solution IL- 1p 1 mg/mL stocksolution. IgG (whole molecule) 1 mg/mL

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7 Enzyme conjugate. streptavidm-alkaline phosphatase conjugate 0.25 mg/mL stock solution (see Note 1) 8. Diluting solutions: appropriate culture medium without serum or serum substitute (incomplete medmm), complete medium, PBS, PBS + 0 05% Tween 20 + 1% BSA 9 Substrate: PNPP (p-mtrophenyl phosphate tablets, 5 mg/tablet) 10 Substrate diluent: 10 mM diethanolamme (DEM) + 0.5 mM MgCl,, pH 9.5.

2.1.2. Equipment and Supplies 1. 96-well ELISA-grade microtiter plates (Dynatech Immunolon, Chantilly, VA; Nunc MaxiSorp, Wlesbaden-Biebrich, Germany; Corning High Binding, Corning, NY) (see Note 2) 2 Microplate reader. 3 Multichannel plpet. 4. 2OOqL pipet tips.

2.2. Direct Sandwich

ELBA

1 Washing solution* PBS + 0.05% Tween 20 2. Blocking solution: PBS + 0.5% bovine serum albumin. 3. Standards: mouse lmmunoglobulins or human immunoglobulm calibration standard (Beckman, Fullerton, CA). 4. Capture antibodies (mouse). a. Affinity-punfled F(ab’)2 fragment goat antimouse IgM (Mu chain specific) or b. Affinity-purified goat antimouse IgA (F(ab’)2 specific or c. Affinity-purified goat antimouse IgA (alpha chain specific). 5. Capture antibodies (human): a Affinity-purified goat antihuman IgM (mu chain specific) or b. Affinity-purified F(ab’)2 fragment goat antihuman IgG whole molecule or c. Affinity-purified goat antlmouse IgA (alpha chain specific). 6. Enzyme-conjugated detecting antibodies: a. Mouse: peroxidase-conjugated affinity-purified goat antimouse immunoglobuhns (IgA, IgM, IgG). b. Human: peroxldase-conjugated affinity-purified goat antihuman immunoglobulms (IgA, IgM, IgG). 7. Peroxldase substrate Kit (Bio-Rad, Richmond, CA). 8. Diluting solutions: PBS or PBS + 5% ultra low IgG fetal bovine serum. 9. Stop solution: 2% oxalic acid m PBS.

3. Methods 3.1. Indirect Sandwich

ELlSA

1. Coat 96-well plates with capture antibody (see Notes 2 and 3): Dilute stock capture antibody to a final concentration of 5 pg/mL in PBS or incomplete medium Add 100 I.LL capture antibody solution per well to 96-well plates and incubate overnight at 4°C

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2 Wash plate with PBS (6 x 250 pL/well) (see Note 4). 3 Add 200 I.IL PBS + 1% BSA or complete RPM1 per well and incubate in a humidified chamber at 37°C for 1 h to block plate. 4 Wash plate with PBS (3 x 250 FL/well). 5 Add 50 pL of standard or test sample per well (see Notes 5 and 6) At least three replicates should be done for each sample and standard. If necessary, samples may be diluted m PBS + 1% BSA or complete medium. Incubate m a humtdifled chamber at 37’C for 2 h 6 Wash plate with PBS + 0.05% Tween 20 (6 x 250 pL/well) 7 &lute stock solution of rabbit antlmouse IL-lb (primary antibody) to a final concentration of 2 yg/mL in PBS + 1% BSA or complete medium. Add 100 pL primary antibody solution per well and incubate at 37°C for 1 h 8 Wash plate with PBS + 0.05% Tween 20 (6 x 250 pL/well). 9. Dilute stock solution of biotmylated goat antIrabbIt IgG to a final concentration of 2 pg/mL m PBS + 1% BSA or complete medium. Add 100 FL detecting antlbody solution per well and mcubate at 37°C for 30 mm 10. Wash plate with PBS + 0 05% Tween 20 (6 x 250 FL/well). 11 Dilute stock solution of streptavldm-alkalme phosphatase to a final concentration of 625 pg/mL m PBS + 0.05% Tween 20 + 1% BSA. Add 100 I.IL enzymeconjugate solution per well and mcubate at 37°C for 30 mm. 12. Wash plate with PBS + 0 05% Tween 20 (4 x 250 FL/well). 13 Wash plate with PBS (2 x 250 FL/well) (see Note 6). 14. No more than 10 mm prior to use prepare substrate solution by dissolving one 5 mg tablet of PNPP per 5 mL of DEM solution for a final concentration of 1 mg/rnL Add 100 p.L of substrate solution per well and incubate for 15 mm at room temperature. 15 Measure optical density at wavelength 405 nm in a mlcroplate reader (see Notes 7 and 8).

3.2. Direct Sandwich

ELBA

1 Coat 96-well plates with capture antibody (see Notes 2 and 3). Dilute capture antibody to a final concentration of 1 pg/mL m PBS Add 150 pL capture antlbody solution per well to 96-well plates and incubate overnight at 4°C. 2 Wash plate with PBS + 0 05% Tween 20 (4 x 250 pL/well) (see Note 4). 3 Add 150 PL PBS + 0 5% bovine serum albumin per well and incubate for 30 mm at room temperature. 4. Decant plates. Do not wash. 5 Add 200 pL of standard or test sample per well (see Notes 4 and 5) At least three replicates should be done for each sample and standard If necessary, samples may be diluted m PBS + 5% ultra low IgG fetal bovine serum. Incubate for 2 h at room temperature m a humldlfled chamber 6 Wash plate with PBS + 0.05% Tween 20 (4 x 250 pL/well) 7. Dilute stock solution of peroxidase-conjugated goat antimouse or antihuman immunoglobulms to a final concentration of 1 yglmL m PBS + 5% ultra low IgG fetal bovine serum (see Note 1) Add 150 yL prnnary antibody solution per well and incubate for 1 h at room temperature in a humidified chamber.

ELISA Quantitatlon of Biological Proteins 8. Wash plate with PBS + 0.05% Tween 20 (4 x 250 pL/well). 9 A maximum of 10 mm prior to use, prepare substrate solution by mixmg nine parts solution A with one part solution B (Peroxidase Substrate Kit-Bio-Rad). Add 100 FL of substrate solution per well and incubate for 15 min at room temperature. 10. Stop reaction after color development (approx 15-30 min) by addition of 100 FL per well 2% oxalic acid (see Note 8) 11 Measure optical density at wavelength 405 nm in a microplate reader (see Notes 7 and 8)

4. Notes Depending on the type of ELISA, either an antigen or antibody conjugated to an enzyme will be used. Antlbody conjugates are more widely available, and relatively nonspeclflc enzyme-conjugated secondary antlbodies (such as goat antirabblt IgG) can be used when the detecting antibodies are in short supply, are difficult to conjugate, or to increase the sensitivity of the reaction (7). In general, highly purified antibody conjugates give the best results m terms of detectlon and background, but are relatively expensive (6). The costs are frequently balanced out by the fact that these purified conjugates may be highly diluted. Optimization by screening multiple concentrations of an appropriate standard using a wide variety of dilutions of different antibody preparations, such as whole polyclonal conjugates, F(ab’)2 fragment conjugates, and affinity-purified conjugates is a critical step. Assays are quantitated by the accumulation of colored product after the addition of a substrate or dye combmatlon appropriate for the particular enzyme Three enzymes are most commonly used m ELISA reactions and the substrates and conditions that are frequently used with the enzymes are summarized in Table 1. The choice of the enzyme/substrate systems 1s dependent on a number of general considerations, as well as factors specific to each EISA method, such as turnover rate, stability of reaction product, lack of endogenous enzyme m the test solution, easy detection, safety issues, and cost 2. The choice of the solid phase to be used 1s highly dependent on the nature and purity of the proteins to be bound. High-capacity materials such as cellulose and nitrocellulose form stable bonds with the coating material, so that they can be used with relatively crude preparations of antigen or antibody and can measure high levels of protein without dilution (3). In addition, they can be stored for extensive periods of time. However, they are more difficult to wash than lowcapacity materials and would not be appropriate when crossreactivity or nonspecific binding may be a problem as the signal-to-background ratio can be quite low. Although it 1sof low-bmdmg capacity, the plastic 96-well microtiter plate 1s certainly the most frequently used solid-phase material. The use of standard tissue-culture microtlter plates 1sgenerally not recommended and a number of companies offer plates specific for ELISA, which have been treated to enhance protein binding and mmlmlze background absorbency. Whereas round-bottomed well

Table 1 Commonly

Used Enzyme/Substrate

Enzyme label Horseradish

peroxrdase

Alkalme phosphatase P-galactosrdase ii

Systems

for ELISA

Substrate

Dye

0 002% H,02

2,2’-Azino drethylbenzothiazohne sulfomc acid (ABTS) Tetra-methylbenzidine (TMB) o-Phenylene dtamme (OPD) 5-Ammosahcyhc acid (5AS) PNPP

0.004% H,Oz 0.004% H,Oz 0.006% H,O, p-Nitrophenyl phosphate (PNPP) 0-Nitrophenyl P-b-Galactopyranoside (ONPG)

ONPG

Buffer Phosphate/citrate

(0.1 M), pH 4 2

Acetate buffer (0.1 M), pH 5 6 Phosphate/citrate (0 1 it4), pH 5.0 Phosphate (0.2 M), pH 6.8 Diethanolamine ( 10 mM) and M&l, (0.5 nnV), pH 9 5 MgCl* and 2-mercaptoethanol (O.OlM)/PBS, pH 7 5

Reading wavelength (nm) Enzyme label Horseradish

peroxtdase

Alkaline phosphatase P-galactosrdase

Nonstopped

Stopped

Stopping solution

Notes

415 655 450 450 405 420

415 450 492 550 405 420

2% oxalic acid 2 A4 H,S04 3 M H,S04 3 N NaOH 3 N NaOH 2 M sodium carbonate

Mutagenic, stable, soluble product Mutagenic, less soluble product. Less stable product, possible carcmogen. Less soluble, stable product Safe, soluble, stable product. Safe, soluble, stable product. Not normally found in brologrcal fluids, so may reduce background

ELISA Quantitation of Biological Proteins

3.

4.

5

6.

plates can be used when vrsual assessment of colorrmetric changes is used, flatbottomed wells are recommended when spectrophotometnc quantitation is required. The simplest method for attaching antigens or antibodies to solid-phase materials, such as plastic 96-well plates, is passive adsorption. Because a majority of proteins bmd through hydrophobic interactions, some proteins may bind to the plate m specific orientations, and care must be taken to ensure that specific antigenie determinants are not masked by solid-phase binding. How well the coating material will attach to the plate is dependent on a number of factors, including the concentration and type of antigen being used, surface characteristics of the plastic, time, and temperature of adsorption (5). Although it is important to determine the optimum concentration for the antigen or antibody m a specific test system, a concentration range of l-50 yg/mL in a volume of 50 pL will generally saturate available binding sites. Clearly, it is best to use purified preparations of coating materials whenever possible as contaminating proteins will compete for available binding sites Thus it follows that solutions containing high concentrations of serum proteins should not be used as diluents for coatmg Many protocols use high-pH buffers, such as 50 mM carbonate, pH 9.6, or 20 mM Tris-HCl, pH 8.5, as the diluent; however, in many instances simple buffers, such as 10 n&f PBS, pH 7.2, work well (3). As with protein concentration, the coating buffer should be optimized for each specific test system In some instances, passive adsorption does not result in effective bmdmg, and a number of protocols using chemicals, such as glutaraldehyde, carbodrmmes, and so on, to covalently couple protems to plastic have been developed (5). Washmg the plates 3-5 times to remove unbound or desorbed proteins is critical prior to beginning any test assay, and all ELISA procedures should include a blocking step after coating to ensure saturation of all available binding sites. It is possible to store coated plates at 4”C, and they may be stable for as long as several months. A common problem in ELISA assays is a high background absorbance. This can often be corrected by increasing the number and efficiency of the washing steps. In many ELISAs the affinity of the reactants are such that the fill and “garbage dump” technique of washing is not rigorous enough to separate unbound from bound reactants, and stronger methods must be used. Preparation of mIL-l/3 standards: Add 5 PL mIL-1B standard stock solution to 50 mL for a 1000 pg/mL solutton. This diluted stock can be aliquoted and frozen for later use. Dilute 1 mL 1000 pg/mL solution with 1 mL complete medium for 500 pg/mL. Dilute 1 mL 500 pg/mL solution with 1 mL complete medium for 250 pg/mL. Dilute 0.8 mL 250 pg/mL solution with 1.2 mL complete medium for 100 pg/mL. Dilute 0.5 mL 100 pg/mL solution with 1 5 mL complete medium for 25 pg/mL Use complete medium only for 0 pg/mL. Assay optimization is the most critical factor for successful ELISA design. Each batch of ELISA plates and new lots of reagents should be tested to ensure acceptable performance Accuracy and consrstency m prpetmg 1s also of considerable importance. Data interpretation is easiest when the absorbance of the highest

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dilution of the standards 1s 1.0 or greater and the background absorbance of a diluent only control is co.05 (6) 7 Nonspecific bmdmg and antibody crossreactivity also may lead to high background. One suggestion to reduce this effect is to preabsorb capture antibodies agamst other antigens with which they might bmd by mcubatton with serumcoated agarose beads or diluted normal sera (3,s). 8 Lack of color development may suggest problems with enzyme conmgates or substrate, it can also be an mdication of defects m bmdmg of the capture protems. It may also mean that sample concentration is below the limits of detection for the assay (generally l-1000 ng/mL for most assays), which is frequently a problem when measuring tissue-culture supernatants. To remedy this problem, samples may be concentrated using a variety of techniques, including centrifugal microconcentration, microdialysis, and ammonmm sulfate precipitation. Unfortunately, when ELISAs fail, it is often necessary to break the entire system down and examine each step

References 1 Rubenstem, K E , Schneider, R. S , and Ullman, E. F (1972) Homogeneous enzyme-immunoassay. A new immunochemical technique. Blochem. Bzophys. Res. Commun. 47,846-858

2. Ullman, E F. and Maggie, E. T. (1980) Principles of homogeneous enzymeimmunoassay, m Enzyme-Immunoassay (Maggio, E. T., ed.), CRC, Boca Raton, FL, pp. 106-13 1. 3. Crowther, J. R. (1995) ELZSA: Theory and Practzce Humana, Totowa, NJ 4. Pollock, P. L., Germolec, D. R., Comment, C E , Rosenthal, G. J , and Luster, M. I. (1994) Human lymphocyte engrafted SCID mice as a model for immunotoxicity assessment. studies with cyclosporme A and TCDD. Fundam Appl Toxic01 22, 130-138. 5. Kemeny, D. M (1990) A Practzcal Guide to ELZSA. Pergamon, Oxford 6. Carpenter, A. B. (1992). Enzyme-linked immunoassays, m Manual of Clinical Laboratory Immunology, 4th ed. (Rose, N. R., de Macario, E. C , Fahey, J L., Friedman, H , and Penn, G. M., eds ), American Society for Microbiology, Washington, DC, pp 2-9 7. Macy, E., Kemeny, M., and Saxon, A. (1988) Enhanced ELISA. how to measure less than 10 picograms of a specific protein (immunoglobulin) m less than 8 hours FASEB J 2,3003-3009

Assays for the Analysis of Synaptic Proteins in Neurodegenerative Disorders Eliezer Masliah and Michael Alford 1. Introduction Neurodegenerative disorders are characterized by damage to selective neuronal populations (I) that could be followed or proceeded by synaptic mJury (2). The mechanisms triggering cell death and synaptic damage in these disorders might be related to gam of a toxic property and/or loss of neuroprotective capacity of a specific neuronal protein (3). Many of these neuronal molecules play an important role in the maintenance and functioning of the synaptic apparatus (4,5). Therefore, specific mutations and other alterations of synaptic proteins might result in particular neurodegenerative diseases. In this regard, recent studies have shown that molecules involved in the pathogenesis of Alzheimer’s disease (AD) m fact have a synaptic location. Examples of such proteins include amyloid precursor protein (APP) (6-8), nonamyloid-P component precursor (NACP) (9), and presemlin 1 and 2 (10-12). Furthermore, other neurodegenerative disorders such as Huntington’s disease (HD) (13,14), Parkinson’s disease (PD) (1%17), Creutzfeldt-Jakob disease (CJD) (la), and myotonic dystrophy (19) have turned out to be the result of mutations m proteins that concentrate at the synaptic site. Therefore, in order to better understand the mechanisms of central nervous system (CNS) dysfunction m neurodegenerative disorders, it 1simportant to determine the concentratrons of specific synaptic proteins at the synaptic site, as well as to determine overall synaptic density in specific brain regions. For example, in AD, memory deficits in early stages of the disease are associated with synaptic loss in the molecular layer of the hippocampal dentate gyrus, which reflects degeneration of the perforant pathway (2,20). In contrast, m later stages of the disease, synaptic loss in the neocortex is associated with deficits in higher cogmtive funcFrom

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tion (21,221. Loss of synapses has now been recognized as a very Important component of the mechanisms of CNS dysfunction not only m AD (2,21-26) but, among others, in Pick’s disease (27), PD (Id), CJD (28), HD (13), and motor neuron disease (29). Measurements of synaptic density are not only important for the study of neurodegenerative diseases, but are also important to understand changes m brain function during development and aging, as well as for the evaluation of the role of neurotrophlc factors and neurotoxins. Synaptic density measurements m brain sections can be made directly by identification of individual synapses with electron microscopy (30), and mdirectly by lmmunocytochemlcal labeling of synapse associated proteins (such as synaptophysin) coupled with quantification by laser confocal imaging (3134), or optical-density measurements (35). Similarly, lmmunochemical techniques m brain homogenates and fractions can indirectly estimate the density of synapses by Western blot (35), dot blot (36,37), radlolmmunoassay (38), and enzyme-linked immunoadsorbent assay (ELISA) (39,40). A recent study by Calhoun et al. (41) compared three quantitative methods to quantify synaptophysinlmmunoreactivity: Western blot, optical densitometry, and stereology m the molecular layer of the rat hippocampus. Although they found that each method presented its own advantages and disadvantages, the study concluded that stereology provided the most reliable and efficient method of detecting synapses, Therefore, immunodetection techniques to identify synaptic proteins (e.g., synaptophysin, a 38-kDa calcium-binding glycoprotem of presynaptic vesicles) have been shown to exhibit sufficient specificity and selectivity for accurate quantitation of synapses (41). The objective of the present manuscript is to describe a sensitive yet easy to use dot-immunobinding assay (37) for quantification of synaptophysin-like immunoreactivity to complement other, more complicated direct measures of synaptic density, such as synaptic quantification by laser scanning confocal imaging and/or stereology.

2. Materials 2.1. Subcellular

Fractionation

1 Homogenization buffer. 10 25 I&! phosphate, NaH,P04 (monobasic 1.85 mM), Na,HP04 (dlbanc 8.40 mM), 150 mM sodmm chloride, 5 mM benzamldme, 3 mM EDTA, 1 mM magnesium sulfate, pH 8.0 Store at 4°C. 2 Phosphate-buffered salme (PBS) diluted from a 25X stock solution (ScyTek, cat no. ABA999). Store at room temperature.

2.2. lmmunoblofting 1. Blockmg and wash solution: PBS with Tween 20. 1X PBS, Tween 20 (O.l%, v/v), sodium azlde (0.05%, v/v). Store at 4°C

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2. PBS with bovine serum albumin (PBS-BSA), pH 7 4: 1X PBS, BSA fraction V (3%, w/v), sodium azide (0.05%, v/w). Filter through a 0.45km filter Store at 4°C. 3 Primary antibody solution: mouse monoclonal antisynaptophysm (0.1 pg/mL, Boehrmger Mannhelm, cat no. 902 322) diluted m PBS-BSA. 4. Secondary antibody solution. antimouse IgG made in rabbit (0 85 yglmL, Accurate Chemical and Sclentlflc Corporation, cat. no JZM005045) diluted in PBS-BSA. 5. [lz51] Protein A solution. > 30 pCi/pg (ICN, cat. no 68038).

2.3. Consumable 1 Whatman chromatography paper (Schleicher & Schuell, Keene, NH, Protran, cat. no 31540). 2. Nltrocellulose membrane (Schleicher & Schuell, Protran, cat. no. 00860) 3. Thick-walled ultracentrlfuge tubes (11 x 34-mm polycarbonate) (Beckman, Fullerton, CA, cat no. 343778)

2.4. Instrumentation 1 2 3. 4

and Software

Somcator (Kontes, Vmeland, NJ) Dot blotter, Mmlfold I Microfiltratlon Manifold (Schleicher & Schuell) PhosphorImager, SF (Molecular Dynamics, Sunnyvale, CA) ImageQuant Software (Molecular Dynamics).

3. Methods 3.1. Tissue Preparation 1 Weigh out 0.1 g (wet wt) frozen tissue; place immediately mto 900 pL of cold brain homogenization buffer (see Note 1). 2. Disrupt by somcation (use lowest power settmg, 30 s to 1 mm). 3. Centrifuge at 5000g for 8 mm at 4°C (low speed spin). 4. Carefully remove the supernatant and transfer to thick-walled ultracentnfuge tubes. 5. Centrifuge at 274,000g for 1 h at 4’C (high speed spin) 6 Resuspend the pellet (particulate fraction) in homogenization buffer and take two 10 pL ahquots for the Lowry assay. For human tissue, use approx 300 pL of homogenization buffer Since rodent tissue tends to yield more total protem, samples should be resuspended m 350-400 pL (see Note 2).

3.2. Preparation

of Standards

1. Standards are relative The curve 1sconstructed by blotting dots from serial chlutlons of particulate fraction prepared from control tissue. 2. The curve provides a standard reference for comparison between cases and assays. Points on the curve are. 0 25, 0.5, 1, 2, 4, and 8 yg of total protein per dot. Each point 1s assigned an arbitrary value of 100 U per microgram of total protein (see Note 3).

Masliah and A/ford 3.3. Test Sample Preparation 1 Dilute each sample to 0.04 mg/mL m homogemzation buffer (1 e., 2 pg/50 pL) 2 Do two serial dilutions of the sample to yield 1 pg and 0 5 l,tg/50 yL dilutions.

3.4. Transfer

Procedure

1 Cut one piece each (8 x 11 5 cm) of Whatman chromatography paper and mtrocellulose (NC) membrane Wet both m disttlled water (the NC membrane should be “floated” for 5-10 mm prior to use) Be sure that both remain wet at all times 2. Place the NC membrane on top of the filter paper to form a sandwich, mount this m the dot blot apparatus and clamp down. 3 Seal the vacuum line mpple after clamping; this prevents the sample from ftltering down through the NC while other samples are being pipetted Add 200 /tL of homogenization buffer to all wells 4. Add standards and samples mto appropriate wells. 10 FL/well for standards, 50 pL/well for the dilutions of test samples 5. After all samples are pipetted, attach vacuum line, and allow all fluid to be drawn through all the wells Follow up with 2 x 200 pL washes wtth homogenization buffer. 6 Disassemble the dot-blot apparatus and cut a notch mto the lower left corner of the blot to help orient it during subsequent steps. 7. Dry blot under a light (heat lamp) for 5 min.

3.5. lmmunochemical

Procedure

1 Blocking* a Place blot m plasttc box. Incubate in PBS-Tween to cover the blot. This mcubation blocks nonspecific binding b Cover and shake on an orbital shaker for 2 h at 4°C 2. Binding of primary anttbody: a. While blot 1sin blocking solutton, prepare the primary antibody m PBS-BSA For each blot a mnumum of 18 mL must be used b. Discard the PBS-Tween blocking solutton c Incubate overnight in primary antibody at 4°C on rocking table. 3. Bmdmg of secondary antibody. a. Pour off primary antibody (reuse 1s not recommended). b. Wash the blot three times (15 mm each) m PBS-Tween at room temperature on a shaker c Add secondary antibody diluted to 0.85 pg/mL m PBS-BSA Incubate for 2 h at 4°C on rocking table. d. Wash blot as m step 2 above 4 Incubation m [125I] protein A a. Determine the spectfic activity of [ t2sI] protein A (specific acttvity > 30 pCt/pg) b. Following standard procedures for handling radioactive materials, dilute to 0 1 @/mL m PBS-BSA m a 50-mL centrifuge tube

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c. Working under a hood, filter through a 0.45 Mlllex syringe filter (cat. no. SLHVR25 LS). d. Incubate blot for 2 h at 4°C on rocking table. Make sure a lead shield 1splaced around the rocker or that the incubation tray itself is shielded with lead foil. e Discard the [ 1251]protein A solution f Wash as above, remembering that washes are hot 5. Autoradiography (on phosphor screen) a Place blot on an appropriately sized rectangle of Whatman paper and seal with Saran Wrap. b. Make the autoradlographlc exposure usmg the phorphor-imaging screen-a good image can usually be obtained by exposing the blot for 2-6 h.

3.6. Blot Analysis 1 Scan the blot and adJust the threshold (see Note 4) 2 Radioactivlty of dots is quantified by application of grid obJects. a 2 x 7 grid for the normalization standards (including blank), and a 2 x 3 grid for each of the test samples (three dllutlons, each in duplicate). 3 Integrate the areas within the grids for volume (i.e , pixel values are summed) 4. Transfer these values (by cut and paste) to a program (Visual Basic, m-house) that fits a cubic-spline to the standard volumes to form the standard curve 5. Estimate test sample values by mterpolatlon (curve fit and interpolation algorithms are taken from Algorithms m C by Robert SedgewIck, AddlsonWesley, 1990) 6. Final data is expressed m arbitrary units per pg protein.

4. Notes 1 The mam advantages of this approach IS that it can be used to assay in a very fast and reliable manner frozen material for which no adequate tissue 1s available for electron microscopy or confocal microscopy. 2 This method also allows the testing of several samples simultaneously. 3 Special emphasis needs to be placed m always running the appropriate standard curve according to the species from which the brain 1s derived 4. A disadvantage of the dot-blot method 1s the inabllity to obtain mformatlon regarding regional distribution of synaptophysm m specific layers of the cortex or in selected nuclei m subcortical regions, for this purpose confocal microscopy and electron microscopy are more appropriate.

References 1. Hof, P. R. and Morrison, J. H. (1994) The cellular basis of cortical dlsconnectlon m Alzhelmer disease and related dementing condltlons, in Alzheimer Disease (Terry, R. D., Katzman, R , and Bick, K. L., eds.), Raven, New York, pp. 197-230. 2 Masliah, E., Mallory, M , Hansen, L , DeTeresa, R., Alford, M., and Terry, R. (1994) Synaptic and neurltlc alterations during the progression of Alzhelmer’s disease Neuroscz Lett. 174,67-72.

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3. Slsodla, S. S and Price, D. L. (1995) Role of the beta-amylord protein m Alzhelmer’s disease. FASEB J. 9, 366-370 4. Maslrah, E. and Terry, R (1993) The role of synaptic protems in the pathogenesis of drsorders of the central nervous system. Brain Pathol. 3,77-85. 5. Mashah, E., Mallory, M , Alford, M , DeTeresa, R., Iwai, A., and Saitoh, T. (1997) Molecular mechanisms of synaptic disconnection m Alzheimer’s disease, m Connections, Cognztton andAZzheimer’s Disease (Hyman, B T., Duyckaerts, C , and Christen, Y., eds.), Springer-Verlag, Berlin, pp. 121-140 6. Askanas, V., Engel, W. K , and Alvarez, R. B (1992) Strong lmmunoreactivity of P-amyloid precursor protem, mcludmg the P-amylold protein sequence, at human neuromuscular Junctions. Neurosci. Lett. 143,96-100. 7 Mashah, E , Mallory, M., Hansen, L., Alford, M , DeTeresa, R , Terry, R., Baudler, J., and Sartoh, T. (1992) Locahzatron of amyloid precursor protein m GAP43-lmmunoreactlve aberrant sprouting neurltes m Alzhelmer’s disease. Bram Res 574,312-316 8. Schubert,W., Prior, R., Werdemann,A., Dncksen, H , Multhaup, G., Masters, C L., and Beyreuther, K. (1991) Localization of Alzheimer p//4 amylotd at presynaptlc terminals. Bram Res 563, 184-194 9. Iwal, A., Mashah, E., Yoshlmoto, M., De Sllva, R., Ge, N., Ktttel, A , and Saltoh, T (1994) The precursorprotem of non-AP componentof Alzheimer’s diseaseamylord (NACP) is a presynaptlc protein of the central nervous system.Neuron 14,467-475 10. Kovacs, D. M., Fausett, H. J., Page,K. J., Kim, T. W., Moir, R. D , Merriam, D E., Holhster, R. D., Hallmark, 0. G., Mancmr, R., and Felsenstem, K. M. (1996) Alzhermer-associated presemlms 1 and 2: neuronal expression m brain and localization to intracellular membranesm mammaltan cells Nature Med 2,224-229 11 Moussaom, S , Czech, C., Pradler, L., Blanchard, V., Bomci, B., Gohm, M , Imperato, A., and Revah, F (1996) Immunohlstochemical analysis of presemlm1 expression m the mousebrain. FEBS Lett. 383,219-222. 12. Elder, G. A., Tezapsldis, N , Carter, J., Shloi, J., Bouras, C , Li, H.-C., Johnston, J. M , Efthlmlopoulos, S., Friedrich, V L , Jr, and Robakts, N. K. (1996) Identc fication and neuron specific expression of the S182/PresemlinI protem in human and rodent brams. J. Neurosct Res. 45,308-320 13. Goto, S. and Hirano, A. (1990) Synaptophysm expression m the strtatum m Huntington’s disease.Acta Neuropathol. 80, 88-91 14. Wood, J D., MacMtllan, J. C , Harper, P. S , Lowenstem, P R., and Jones, A L. (1996) Partial characterlzatron of murme huntmgtin and apparent varlattons m the subcellular localization of huntmgtm in human, mouseand rat brain Human Mol Gen. 5,48 l-487 15. Ito, H , Goto, S , Hlrano, A., and Yen, S. H (1991) Immunohistochemlcal study of the hippocampus m parkmsonism-dementiacomplex of Guam J. Ger Psych Neural 4,134-142 16. Wakabayashr, K., Honer, W G , and Masliah, E. (1994) Synapse alterations in the hlppocampal-entorhmal formation in Alzheimer’s diseasewith and without Lewy body disease.Brain Res 667,24-32.

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17. Polymeropoulos, M H , Lavedant, C., Leroy, E., Ide, S. E , DeheJia, A , Dutra, A , Pike, B., Root, H., Rubenstem, J., Boyer, R., Stenroos, E. S., Chandrasekharappa, S., Athanassiadou, A, Papapetropulos, T., Johnson, W. G., Lazzarim, A. M., Duvoism, R. C., Di Iorio, G , Golbe, L. I., and Nussbaum, R L. (1997) Mutation m the alpha-synuclem gene identified in families with Parkinson’s disease Science 276,2&U-2047 18 Kttamoto, T., Shin, R-W , Doh-ura, K., Tomokane, N., Miyazono, M., Muramoto, T., and Tatershr, J. (1992) Abnormal isoform of prron proteins accumulates m the synaptic structures of the central nervous system m patients with CreutzfeldtJakob disease. Am J Path01 140, 1285-1294. 19 Whiting, E. J., Waring, J D., Tamar, K., Somerville, M J., Hmcke, M., Stames, W. A., Ikeda, J E , and Korneluk, R. G (1995) Charactertzation of myotomc dystrophy kinase (DMK) protein m human and rodent muscle and central nervous tissue. Hum Mol. Gen 4, 1063-1072. 20. Hyman, B. T., Van Hoesen, G. W , Kromer, L. J., and Damasio, A R. (1986) Perforant pathway changes in the memory impairment of Alzhermer’s disease. Ann Neurol. 20,472-481 21. DeKosky, S T and Scheff, S. W (1990) Synapse loss m frontal cortex biopsies m Alzhermer’s disease: correlation with cognitive severity. Ann. Neurol. 27,457-464. 22. Terry, R. D., Masliah, E , Salmon, D P , Butters, N , DeTeresa, R., Hill, R , Hansen, L A., and Katzman, R. (1991) Physical basis of cognitive alterations m Alzhelmer disease* synapse loss is the maJor correlate of cognitive impairment. Ann. New-01 30,572-580. 23 Davies, C. A., Mann, D. M A., Sumpter, P. Q., and Yates, P 0. (1987) A quantltatlve morphometric analysis of the neuronal and synaptic content of the frontal and temporal cortex m patients with Alzheimer’s disease. J. Neurol. Sci 78,15 1-164 24. Hamos, J E , DeGennaro, L J., and Drachman, D. A (1989) Synaptic loss in Alzheimer’s disease and other dementias Neurology 39, 355-36 1. 25. Honer, W. G , Dickson, D. W , Gleeson, J., and Davies, P. (1992) Regional synaptic pathology m Alzheimer’s disease. Neurobiol. Aging 13,375-382 26. Hemonen, O., Soinmen, H , Sorvari, H., Kosunene, O., PalJarvi, L , Korvisto, E., and Riekkinen, P. J (1995) Loss of synaptophysm-like immunoreactivity m the hrppocampal formation is an early phenomenon in Alzheimer’s disease. Neuroscieace64,375-384. 27. Masliah, E., Terry, R. D , DeTeresa, R. M , and Hansen, L. A (1989) Immunohistochemrcal quantification of the synapse-related protein synaptophysin in Alzhermer disease. Neuroscl. Lett. 103,234-239 28. Clinton, J., Forsyth, C , Royston, M. C , and Roberts, G W. (1993) Synaptic degeneration is the primary neuropathological feature m prion disease: a prehminary study NeuroReport 4,65-68. 29. Ince, P. G., Slade, J , Chmnery, R. M , McKenzie, J., Royston, C., Roberts, G. W., and Shaw, P J. (1995) Quantitative study of synaptophysin immunoreactivity of cerebral cortex and spinal cord m motor neuron disease. J. Neuropathol. Exp. Neurol. 54,673-679.

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30. Calverley, R., Bedi, K , and Jones, D. (1988) Esttmation of the numerical density of synapses m rat neocrotex comparison of the “dissector” with an “unfolding” method. J. Neuroscz. Methods 23, 195-205. 3 1. Lichtman, J., Sunderland, J., and Wdkmson, R. (1989) Hugh-resolution imagmg of synaptic structure with a simple confocal microscope New Bzologzst 1,75-82 32 Mashah, E., Elhsman, M., Carragher, B., Mallory, M., Young, S., Hansen, L , DeTeresa, R., and Terry, R. D (1992) Three-dimensional analysis of the relationship between synaptic pathology and neuropil threads m Alzhelmer disease J. Neuropathol. Exp. Neurol. 51,404-414 33. Mashah, E., Terry, R. D., Mallory, M , Alford, M., and Hansen, L. (1990) Diffuse plaques do not accentuate synapse loss in Alzhetmer disease. Am. J Pathol. 137, 1293-1297. 34. Mossberg, K., Arvidsson, U., and Ulfhake, B (1990) Computerized quantification of immunofluorescence-labeled axon terminals and analysts of colocalization of neurochemmals in axon terminals with confocal scanning laser microscope J Hzstochem Cytochem. 38, 179. 35 Mashah, E , Hansen, L., Albright, T., Mallory, M , and Terry, R. D (1991) Immunoelectron microscopic study of synaptic pathology in Alzheimer disease Acta Neuropathol. 81,428-433 36. Jahn, R., Schiebler, W , Quiment, C., and Greengard, P (1985) A 38,000-dalton membrane protem (~38) present m synaptic vesicles Proc. Natl. Acad. Sci USA 82,4137-4141 37. Alford, M. F , Mashah, E., Hansen, L. A , and Terry, R D. (1994) A simple dotimmunobmdmg assay for the quantification of synaptophysm-like immunoreactlvlty m human brain. J Hzstochem. Cytochem 42,283-287 38 Perdahl, E., Adolfsson, R., Alafuzoff, I , Albert, K. A , Nestler, E. J , Greengard, P., and Wmblad, B (1984) Synapsm I (protein I) m different bram regions m senile dementia of Alzhetmer type and m multunfarct dementia J. Neural Transmzss. 60, 133-141. 39. White, C. L., Simmons, A. L., and Btgio, E. H (1993) Determmatton of synapse denstty m Alzheimer disease neocortex usmg an enzyme-linked immunosorbant assay (ELISA) and monoclonal antibody SY38 to synaptophysm. J Neuropathol. Exp Neural 52,263. 40 Wtedenmann, B. and Franke, W W (1985) Identification and localization of synaptophysin, an integral membrane glycoprotem of Mr 38,000 characteristic of presynaptic vesicles Cell 41, 1017-1028 41. Calhoun, M. E., Jucker, M , Martin, L. J., Thmakaran, G., Price, D. L , and Mouton, P. R (1996) Comparative evaluation of synaptophysm-based methods for quantification of synapses J Neurocytol 25,821-828

19 Zymographic of Gelatinase Paul E. Gottschall,

Method for the Measurement Activity in Brain Tissue J. Wenjun Zhang, and Suman Deb

1. Introduction Specific recognition of cell-surface molecules with other cells or extracellular matrix (ECM) is fundamental for cellular motility, reorganization, and prohferatron. To carry out these actrons, cells often displace space previously occupied by cells or the ECM, thus proteolysrs may be required. More functtonally m different model systems,mtegrm-mediated mteraction of cells with ECM influences or directs cell growth, differentiation and survival via specific intracellular signaling pathways (I-3). Thus, the interplay between binding of mtegrms (and other surface molecules) with ECM and the proteolysis of ECM must be highly orchestrated. The mechanism of degradation of ECM for these physiological purposes IS under stringent control, turning on only when appropriate srgnals are in place for a subsequent function. The importance of ECMdegrading proteases m such interactions was shown recently in transgenic animals expressing an autoactivated, ECM-degrading metalloprotease targeted to mammary eprthelial cells. These epithelial cells underwent early apoptosis near the end of pregnancy. When these transgemc mice were crossed with mice overexpressing an endogenous inhibitor of the protease, early apoptosis was not observed (4). These results emphasize the importance of proteinases and their mhibrtors in regulating the functions of cell-ECM interactions. The matrrx metalloprotemases (MMPs) are a family of proteases that function mainly m the degradation of ECM. There are at least 16 proteins m this famrly of calcium-requiring, zinc-containing endopeptidases that act to degrade matrix components such as basement membrane and interstitial collagen, frbronectm, laminin, and proteoglycan. Several of these enzymes act m a wide range of physiological and pathologrcal conditions. The common link among From

Melhods in Molecular Edlted by J Harry

Medmne, and

vol 22 Neurodegenerabon

H A Tllson

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and Protocols NJ

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these putative functions IS that the MMPs are expressed and become active at times of tissue reorganization or cellular movement. At least three mechanisms control the activity of the MMPs: gene expression, activation of the latent zymogen by proteolytic cleavage, and production of specific, endogenous protease inhibitors, termed the tissue inhibitors of metalloproteinases (TIMPs) Evidence suggesting that the MMPs, along with other ECM-degrading enzymes, participate in neuronal physiology and neurological pathology has grown rapidly in recent years. Early work demonstrated that the plasmmogen activators and the MMPs are located near the growth cone in a growing neurite in vitro, suggesting that ECM-degrading enzymes play a role m neunte outgrowth during development or regeneration (5-7). More recently, it has been shown that mice deficient in the production of tissue plasmmogen activator resist excitotoxin-induced seizure and neuronal degeneration (8). Admmistration of a hydroxamate metalloprotemase inhibitor m mice inhibited or reversed the symptoms of experimental autoimmune encephalitis, an animal model of multiple sclerosis (9,10). In addition, MMPs appear to be overexpressed m nervous systemtissue of distinct neurological pathologies including Alzheimer’s (II), amyotrophic lateral sclerosis (12), multiple sclerosis (13), and a rodent peripheral nerve crush model (14). MMPs have been implicated in neoplastic pathologies of the nervous system, especially m the invasive character of gliomas (1516). MMP activity and protein expressed by nervous system tissues has been measured by activity assays(9,17), cell-activity assays(18), zymography and (11,19,20), immunocytochemistry (21,22). mRNA levels for the individual MMPs has been assayed as an indicator of enzyme activity (23). There are obvious advantages and disadvantages to each of these techniques. Immunocytochemistry is the least quantitative of the techniques, and gives no indication as to the activational state of the enzyme, but has the advantage of identifying a particular cell type or tissue in which the enzyme is expressed. Activity assays and cell-activity assays provide information about the activational state of a crude sample, but do not distinguish which particular enzyme (or sometimes even enzyme family) is active. Depending on the sensitivity, zymography detects both latent and active species, and individual enzymes may often be identified based on their molecular weight. Latent species are detected on a zymogram because SDS treatment dissociates the critical Cys-Zn2+ interaction that maintains the enzyme in its latent form. The use of gelatin zymography to quantitate the activity of the gelatinases (MMP-2 and MMP-9) is an elegant technique that is sensitive to lo-30 pg (138-417 attamol of MMP-2) of enzyme (24) and the assay is linear over a IO-20-fold range of enzyme activity. The use of immobilized gelatin in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel that acts as a substrate for samples that con-

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taining gelatin-degrading enzymes was first developed by Heussen and Dowdle m 1980 (25). The zymographic assay of the gelatinases, MMP-2 and MMP-9, in tissue samples 1s confounded by several factors. The enzymes may be present in serum and extraction of serum with the tissue may contribute to total tissue activity (26). Often detergent is used to obtain good recovery of the enzymes since they bind avidly to ECM. Large amounts of total protein recovered from detergent-extracted samples stain with Coomassie blue, and may mask clearmg (if this protein migrates at a similar molecular weight) that would be observed in samples that contain less total protein. This factor is a major problem when enzyme activity 1s low, for example in nontransformed tissue. Reducing the amount of total protein in the extracted tissue samples, i.e., partially purifying the enzymes, would enhance the observation of the cleared bands after degradation of gelatin. Thus, we developed a zymographic assay that uses gelatin-support purification of a tissue sample prior to zymography (27) This method greatly enhanced our ability to quantitate gelatmase (MMP-2 and MMP-9) activity m brain tissue samples. What follows is a description of our simple, small, sample purification technique and the method for quantitating this activity using zymography and densitometric analysis. This method allows concentration of the activity while at the same time purifying the activity away from much of total cellular protein. It may be employed with conditioned media samples (or other samples) as well tissue samples, where activity of the sample is too low for detection with zymography. A previous paper has detailed how zymography can be used to quantitate gelatmase activity (12). 2. Materials 2.1. Extraction 1. Extraction buffer 50 mM Tris-HCl, pH 7.6, 150 n&I NaCl, 5 miI4 CaCl,, 0.05% Bq-35 (v/v), 0 02% NaN3 2. Extraction buffer with Triton X-100: Buffer in 1 containing 1% Trlton X-100 (reagent grade) 3 Elutlon buffer: Buffer contaming 10% dlmethylsulfoxide (DMSO) (reagent grade). 4. Gelatm-Sepharose 4B (Pharmacla Blotech, Uppsala, Sweden).

2.2. Preparation

of SDS-PAGE Gels with Gelatin

1 1.5 M Tris-HCl, pH 8.8 2. 0.5 M Tns-HCl, pH 6 8 3. 1% Gelatin; prepare fresh (Sigma, St. Louis, MO); boll for dissolving gelatin, be sure to cool before adding to acrylamlde solution. 4. 10% SDS

5. Acrylamlde/bls stock (toxic) (30%; 2.6% C) (seeNote 1). 6. 10% Ammonium

persulfate (prepare fresh dally)

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7. TEMED (iV,iV,N’,N’-tetra-methylenedlamlne). 8. Electrode buffer Tris base (15.0 g), glycme (72.0 g), SDS (5 0 g), brmg up to 1.O L.

2.3. Zymography 1. Sample buffer (5X), consists of 0.4 M Tns-HCl, pH 6.8,5% SDS, 20% glycerol, 0 03% bromphenol blue (12) 2 Collagenase from Clostridzum hutoZytzca; for general use, cat no. CO130 from Sigma 3. Molecular weight markers.

2.4. Gel Apparatus 1 Mmigel 2 0 75-mm spacers and comb.

2.5. Post-SDS-PAGE 1. 2. 3 4. 5.

Substrate

Degradation

2.5% Tnton-X-100 Incubation buffer: 21 mM Tns-HCl, pH 7 6, 10 mM CaCl,, 0.04% NaN,. 0.1% Coomassle blue dissolved m 40% methanol and 10% acetic acid Destain solution: 40% methanol, 10% acetic acid. Apparatus for densitometrlc analysis of gels (see Note 2)

3. Methods 3.1. Tissue Extraction 1 All procedures are performed at 4°C. Brain &sues are frozen prior to use. For dissection, the entire brain 1s placed m phosphate-buffered saline (PBS) after careful removal of the menmges The brain regions are then dlssected on ice, frozen on dry ice, and stored at -80°C until extraction (see Note 3). 2. Individual brain regions are thawed, and homogenized by hand m 0.5 mL of extraction buffer containing 1% Triton X-100 (see Note 4) usmg a Teflon-glass homogemzer (15 strokes) A lo-p,L aliqout of the homogenate was saved and assayed for total protein (see Note 5) The 0.5-mL sample 1s then transferred to a microfuge tube. 3 The homogenate is centrifuged at 12,OOOg and the supernatant recovered (see Note 6). Fifty mlcrohters of prewashed and diluted gelatin Sepharose 4B IS then added to the sample and incubated with the sample for 60 mm with constant shaking. Prior to begmnmg the extraction, an appropriate volume of gelatmSepharose 4B 1s washed with extraction buffer three times before use. The pelleted Sepharose of required volume 1s diluted 1 1 with extraction buffer and 50 pL of this suspension 1s added to each sample 4 The sample 1s then centrifuged at 5000g for 5 mm The Sepharose 4B pellet 1s resuspended m 500 pL of extraction buffer and centrifuged again at 5000g for 5 min. Next, 150 pL of elutlon buffer (see Note 7) IS added to the pellet and the sample incubated for 30 mm. The sample 1s centrifuged at 5000g for 5 mm, the supernatant collected and frozen at -80°C until assayed for gelatmase activity.

Zymographic

Gelatmase Measurement

3.2. SDS-PAGE in Gel Containing

213

0.1% Gelatin

1. 7.5% SDS-PAGE gels (see Note 8) are prepared m a standard manner except that 1 mL of water is replaced by 1 mL of a 1% gelatin solutton when using a 10-mL solutron to prepare the running gel. Great care 1s taken to measure volumes to prepare these gels for consistency purposes. 2. To a 30-mL polypropylene centrifuge tube add: 3 85 mL of distilled, deionized water (at least 18 MR), 2 5 mL of 1.5 M Trrs-HCl, pH 8.8, 1 .OOmL 1% gelatin, 100 PL of 10% SDS stock, and 2 50 mL of 30% acrylamtde/bts acrylamrde. Degas for 5 mm (gel polymerizes faster if solution is degassed). 3 Add 50 PL of 10% ammomum persulfate and 5 PL of TEMED, swtrl the solution, and cast into a preassembled, glass plate sandwich-holder apparatus. The lo-mL volume is sufficient to cast two mnngels. Cover the solutron with a mnuma1 volume of water-saturated isobutanol and allow to polymerme (see Note 9). 4 Once polymertzed, wash gel three times with water. After the final wash, blot the top of the gel with filter paper To prepare the stacking gel, combme 6.10 mL water, 2.50 mL Trrs-HCl, pH 6 8, 100 FL 10% SDS, 1 30 mL 30% acrylamtde/ bts acrylamrde, and degas. Add 50 yL of 10% ammomum persulfate and 10 PL of TEMED, swirl, and pour mto apparatus with preplaced comb (lo-well) Adjust comb, remove any bubbles, allow 2-3 mm between the teeth and the runnmg gel, and allow to polymerize 5. Once the stacking gel IS polymerrzed, remove the comb, and place gels on electrode holder Wash the wells with water three times, add electrode buffer to the reservoir, and begin to load samples. 6. A molecular weight marker set and 2 ng of bacterial collagenase as a posmve control sample are loaded onto every gel (see Note 10). Samples volumes may be as high as 30 PL dependmg on the quahty of the sample wells Either 5X or 2X sample buffer may be used to prepare the samples depending on the activity of the sample 7 The samples are electrophoresed at 180 V constant voltage, generatmg approx 40 mA/gel that ~111 decrease over the run time. Runnmg time on a mmtgel IS 35-45 mm Once the bromphenol blue reaches the bottom of the gel, stop the current and turn off the power supply.

3.3. Post-SDS-PAGE

Processing

1, Pour off electrode buffer m the reservoir, remove gel(s) from the electrode holder, remove spacers, and wnh a spatula m the spacer locatton, pry open the glass plates Use a spacer to remove the stacking gel, and roll the gel into 100 mL of 2 5% Trtton X-100. Swrrl slowly on a rotary shaker at room temperature Pour off the Trtton X-100, add another 100 mL, and swirl for 20 min. Pour off the Trtton X-100 completely, and add 100 mL of incubation buffer. Place a top on the container, place m a shaking, 37°C water bath (approx 32-35 rpm), and mcubate for 16 h (overnight) (see Note 11) 2 The next morning, remove the gels and place m 0.1% Coomasste blue (75 mL). Stain the gels at room temperature for 1 h, remove stain, and add destammg solu-

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tion (100 mL). Destain with two changes of destaining solution for 30 min each or until clear proteolytic bands appear on a contrasting blue background. Consistency of staining and destaining improves reproducibility 3. The gels are covered m plastic wrap, and placed m the refrigerator overnight The plastic wrap is changed the following mornmg and gels are then scanned and photographed.

3.4. Densitometry

and Analysis

of Zymograms

1. Gels or photographs may be used for densitometric scannmg. Gels are scanned at the highest resolution, the analysis inverted since the scan generates white bands on a black background, and analyzed with area or volume integration (see Note 12) 2. Sample values are expressed as a fraction of one of the bacterial collagenase bands (within the linear range of the assay) and corrected for protein in the original homogenate (see Note 13).

4. Notes 1 In our laboratory, liquid acrylamide/bis is purchased from Bio-Rad (Hercules, CA) for the preparation of gels. We obtain good gel-to-gel consistency with this reagent. Precast 10% Tris-glycine gels containing 0 1% gelatin for zymography are available from Novex (San Diego, CA). 2 We use a Bio-Rad Personal Densitometer 670 and Molecular Analyst software for analysis of the gels. 3. We dissect tissues and freeze bilateral regions in separate tubes Typically, tissues are not stored for more than 1 wk in the freezer. Only a unilateral region is required for the assay. Regions that we have assayed are frontal cortex (approx 100 mg piece), hippocampus, striatum, diencephalon, midbrain, and cerebellum (approx 100 mg piece); the other half of the brain region may then be used as a control tissue if the animal was injected mtracerebrally or it may be used m another assay 4. CHAPS(3-([3-cholam~dopropyl)d~methyl-ammon~o]-1-propane-sulfonate) is as effective, but not more effective, than Triton X-100 m extracting gelatmases from brain tissue (26). Activity is not different m the presence or absence of a cocktail of protease inhibitors. 5. The bicmchonmic acid protein assay performs reasonably well with samples contammg 1% Triton X-100 6. After homogenization m buffer contammg 1% Triton X- 100 and centrifugation, negligible MMP-9 activity is retamed within the pellet and approx 10% of the total MMP-2 activity remains in the pellet after re-extraction (26). 7 Depending on the degree of sample concentration required for detection on the zymogram, smaller volumes of elutlon buffer may be used for elutmg the enzymes from the gelatm-Sepharose. 8. Running gels (7.5%) are employed because these gels provide the good separation of the active and latent forms of MMP-2 and MMP-9. In addition, dimers of MMP-9 often will appear (at approx 200 kDa) and the 7.5% gel allows vtsuahzation of this activity

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9. The presence of gelatm does not appear to affect polymerization time of acrylamrde. When leftover solutron m 30-mL tube has polymerized, the gel has polymerized. 10. It 1s convenient if the molecular weight marker set includes bovine serum albumin as one of the markers since the 66-kDa protein is a good marker for identifymg MMP-2. Be sure to sktp a lane between the molecular-weight marker and any brological sample, smce it 1spossible that drffusron of the mercaptoethanol may affect the actrvrty of a sample. We use molecular weight markers subjected to reducing condmons to estrmate the molecular weight of the MMPs. However, smce the markers are run under reducing conditions and the samples under nonreducmg condmons, this is, at best, an estimate of the molecular weights. A more appropriate method would be to estimate molecular weights of the MMPs on a Western blot. In addmon, 2 ng of bacterial collagenase are run on every gel as an internal, positrve standard This allows correctron for mtergel variation when samples are analyzed by densitometry. 11. Increasing the incubation time increases the sensitivrty of detection but tends to decrease the lmearrty of the actrvrty. 12. Gels are photographed with Polaroid 59 Instant Color Film. We employ area mtegratron of a curve generated from OD and length of the gel using the highest resolution on the densitometer. Background is subtracted by scanmng an empty, adjacent lane and subtracting these values from values m the sample lanes Volume integration is just as linear and useful. 13 Three distinct bands with varying activity are observed with the bacterial collagenase posmve Internal standard. Use the band that is within the linear range of the assay (as determined previously). 14. MMP-2 and MMP-9 bmd avidly to gelatm and are efficient degraders of this protein However, rt 1spossrble that other gelatin-degrading enzymes are present m a sample. Identificatron of each activity on a zymogram may be venfred by rmmunopreciprtation of a sample using a specrfrc antibody, followed by zymography or employing a Western blot. Antibodies to the human enzymes are commercially available 15. One would expect that thus method may be extrapolated to other enzymes that bind avidly to their substrate For example, it may be possible to measure the casemolytrc MMPs (MMP-3 and MMP-7) using immobilized casem to concentrate and partially purify the enzyme from tissue prior to casein zyography.

References 1 Aoshiba, K., Rennard, S I , and Spurzem, J. R. (1997) Cell-matrix and cell-cell mteractrons modulate apoptosrs of bronchial eprthelial cells Am J. Physzol. 272, L28-L37 2 Frlsch, S M. and Francis, H (1994) DisruptIon of epithehal cell-matrix mteractrons induces apoptosis. .I. Cell Bzol 124,619-626. 3. Werb, Z , Sympson, C. J., Alexander, C. M., Thomasset, N , Lund, L. R., MacAuley, A , Ashkenas, J., and Bissell, M. J. (1996) Extracellular matrix

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7 8

9

10.

11.

12.

13.

14.

15. 16

17

Gottschall, Zhang, and Deb remodeling and the regulation of epnhehal-stromal mteractions during differentiation and involution [Review]. Kidney Int. Suppl. 54, S68-S74. Alexander, C M , Howard, E W , Bissell, M J , and Werb, Z (1996) Rescue of mammary epithehal cell apoptosis and entactm degradation by a tissue mhibitor of metalloprotemases-1 transgene J. CeZEBzol 135, 1669-1677 Krystosek, A. and Seeds, N W. (1981) Plasmmogen activator secretion by granule neurons m cultures of developmg cerebellum. Sczence 213, 7810-7814. Pittman, R N. (1985) Release of plasmmogen activator and a calcmm-dependent metalloprotease from cultured sympathetic and sensory neurons Dev Bzol 110, 91-101 Pittman, R N and Williams, A G (1988) Neurite penetration mto collagen gels requires Ca++-dependent metalloprotemase activity Dev Neuroscz. 11,41-5 1 Tsnka, S E , Gualandris, A , Amara, D G , and Strickland, S (1995) Excitotoxminduced degeneration and seizure are mediated by tissue plasmmogen activator Nature 371,340-344. Clements, J M , Cossms, J A., Wells, G M A., Corkill, D J., Helfrich, K , Wood, L M., Pigott, R , Stabler, G , Ward, G A , Gearing, A. J H., and Miller, K M (1997) Matrix metalloprotemase expression during experimental autoimmune encephalitis and effects of a combined matrix metalloprotemase and tumour necrosis factor inhibitor. J. Neuroimmunol 74, 85-94 Gijbels, K., Galardy, R. E., and Steinman, L. (1994) Reversal of experrmental autoimmune encephalomyelms with a hydroxamate inhibitor of matrix metalloproteases. J. CZm. Invest. 94,2177-2182 Backstrom, J. R., Miller, C. A, and Tokes, Z. A. (1992) Characterization of neutral protemases from Alzheimer-affected and control bram specimens identification of calcium-dependent metalloprotemases from the hippocampus J Neurochem. 58,983-992 Lim, G. P., Backstrom, J R., Cullen, M J., Miller, C. A., Atkinson, R D , and Tokes, Z. A. (1996) Matrix metalloproteinases m the neocortex and spinal cord of amyotrophic lateral sclerosis patients. J. Neurochem. 67, 25 l-259. Gijbels, K., Masure, S., Carton, H., and Opdenakker, G. (1992) Gelatmase in the cerebrospmal fluid of patients with multiple sclerosis and other inflammatory neurological disorders J Neuroimmunol. 41,29-34 LaFleur, M., Underwood, J. L., Rappolee, D. A., and Werb, A. (1996) Basement membrane and repair of mJury to peripheral nerve defining a potential role for macrophages, matrix metalloprotemases, and tissue mhibitor of metalloprotemase- 1 J Exp Med 184,231 l-2326 DeClerk, Y. A., Shimada, H., Gonzalez-Gomez, I., and Raffel, C. (1994) Tumoral mvasion in the central nervous system. J. Neuro-Oncology 18, 111-121. Rao, J S , Steck, P A , Mohanam, S , Stetler-Stevenson, W. G., Liotta, L A , and Sawaya, R. (1993) Elevated levels of Mr 92,000 type IV collagenase m human brain tumors. Cunc Res. 53,2208-2211. Backstrom, J R , Lim, G P , Cullen, M J , and Tokes, Z A (1996) Matrix metalloproteinase-9 (MMP-9) is synthesized m neurons of the human hippocampus and is capable of degrading the amyloid-l) peptide J. Neurosci 16,79 IO-791 9

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18. Colton, C. A., Kerr, J. E., Chen, W T., and Monsky, W. L. (1993) Protease production by cultured microgha-substrate gel analysis and immobihzed matrix degradation J. Neuroscl. Res. 35,297-3&I 19. Nelson, R B and Sirnan, R (1989) Identification and charactertzatron of calcium-dependent metalloproteases in rat brain, J. Neurochem 53,641-647. 20. Rosenberg, G. A., Dencoff, J E., Mcguire, P. G., Liotta, L A , and StetlerStevenson, W. G. (1994) Injury-induced 92-kilodalton gelatinase and urokinase expression m rat brain. Lab. Znvest. 71,417-422. 21. Maeda, A. and Sobel, R A (1996) Matrix metalloproteinases m the normal central nervous system, microghal nodules, and multiple sclerosis lesions J. Neuropathol. Exp. Neurol. 55,300-309 22. Yamada, T , Yoshiama, Y , Sato, H., Seiko, M., Shmagawa, A , and Takahashi, M (1995) White matter microgha produce membrane-type matrix metalloprotease, and activator of gelatmase A, in human brain tissues. Acta Neuropathol 90,421-424 23. Sato, H., Kida, Y , Mai, M E., Sasakr, T., Tanaka, J., and Seiko, M. (1992) Expression of gene encoding type IV collagen-degrading metalloprotemases m various human tumor cells. Oncogene 7,77-83. 24. Kleiner, D. E. and Stetlerstevenson, W G. (1994) Quantitative zymographydetection of picogram quantities of gelatmases Anal. Bzochem. 218,325-329. 25. Heussen, C. and Dowdle, E B (1980) Electrophoretic analysis of plasmmogen activators m polyacrylamide gels contammg sodmm dodecyl sulfate and copolymerized substrates. Anal. Blochem. 102, 196-202. 26 Zucker, S , Lysik, R. M., Zarrabl, H. M., Moll, U., Tickle, S P , Stetler-Stevenson,W , Baker, T. S., and Docherty, A. J. (1994) Plasmaassayof matrix metalloprotemases. MMPs andMMP-inhibitor complexesin cancer.Ann. NYAcad. Scz.732,248-262. 27 Zhang, J W and Gottschall, P. E. (1998) Zymographic measurementof gelatmase activity m brain tissueafter detergent extraction and affinity-support purification. J. Neurosci. Meth , in press.

The Measurement of Monoamine Neurotransmitters in Microdialysis Perfusates Using HPLC-ECD Ian Acworth

and Michael L. Cunningham

1. Introduction Microdialysis is a perfusion-based sampling procedure that IS now used routinely to study the chemistry of the extracellular environment of a variety of tissues in the living organism (1,2). A microdialysis probe (consrsting of an inlet and outlet tube connected by an area of semipermeable membrane) IS perfused at a constant flow rate with a medium (arttficial cerebrospinal fluid, CSF; aCSF) that is iso-osmotic to and contains ions at the same concentration as the extracellular fluid. Analytes pass through the semipermeable membrane down their concentration gradient and are swept through the probe by the constantly moving perfusron medium. Once collected, samples can then be analyzed by a variety of different analytical procedures. Prior to microdialysis perfusion, neurochemical studies often involved the use of tissue homogenates. With this technique, changes in neurotransmrtter release were inferred mduectly by changes in the ratio of metabolites to parent neurotransmitter levels (met/nt ratio) following a physiological or pharmacologrcal manipulation. Unfortunately, homogenization destroys neurotransmitter compartmentalization and it cannot readily differentiate between released neurotransmttter and intraneuronal stores. Similarly, neurotransmitter metabohtes may be derived from both released neurotransmitter and from intraneuronal breakdown of both released and nonreleased neurotransmitter pools. Consequently, some researchers are questioning the validity of using changes in the metint ratio as an accurate predictor of altered neurotransmitter release. Furthermore, although the metint ratio is readily affected by gross pharmacological intervention (e.g , inhibition of neurotransmitter synthesis; inhibition of From

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catabolic enzymes) it is much less affected by more subtle pharmacological manipulatron or by changes in behavior. The use of tissue homogenates also precludes pharmacokinetic and pharmacodynamic studies in a single ammal and only permits the researcher a single metabohc snapshot at one time point. Microdialysis perfusion overcomes many of the hmitations of tissue homogenates and can sample released neurotransmitter directly, thus allowing a single animal to be used in dose-response and pharmacokmettc studies Furthermore, since microdialysis can be used in the awake, freely movmg animal rt is readily applicable to behavioral studies. Microdialysis perfusion is not without its own issues. The typical membranes used in the construction of microdialysis probes often limit diffusion to lower molecular weight solutes (typically cl kDa). This sample “filtration” 1s an advantage when trying to measure monoammes m the presence of larger molecular weight coelutants, but is a major disadvantage when some of the higher molecular weight compounds, such as neuropeptldes, are of interest. The design of the microdialysis experiment (perfusion and analysis) 1salso critical For example, rapid (sub-min) sampling IS desired when using microdialysis to relate any changes in behavior to altered extracellular levels of neurotransmitter, as behaviors are usually completed very quickly. Although it is physically possible to collect microdialysis perfusates at this rate, other issues may arise. Rapid sampling at slow perfusion flow rates produces low volume samples that can be problematic to handle. Rapid sampling at fast flow rates may produce a large but very dilute sample, with the level of the analyte far below the hmtt of detection of the analytical equipment. In each of these casessamples still have to be analyzed. Because most high-performance liquid chromatography (HPLC)-based techniques require lo-30 mm to complete an analysis, unstable solutes (e.g., catecholammes) may undergo variable rates of autooxidation if not properly preserved. Thus, the sampling rate is a compromise between the perfusion flow rate, the sample volume obtained, the amount of analyte m the sample, and the analysis time. Conventtonal microdtalysls balances these factors, so that the flow rate chosen provides sufficient recovery of the analyte by the microdialysls probe for analysis, and matches the collection time to the analysis time. If all these criteria are matched then samples can be measured “online.” A variety of techniques have previously been used to analyze microdialysis perfusates, but HPLC-based methods are by far the most common. The concentration of monoamines in brain microdialysls perfusates are typically m the sub-to-low pg/collection range. For example, literature values for dopamme m the striatum is 15-50 pg/20 PL but ~500 fg/20 PL in the prefrontal cortex (3,4). Of all the detectors currently available, only electrochemical detection (ECD) offers the greatest sensitivity and selectivity to directly measure the low levels

HPLC-ECD Measurement

of Monoamme Neurotransmitters

of monoamines m microdialysis perfusates. Two types of electrochemical detectors are currently used (5). The most common and rehable approach favored for the analysis of microdialysis perfusates 1sthe use of normal-bore (columns with internal diameters of 3 O-4.6 mm) HPLC with coulometric detection. Coulometrlc detection is a concentration-dependent technique readily capable of measuring as little as 350 fg of analyte in sample volumes of >lO yL. Amperometric detection is a less desirable approach and suffers from a low electrolytic efficiency, poor sensitivity, and instabrlity. This approach can be improved by coupling amperometrlc detection to mlcrobore HPLC (forcing the amperometrlc detector to become more coulometric in nature) (5). Microbore HPLC with amperometric detection is mass-sensitive with practical limits of detection of approx 100 fg of analyte but with restrlcted sample volumes of typically ~2 yL. Furthermore, microbore HPLC is less practical, requiring a specialized pump and injector, the column is readily blocked and the thin-layer amperometric electrode needs frequent maintenance. A common misconception is that since microbore can only handle a small sample volume, a microdialysis sample can readily be split for analysis of different analytes on different microbore systems.This is only true if the microdialysis sample contains levels of analyte above the limit of detection of the different microbore systems. In this chapter we present the use of in vivo microdialysis perfusion coupled to normal-bore HPLC with coulometric detection for the measurement of extracellular

fluid monoamme

levels in different brain regions.

2. Materials Glasswareusedfor mobile-phaseproduction should not be cleanedin soapsolution. Instead only dedicated glassware should be used. Soap dried onto the inner surface of glassware may dissolve mto the mobile phase an act as an ton-paumg reagent causing a shift m a monoamine’s retention time. Furthermore, soap may form undissolved flakes m the mobile phase that can lead to increased system back pressure. Store glassware covered to eliminate contamination with dust (partlculates can lead both to increased system noise and back pressure). Glassware should be briefly rinsed with 100-200 mL purified water before use 2. All water employed should be of HPLC grade, either commercially avallable or produced from a purpose-designed HPLC water system (e.g., M&-Q, Mlllipore, Bedford, MA). Do not use water with a reslstlvity of 48.0 MR cm. Water must then be passed through a Cl8 solid-phase extraction cartridge (e.g., Sep-pak, Waters, MIllford, MA) using a vacuum pump. This step removes trace organic metabohtes that may Interfere with analysis (e.g., ghost peaks, prolonged voids) and also helps to greatly reduce background currents and noise within the HPLC-ECD system Each l-cm3 extraction cartridge can purify approx 2 L of water. Discard the first 200 mL of water. Periodic checks should be made on the

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Acworth and Cunningham quality of the water by analyzing it on the HPLC-ECD system at the gain range used for mlcrodlalysls perfusate analysis. (Some spurious reagent peaks or system peaks may occur during the analytical run. These can be ignored d they do not interfere with the analyte peaks of interest.) All salts used m the production of aCSF, preservative solution, standards and mobile phase, should be of the purest reagent grade available with minimum amounts (typically l G&2) 1s formed between the pipet tip and the cell membrane; again, this is momtored on an oscilloscope or computer as described above Seal formation will be aided by applying negative voltage to the pipet tip After obtaming a GQ seal, and ensurmg that the membrane potential 1sset close to the resting membrane potential of neurons (l,e., close to -70 mV) if m voltage-clamp mode, or zero current if m current-clamp, the patch of membrane directly under the pipet can be ruptured by a short burst of negative pressure (suction) to achieve the “whole-cell” recording mode This will be apparent from the sudden appearance of capacltative transients (because of the capacltative nature of the lipid bilayer) d using a patch-clamp amplifier in voltage-clamp mode. Compensation of capacitance and series reststance is then done. This procedure should be outlined in detail in the manual provided with the amplifier An appropriate gain and filtermg frequency should be chosen based on the expected amplitude and speed of acquisition (see Note 6). 9. Once the cell is m whole-cell recording mode, and the membrane current and series resistance are stable (see Note 7), the experimenter can begin testing for the responses of interest Remaining technical challenges include the posltiomng of the perfusion tube for the application of exogenous agonists (see Note 8) or of the stimulating electrode for evoking synaptic currents. A second mlcromanipulator to control the movement of these, and the use of a camera/monitor, will make these relatively simple. 4. Notes 1. Blades are supplied with certain tissue slicers, and blades specifically designed for this purpose are available commercially (e.g , Campden Vibroshce). We use

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3.

4

5.

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ordinary double-edged razor blades, which often have an oily coat and need to be wiped wrth ethanol, then rmsed, before use. A critical factor in preparing healthy brain slices that are amenable to good, stable whole-cell recordings 1s speed. Using younger animals with soft craniums and small brains (which cool down more rapidly) can help It is also helpful to have a well-organized routme for the preparation. Many good reviews are available for guidance (2,7) The equipment for preparmg bram slices should be arranged m such a way that the experimenter can prepare brain slices quickly. Space for the dissection, and close positioning of the tissue slicer and mcubatton chamber, should definitely be priortties In cases m whtch the debris is overwhelming, or where very raped apphcation of agonists 1s required, it may be helpful to remove the debrts and neuroptl above target cells prior to recordmgs (7). This can be achieved by using a pipet of larger diameter and filled with aCSF. The pipet is positioned above and to the side of the vrsuahzed target cell, and then positive pressure is applied to the pipet. The pipet is then moved across the surface of the cell to blow away any debris and neuropil. Suction can then be applied to pull the removed debrts up into the pipet tip. Assessmg the health of the slice: When recording from vtsuahzed neurons, a good mdicator of the health of the slice 1s the condition of cells, as described in Subheading 3.1., step 5. In particular, when using hippocampal slices, healthy lookmg interneurons and dentate gyrus neurons are good indicators of healthy slices. Extracellular dendrmc field potential recordings can be used to demonstrate healthy synaptic connections m slices. In a healthy hippocampal shce, field potentials should be approx 0.5 mV wtth a small fiber volley, and have a stable rate of rise and decay over time. Assessing the health of the cell and quality of the electrical recordings: Cells should have a stable resting potential (comparable to known charactertstlcs of the cells), with a low and stable leak current and series resistance (if under voltageclamp condmons). Vartous qualitative parameters of cell viability could include the presence of action potentials, consistent shape and frequency of firing, and the threshold for action potential firing. Evoked synaptic currents: The position of the electrode and size of sttmulus may have to be experimented with until optimum sttmulation is achieved. Again, the stablhty over time should be monitored. Specific components of synaptic currents (for example, mhibitory or excitatory components) can be isolated pharmacologically Rapid perfusion of drugs. We have found that the angle and position of the tube IS absolutely critical for obtaming responses to agonists that cause pronounced desensitization. Where possible, a perfusion system should always be tested and optimized first with a nondesensitizmg agonist Arttfacts should be tested for by applying aCSF Once responses are obtained to the agonist of Interest, the stability of the response over time should be monitored before antagonist effects are monitored. Series resistance should be monitored throughout the experiment as an mdication of the quality of the recording.

Glutamate Receptor Channels 9. There is a strong lmk between the activatton of glutamate receptors, the alteration m cytoplasmic Ca*+ levels, and neuronal cell death. However, it is not clear whether the alteration in glutamate responses causes, or are altered as a result of, the subsequent steps leading to cell death. In addition, the glutamatergic synaptic responses could be affected both by changes m the presynaptic release of glutamate, and/or the postsynaptrc response. The techniques described will provide a method to determine whether an experimental compound or condition will affect the glutamate response, and whether it is caused by a pre- or postsynaptic effect. 10 When recordmg from a postsynaptic cell and stimulating presynapttc mputs to evoke synaptic currents, glutamate release by presynaptic terminals will open the glutamate-gated ion channels on the postsynaptic cell and induce an electrical response; this electrical response is measured directly as described above. The amphtude and duration of the giutamatergic synaptic response will be determmed mostly by the amount and time course of glutamate release into the synaptic cleft, the time course of its presence in the cleft, and the characteristics of the postsynaptic response (e.g., the kmetics of the channels opened by glutamate on the postsynaptic cell) Therefore if a particular compound (e.g., glutamate antagomst) or experimental condition (e.g , oxidative stress) was suspected to somehow regulate the release of glutamate or its actions m conJunction with either the induction or prevention of neuronal cell death, then testing the ability of thts compound or condition to alter evoked glutamatergic synaptic currents would be a first step to unravelmg the molecular details of this actton. If alterations m these synapttc currents were observed, then it is hkely to be because of an alteration m either the release of glutamate or postsynaptic responsiveness. The simplest way to distinguish between a pre- vs postsynaptic site of action is to compare the amplitude and time course of responses to exogenously applied glutamate under the same experimental conditions; this would then be a test of postsynaptic responsiveness. 11. As mentioned in Subheading l., the utilization of the patch-clamp techniques described here provides direct access to the intracellular miheu through the patch electrode. As the mflux of Ca*+ through the glutamate receptor channels and the subsequent activation of Ca*+-dependent signal transduction cascades may be involved m neuronal cell death, these techniques will also provide a method to test if Ca2+-dependent enzymes (e.g , calcmeurin and PKC), that may be involved m neurotoxic events, regulate either the presynaptic release of glutamate or the function of the glutamate receptor channels. 12. For experiments to investigate the involvement of signal-transduction mechanisms, the perforated-patch method (8) may be preferable to the classical wholecell method for recording responses from receptor ion channels m the entire cell membrane. The perforated-patch techmque, by using pore-formmg anttbtotics, helps to prevent the loss or dilutton of larger molecules and proteins which may be important for signal-transduction cascades. For this purpose, a stock solutton of nystatin, a pore-forming antrbiotic, is made up using DMSO (50 mg/mL, sonicate approx 20 s) Thus IS then diluted into the intracellular solution consistmg of

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Jones and Yakel the mam ingredients but without Ca2+ or the Ca2+ buffer, as these will not permeate the nystatm pores. Approximately 2-3 J.~Lof this stock solutron diluted m 0.5 mL of intracellular solution gives a final concentration of nystatm of 0.25 mg/mL (and 0 5 % DMSO)

References 1. Alger, B. E., DhanJal, S. S , Dmgledine, R., Garthwatte, J , Henderson, G , King, G. L., Lipton, P., North, A., Schwartzkrom, P A , Sears, T A., Segal, M., Whrttingham, T. S., and Wrllrams, J. (1984) Brain shce methods, m Bruzn Slices (Dmgledme, R., ed ), Plenum, New York, pp. 381-437 2. Edwards, F A. and Konnerth, A. (1992) Patch-clampmg cells in shced tissue preparatrons. Methods Enzymol 207,208-222. 3 Blanton, M. G., Lo Turco, J J., and Krregstem, A. R (1989) Whole cell recording from neurons m shces of reptrban and mammahan cerebral cortex. J Neuroscl Methods 30,203-2 10. 4. Stuart, G. J., Dodt, H -U , and Sakmann, B. (1993) Patch-clamp recordings from the soma and dendrrtes of neurons m brain slices using infrared video mrcroscopy. Pflugers Arch. 423,5 1 I-5 18 5 Rae, J. L. and Levis, R A (1992) Glass technology for patch clamp electrodes Methods Enzymol 207,66-92 6. Gibb, A. J. and Edwards, F A (1994) Patch clamp recordings from cells m sliced tissues, m Mtcroelectrode Techniques The Plymouth Workshop Handbook (Ogden, D., ed.), The Company of Btologtsts Limited, Cambridge, UK, pp. 255-274 7. Edwards, F. A., Konnerth, A , Sakmann, B., and Takahasht, T (1989) A thm slice preparation for patch clamp recordings from neurones of the mammahan central nervous system. Pjltigers Arch. 414,600-612. 8 Horn, R. and Marty, A (1988) Muscarmtc acttvatron of ionic currents measured by a new whole-cell recordmg method. J. Gen. Physiol. 92, 145-159.

23 Selective Jeanene

Silver Staining for Neural Degeneration

K. Olin and Karl F. Jensen

1. Introduction Silver degeneration stains are typically considered the domain of studies of neural circuitry, but have also proven useful m studies of neurotoxicity. They are particularly well suited to localizing sites of injury, and determining the extent and time-course of degeneration. A variety of approaches have been used to selectively stain degenerating neurons and avoid the staining of intact tissue, including suppression of staining of normal fibers (1,2), the use of physical rather than chemical developers (3-7), and the mclusion of enhancers, such as copper (8-13). These methods provide exquisitely detailed images of neuronal morphology and a number of reviews discuss the relative advantages and disadvantages of the various methods and important considerations for their interpretation (8,9,1418). The mechanism by which particular tissue components become stained with silver is only partially understood and may vary with procedure. Consequently, most procedures have been developed empirically by trial and error in an attempt to optimize results for detection, resolution, contrast, and reproducibility. Different variations represent optimal procedures for specific conditions of fixation, embedding, and sectioning, as well as particular tissues and their cellular constituents. In addition, labeling degenerating neurons is dependent on a variety of experimental variables, such as fixation, water, reagent quality, and even relatively minor contaminants on glassware. Although a number of methods specify aldehyde fixation by vascular perfusion, there are also methods adapted for use with immersion-fixed tissue from human autopsy (19,20).

Although most procedures have been developed to minimize the occurrence of artifacts, it is still necessary to be aware that certain kinds of artifacts can be From

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Olin and Jensen dependent on species,age, and methodological variables, such as fixation. Such considerations are therefore particularly important when trying to assessthe differential vulnerability of sensitive subpopulations, such as the developing organism c&21-23). de Olmos and coworkers (8) have described a variety of artifacts, includmg deposits, that may appear as terminal degeneration and partial staining of normal fibers and myelm artifacts that may give an appearance similar to degenerating axons. The survival time for the optimal demonstration of neuronal mJury varies with the silver method employed, the insult, and the type of neurons inJured. In addition, not all the inJured neural elements will be stained at the same time. The time-course for the stammg followmg a particular insult must be emprritally determined. The temporal and spatial pattern of stammg can reflect differences in inherent vulnerability of the neurons, regional difference m the time-course of development of argyrophiha, as well as transneuronal degenerative effects. One of the drawbacks that silver degeneration stains share with other specialized stains is that the absence of staining can not be construed as the absence of damage. The converse is also true. The fact that a neuron is unambiguously stained may not necessarily indicate that the neuron is committed to die. Nonetheless, consistent dose- and time-dependent patterns of silver degeneration provide compellmg evidence of toxicant-induced injury. Silver degeneration stains have been used to demonstrate the patterns of injury induced by a variety of toxicants, including organometals, 6-hydroxydopamine, pyridme compounds, organophosphates, cocame amphetamine analogs, capsaicm, and excitotoxicants (9,15,20,23-50). The method we describe m this chapter is one of several versions of the cupric silver stain that was taught to us by Dr. Jose de Olmos. A detailed description of a more recent version of this method has been published (12). The results obtained when this method is applied to sections of brain from animals administered various toxicants illustrate some characteristic patterns of degeneration. In coronal sections from animals administered trimethyltm, evidence of perikaryal, axonal, and termmal degeneration can be seen as drstinct patterns in the cerebral cortex and hippocampus (Fig. 1). In sagittal sections from mice administered MPTP (Fig. 2) terminal degeneration of nigrostriatal proJections is evident, as is perikaryal degeneration m substantia nigra. The dose-dependence of the area1 distribution and intensity of axonal and terminal staining following administration of MDMA can also be visualized with this method (Fig. 3). Reliable quantification can be achieved by area1 measurements of regions showing intense stammg and can be used to demonstrate both the time-course and dose-dependence of the extent of degeneration (Fig. 4).

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2. Materials 2.7. Reagents Reagents

Source

Acetone DL-Amino-butyric acid DL-Alanine Ammomum hydroxide, concentrated Cacodyllc acid Cadmium nitrate Citric acid Cupric nitrate Ethanol, 200 proof Ethanol, 190 proof Formalm, 10% Glacial acetic acid, concentrated D-(+)-Glucose Hydrochloric acid, concentrated Isopropanol Kodak rapid fix with hardener Lactic acid Lanthanum nitrate Lithium hydroxide Neutral red Paraformaldehyde Potassium chlorate Potassium ferricyanide Potassium permanganate Pyrldine Silver nitrate Sodium chloride Sodium thlosulfate Sucrose Sulfuric acid, concentrated Triethanolamme Xylenes

2.2. Solutions

Burdick & Jackson Sigma Sigma Fisher Sigma Fluka Sigma Sigma McCormick Distilling McCormick Distilling Fisher Fisher Sigma Fisher EM Science Eastman Kodak Sigma EM Science Sigma Aldrich Fisher Fluka Sigma Sigma Sigma Accurate Fisher Sigma Fisher Fisher Sigma Fisher

Caution* I

T H, T, PC, PM H, I, PC I I

I, I’, C, M Cor I PM I I I I H I H D P, T I, T I I I Cor I I

(see Note 1)

1. Stock solutions: 0.5% cadmium nitrate, 1% citric acid, 0.5% cupric nitrate, 10% formalin, 0.5% lanthanum nitrate, 0 4% lithium hydroxide, 2% sodium thlosulfate. These solutions are stable at room temperature up to 2 mo. “Cautions C, carcinogen, Cor, corrosive. D, destructive to tissue, H, harmful, I, lrntant, M, mutagen, P, poison, PC, possible carcinogen, PM, possible mutagen, T, toxic

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Fig. 1. Silver degeneration staining following administration of trimethyl tin. (A) Coronal section from the brain of a rat administered 8 mg/kg trimethyl tin. (B) Coronal section from the brain of a rat administered saline. Note: The section from the control rat was outlined to indicate its perimeter. Scale bar = 1 mm. 2. Saline: 1000 mL dH,O, 9 g sodium chloride, 8 g sucrose, 4 g glucose. 3. Fixative (4% paraformaldehyde in 0.1 M cacodylate buffer): a. 0.2 M Cacodylate buffer: 1000 mL dH,O, 42 g cacodylic acid, adjust pH to 7.3. b. 8% Paraformaldehyde: Heat 1000 mL dH,O to 6O”C, add 80 g paraformaldehyde, stir 15-20 min, then add 10 M NaOH dropwise until clear; let cool. c. 4% Paraformaldehyde fixative: add 0.2 M cacodylate buffer to 8% paraformaldehyde, filter, adjust pH to 7.3. 4. Cupric silver preimpregnation solution (see Notes 2 and 3): 300 mL dH,O, 300 mg AgN03, 150 mg aminobutyric acid, 150 mg DL-alanine, 6 mL 0.5% cupric nitrate, 0.6 mL 0.5% cadmium nitrate, 4.5 mL 0.5% lanthanum nitrate, 4.5 mL 0.5% neutral red, 4 mL pyridine, 3 mL isopropanol, and 3 mL triethanolamine. Add reagents in the order listed. Dissolve each reagent before adding the next. Prepare solution at least 12 h prior to use. Keep this solution in the dark. Filter solution through glass wool immediately before use.

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Fig. 2. Silver degeneration staining following administration of MPTP. (A) Saggital section from the brain a mouse administered saline. (B) Saggital section from the brain a mouse administered MPTP (48 survival, 40 mg/kg MPTP). Scale bar = 1 mm. Note: The section from the control rat was outlined to indicate its perimeter.

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Fig. 3. Silver degeneration staining following administration of MDMA. Photomicrographs from sections of the forebrain of rats sacrificed 48 h after a total of four doses of MDMA administered over 2 d. (A-D) High magnification of upper layers of neocortex. Scale = 100 pm. (E-H) Lower magnification of frontoparietal cortex. Scale bar = 1 mm. (A, E) saline, (B, F) 25 mg/kg MDMA, (C, G) 50 mg/kg MDMA, (D, H) 150 mg/kg MDMA. From ref. 24. 5. Diamine silver impregnation solution (see Note 4): 50 mL dH,O, 4.12 g AgNOs, 40 mL 100% ethanol, 500 yL acetone, 6.5 mL ammonium hydroxide (cont.), and 30 mL 0.4% lithium hydroxide. Make immediately before use, adding chemicals in the order listed. 6. Reducing solution: 800 mL dH,O, 11 mL 10% formalin, 6.5 mL 1% citric acid, and 90 mL 100% EtOH. It is critical that this solution be used at 32°C. 7. Bleaching solutions: a. First bleach: 10 mL 4% potassium chlorate, 600 mg potassium ferricyanide, 200 pL pure lactic acid. b. Second bleach: 30 mL 0.6% potassium permanganate, 1 mL 5% sulfuric acid. 8. Stock rapid fix: 125 mL dH20, 31 mL rapid fix solution. A, 3.5 mL rapid fix solution; B, 90.5 mL dH,O.

3. Methods 3.1. Cardiac Perfusion 1. Flush with saline 1 min, then perfuse for 30 min or with 1 L of fixative. Remove brain and postfix overnight. 2. Sink brains in 20% sucrose in 0.1 M cacodylate buffer at 4°C.

Neurodegeneration

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Dow-Dependence of MDMA Induced Degeneration In Neoeortex and Strlatum 11 to 0 8 7

0

0

28

80

75

loo

160

Dose(make) Mwka)

Dose

B

Time Course Course of MDMA Induced

Degeneration in Neocotiex and Striatum II cc ”” II

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t

[IiiE 1.

.,

18hm(2~)

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Fig. 4. Quantification of silver degeneration staming followmg admmistratton of MDMA The volume of tissue in which silver degeneration staining was present was determined by vrsually outlinmg staining areas from homologous sections from a 0.72 mm thick sagtttal block. (A) Dose response. Animals received each indicated dose of MDMA twice a day for 2 d and were sacrificed 48 h after the last dose. Vertical bars indicate standard errors. (B) Time-course. Animals received 4 x 100 mg/kg MDMA and were sacrificed 18 h later, with the exception of the group indicated with 2x, which only received 2 x 100 mg/kg From ref. 24.

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3.2. Sectioning 1. Cut 40-y frozen sections into 24-well plates containing fixative. 2. Store sections m refrigerator for at least 48 h prior to prelmpregnatlon (see Note 5).

3.3. Preimpregna

tion

1. Prepare prelmpregnatlon solution as described above (see Notes 6 and 7). 2 Rinse sections briefly m two changes of dH20 3 Preimpregnate sections m mlcrowave to 45°C (see Note 8). Remove from oven; temperature should continue rising to 50°C 4 Let stand at least 1 h. Sectlons can be left overnight at this point (see Notes 9 and 10)

3.4. impregnation 1 After cooling for 1 h, transfer the sectlons to crucibles wrth the aid of a glass hook and rinse briefly in dH,O (see Note 11) 2 Rinse m two changes of acetone, 15 s each. 3. The sections should then be rinsed m the impregnation solution to avoid acetone carryover. Transfer sections to the impregnation solution for 1 h on a shaker.

3.5. Reduction 1. While sections are incubating m the impregnation solution, place 300 mL preparation dishes m a 32°C water bath Add 60 mL reducer to each Jar and heat to 32°C. 2. Transfer sections without washmg directly to reducer solution for 25 min 3. Add 150 FL impregnation solution to each 60 mL dish of reducer after the sections have been transferred. (Sections should appear a dark reddish brown.) 4 Transfer sections rapidly to dH,O for 1 mm m order to stop the reduction. 5. Place sections m 0.5% glacial acetic acid for 1 mm 6 Rinse m dH20 for at least 1 mm prior to bleachmg. Sections may be stored In dH,O overnight at this point

3.6. Bleaching

and Stabilization

1 Transfer to first bleach for 30 s to 1 mm (see Note 12) stop bleachmg by rinsing m dH20. 2. Transfer to the second bleach for 10 s, then to dH20 briefly (see Note 13). 3. Place sections m 2% sodium thlosulfate for 3-5 mm (until transparent). 4. Rinse m dH20, then transfer to Kodak rapld fix working solution (dilute stock solution 1:6 with dH,O) for l-5 mm. 5 Rinse in two changes of dHzO 6 Sections may now be mounted, cleared (see Note 14), and covershpped

4. Notes 1 All water must be extremely clean and free of alummum. All glassware should be cleaned in nitric acid and rinsed with dHzO (double dlstllled). Any contaminants

Neurodegeneration

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6

7.

8. 9 10. 11 12. 13. 14

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Sliver Staming

may lead to artifactual deposits, patchy staimng, or failure to impregnate degenerating fibers. Pyridme should be sulfur free. It aids in penetration mto the section. Too much pyrtdme will increase nuclear staining. Neutral red speeds the reaction The source of the neutral red does make a difference. If the impregnation solution turns black, tt has been contaminated with acid. Sections may be kept m fixative under refrigeration for several months This postfixation suppresses normal fiber staining With extended post-fixation, impregnatton of degenerating fibers might be reduced. The age of the animal and the thickness of the section affect the final result to varying degrees. In general, brain sections from younger animals will show more nuclear stammg. This can be reduced by decreasing the amount of pyridine m the preimpregnation step. The solutions presented here have been fine-tuned for adult rat tissue Solutions and times for young rat or mouse tissue are the same with one exception. Reduce the volume of pyridme m the preimpregnatton solutton to 1 mL Other species may require some adJustments Prermpregnation is carried out m 60 mL specimen Jars. Sections viewed under a stereoscope after the preimpregnatron step should appear red. A green color indicates that the preimpregnation was too long. After preimpregnation, sections are transferred throughout the procedure m porcelain crucibles A plastic rack cut to hold the crucibles aids m transferring several at one time. Two-quart Pyrex casserole dishes are used for solutions and rinses, except where noted A stereoscope can aid in the first bleaching. The second bleach can eliminate edge artifact to some degree as well as patchy nonspecific staining. Use alcohols to clear sections before covershppmg.

Acknowledgment This chapter has been reviewed by the National Health and Environmental Effects Research Laboratory

of the US Environmental

Protection

Agency, but

the views expressed may not necessarrly reflect agency policy and mention of trade names or commercral products does not constitute endorsement or recommendatron for use. We would like to thank Dr. de Olmos for teaching us this version of his cupric silver staining method. References 1. Nauta, W J. H. and Gygax, P. A. (1954) Silver impregnation of degenerating axons in the central nervous system. a modified technique. Stam Technol. 29,91-93. 2. Fink, R. P. and Hemmer, L. (1967) Two methods for selective silver impregnation of degenerating axons and their synaptic endings m the central nervous system Bram Res. 4, 369-374.

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3 Gallyas, F. (1980) Chemical nature of the first products (nuclei) of the argyrophil staining. Acta Histochem. 67, 145-l 5 8. 4. Gallyas, F., Zaborszky, L., and Wolff, J R. (1980) Experimental studies of mechanisms involved m methods demonstrating axonal and terminal degeneration. Stazn Technol 55,28 I-290.

5. Gallyas, F. (1971) A prmctple for stlver stammg of &sue elements by physical development. Acta Morphol. Acad. Set. Hung. 19,57-7 1, 6. Gallyas, F. (1982) Suppression of the argyrophil III reaction by mercapto compounds (a prerequisite for the mtensificatton of certain histochemtcal reactions by physical developers). Actu Histochem. 70,99-105. 7. Gallyas, F. (1982) Physico-chemical mechanism of the argyrophil III reaction. Histochemistry

74,409-421

8. de Olmos, J. S., Ebbesson, S. 0. E., and Heimer, L. (1991) Silver methods for the tmpregnatton of degeneratmg axoplasm, in Neuroanatomtcal Tract-Tracang Methods (Heimer, L. and RoBards, M. J., eds.), Plenum, New York, pp 117-170 9. Beltramino, C A , de Olmos, J. S , Gallyas, F., Hemmer, L., and Zaborszky, L (1991) Silver stammg as a tool for neurotoxlc assessment. NZDA Res Monogr. 136,101-132. 10. de Olmos, J. S. (1969) A cupric-silver method for impregnation of terminal axon degeneration and its further use in staining granular argyrophilic neurons. Brazn Behav. Evol. 2,21 O-237.

11. de Olmos, J. S. and Ingram, W. R. (1971) An improved cupric-silver method for impregnation of axonal and terminal degeneration. Brain Res. 33,523-529. 12 de Olmos, J. S., Beltrammo, C. A., and de Olmos de Lorenzo, S. (1994) Use of an ammo-cupric-stlver technique for the detection of early and semiacute neuronal degeneration caused by neurotoxicants, hypoxia, and physical trauma. Neurotoxicol Teratol. 16,545-561

13 Carlsen, J. and de Olmos, J. S. (1981) A modified cupric-silver technique for the tmpregnatton of degenerating neurons and their processes Brutn Res. 208, 426-43 1.

14. Heimer, L (1967) Silver impregnation of termmal degeneration m some forebrain fibers systems: a comparative evaluation of current methods. Brutn Res 5, 86-108. 15 Balaban, C. D (1992) The use of selective silver degeneration stains in neurotoxicology lessons from studies of selective neurotoxicants, m The Vulnerable Bratn and Environmental

Risks, vol I Malnutrttion

and HazardAssessment

(Isaacson, R. L. and Jensen, K. F., eds.), Plenum, New York, pp. 223-238. 16. Nauta, W. J H and Ebbesson, S 0. E. (1970) Contemporary Research Methods in Neuroanatomy Springer-Verlag, New York 17. Jensen, K. F. (1995) Neuroanatomical techniques for labeling neurons and then utility m neurotoxicology, m Neurotoxtcology Approaches and Methods (Chang, L. W. and Slikker, W., eds.), Academic, New York, pp. 27-66. 18. Voogd, J. and Feu-adbend, H. (1981) Clusstcal Methods in Neuroanatomy Volume 2, Methods m NeurobzoEogy (Lahue, R., ed.), Plenum, New York, pp 301-364.

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19. Grafe, M. R and Leonard, C. M. (1982) Developmental changes in the topographical distribution of cells contributing to the lateral olfactory tract. Brazn Res. 255, 387-400 20. Harvey,

J. A. and McMaster, S. E. (1976) Neurotoxrc action of parachloroamphetamme in the rat as revealed by Nissl and silver stams. Psychopharmacol. Bull 12,62-64

21 Leonard, C. M. (1975) Developmental changes m olfactory bulb projections revealed by degeneration argyrophrha. J. Comp Neural. 162,467-486. 22. Janssen, R., Schweitzer, L., and Jensen, IS. F. (1991) Glutamate neurotoxrcrty in the developing rat cochlea: physiological and morphological approaches Brain Res. 552,255-264.

23. Schweitzer, L., Jensen, K. F , and Janssen, R. (1991) Glutamate neurotoxicrty u-r rat auditory system: cochlear nuclear complex. Neurotoxicol Teratol 13,189-193. 24. Jensen, K. F., Olin, J., Haykal-Coates, N., O’Callaghan, J., Miller, D. B , and de Olmos, J. S (1993) Mapping toxicant-induced nervous system damage with a cupric silver stain: a quantitative analysis of neural degeneration induced by 3,4-methylene-dtoxymethamphetamme. NINA Res. Monogr. 136, 133-149. 25. O’Callaghan, J. P and Jensen, K F. (1992) Enhanced expression of glial fibrillary acrdic protem and the cupric silver degeneration reaction can be used as sensitive and early indicators of neurotoxicity. Neurotoxicology 13, 113-l 22. 26 Tanaka, D., Jr. (1989) Degeneration patterns m the chicken central nervous system induced by ingestion of the organophosphorus delayed neurotoxin triorthotolyl phosphate A silver impregnation study. Bram Res. 484,240-256 27. Tanaka, D., Jr., Bursran, S. J., and Lehning, E. J. (1992) Silver impregnation of organophosphorous-induced delayed neuropathy m the central nervous system, in The Vulnerable Brain Volume 2. Toxins in Food (Isaacson, R. L. and Jensen, K. F , eds ), Plenum, New York, pp 215-234. 28 Maler, L , Fibiger, H C., and McGeer, P. L. (1973) Demonstration of the nigrostrratal projection by silver staining after nigral injection of 6-hydroxydopamine. Exp. Neural. 40,505-5 15. 29. Desclin, J. C. and Escubi, J. (1974) Effects of 3-acetylpyridme on the central nervous system of the rat, as demonstrated by silver methods. Bram Res. 77,349-364. 30. Balaban, C. D., O’Callaghan, J. P., and Billingsley, M. L. (1988) Trrmethyltininduced neuronal damage in the rat brain: comparative studies using silver degeneration stains, immunocytochemistry and immunoassay for neuronotypic and gliotypic proteins Neurosci 26,337-361 31. Varghese, R G , Bursian, S J , Tobias, C , and Tanaka, D., Jr. (1995) Organophosphorus-induced delayed neurotoxrcity: a comparative study of the effects of triorthotolyl phosphate and triphenyl phosphite on the central nervous system of the Japanese quail. Neurotoxlcology 16,45-54. 32 Varghese, R G., Bursian, S. J., Tobias, C., and Tanaka, D., Jr (1995) Triphenyl phosphate-induced neuropathy m the avian forebrain: a silver impregnation study of the visual and auditory systems of the Japanese quail. Neurotoxlcology 16, 105-l 13

Olin and Jensen 33 Tanaka, D., Jr and Burstan, S J (1989) Degeneratton patterns m the chicken central nervous system Induced by ingestion of the organophosphorus delayed neurotoxin trrorthotolyl phosphate. A silver impregnation study Bruzn Res. 484,240-256 34 Scallet, A. C., Bmtenda, Z., Caputo, F A, Hall, S , Paule, M G., Rountree, R. L , Schmued, L., Sobotka, T , and Shkker, W , Jr. (1993) Domoic acid-treated cynomolgus monkeys (M. fascicularts) effects of dose on htppocampal neuronal and terminal degeneration. Brain Res 627,307-3 13. 35. Tanaka, D., Jr., Burslan, S. J., and Lehnmg, E J. (1992) Neuropathologtcal effects of triphenyl phosphite on the central nervous system of the hen (Gallus domesttcus) Fundam. Appl Toxzcol l&72-78 36. Tanaka, D., Jr., Burstan, S. J., and Lehnmg, E. (1990) Selective axonal and terminal degeneration m the chicken bramstem and cerebellum followmg exposure to bls(l-methylethyl)phosphorofluoridate (DFP). Brucn Res. 519,200-208. 37. Rlcaurte, G. A, Gmllery, R. W., Setden, L S., Schuster, C. R , and Moore, R. Y (1982) Dopamme nerve termmal degeneratron produced by high doses of methylamphetamme in the rat brain. Brazn Res 235,93-103 38. Ricaurte, G A , Gurllery, R. W , Seiden, L S., and Schuster, C R. (1984) Nerve terminal degeneration after a single inJection of D-amphetamine m iprmdoletreated rats: relatron to selective long-lastmg dopamme depletion. Brain Res. 291, 378-382 39 Tanaka, D , Jr., Bursian, S. J., and Aulerich, R. J. (1994) Age-related effects of trtphenyl phosphate-induced delayed neuropathy on central visual pathways m the European ferret (Mustela putorius furo). Fundum. Appl. Toxzcol. 22,577-587. 40 Rlcaurte, G. A., Forno, L S., Wilson, M. A , DeLanney, L. E., Irwin, I., Molhver, M E., and Langston, J. W. (1988) (+/-)3,4-Methylenedtoxymethamphetamine selecttvely damages central serotonergic neurons m nonhuman primates. JAMA 260,5 l-55. 41. Scallet, A C., Lope, G W., Ah, S F., Holson, R. R , Froth, C. H., and Shkker, W., Jr. (1988) Neuropathologtcal evaluation by combined tmmunohtstochemistry and degeneration-specific methods: application to methylenedioxymethamphetamme. Neurotoxzcology 9,529-538. 42. Shkker, W., Jr , Ah, S. F., Scallet, A C., Froth, C H., Newport, G. D., and Bailey, J R. (1988) Neurochemical and neurohrstologrcal alterations m the rat and mokey produced by orally admmistered methylenedtoxymethamphetamme (MDMA). Toxicol. Appl. Pharmacol. 94,448-457 43 Ricaurte, G. A., Bryan, G , Strauss, L , Seiden, L S , and Schuster, C R (1985) Hallucmogemc amphetamine selecttvely destroys brain serotomn nerve terminals. Sczence 229,986-988. 44. Tanaka, D., Jr., Burnan, S. J., Lehnmg, E J., and Aulerich, R J. (1991) Delayed neurotoxic effects of bts (1 -methylethyl) phosphorofluoridate (DFP) in the European ferret a possible mammalian model for organophosphorus-induced delayed neurotoxrcity Neurotoxzcology 12,209-224 45 Ritter, S. and Dinh, T T. (1988) Capsalcin-induced neuronal degeneration: silver impregnatton of cell bodies, axons, and terminals m the central nervous system of the adult rat J. Comp. Neurol 271,79-90.

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46. Inui, K , Mitsumori, K , Harada, T., and Maita, K (1994) Quantitative analysis of neuronal damage induced by tri-ortho-cresyl phosphate m wistar rats. Fundam Appl Toxicol. 20,111-l 19 47 Balaban, C D (1985) Central neurotoxic effects of mtraperitoneally admmistered 3-acetylpyridme. harmaline and macmamide in Sprague-Dawley and LongEvans rats. a critical review of central 3-acetylpyridme nemotoxicity Brain Res. Rev. 9,21-42. 48 Turner, B H , Wilson, J. S , McKenzie, J. C., and Richtand, N (1988) MPTP produces a pattern of mgrostriatal degeneration which comcides with the mosaic organization of the caudate nucleus Brain Res 473, 60-64 49 Fix, A S., Ross, J. F., Stitzel, S. R., and Switzer, R C (1996) Integrated evaluation of central nervous system lesions: stams for neurons, astrocytes, and microgha reveal the spatial and temporal features of MK-80 1-induced neuronal necrosis m the rat cerebral cortex Toxlcol Path01 24, 291-304 50. Glees, P. and Jansik, H. (1965) Chemically (TCP) Induced fiber degeneration in the central nervous system, with reference to clinical and neuropharmacological aspects Prog. Bram Res 14, 97-121.

lmmunohistological of Cerebrovascular

Markers Integrity

Karl F. Jensen, Jeanene and Robert L. lsaacson

K. Olin, Julie A. Varner,

1. Introduction The bram depends on other organ systemsof the body for oxygen, nutrients, and the elimination of metabolic byproducts. The primary route for such transfer of these essentials is the cerebrovasculature. The cerebrovasculature also participates in metabolizing or excluding xenobiotics, segregating components of the immune response, regulating pH and osmolarity of the cerebrospinal fluid, selectively distrrbutmg hormones, and impeding pathogenic invasion. Various aspects of these diverse functions are attributed to the complex structural and molecular properties of cerebral endothelial cells collectively referred to as the blood-brain barrier (I-7). The hallmark structural specialization of the blood-brain barrier is the tight junction between the endothelial cells, which prevents diffusion of plasma proteins and molecules of a similar size or larger (8-12). Other structural specializations include close apposition of astrocytic endfeet, sparsely drstributed pericytes, and extensive association with mrcroglia. Molecular specializations include endothelial expression of transporters and enzymes, such as those involved in xenobiotic metabolism (12-27). The vasculature of certain brain regions lack some of these specializations, presumably because of then function in chemoreception or neurohumoral regulation. These regions are referred to as the circumventricular organs and include area postremta, lamma terminalis, median eminence, neurohypophypsrs, the subfornical organ, and the choroid plexus. The vasculature of several other regions of nervous system also exhibit greater permeability, most notably the arcuate nucleus of the hypothalamus and the dorsal root ganglia (1,4,28-30). From

Methods m Molecular Medune, vol 22 Neurodegenerahon Methods and Protocols EcMed by J Harry and H A Tdson 0 Humana Press Inc , Totowa, NJ

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There are regional variattons in density and complexity of the cerebrovasculature. Capillary density ranges from 2000 mm of capillaries per cubic millimeter of tissue in regions of the hypothalamus to cl50 mm of capillaries per cubic millimeter of tissue in regions of the spinal cord (31). Synaptic density m the gray matter parallels local differences m the vascular density. This relationship may be a requisite for the link among blood flow, metabolism, and regional neural activity. Although mechamsms of such neurovascular coupling remain controversial, blood flow and vascular permeability can be modified by the cholinergic, adrenergic, and serotonergic innervation of blood vessels as well as autoregulatory pathways within the endothelium (21,22,32-37). The cerebrovasculature 1s a site of injury in a number of neurological diseases and its mvolvement in toxicant-induced injury to the nervous system derives from alterations m blood flow, damage to the blood-brain barrier, and its role m xenobiotic metabohsm (28,38-57). Because of the diverse aspects of the cerebrovasculature that may be altered as a result of toxicant exposure, morphological assessmentof its integrity is a complex challenge. Our current approach to evaluatmg the cerebrovasculature is to use lectin labelmg of microgha, antisera that recogmze endothehal cell antigens, and antisera that recognize antigens that are indirect mdicators of the disruption of vascular integrity. 1.1. Lectin Labeling of Microglia Rapid hypertrophy of microglia cells follows physical disruption of the blood-brain barrier as well as toxicant-induced neurodegeneration (S-60). We use the Isolectin B, from Griffoma, which has been demonstrated to be a reliable way of labeling microglia (61-64). This method can demonstrate the dramatic hypertrophy of the microglia 48 h after a knife cut to the neocortex (Fig. 1). Microgha activation can be widespread following such an injury, as seen the hippocampus contralateral to the knife cut (Fig. 2). Lectin stammg can also detect rapid changes m neurotoxicant-induced alteration m the bloodbrain barrier, such as the response observed 24 h after systemic administration of AlCls (Fig. 3). 1.2. Endotheliai Cell Antigens Immunohistochemical labeling of P-glycoprotem, a product of the multidrug resistance genes that is expressed by endothehal cells of the cerebrovasculature, can be altered by acute treatments known to disrupt the blood-brain barrier (20) As an additional confirmation of alterations observed m P-glycoprotem labeling, we employ a commercially available antisera, EBA, that recognizes a blood-brain barrier-related antigen (65,66). Alterations m the staining pattern with these two antisera could be the result of a number of types of alterations ranging from an overt tissue disruption and subsequent remodeling of the vas-

Cerebrovascular

Integrity

Fig. 1. Lectin labeling of microglia in the neocortex. (A) Neocortex of a control rat. (B) Neocortex from a rat sacrificed 48 h after a neocortical knife cut. Scale bar = 5 urn. culature to more subtle changes, such as degradation of the antigen or postranslational alterations influencing epitope availability. We compare the labeling patterns of these two antisera to one that recognizes laminin, a robust marker providing a clear and comprehensive image of cerebral vessels (66,67). A dramatic reduction in the staining with the EBA antibody is indicative of an alteration in the blood-brain barrier 48 h after a neocortical knife cut (Fig. 4). The structural presence of the vasculature is demonstrated in an adjacent section with immunostaining for laminin. A similar alteration is observed in the pattern of immunostaining for P-glycoprotein (Fig. 5).

1.3. Antigens

Indicative

of the Disruption

of Vascular Integrity

Serum proteins, such as IgM and other antibodies, are typically excluded from neuronal tissue. The presence of IgM in brain parenchyma can be regarded as an indicator of the disruption of cerebrovascular barrier function. We use an antisera recognizing rat IgM as indicator of the leakage of such plasma proteins (68-70). There is a virtual absence of immunostaining for IgM in the parenchyma of the brain of normal animals. Following neocortical knife cut, however, IgM accumulation within the parenchyma as well as the vasculature can be detected (Fig. 6). Since the acute alterations in barrier function may not be detectable under conditions of long-term survival, in chronic studies we use antisera recognizing amyloid A and beta-amyloid to detect accumulations associated with the cerebrovasculature. Vascular tissue produces P-amyloid, and the vascular

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Fig. 2. Lectin labeling of microglia in the neocortex and hippocampus. (A) Low power photomicrograph of lectin labeling in neocortex and hippocampus in an animal sacrificed 48 h after a knife cut to the contralateral neocortex. Scale bar = 500 pm. (B) Higher power photomicrograph of hippocampus. Scale bar = 5 urn. deposition of amyloid may result from alterations that frequently occur with aging in the basement membrane of blood vessels. Alternatively, amyloid deposition may be a consequence of the development of blood vessel abnormalities. Hypotheses have been put forth proposing amyloid as a causative factor in neurodegeneration; others suggest its accumulation is a secondary consequence of injury. Certain toxicants, such as aluminum, can influence the distribution amyloid (55,71-83). We have been able to detect immunostaining of both amyloid A and P-amyloid associated with the cerebrovasculature in animals chronically administered 5 ppm AlFs in their drinking water (Fig. 7).

Fig. 3. Lectin labeling of microglia following systemic administration of AlCl,. (A) Low power photomicrograph of the hippocampus from a rat sacrificed 48 h following administration of 100 mg/kg AlCI,. Scale bar = 500 pm. (B) High power photomicrograph of hippocampus. Scale bar = 5 pm.

2. Materials (see Table 1) 2.1. Antisera See Table 2 for a list of antisera.

2.2. Stock Solutions 1. 50% ethanol. 70% ethanol. 0.5% cobalt chloride. 1% nickel ammonium sulfate. Acid water (4% glacial acetic acid). Xylene.

2. 3. 4. 5. 6.

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Fig. 4. Immunostaining for laminin and the EBA antigen following neocortical knife cut. (A,B) Coronal sections of brain from a control animal. (C,D) Coronal sections of brain from an animal sacrificed 48 h following a knife cut to the contralateral neocortex. (AC) Immunostaining for laminin. (B,D) Immunostaining for EBA. Scale bar = 100 pm.

2.3. Buffers 1. 0.05 M Tris-buffered saline (TBS): 800 mL dH20, 6.61 g Trizma hydrochloride, 0.91 g Trizma base, 8.5 g sodium chloride. Bring volume to 1 L with dH,O. Adjust pH to 7.4. 2. 0.1 M Phosphatebuffer (PB): 800 mL dH,O, 28.4 g dibasicsodiumphosphate,6.9 g monobasicsodiumphosphate.Bring volume to 2 L with dH,O. Adjust pH to 7.4.

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Fig. 5. Immunostaining for P-glycoprotein following neocortical knife cut. Immunostaining with a monoclonal antibody (clone JSB-1) for P-glycoprotein of a coronal section from the brain of a rat sacrificed 48 h after a knife cut to the contralateral neocortex. Note the correspondence in this pattern of staining to the one for immunostaining for EBA in Fig. 4D. Scale bar = 100 pm.

3. TBS with cations: 1 L 0.05 M TBS (see above), 0.11 g calcium chloride, 0.125 g manganese chloride, 0.095 g magnesium chloride, 1 mL Triton X-100 (see Notes 1 and 2).

3. Methods

3.1. Depara ffiniza tion, Hydration and Dehydration 3.1.7. Deparaffinization of Sections (see Note 3) 1. 2. 3. 4. 5. 6.

3 changes of xylene, 10 min each change. 3 changes of 100% ethanol, 3 min each change. 3 changes of 95% ethanol, 3 min each change. 70% ethanol, 3 min. dH20, 5 min. Buffer, 20 min (unless noted).

3.1.2. Dehydration of Sections Prior to Coverslipping 1. 2. 3. 4.

70% ethanol, 2 changes of 3 changes of 4 changes of

3 min. 95% ethanol, 3 min each change. 100% ethanol, 3 min each change. xylene, 5 min each change.

of Sections

Fig. 6. Immunostaining for IgM following neocortical knife cut. Immunostaining with a polyclonal antibody recognizing rat IgM of a coronal section from the brain of rat sacrificed 48 h after a knife cut to the contralateral neocortex. (A) Low power photomicrograph of thalamic region. Scale bar = 500 pm. (B) High power photomicrograph of thalamic region illustrating both the intravascular and intraparenchymal labeling of IgM. Scale bar = 5 pm.

3.1.3. Hydration of Sections 1. 2. 3. 4.

3 changes of 3 changes of 75% ethanol, 2 changes of

100% ethanol, 10 dips each change. 95% ethanol, 10 dips each change. 10 dips. dH,O, 10 dips each change.

3.2. Staining Procedure

for Paraffin

Sections (see Note 4)

1. Deparaffinize sections. 2. Blot area around sections and carefully circle sections with a PAP pen hydrophobic slide marker. Do not touch edges of the section with the marker (see Note 5).

278

Cerebrovascuiar

Integrity

Fig. 7. Immunostaining for amyloid in the brains of animals chronically administered AlF,. (A) Immunostaining for amyloid A in a coronal section of thalamus from animal chronically administered 5 ppm AlF, in their drinking water. (B) Adjacent section immunostained for P-amyloid, displaying significant immunoreactivity associated with blood vessels. Note the different patterns of distribution of the two amyloids. Scale bar = 100 pm. 3. Block with 10% normal serum in buffer for 30 min. 4. Blot serum from sections. 5. Cover sections with primary antisera diluted in buffer with 1% normal serum (see Notes 6 and 7). Incubate overnight at room temperature. 6. Rinse in three changes of buffer for 5 min each. 7. Incubate sections with biotinylated secondary antibody (see Notes 8 and 9) diluted in buffer (1:200) for 1 h. 8. Prepare the ABC reagent approx 45 min after starting the incubation with the secondary antibody. Mix and let stand for at least 30 min prior to use. 9. Rinse in three changes of buffer for 5 min each.

Jensen et al.

280 Table 1 List of Materials Reagents 3,3’-DJaminobenzJdJne tetrahydrochlonde Bluing reagent Calcium chloride Cobalt chloride Eosln-Y, alcoholJc Ethanol, 200 proof Ethanol, 190 proof Gill-3 Hematoxylln Glacial acetic acid, concentrated Hydrochloric acid, concentrated Hydrogen peroxide, 30% Manganese (II) chloride, anhydrous Magnesium chloride, anhydrous Nickel ammonium sulfate Normal goat serum Normal horse serum Normal rabbit serum PAP pen hydrophobic slide marker Permount Sodium chlorJde Sodium phosphate, dJbasJc, anhydrous Sodium phosphate, monobasic, anhydrous Tr1ton X-100 Trlzma base Trizma hydrochloride Vectastam ABC KJt, Elite Goat IgG Vectastain ABC Kit, Elite Mouse IgG VectastaJn ABC Kit, Rabbit IgG Xylenes

Source PolyscJences Shandon FJsher Fisher Shandon McCormJck Distilling McCormJck DistJllJng Shandon Fisher Fisher Sigma Fluka Sigma FJsher Vector Vet tor Vector Res. Products InternatJonal Fisher Fisher Fisher Fisher Sigma SJgma Sigma Vector Vector Vector FJsher

CautJon@ C I I SC-PT I

I Cor I I I C

aCautlons C - carcmogen, Cor - corrosive, I - Irritant, PT - possible teratogen, SC - suspected carcmogen 10 11 12. 13

Incubate sections with ABC reagent for 1 h. Prepare 0 03% hydrogen peroxide from 30% stock solution Rinse Jn three changes of buffer for 5 m1n each Prepare the DAB substrate as follows* a Inject 10 mL of dH20 Jnto vial with 10 mg 3,3’-dlam1nobenzadJne ride and shake to m1x b Inject 10 mL buffer (0 1 M TBS or 0.2 M PB) and m1x c Add 0.5 mL of 0 5% cobalt chloride and mix

tetrachlo-

Table 2 Antisera Antisera Anti-Isolectm I Gnffoma (Banden-aea) polyclonal Anti-EBA monoclonal, SMI 17 1 Anti-Amyloid A monoclonal, clone MC 1 Anti-P-Amylotd monoclonal, clone 6F/3D Anti-Lammm Anti-IgM polyclonal Gnffonia (Bandeuaea) Slmplmrfolra Isolectm B, Griffoma (Bandenaea) Slmplicrfolia Isolectin B,, peroxidase labeled Anti-P-glycoprotem (pepttde) monoclonal, clone JSB-1 Anti-P-glycoprotem (peptide) monoclonal, clone C2 19

Source

Buffer

Host

Normal sera

Dllutton

Vector

TBS

Goat

Rabbit

1 500

Stemberger

TBS

Mouse

Horse

1-1000

Dako

TBS

Mouse

Horse

1:lOO

Dako

TBS

Mouse

Horse

Grbco Jackson

TBS PB

Mouse Rabbit

Vector

TBS

Goat

Sigma

TBS

Signet

TBS

Mouse

Horse

Signet

TBS

Mouse

Horse

Pretreatment

Procedure variations

Buffer with cations

3.3.1

1:lOO

Fomuc acid

3.3.3

Horse Goat

1:lOOO 1:lOOO

Pepsm m HCl’

3 3.4

Rabbit

1:lSOO 1:20

3.3.1 3.3.2

1:40

Buffer with cations Pepsin m HCl

1.80

Pepsin m HCl

3 3.4

3.3.4

Jensen et al.

282

14. 15 16 17 18.

d Add 0.4 mL of 1% nickel ammonium sulfate and mrx e Add 0.6 mL of 0.03% hydrogen peroxide and mix. React sections with DAB solution for 10 mm. Rinse in three changes of buffer for 5 mm each Rinse in dHz0 for 5 mm. Dehydrate sections Coverslip using permount.

3.3. Procedure Variations 3.3.1. lsolectin BdAntilectin 1. Deparaffimze sections. 2. Immerse slides m 0.05 M TBS buffer containing cations for 30 mm 3. Blot area around sections and carefully circle sections with a PAP pen hydrophobrc slide marker Do not touch edges of the section with the marker 4 Incubate with Isolectin B4 with 0.1% Triton X-100 for 48 h at 4°C 5 Rinse m three changes of buffer for 5 mm each. 6 Block in 10% normal serum for 30 mm 7 Incubate with antrlectin diluted m buffer with 1% normal serum and 0 1% Truon X-100 for 2 h. 8. Proceed as m steps 6-18 m Subheading 3.2.

3.3.2. Peroxidase-Labeled

Lectin

1. Deparaffmrze sections. 2. Immerse slides m 0.05 M TBS buffer contammg cations for 30 mm 3. Blot area around sections and carefully circle sections wtth a PAP pen hydrophobic slide marker. Do not touch edges of the section with the marker 4. Incubate with peroxrdase-labeled lectm with 0 1% Trrton X-100 overnight at room temperature 5 Proceed as in steps 11-18 m Subheading 3.2.

3.3.3. Formic Acid Pretreatment (see Note 70) 1 2 3 4

Deparaffmrze sections. Treat secuons with concentrated fomc acid for 3 min, then rinse thoroughly with buffer. Rinse m buffer for 5 mm. Proceed as m steps 2-18 m Subheading 3.2.

3.3.4. Pretreatment with Pepsin in 0.1 N HCI 1. Deparaffllze sections (as m Subheading 3.1.1., except only nnse 111buffer for 10 mm) 2 Immerse slides m 5 l.tg/mL pepsin m 0 1 N HCl, pH 2.5, for 30 mm, then rinse several times m dH,O. 3 Rinse m for 10 mm in buffer. 4. Preincubate sections m 0.3% Trlton X-100 m buffer for 10 mm. 5. Block with 5% normal serum for 30 mm.

283

Cerebrovascular Integrity

6. Incubate with prtmary antisera diluted in buffer with 1% normal serum and 0.3% Trtton X- 100 overnight at room temperature. 7 Follow steps 6-8 m Subheading 3.2. 8. Incubate sections with diluted secondary antibody (1:400) for 2 h. 9. Proceed as m steps 9-18 m Subheading 3.2.

3.4. Hematoxylin 1. 2. 3 4. 5. 6. 7 8 9. 10. 11. 12. 13 14.

and Eosin-Y Staining

for Paraffin

Sections

Deparaffimze sections (as in Subheading 3.1.1.). Immerse m freshly filtered Gill-3 hematoxylm for 2 mm. Rinse by dippmg slides m dHzO 10 trmes. Immerse m acid water for 1 min Rmse by dipping slides m dH,O 10 ttmes. Immerse m Bluing reagent for 1 min. Rinse by dipping shdes in two changes of dH,O 10 times each. Transfer through two changes of 95% ethanol, dipping in each 10 times. Immerse slides m Eosin-Y for 15 s. Rmse by dipping shdes m two changes of 95% ethanol for 1 mm each Transfer through three changes of 100% ethanol, dipping in each 10 times Transfer through two changes of xylenes, dipping in each 10 ttmes. Clear sections m xylenes for 5 min. Coverslip using permount

4. Notes 1. All buffers should be freshly prepared. 2 Avoid contammation of solutions with detergents and hand lotions. 3. Change alcohols and xylenes used for deparaffmlzation and dehydration frequently. 4. Slides were treated with Vectabond Reagent (Vector Laboratories) prior to mounting sections to prevent them from detaching during staming 5. Do not let the secttons dry out during the staining procedure. Incubate sections in a humtdified chamber Glass casserole dishes with moist paper towels m the bottom work well. 6. Check that the selected antisera crossreacts with the species from which section were taken 7 Attempts to stain secttons pretreated with heat in a 10 mA4 citrate buffer (pH 6.0) with lammin antisera were unsuccessful 8. Vectastain kit should match the host species of the primary antisera. 9. Do not mix contents from two dtfferent kits. If one kit has msufftctent stock solutions for the number of sections, use a new ktt for the entire staining procedure. 10. Expect to lose approximately one-thud of the sections pretreated with formic acid.

Acknowledgment This chapter has been reviewed by the National Health and Environmental Effects Research Laboratory of the US Environmental Protection Agency, but

284

Jensen et al.

the views expressed may not necessarily reflect agency policy and mention of trade names or commercial products does not constitute endorsement or recommendatron for use.

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25 Electron Probe X-Ray Microanalysis A Quantitative Electron Microscopy Technique for Measurement of Elements and Water in Nervous Tissue Cells Richard M. LoPachin

and Christopher

L. Gaughan

1. Introduction Although structure and function of the major cells comprising nervous tissue have been studied extensively, very little detailed information exists concerning subcellular distributions of water and such elements as Na, K, Cl, and Ca. This mformation gap limits our understanding of cell physiology since transmembrane gradients of corresponding ionic species are critically involved in modulating the metabolic and signaling behavior of neuronal and glial cells (I). Moreover, substantial evidence indicates that neuropathic conditions induced by a variety of injury events (e.g., xenobiotlc intoxicatron, disease processes, trauma) involve shifts in subcellular ion composition and volume regulation (2-4). Several techniques (e.g., atomic absorption spectrophotometry, ion selective microelectrodes, ion-sensitive fluorescent dyes) have been used to measure tissue or cellular levels of elements (ions) in normal and injured nervous tissue. In our laboratory, we have used electron probe X-ray microanalysis (EPMA) to investigate the role of ion and water deregulation in different central and peripheral neuropathies (5-7). EPMA is a quantitative electron microscope technique that measures both water content (percentage water) and total (free plus bound) concentrations of biologically relevant elements (e.g., Na, K, S, P, Cl, Ca, and Mg in mmol/kg dry or wet weight) in cellular morphological compartments. Unlike other methods of ion/element measurements, EPMA permits simultaneous determinations of multiple elements and allows optical differentiation of nervous tissue cell types and then processes (e.g., nerve and glial cell bodies, dendrites, axons) with subsequent From

Methods In Molecular Edlted by J Harry

Medmne, vol 22 Neurodegenerabon and H A T~lson 0 Humana Press

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LoPachrn and Gaughan analyses of submembrane regions or organelles (e.g., axoplasm, mitochondria). The ability to measure subcellular drstributrons of elements and water permrts the functional status of neuronal and gllal cells to be assessed.Since compensatory changes in cell water are an important consequence of injury-induced elemental (ionic) flux, the capacity to measure water content IS critical for Interpreting the pathophysiologlcal relevance of elemental alterations (8). Thus, EPMA provides a combination of morphological and quantitative analyses that cannot be achieved by other chemical or electrophysiological methods. EPMA is based on the collection of X-rays that result when electrons of an electron mtcroscope beam interact wrth elemental atoms comprismg the tissue section (9,IO). Two types of X-rays are produced by electron-electron interactions: characteristic X-rays and continuum radiatron. Characteristic X-rays are produced when high-energy electrons of the microscope beam strike and eject inner shell electrons of an atom. Resulting inner orbital romzatton IS a labile state that IS stabilized by decay of an outer shell electron into the lower energy inner orbital. This transition requires release of energy in the form of an X-ray photon. The energy of this photon is equivalent to the energy difference between the two shells and IS characteristic for that element since the atomrc number of the element determines the discreet bindmg energies of each shell. Continuum radiation, also called bremsstrahlung or white radiation, occurs when incident beam electrons are inelastically scattered by the electromagnetic field of resident atomic nuclei. Because of deceleratron, the incident electron looses an amount of energy whrch ranges from zero to the initial ronizatron energy of primary electrons. Continuum is a function of the total number of all atoms in the analyzed compartment and is, therefore, a measure of corresponding mass. Both types of X-rays are collected by an energy-dispersive spectrometer (EDS system) that can perform srmultaneous multielemental analyses (Z > 10). The EDS system can be interfaced with either a scanning or transmrssion electron microscope and IS connected to a PC-based multichannel analyzer for collection and processing of X-rays. To determine subcellular elemental distribution by EPMA, nervous tissue is collected by one of several cryopreservative methods and then sectioned in the cryochamber of a specially designed mrcrotome. Cryosectrons are transferred under vacuum to the column of an electron mrcroscope where the frozen hydrated state IS maintained by a cold stage. For calculatron of compartmental water content, wet werght specrmen mass is measured in hydrated cryosectrons by determining contmuum generation rates. Cryosections are then dehydrated m the vacuum column by raising the temperature of the cold stage. Morphological compartments (e.g., mitochondria) can be visuahzed and analyzed m dehydrated sections by rastering the electron beam within correspondmg anatomical boundaries. Generated X-rays are collected by the EDS system and

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Probe

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converted to dry weight concentrattons (mmol element/kg dry weight) by standardization. Water content (% water) of each morphological compartment is determined by the ratio of continuum counts in the hydrated and dried statesand is used to calculate wet weight concentrations of elements (11,12). It is important to note that EPMA measures total (i.e., free plus bound) element concentrations. However, by making reasonable assumptions, ionized concentrations of elements can be calculated using the dry weight and percent water data (13). 2. Materials 2.1. Cryopreparation and Cryomicrotomy Although early EPMA studies used tissue prepared by conventional wet chemical methods (e.g., glutaraldehyde fixation and osmium staining), more recent studies demonstrated that such fixation promotes artifactual translocanon of elements (14,15). As a result, cryopreparative methods were developed to ensure rapid tissue freezing with preservation of normal subcellular elemental drstribution and morphological structure (16). Tissue can be rapidly frozen by a variety of methods, e.g , clamp freezing using liquid nitrogen-cooled polished metal surfaces (17), quench freezing with super-cooled liquids (e.g., Freon-12, isopentane; ref. IS) or by slam freezing on helmmcooled polished metal (19). Regardless, the goals of cryopreparation are to remove cellular heat rapidly and thereby minimize the growth of ice crystals that interfere with analysis and morphological identification, Once the tissue is frozen, it can be sectioned in a cryoultramicrotome (e.g., Reichert FC4; Leica Inc., Deerfield, IL) at selected ambient temperatures which range from -140°C (20) to -45°C (17). Frozen tissue samples can be sectioned using diamond, tungsten, or glass knives with placement of subsequent cryosections on nickel, copper, or berylhum grids. The grids are generally precoated with a conductive carbon film or a carbon-coated nylon film. 2.2. Energy Dispersive Spectrometer (EDS) In biological studies, X-rays (characteristic and continuum) are most frequently detected using an energy-dispersive spectrometer (e.g., Microtrace 5500 System; Noran Instruments, Middleton, WI) that can perform simultaneous multielemental analyses (Z > 10) with low, nondamaging beam currents (e.g., 1 nA). An EDS system consrsts of a lithium-drifted sillcon (Si[Li]) semiconductor that can collect X-rays m the form of charge pulses. Each charge pulse 1s converted to a voltage pulse that is proportronal to the energy of the arriving X-ray. A multrchannel analyzer converts the analog signal from the detector into a digital signal that can be sorted based on predetermined energybands(e.g.,Na=0.96-1.12keV,K=3.24-3.40keV,Ca=3.6~3.76keV). The resultmg spectrum relating the number of counts per channel (energy band)

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can be displayed on a video monitor with the characteristic peaks for each element of interest marked. Si(L1) detectors are not stable at room temperature and, therefore, must be cooled by a cold finger attached to a liquid nitrogen Dewar. The detector is isolated from the microscope chamber by a beryllmm window which, unfortunately, absorbs low-energy X-rays from elements lighter than Na (Z = 11). This problem has been circumvented by the recent development of thin plastic windows and wmdowless detectors that permit measurements of elements as light as boron (Z = 5). 2.3. Electron Microscope EDS systemscan be mounted on either transnussion (TEM) or scanning (SEM) electron nncroscopes.TEM/EDS systems(e.g., 1200EX TEMSCAN; JOEL, Boston, MA) are generally used in studies requirmg high spatial resolution (~20 nm). This type of nucroanalytical systemrequires addmon of a scanning attachmentthat allows the electron beam to be rastered withm chosen cell regions. For TEM/EDS systems, the cold-stage unit (e.g., gatan cryotransfer system, Gatan, Inc., Warrendale, PA) with specimen gnd holder is separateand removable. The liquidnitrogen-cooled unit is inserted mto the microtome cryochamber accessport and frozen tissue sectionsare loaded onto the grid. When the grid is full, the cold stage is simply transported to the microscope and is inserted mto the vacuum column, In studiesin which low spatial resolution is acceptable,cellular elemental concentrations can be measured usmg an SEM/EDS system (e.g., AMRay 1000 SEM; Bedford, MA). To identify ultrastructural compartments for analyses, scanning microscopes can be fitted with transmuted electron detectors that provide scanning-transmission electron microscopy (STEM). For SElVUEDSsystems,the speclmen grid is generally held by a separateshuttle unit contained within the microtome cryochamber. When the grid is full, the shuttle can be removed from the cryochamber and transferred in an evacuated device (Dehin transporter) directly onto a cold stage in the SEM vacuum column (21). 2.4. Computational Algorithms and Element Standards For quantitation of dry weight concentrations m thin biological sections,a modification of the Hall et al. (22) method of continuum normalizatton is used (23). This method is based on the finding that continuum is directly related to the mass of the specimen and, therefore, the ratio of characteristic counts to contmuum counts is proportional to the massfraction (mass of element/massof specimen) of element m the analyzed volume. This is expressedby the following equation: Rx=Px-bxlWt-

We

(1)

where Rx is the mass fraction of element x, Px is the characteristic X-ray counts for element x, bx is the background counts under the characteristic peak, Wt is

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the continuum counts in a selected region, and We is the extraneous contmuum counts. The absolute dry weight concentration of element x (mmol/kg dry weight) can be derived by standardization where an Rx value from an unknown concentration of x in a given morphological compartment is compared to a previously derived standard curve of mass fraction ratios from known concentrations of x. Typical standards contain incremental concentrations of element dissolved in a matrix, such as albumin, gelatin, or polyvmyl pyrrolidone. Each matrix is presumed to mimic the dry weight mass of the specimen and experience similar beam-induced massloss (12). Previous analytical algorithms proposed the use of albumin peripheral standards where, prior to freezing, the tissue was dipped in a isotonic albumin-Ringer’s solution, which forms an outer layer and is frozen along with the tissue (24). The adherent solution provides a ready standard for calculation of dry weight element fractions in a selected cell compartment. However, later studies showed that this procedure was unrehable since it modified the elemental composition of both the applied standard and tissue cells (25). Compartmental water content (% water) and wet weight concentrations (mmol element/kg dry weight) can be determined by the followmg equations: % Hz0 = (1 - WdlWh) x 100%

(2)

absolute wet weight mass fraction: (3) Where Wd and Wh are the corrected continuum counts for dried and hydrated section areas, respectively, and Cx, and Cx, are the wet and dry weight concentrations for element x, respectively. Cx, = Cx,( 1 - %H20)

3. Methods 3.1. Cryopreparation In our laboratory, EPMA has been used to examine element and water distribution in rat peripheral nerve, dorsal root ganglion, optic nerve, and spinal cord. In most cases,the tissue was rapidly excised and quench frozen in liquid nitrogen cooled (-185°C) Freon or isopentane. Clamp freezing using hquidnitrogen-cooled polished metal surfaces (e.g., copper blocks attached to pliers) has also been used successfully (17). 3.2. Cryomicrotomy Within the cryochamber of the microtome, the tissue is mounted on an appropriate sample chuck and sectioning is initiated. We use glass or tungsten knives and section at a nominal thickness of 500 pm and an ambient cryochamber temperature of -45°C. The frozen, hydrated sections are removed from the

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knife using an eyelash attached to a wooden dowel and are transferred to a carbon-coated, nylon covered beryllium grid located on a beryllmm shuttle within the cryochamber. When the grid is full, the shuttle IS withdrawn through a cryochamber port into an evacuated transport device. The shuttle and grid are transferred onto the cold stage (-185OC) located within the vacuum column of an SEM. 3.3. Hydrated Measurements Wet weight specimen mass is measured in hydrated cryosections by determining continuum generation rates. The beam is rastered within large section areas (60 x 60-pm) and the correspondmg contmuum data are stored on a computer disk for later retrieval. Elemental analyses of mdivrdual anatomrcal compartments

are not performed

m the hydrated

state because morphological

rdentrficatron is difficult and hydrated samples are sensitive to beam-induced radiation damage. Once the hydrated contmuum data are collected, the cryosectrons are dehydrated in the vacuum column by raising the temperature of the cold stage from -185°C to -60°C for 30 min. Once dehydratron is complete, cold stage temperature is returned to -185°C. 3.4. Dry Weight Measurements Morphological compartments are visualized in dehydrated cryosection using scanning transmission electron microscopy (STEM). EPMA data can be collected by two methods. static probe analysis and digital X-ray imaging. For static analysis, the operator manually places the beam over a morphologrcal compartment and then initiates collection of X-rays by the EDS system. The electron beam (20 keV accelerating voltage, 0.4 nA current) is rastered within anatomical boundaries of a chosen structure (e.g., mitochondria, axoplasm). X-ray spectra are collected over a given time period (e.g., 100 s hve countmg time) and elemental concentrations (mmol element/kg dry weight) are determined using software applying the Hall et al. (22) method of contmuum normalization (see Subheading 2.4.). Chemical maps of subcellular elemental distribution can be generated using digital X-ray imaging (26). Here the beam (1.4 nA; 20 keV) IS computer controlled and is moved point-by-point across a specimen region in accordance with a preselected analysis matrix (e.g., 64 x 64 point matrix, 2-s dwell time/pixel). The collected data are used to form digital images m which each point or pixel is fully quantitative wrth respect to elemental concentrations and water content. Digital imaging has been used to study the distribution

of elements

and water in normal

leech ganglion

(8,26-

28), myelinated axons, cell bodies, and glia in rat CNS and PNS (17,29) and cellular areas in mouse cerebellar cortex (30). X-ray maps are especially useful in studies of nerve-cell injury since the quantitative and spatial mformatron

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provided can show precisely how damage causes decompartmentalization of both water and elements (6,8,29). 3.5. Data Analyses Table 1 shows water contents and dry weight elemental data of large myehnated axons and respectrve mltochondria from rat tibia1 nerve. Because EPMA permits multiple compartmental measurements from a sample, one-way analysis of variance (ANOVA) is used to demonstrate that analyses from individual peripheral nerves of an experrmental or control group can be pooled as independent data to derive a group mean. Therefore, descriptive parameters, such as group means and variances, are not based on the number of tibia1 nerves (I.e., 34time point), but rather are derived from pooled axon data. In previous studies (e.g., ref. 6), two types of statistically significant changes were determined. increases in variance (i.e., heterogeneity or increased dispersion) and

shifts in group mean data. Consequently, where appropriate, parametric (for mean shifts) and nonparametrlc (for variance) statrstics are applied to the data. For example, to compare nonparametrrc data, squared deviates from wrthm groups can be calculated and a Kruskal-Wallis test can be applied among groups. A Mann-Whitney U test with Bonferrom correctron can be used to determine differences between control and treatment group data (variance). For parametric analyses, statistical differences among group means can be determined using one-way ANOVA followed by a Dunnett’s t-test.

4. Notes 1. Cryoultramrcrotomy controversres: Cryosectioning of frozen biological material IS a complex and poorly understood process. Controversy surrounds the temperature at whrch tissues are sectioned (e.g , refs. 27,31). Many research groups cut at very low temperatures (

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