No title

international Review of

NEUROBIOLOGY VOLUME 33

Editorial Board W. Ross ADEY

PAULJANSSEN

JULIUSAXELROD

KETY SEYMOUR

Ross BALDESSAKINI

KEITH KILLAM

SIRROGERBANNISTER

CONANKORNETSKY

FLOYDBLOOM

ABELLAJTHA

DANIELBOVET

BORISLEBEDEV

PHILLIP BKADLEY

PAULMANDEL

YURI BUROV

HUMPHRY OSMOND

Josf DELGADO

RODOLFOPAOLETTI

SIRJOHN ECCLES

SOLOMON SNYDER

JOELELKES

STEPHENSZARA

H . J. EYSENCK

MARATVARTANIAN

KJELLFUXE

STEPHENWAXMAN

Bo HOLMSTEDT

RICHARDWYATT

International Review of

NEUROBIOLOGY Editedby JOHN R. SMYTHIES Department of Neuropsychiatry Institute of Neurology National Hospital London, England

RONALD J. BRADLEY Department of Psychiatry and Behavioral Neurobiology The Medical Center University of Alabama Birmingham, Alabama

VOLUME 33

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PRINTED IN THE UNITED STATES OF AMERICA

9 2 9 3 9 4 9 5 9 6 9 1

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9 8 1 6 5 4 3 2 1

CONTENTS

Olfaction

S. G. SHIRLEY I. Introduction . . . . . . . . . . . . . . . . ........................ 11. Biochemistry of Transduction ........................ 111. Physiology of Receptor Cells.. .................................. IV. Receptors and Patterns of Response.. ............................. V. Transfer of Information ......................................... VI. Perireceptor Events. ................ .......................... VII. Conclusion.. .................................................... References. . . . ......................................

1

7 13 16 25 35 40 41

Neuropharmacologic and Behavioral Actions of Clonidine: Interactions with Central Neurotransmitters

JERRYJ. BUCCAFUSCO I. 11. 111. IV. V. V1. VII.

Introduction .................................................... Receptor Specificity.. ........................... ........... Role of Brain Neurotransmitters in the Antihyperte Antiwithdrawal Effects. ......................... Other Pharmacological Actions ........................ Summary and Conclusions ....................................... Future Directions.. .. ......................... References . . . . . . . . . . . . . . . . . . . ..............................

56 59 63 73 85 95 98 100

Development of the Leech Nervous System

GUNTHERS. STENT,WILLIAMB. KRISTAN, JR., STEVENA. TORRENCE, KATHLEEN A. FRENCH, AND DAVIDA. WEISBLAT I. Introduction to the Leech.. ...................................... 109 11. Morphological Development and Staging .......................... 127 V

vi

CONTENTS

111. Behavioral Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Developmental Cell Lineage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Myogenesis and Neurogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I\’.

I34 137 151

183 187

GABA, Receptors Control the Excitability of Neuronal Populations

ARMIKSTELZER I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I95

11. GABAergic Inhibition: 111. GABAergic inhibition:

197 202

GABAA Receptor Function: Control of the Excitability o Populations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. GABA,\ Receptor Function: Tetanization .......................... VI. GABA;\ Receptor Function: Synchronization. ...................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

207 218 263 278

I\’.

Cellular and Molecular Physiology of Alcohol Actions in the Nervous System

FORREST F. WEIGHT ................................................ ............................... 111. Alcohol Effects on Neuronal Firing . . I\‘. Alcohol Effects on Cellular Mechanisms ........................... V. Summary and Conclusions . . . . . . . . . . ............ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF RECENTVOLUMES ...........................

289 290 292 303 336 342

349 365

0LFACTl0 N S.

G.Shirley

Department of Chemistry University of Warwick Coventry, CV47AL, England

I. Introduction 11.

111.

IV.

V.

VI.

VII.

A. Scope B. Structure of the Peripheral Olfactory System Biochemistry of Transduction A. Cyclic AMP B. Phosphoinositides C. Direct Gating D. Lipid Responses Physiology of Receptor Cells A. Non-Odor-Induced Activity B. Odor- and Cyclic Nucleotide-Induced Activity Receptors and Patterns of Response A. Receptor Molecules B. Receptor Cells C. Responses in the Olfactory Bulb Transfer of Information A. The Vectorial Representation B. Odor Concentration and Signal/Noise C. Information Transfer from Mucus to Receptor Molecule D. Information Transfer from Receptor Molecule to Cell Perireceptor Events A. The Olfactory Mucus B. Control of Secretion C. Central Control of the Sensory Cells D. Xenobiotic-Metabolizing Enzymes E. Odorant-Binding Protein Conclusion References

1. Introduction

A. SCOPE The last decade has seen much progress in the understanding of olfaction. The purpose of this article is to review the molecular and cellular processes by which information is extracted from an incoming I INTERNATIONAL REVIEW OF NEUROBIOIDGY, VOL. 33

Copyright 0 1992 by Academic Press, lnc. All rights of reproduction in any form reserved.

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S. C . SHIRLEY

olfactory stimulus and passed to the central nervous system. T h e mechanisms of insect olfaction are excluded and readers are directed to the works of Kaissling (1986, 1987), O’Connel (1986), and Payne et al. (1986). Although work on invertebrates and fish is mentioned, this article concentrates mainly on the processes occurring in the air-breathing vertebrates.

€3. STRUCTURE OF THE PERIPHERAL OLFACTORY SYSTEM

1 . Oueroiew I n mammals and amphibia, the sensory cells of olfaction lie within the olfactory mucosa. In mammals, the mucosa covers part of the ethnioturbinates and part of the nasal septum, so forming the lining of airfilled cavities adjacent to the main air passages. During inhalation, eddies break away from the main airstream and enter these cavities, where airborne organic molecules can interact with the olfactory receptors. T h e superficial layer of the mucosa is a pseudostratified columnar epithelium. It is avascular and contains the sensory cells, supporting cells, and basal cells. A thin layer of mucus covers the olfactory epithelium, separating it from the air space. T h e mucus derives from the supporting cells and olfactory glands. T h e deeper part of the mucosa, the lamina propria, contains blood vessels, connective tissue, secretory glands, and bundles of sensory cell axons. The ultrastructure of the olfactory mucosa has been reviewed by Menco (1983). Morphologically and functionally the sensory cells are bipolar neurons, although they are, embryologically, of epithelial origin (Cuschieri and Bannister, 1975). Axons from these cells run in bundles or fasciculi through fine holes in the cribiform plate and into the brain. T h e axons form synapses within the glomeruli of the olfactory bulb. T h e glomeruli are large structures (loo+ pm) where axons from several tens of thousands of sensory cells form en passant synapses with about 100 secondary (mitral and tufted) cells. T h e neurotransmitter at these synapses is the dipeptide carnosine (P-alanyl- 1-histidine) (Neidle and Kandera, 1974; Gonzales-Estrada and Freeman, 1980; Burd et al., 1982; Margolis et al., 1985, 1987; Sakai et al., 1987, 1988). The axons of the mitral cells and of some tufted cells are the main output of the olfactory bulb and run ipsilaterally to the olfactory cortex. In addition to the olfactory system outlined above, most air-breathing animals (excluding man) have an additional chemical sense, the vomeronasal system. This usually takes the form of a tube, lined with

OLFACTION

3

sensory cells, linking the oral and nasal cavities. The axons of these cells project to the accessory olfactory bulb; this structure is generally similar to and contiguous with the olfactory bulb. Whereas the vomeronasal system is usually stimulated by materials in solution, it can be stimulated by airborne substances, probably after solution in saliva. The importance of this system has been discussed by Wysocki (1979) and Doty (1988). There is also innervation of the nasal cavities by the trigeminal nerve, and it plays a role in chemosensation (e.g., Silver et al., 1986). 2. The Primary Cells The sensory cell bodies are fusiform, small (typically 15 X 4 pm), and generally lie at depths of greater than about 50 pm within the olfactory epithelium. From each, a single dendrite extends between the supporting cell to the tissue surface and ends in a terminal swelling about 1.5 pm in diameter. The terminal swellings carry modified cilia. T h e basal bodies of these cilia appear normal in the electron microscope. Each cilium has a proximal segment about 1.5 pm in length and 200 nm in diameter containing the usual 9(2) + 2 pattern of filaments. The cilia then taper to about 90 nm in diameter and the filaments become fewer in number and less regular in organization. In mammals, the distal parts of the cilia are typically 30 pm in length; in amphibia they may be up to 100 pm long. Mature olfactory cilia are immotile, although immature forms of amphibian cilia may be motile (Mair et al., 1982). Scanning electron microscopy has proved valuable in revealing details of the olfactory surface in fish (Breipohl et al., 1973; Doroshenko and Motavkin, 1986), amphibians (Mair et al., 1982; Menco, 1977; Klein and Graziadei, 1983), birds (Breipohl and Fernandez, 1977), reptiles (Wang and Halpern, 1980), and mammals (Menco, 1977). The technique can also be used to reveal the structure of the mucosa in depth (Costanzo and Morrison, 1989; Morrison and Costanzo, 1989). Measurements of the period between odor application and response, coupled with a knowledge of the diffusion coefficients of the odorant, imply that the transductory apparatus is very close to the tissue surface, in the cilia, terminal swellings, or apical part of the dendrite (Getchell et al., 1980). Other evidence reviewed by Getchell et al. (1984) also suggests that the transductory apparatus is located in the cilia. At the level of the ciliary taper there is a prominent necklace of particles in the membrane. The necklace particles are firmly anchored to the cytoskeleton. They form a region of membrane attachment and a barrier to lateral diffusion in the membrane (Menco, 1988b). T h e distal parts of the cilium have a high density of membrane-embedded particles (Menco et al., 1976). Particle densities of 1000 pm-* (frog) to 2300

4

S . G. SHIRLEY

pm-2 (dog) were found in a series of freeze-fracture studies by Menco (198Oa-d). It is hypothesized that these particles form part of the transductory mechanism. T h e cilia1 membranes of the receptor cells are also more electron opaque than those of the respiratory cells (Menco, 1989b). Freeze-fracture techniques have also been used to study the monkey epithelium by Engstriim et al. (1989). The olfactory receptor neurons are unusual in that they have a finite lifetime and they are replaced by new cells derived from the basal cells. Breipohl et al. (1986) have reviewed the control mechanisms of this replacement. I t is possible that the availability of space for the developing neuron is a controlling factor (Mackdy-Sim et al., 1988). It is likely under natural circumstances that each receptor cell has a lifetime of many months (Hinds et al., 1984), but this can be artificially reduced. Following transection of the axons, the receptor cells degenerate. New cells appear at about the tenth day and replacement is complete at about a month. Simmons and Getchell(l981) found that the newly differentiated neurons had spontaneous electrical activity, odor specificity, and concentration-response profiles as normal neurons before the axons made contact with the olfactory bulb. However, Masukawa et al. (1985) found that the newly differentiated cells were generally nonspiking. Immature cells have been found to be less responsive to odor than are mature ones and they show few action potentials (Lidow et al., 1987a,b); they also have smaller resting potentials and are less responsive to injected current (Hedlund et al., 1987; Masukawa et al., 1987). The axons of the sensory cells are nonmyelinated; Schwann cells envelop bundles of axons and extend tongues of cytoplasm between the fibers (Barber and Lindsay, 1982; Rafols and Getchell, 1983). There are conflicting reports as to whether the Schwann cells are epithelial or glial. Vollrath et al. (1985), Ophir (1987), and Ophir and Lancet (1988) found keratin as the filament protein. Whereas glial fibrillary acidic protein (GFAP) was found by Barber and Lindsay (1982) and Barber and Dahl (1987), who reported that there were low levels of the central type of GFAP for much of the length of the fasciculi but higher levels at the receptor cell end and the possibility that peripheral-type GFAP is also present. I n view of the continuous turnover of the olfactory neurons, several groups have looked for signs of immaturity in these cells. T h e cells have immature versions of microtubule-associated proteins (Viereck et al., 1989) and cell adhesion molecules (Miragall et al., 1988). It is rare for olfactory axons to show staining with antibodies to neurofilament protein (Vollrath et al., 1985), but there is disagreement as to the presence of vimentin. This intermediate filament protein has been reported by

OLFACTION

5

Schwob et al. (1986) but not by Vollrath et al. (1985), Ophir (1987), or Ophir and Lancet (1988) (see also Yamagishi et al., 1989a,b). Antigens associated with neuritic outgrowth continue to be expressed in the adult animal (Stallcup et al., 1985; Wallis et al., 1985). The growth-associated protein B50/GAP43 can be found in the epithelium of adult rats (Verhaagen et al., 1989). It seems to be associated with the newly differentiated subpopulation of cells and its expression ceases when that of olfactory marker protein (OMP) starts. OMP is a protein of unknown function, taken to be a marker of mature olfactory receptor neurons (Margolis, 1972, 1988), but is also found at very much lower levels in other brain regions (Baker et al., 1989). The expression of OMP is thought to coincide with ciliogenesis (Menco, 1989a). The olfactory receptor neurons also carry receptors for nerve growth factor (Taniuchi et al., 1986) and high levels of nerve growth factor can be found within the neurons of the developing epithelium (Williams and Rush, 1988). Regenerative neurogenesis is not a recapitulation of prenatal development (Menco, 1985). Development processes have been reviewed by Brunjes and Frazier (1986), Penderson et al. (1986a), Farbman and Menco (1986), Farbman (1988), and Mair (1988). Regeneration following transplant or bulbectomy has been reviewed by Graziadei and MontiGraziadei (1986a) and Barber and Jensen (1988). It is likely that under normal circumstances the axons reach their targets along preexisting glial channels (Barber, 1982a,b; Barber and Dahl, 1987; MontiGraziadei and Morrison, 1988; Monti-Graziadei and Graziadei, 1989). But it has been hypothesized by Graziadei and Monti-Graziadei (1986b) that no target is necessary for glomerulus formation, the receptor cell axons being capable of self-selection. In addition to the ciliated cells already described, there are also some microvilli-bearing cells with distal processes (e.g., Menco, 1980a; Moran et al., 1982; see also Akerson, 1988) and it has been demonstrated by Rowley et al. (1989) that these send axons to the olfactory bulb. The function of these cells is unknown, but in the human 10% of axons from the olfactory region derive from such cells.

3. Connections between the Epithelium and Bulb There is a relationship between the position of a glomerulus and the positions of its sensory cells. To each glomerulus corresponds a field that is a set of strips running anterior-posterior in the epithelium. By no means do all sensory cells in such a strip project to the same glomerulus, but there is a tendency for them to project to neighboring ones. This has been demonstrated by horseradish peroxidase injection or lavage Uastreboff et al., 1984; Stewart, 1985; Penderson et al., 1986b; Astic and

6

S. G . SHIRLEY

Saucier, 1986, 1988; Saucier and Astic, 1986; Astic et al., 1987; Stewart and Penderson, 1987; Zheng and Jourdan, 1988), radio-leucine injection (Mackay-Sim and Nathan, 1984), and physiological methods (Costanzo and Mozell, 1976; Costanzo and O’Connel, 1980). Any particular bulb neuron has a field of receptor cells that are excitatory and a field of cells that are suppressive and there is a tendency for the excitatory field to be more compact (Kauer and Moulton, 1974). There is electrophysiologicdl evidence that the excitatory and inhibitory components of the receptive field of an olfactory bulb neuron are not well segregated Uiang and Holley, 1987). I n addition to dense projections to neighboring glomeruli, any given region of epithelium has diffuse projections to the whole olfactory bulb (Kauer, 1981) and there are diffuse projections from the whole epithelium to any part of the bulb (Kauer, 1980). There are monoclonal antibodies capable of distinguishing subclasses of olfactory receptor cells and there is a tendency for any given glomerulus to receive its input from like cells (Fujita et al., 1985; Mori et al., 1985; Mori, 1987b; Schwob and Gottlieb, 1986, 1987, 1988) o r for there to be a general relationship between the position of the positive cells and the positive glomeruli (Shinoda et al., 1989). T h e same kind of relationship may be observed with lectin binding (Key and Giorgi, 1986; Barber, 1989). 4. The Olfclctoq Bulb

The structure of the olfactory bulb has been reviewed by Halasz and Shepherd (1983), Macrides and Davis (1983), Scott (1986), Mori (1987a), Scott and Harrison (1987), and Dawson et al. (1988). T h e principal cells are the mitral and the tufted cells. Each extends a single primary dendrite that arborizes within a single glomerulus and receives input from the sensory cell axons. Each side of the rabbit olfactory bulb has about 1900 glomeruli with about 24 mitral and 70 tufted cells per glomerulus. Each glomerulus receives the axons of about 25,000 sensory cells and the en p a m n l synapses are such that each mitral cell receives input from 1300-6500 sensory cells. This extremely high degree of convergence is one of the characteristic features of the olfactory system. In addition, each mitral or tufted cell extends secondary dendrites that make many reciprocal dendrodendritic synapses with the axonless granule cells. The olfactory bulb is very rich in dendrodendritic synapses and is a useful source of material for their study (Chiflikian et al., 1986). I‘he granule-to-mitral synapse is inhibitory, using y-aniinobutyric acid as transmitter. T h e niitral-to-granule transmission, which includes some axodendritic synapses, is excitatory. T h e main axons of the mitral and

OLFACTION

7

some tufted cells project to the ipsilateral olfactory cortex. Collaterals branch, forming synapses with granule cells. These collaterals extend further than do the secondary dendrites of the same cell and some project to the other side of the olfactory bulb, making synapses with granule cells. There are subtypes of mitral and tufted cells distinguishable on morphological and functional grounds (Macrides et al., 1985; Scott, 1986, 1987). Within the glomeruli; the sensory cell axons also form synapses with the dendrites of periglomerular cells. There are indications that the sensory cells induce a dopaminergic phenotype in the periglomerular cells via a calcitonin gene-related peptide (Denis-Donini, 1989; see also Baker, 1988). T h e periglomerular cells also form reciprocal dendrodendritic synapses with the mitral and tufted cells. The periglomerular cells have short axons, which make synapses with the primary dendrites of the mitral and tufted cells and with the dendrites and somata of other periglomerular cells. There are also several other types of short axon cells within the olfactory bulb. The main centrifugal input to the olfactory bulb is to the granule cells. Many neuropeptides, hormones, or their receptors have been identified in the olfactory bulb. These include insulin (Matsumoto and Rhoads, 1990; see also Baskin et al., 1988), atrial natriuretic peptide (Katawa et al., 1986; Gibson et al., 1988; Glembotski et al., 1989), neuropeptide Y (Scott et al., 1987; Ohm et al., 1988), somatostatin (Scott et al., 1987), enkephalins (Kosaka et al., 1987), substance P (Baker, 1986a,b), vasoactive intestinal polypeptide (Alonso et al., 1989), luteinizing hormone-releasing hormone (Zheng et al., 1988), and cholecystokinin-8 (Seroogy et al., 1985; Matsutani et al., 1989a). The distributions of many of these substances define subpopulations of cells in the bulb and the distributions of several neuropeptides have been studied in the guinea pig (Matsutani et al., 1989b) and in the developing rat (Matsutani et al., 1988).

11. Biochemistry of Transduction

This subject has been reviewed (Lancet, 1986, 1988; Lancet et al., 1988; Bruch et al., 1988; Snyder et al., 1988b; Shirley and Persaud, 1990) and there have been several minireviews (Anholt, 1987; Lancet and Pace, 1987; Snyder et al., 1988a, 1989). Four kinds of transduction mechanisms have been proposed. These are mechanisms based on cyclic

8

S. G . SHIRLEY

AMP, phosphoinositides, the direct opening of ion gates by odorants, and the inherent response of lipids to odorants. These will be discussed in turn.

AMP A. CYCLIC In general, adenylate cyclase systems consist of a number of components: one or more types of receptor, one or more guanine nucleotidebinding proteins (G proteins), the adenylate cyclase catalytic unit, and one o r more phosphodiesterases. Normally the G protein carries tightly bound guanosine diphosphate (GDP). T h e binding of a ligand to a receptor allows the G protein to exchange this for cytosolic guanosine triphosphate (GTP). If the G protein is of the stimulatory type (G,), this activated form of the G protein interacts with the catalytic unit to increase catalytic activity. T h e G protein hydrolyzes its bound GTP to GDP and returns to the inactive form. Inhibitory G proteins (G,) are believed to act by deactivating G , (e.g., Gilman, 1987), but to date there is no evidence for the involvement of an inhibitory G protein in olfactory transduction. The adenylate cyclase catalytic unit converts ATP to cyclic AMP; this is the only known mechanism for the generation of this species. Cyclic AMP has a controlling function for many cellular processes and it is converted to AMP by the action of the phosphodiesterases. In addition to its transductory function, cyclic AMP has been implicated in the control of neuritic outgrowth in developing cells. An analog of cyclic AMP, phosphodiesterase inhibitors, and forskolin (a potent stimulant of adenylate cyclase) all promote outgrowth in explanted developing olfactory epithelia (Johnson et al., 1988b). 1. E v d e i t c e f o r the Inzdvement of A d e y late Cyclase

Kurihara and Koyania (1972) demonstrated the presence of high levels of adenylate cyclase in the olfactory mucosa. The olfactory cilia of mammals and amphibia can be detached by calcium shock (Anholt et al., 1986; Chen et al., 1986a; Lazard et al., 1989) o r by sonication (Shirley et al., 1986). Such preparations show a very high level of adenylate cyclase activity, which can be stimulated by the addition of odorants (Pace et al., 1985; Shirley et al., 1986; Sklar et al., 1986). The dose-response curves for odorant activation of adenylate cyclase are fairly flat, having Hill coefficients of about 0.3-0.6, and so are similar in shape to overall olfactory dose-response curves from psychophysical measurements. The odorant concentration range in which stimulation of the adenyl-

OLFACTION

9

ate cyclase occurs is typically micro- to millimolar. It is important to remember that despite their generally hydrophobic nature, most odorants partition so that their concentration in water (or mucus) is much higher than that in air (see Section V1,A). This concentration range is comparable with that which will elicit an electrophysiological response from the tissue (Shirley et al., 1987b) and, when corrected for partitioning, that which can be smelled. There is a good correlation between an odorant’s potency in stimulating adenylate cyclase and its potency in stimulating the electroolfactogram (Lowe et al., 1989). The electroolfactogram is a measure of the combined generator currents of the receptor cells (Ottoson, 1956; Gesteland, 1975). The report by Sklar et al. (1987) that some classes of odorant do not stimulate adenylate cyclase is based on measurements at a single odor concentration, which may have been subthreshold for some compounds. Some materials classified as nonstimulants by Sklar et al. (1987) have been found to stimulate at higher concentrations (Lowe et al., 1989). Adenylate cyclase has been histochemically localized to the cilia, terminal swellings, and dendritic shafts of the primary cells (Asanuma and Nomura, 1989). Jones et al. (1988) have shown that both the enzymatic activity and the mRNA for adenylate cyclase are predominantly expressed in the receptor cells. Wheat germ agglutinin, which inhibits the electrophysiological response to odorants, also inhibits the odorant activation of adenylate cyclase (Lancet et al., 1987). Cyclic AMP analogs and phosphodiesterase inhibitors have been shown to interfere with olfactory transduction by Minor and Sakina (1973), Menevse et al. (1977), and Persaud et al. (1988a). There is good evidence (Sections III,B,2 and III,B,3) for the existence of cyclic nucleotide-controlled ion gates in the sensory cells. There have been no reports of measurement of cyclic AMP levels within the olfactory tissue, but neuroblasts from the olfactory epithelium have been shown to accumulate cyclic AMP when challenged with odorants in culture (Coon et al., 1989). Preparations of olfactory cilia also contain proteins that are phosphorylable in a cyclic nucleotide-dependent manner (Heldman and Lancet, 1986; Kropf et al., 1987). T h e adenylate cyclase catalytic unit of the olfactory system has been isolated by Pfeuffer et al. (1989). It is a novel form of the enzyme, having a high molecular weight (180,000), and it is highly active, with a turnover number of about 8000Imin.

2. G Protein Guanosine triphosphate is essential for the activation of the olfactory adenylate cyclase (Pace et al., 1985; Shirley et al., 1986), implying that the

10

S. G. SHIRLEY

cyclase is coupled to the receptor via a G protein. This is confirmed by the observation of Vodyanoy and Vodyanoy (1987a,b) that lipid bilayers incorporating olfactory membrane fragments require GTP in order to display conductance changes; Weinstock et al. (1986) and Wright et al. (1987) also confirmed that humans with a G protein deficiency have an impaired sense of smell. Further evidence for the involvement of a G protein in transduction comes from the observation that odorants can cause small modulations in the binding of guanine nucleotide analogs in olfactory preparations (Lancet, 1988) and by guanine nucleotide-induced changes in the affinity of olfactory receptors for ligdnds (Bruch and Kalinoski, 1987). NotJoselov et al. (l988a,b) have isolated a protein complex from the skate. T h e complex can be dissociated by GTP or analogs and its GTPase activity can be increased by amino acids that are olfactory stimuli for this fish. This could be a receptor-(; protein complex. Interaction between G protein and the cyclase catalytic unit can be demonstrated in reconstituted detergent extracts of olfactory material (Anholt, 1988). The involvement of G proteins in olfaction has also been studied by Parfenova and Etinghof (1988). Several G proteins can be found in the olfactory mucosa. T h e stimulatory (GJ, inhibitory (G,), and other (Go) varieties are present and G, is enriched in cilia1 preparations (Anholt et al., 1987a,b). However, the G protein involved in olfactory transduction is a variant (Pace and Lancet, 1986). It has now been cloned and is called G 1 (Nakamura and Gold, 1987; Persaud et al., 1988a; DeSimone et al., 1988) and the relationship between depolarizing current and spike rate is sharp (Hedlund et al., 1987; Masukawa et al., 1987). This suggests that any particular cell signals useful information over a quite narrow concentration range, as suggested by Lidow et al. (1987b), although it may remain maximally activated at higher concentrations (Duchamp-Viret et al., 1989). But there is no reason to suppose that this range stays constant in the face of a changing background. 3. The Arrangement of Receptor Molecules on Cells

A key question is of the arrangement of receptor molecules on cells, because this arrangement relates the response spectra of the molecules to the response spectra of the cells. The response spectra of cells are stable and tend to be broad, but have so far resisted attempts at classifica-

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S. C;. SHIRLEY

tion. 'The remarks of Section V,C,S apply to the classification of cells as well as to the classification of molecules, with the added complication that cells may show variations of sensitivity that are not of receptor molecule origin. Perhaps the classification scheme most likely to succeed in these circumstances is one based on the ratios of the concentrations at which two or more different odorants elicit the same response. This kind of approach has been used successfully to classify olfactory cells in insects (Kafka, 1987). Ion gates, adenylate cyclase, G protein, and, presumably, receptors are membrane-bound molecules and as there are diffusion barriers in the membrane these components could be compartmentalized within the cell. Cyclic AMP is soluble, but on the time scale of transduction could only difFuse for a distance of a few micrometers, not, for instance, for the entire length of a cilium. So it is likely that any given ion gate responds only to events occurring at local receptor molecules and conceivable that different kinds of receptor molecule could be compartmentalized within the same cell to control different ion gates. But neither odorants nor second messengers could diffuse from the trdnsductory apparatus to the cell body at the speed of transduction, and this signaling is believed to be purely electrical. T h e spike-generating apparatus receives only a summated signal from the receptor molecules. If a cell carries more than one type of receptor molecule and odor-quality information resides in the differences in an odorant's ability to stimulate the different receptor types, then addition of the signals will destroy the infbrniation. If a cell carries more than one kind of receptor molecule, then the mechanism must involve both addition and subtraction and it will be necessary to demonstrate odor-specific excitations and inhibitions occurring within the same cell. Within the olfactory bulb, there is ample evidence for both excitatory and inhibitory pathways and no conceptual difficulty in understanding the extraction of qualitative information.

4. Conuergence T h e effect of the high degree of convergence between receptor and secondary cells has been discussed by van Drongelen et al. (1978), who pointed out that the secondary cells should be more sensitive to odorants than are the receptors. If a function of the secondary cells is to average noisy signals, then the sensitivity should improve roughly with the square root of the number of receptors connected to each secondary. For a convergence ratio of about 1000, the sensitivity of a secondary cell should be about one and a half orders of magnitude greater than a receptor. This is close to what has been observed by Duchamp-Viret et al.

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35

(1989). These authors also point out that a bulb can respond to odorants with many of its peripheral connections broken, but that its sensitivity is drastically reduced under these conditions.

VI. Perireceptor Events

T h e events that occur in the olfactory mucus prior to, during, and after transduction are of great significance and have been reviewed by Getchell et al. (1984).

A. THEOLFACTORY Mucus Very little is known about the olfactory mucus. The mucus over the respiratory region has two layers of different consistency, but that over the olfactory region is a single layer (Menco, 1989b). A histochemical study by Gladysheva et al. (1986) revealed acidic, sulfated, and neutral mucopolysaccharides in vertebrate mucus. T h e mucus is also sodium poor and potassium rich compared with most intercellular fluid (Joshi et al., 1987). Hornung et al. (1987) have studied the equilibrium partitioning of odorants between air and the olfactory mucosa. It must be emphasized that the mucus (which bathes the receptor apparatus) is not the major component of the mucosa and that dynamic considerations (deviations from equilibrium) may be important in viva However, the conclusion of these authors is that for most odorants the mucosa/air partition coefficient is within a factor of two or three of the water/air partition coefficient, which can therefore be used as a rough guide. As the concentration of an odorant partitioning into the mucus is of fundamental importance to an olfactory biochemist studying receptor affinities, etc., it is worth reiterating the physical chemistry of the situation. T h e equilibrium distribution of an odorant between water and air is usually described by a partition coefficient (molar concentration in water/molar concentration in air) or a Henry coefficient (the limit at low concentration of the mole fraction in air/mole fraction in water). Both are temperature sensitive, as is the ratio of molar volumes of water and air that relates the two. Typically, odorants have partition coefficients of between 50 and 10,000, although examples can be found outside this range, but for virtually all odorants the molar concentration in water is higher than that in air. By elementary thermodynamics, the air above a

36

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saturated solution of a sparingly soluble material contains vapor at the saturated vapor pressure. So a knowledge of the saturated vapor pressure, which is related to the molar concentration in air, and the solubility allows an estimate of the partition coefficient if no better data are available. 'The presence of a cosolute in the water affects the distribution only insofar as it changes the solution properties (dielectric constant or structure, etc.) of the water. i n this respect, probably the most significant solutes in mucus are the salts, and saltwater/air partition coefficients should provide a better approximation to the olfactory situation. For biochemical purposes, the significant variable is the concentration of material free in solution, because this is the thermodynamic driving force determining binding to a receptor and it is this concentration that is related via the partition coefficient to the concentration of vapor above the liquid. The ability of a cosolute to bind odorant is irrelevant, as it simply creates a third compartment whose contents come to equilibrium without disturbing the relationship between free solution concentration and vapor concentration. T h e same is true for any other entity that can absorb odorant. At equilibrium, the free solution concentration is simply related to the vapor concentration despite binding, the solution of odorant into membranes, and monolayers formed on equipment surfaces, etc. Such compartments do affect the time taken to reach equilibrium when the tissue is stimulated via the gas phase, as more material must be transferred. But the finding that mucosa/air partition coefficients are fairly close to water/air coefficients means that this delay is neither large nor unpredictable. The olfactory receptor molecules are very superficial in the mucosa and the mucus at their depth will equilibrate with the air more quickly than will deeper material. if the tissue is stimulated by an odorant in moving air, then the time to achieve equilibrium should be little greater than the longer of two characteristic times. T h e first is the time for an odorant to diffuse to equilibrium in the mucus (tens to a few hundred milliseconds, depending on the mucus thickness). The second is the time for sufficient air to pass over the tissue to deliver enough material to bring the superficial layers to their equilibrium concentration; this time depends on the partition coefficient of the odorant, as this determines how much material must be transferred, but under typical experimental conditions and for odorants of partition coefficients up to a few thousand, this time should not exceed 1 sec. So for odorants of low and moderate partition coefficients, the free solution concentration in the vicinity of the receptor molecules should, under experimental conditions on a time scale of seconds, be close to the equilibrium value. But during a sniff zn vivo, on a 100-msec time scale, concentrations are likely

OLFACTION

37

to be changing. Prediction of these changes requires detailed knowledge of the aerodynamics of the cavity and physical chemistry of the particular odorant. Odorants are delivered to the olfactory epithelium in a moving airstream that passes over the relatively stationary mucus layer. This, coupled with the ability of the mucus to absorb odorants differentially, could result in some measure of chromatographic separation, possibly leading to characteristic concentration gradients of odorants across the epithelium and variations of concentration with time. This “imposed” spatial patterning has been proposed as a contributory mechanism for odorant recognition (Mozell, 1970; Moulton, 1976; Hornung and Mozell, 1981; Mozell et al., 1987). However, Laing (1988) found no correlation between the absorption properties of an odorant and the human ability to recognize that odorant in mixtures.

B. CONTROL OF SECRETION The control of secretion in the olfactory mucosa has been reviewed by M. L. Getchell et al. (1988). The olfactory mucosa is innervated by the trigeminal nerve. T h e sensory role of this nerve (e.g., Silver et al., 1986) is beyond the scope of this review, but the secretomotor function may play an important modifying role in olfaction. Substance P immunoreactivity is found near the Bowman’s glands and blood vessels of the lamina propria (Papka and Matulionis, 1983); also, fibers extend to near the epithelial surface (Bouvet et al., 1987a; M. L. Getchell et al., 1989; Zielinski et al., 1989a). These fibers terminate in varicosities mainly between the sustentacular cells with no morphological sign of synaptic contact. The fibers are present at a ratio of about one fiber to 30-300 sensory neurons in different amphibian species. Stimulation of the trigeminal nerve or topical application of substance P gives morphological signs of secretion (M. L. Getchell et al., 1989). T h e olfactory mucosa also receives innervation by the terminal nerve (Wirsig and Getchell, 1986) and there are cholinergic terminals between the gland cells of Bowman’s glands and near the adjacent blood vessels (Zielinski et aE., 198913). Vasoactive intestinal peptide may also play a role in secretory control (M. L. Getchell et al., 1987, 1988). There is evidence for adrenergic fibers in the olfactory mucosa (Kawan0 and Margolis, 1985) and for a-adrenergic (Zielinski et al., 1989b) and P-adrenergic (Getchell and Getchell, 1984) regulation of secretion in the olfactory glands. Luteinizing hormone-releasing hormone immunoreactive fibers can be found around the ducts of the anterior medial

38

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SHIRLEY

glands and close to the blood vessels of the olfactory mucosa in the rat (Zheng ~t al., 1988). Odorants also can induce secretion from the glands of the olfactory mucosa (M. L. Getchell et al., 1987) and this effect can be blocked by a muscarinic cholinergic antagonist, probably indicating a secretomotor reflex. In addition, odorants can stimulate secretion from sustentacular cells in some species (Ekblom et al., 1984; M. L. Getchell et al., 1987), although in man (Moran et al., 1982;Jafek, 1983) and some other species (Yamamoto, 1976) the sustentacular cells seem to be nonsecretory. The control of this process is unclear but it may be relevant that odorants can induce a depolarization of long duration in the supporting cells (Trotier and MacLeod, l986b).

C;. CENTRAL CONTROL OF

THE

SENSORY CELLS

Endocrine processes have an important role in the modulation of olfactory function generally (reviewed in Doty, 1989). This section is concerned only with possible central control of the sensory cells. Application of substance P can evoke a slow electrical potential change from the olfactory tissue (Bouvet et al., 1984), cause changes in the spike activity of receptor neurons (Bouvet ct al., 1988),and modify the responsiveness to odorants (Bouvet et d., 1987b). Stimulation of the trigeminal nerve has the same effects (Bouvet el al., 1987b,c). Substance P immunoreactivity is also found in the olfactory epithelium of fish (Szabo et al., 1987). Acetylcholine also causes slow electrical potentials (Bouvet et al., 1984) and modifies spike activity in the receptors (Bouvet et al., 1988). It seems likely that these events are mediated via receptors on the sensory neurons rather than simply being consequences of changes in mucus secretion. The sensory cells could, therefore, be under some measure of central control. ‘The olfactory nerves d o carry nonodor receptors; peripheral-type benzodiazepine receptors were found by Anholt et al. (1984), and in the sensory cells of the lobster, there is a chloride channel, directly controlled by histamine (McClintock and Ache, 1989a), which seenis to tnodulate spiking activity (Bayer et al., 1989).

I). XENoBIOrIC-METABOLIZIIL’G

ENZYMES

‘Theolfactory mucosa is a rich source of enzymatic activity capable of metabolizing foreign substances. This subject has been reviewed by Dahl (1988)and Dahl et al. (1988). T h e presumed function of these enzymes is the metabolism of odorants to odorless or more soluble compounds,

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39

although it is possible that they might, by metabolism, change odor quality. These enzymes often have a very wide specificity, and there is no reason to suppose that they do not metabolize endogenous material also. T h e olfactory mucosa contains high levels of cytochrome P-450 (Dahl et al., 1982; Voigt et al., 1985; reviewed by Jenner and Dodd, 1988a), which has different substrate specificities and different kinetic parameters compared to the liver enzyme (Brittebo and Ahlman, 1984; Brittebo and Rafter, 1984). The high levels of activity in the olfactory tissue are due in part to intrinsic differences between the olfactory and liver enzymes (Reed et al., 1988) and in part to enhanced electron flow due to the high ratio of NADPH:cytochrome P-450 reductase:cytochrome P-450 in the olfactory tissue (Reed et al., 1986). Among other functions in this tissue, P-450 has been implicated in the metabolism of steroids (Brittebo and Rafter, 1984; Brittebo, 1982), possibly including exogenous odorous molecules (Gower et al., 1981; Persaud et al., 1988b). The olfactory enzymes and those of other tissues differ in their properties. There are mechanistic differences between olfactory and liver enzymes (Reed and De Matteis, 1989). The olfactory tissue contains unique isoenzymes (Ding and Coon, 1988), different isoenzyme profiles than those found in other tissues (Ding et al., 1986;jenner and Dodd, 1988b; Larsson et al., 1989), and there are differences in inducibility (Ding et al., 1986). Sequence data derived from a cDNA clone (Nef et al., 1989) indicate that an olfactory enzyme, although a member of the P-45011 family, has novel features. Other enzymes that have been found in the olfactory region are FAD-monoxygenase (McNulty and Heck, 1983; McNulty et al., 1983), epoxide hydrolases (Bond, 1983), aldehyde dehydrogenase (CasanovaSchmitz et al., 1984; Bogdanffy et al., 1986), carboxylesterases (Stott and McKenna, 1985; Dahl et al., 1987; Bogdanffy et al., 1987), rhodanase (Dahl, 1989), and the “phase 11” enzymes responsible for transferring water-soluble groups (Bond, 1983; Baron et al., 1986). There are reports of a rather more “odor-specific”metabolizing system in the lobster. T h e adenine nucleotides are important olfactory stimuli for these animals and dephosphorylation enzymes and an adenosine uptake system have been found (Trapido-Rosenthal et al., 1987a,b; Gleeson et al., 1989). E. ODORANT-BINDING PROTEIN

Odorant-binding protein was identified and isolated from olfactory tissue on the basis of its ability to bind pyrazines (Pelosi and Pisanelli, 1981; Pelosi et al., 1982; Bignetti et al. 1985; Pevsner et al., 1985)and was

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originally thought to be a receptor molecule. However, the specificity is weaker than originally believed and the protein will bind many odorants at micromolar concentrations (Topazzini et al., 1985; Pevsner et al., 1986; Bignetti et al., 1988). T h e protein is soluble and exists as a dimer of 2 X 19 kDa (Pevsner et al., 1985). In the cow it is found in the glands of the olfactory and respiratory epithelium (Pevsner et al., 1986) but in the rat it is found in the lateral nasal gland, which discharges just behind the external naris at a considerable distance from the olfactory epithelium (Pevsner et al., 1988a). The protein has been cloned and sequenced (Lee et ul., 1987; Pevsner et al., 1988b) and it is a member of the a2-microglobulin family of carrier proteins. T h e properties of this protein have been reviewed by Snyder et al. (1988b). There have been speculations as to the function of this protein. If an odorant partitions between air and an aqueous medium, it achieves a higher concentration in the liquid than in the gas. Hence, the olfactory mucus does not pose a permeability barrier to odorants and a carrier protein cannot facilitate diffusion under these circumstances; in fact, it will retard diffusion by virtue of its high molecular weight. It is unlikely, therefore, that the odorant-binding protein is part of a simple delivery system, as suggested by Schofield (1988). An alternative suggestion is that the protein-containing secretion is atomized near the naris in the rat to scrub the incoming air and act as part of an odorant delivery system, but the physical design of the olfactory cavity is such that the probability of deposition of an aerosol particle is less than that of deposition of an odorant direct from the gas phase. If the protein serves as a prereceptor or as part of an odor-clearance mechanism for the olfactory epithelium, it is difficult to see why it should be produced so far from the epithelium in the rat and have no mechanism for delivering it there (the flow of mucus is away from the epithelium). Perhaps the most likely function is as a scavenger to keep the air passages in general clean, and it is fortuitous that some of the protein is produced in the olfactory epithelium of the cow.

VII. Conclusion

Much work remains to be done for a full understanding of the olfactory system and it requires collaboration across many different disciplines. Aerodynamics and physical chemistry can help define the concentration profiles of materials in the mucus and lead to an understanding of stimulus dynamics. A great goal for the biochemists and

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41

molecular biologists is the separation of the various types of receptor molecules, as this leads to an understanding of the response spectrum of each, definitive structure-activity relationships, and the promise of the understanding of the chemistry of odor-receptor interactions. Beyond this it may lead to the ability to “type” cells according to their receptor content, a technique invaluable in the understanding of system function and of the guidance processes involved in regenerative glomerulus selection and synapse formation. The cell physiologists have the problem of the arrangement of the receptor molecules on cells and cellular control, the stoichiometry of the transductory system, and also the mechanisms whereby and the extent to which primary cells act as information processors rather than as simple transducers. T h e system physiologists and neuroanatomists will contribute an understanding of the neural mechanisms involved in olfactory information processing and their changes with learning. T h e fields of psychology, animal behavior, and information theory also have much to contribute. But above all it is necessary to appreciate the relationships between the various fields of study.

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Snyder, S. H., Sklar, P. B., and Pevsner, J. (1988a).J. B i d . C h m . 263, 13971-13974. Snyder, S. H . , Sklar, P. B., and Pevsner, J. (1988b). In “Molecular Neurobiology of the Olfactory System: Molecular, Membranous and Cytological Studies” (F. L. Margolis and T. V. l o &kg) in young monkeys, but the aged animals were more sensitive in this regard, with the lO-pg/kg dose producing marked sedation and impairment in performance. The sedative action of clonidine itself does not appear to be the cause of the enhanced performance (Arnsten et al., 1988). Again, despite these promising results in nonhuman primates, clinical trials using clonidine and the related drug guanfacine did not improve the neuropsychological rating of intellectual and memory function in Alzheimer’s patients (Schlegel et al., 1989). This disparity in results regarding Korsakoffs patients indicated above may reflect a more consistent or dramatic loss of cortical noradrenergic innervation associated with the latter disease. It is possible that certain subpopulations of dementia patients, including Alzheimer’s dementia, might still benefit from therapy with central a,-adrenergic agonists. Also, it is generally recognized that Alzheimer’s disease involves alterations in several neurotransmitter systems and it may be necessary to address pharmacologically these multiple deficits.

VI. Summary and Conclusions

A. THEDIVERSITY OF PHARMACOLOGICAL ACTIONS T h e considerable number of pharmacological actions reported for clonidine as indicated in Table I and the substantial number of potential and actual clinical uses (Table 11) are probably unprecedented for a single pharmacological entity. Such a diverse pharmacological profile is undoubtedly a reflection of a diverse mechanism of action. Clonidine

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and related drugs have been demonstrated to interact with classical neurotransmitter systems, the catecholamines, indolamines, cholinergic, opioidergic, and amino acid transmitter systems. For each action of clonidine no single mechanism has been clearly identified as mediating the pharmacological effect. Perhaps the only common link is that most, if riot all, of its actions are mediated through stimulation of a,-adrenergic receptors (Table I). However, even this direct mechanism may be complicated. T h e emergence of multiple clonidine-binding sites as well as the discovery of a novel (nonadrenergic) iniidazole receptor allow for an even greater diversity in its mechanism of action. Also to be considered is the strategic location of clonidine-binding sites. The location of such a site on the neuron soma could have a completely different effect in terms of excitability of the cell if the location of the receptors is the nerve terminals. T h e role of clonidine’s presynaptic actions, particularly regarding central catecholamine systems, has been addressed several times. In many of clonidine’s actions such an inhibitory effect on catecholamine release has not appeared to play an important role. Nevertheless, inhibition of catecholamine release cannot be discounted as an important contributor to clonidine’s long-term actions. It is not yet clear whether “postsynaptic” clonidine-binding sites are innervated by catecholamine nerve terminals. For example, although clonidine’s inhibitory action on central cholinergic transmission is inhibited by a,-adrenergic antagonists, blockade of these receptors in the absence of clonidine does not lead to increased acetylcholine release (Buccafusco and Spector, 1980a). Therefore, either the clonidine receptors on cholinergic neurons are not innervated or the putative noradrenergic tone is quiescent under normal circumstances. In either case, it is possible that such receptors, located on cholinergic or on other systems, continually sample and respond to changes in catecholamine levels in the cerebrospinal fluid. The mutually antagonistic action of norepinephrine and acetylcholine in dually innervated autonomic effector organs is enhanced through presynaptic modulation. That is, norepinephrine overflow during periods of intense sympathetic stimulation can reduce the release of acetylcholine from parasympathetic nerve endings. This relationship between adrenergic and cholinergic neurons may be further amplified in the CNS, which is perhaps more of a closed system than the peripheral circulation. This concept of neurotransmission via the brain extracellular fluid has more recently been termed volume transmission (Fuxe and Agnati, 1991). Volume transmission was originally invoked in part to explain the presence of neurotransmitter receptors at extrasynaptic sites, but the concept may also help to explain the lack of effect of ag-

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adrenergic blocking drugs on cholinergic function indicated above. For example, if central cholinergic activity is indeed modulated by extracellular levels of catecholamines, it is possible that after chronic treatment with clonidine, decreased levels of catecholamines in the cerebrospinal fluid could have an impact on cholinergic neurotransmission. Another factor contributing to the diversity of clonidine’s actions is the nature of the signal transduction processes reported to be activated by the drug. Consistent actions on neuronal cAMP and related systems have been difficult to obtain. Clonidine’s effect on brain cAMP have been demonstrated to be dependent upon the brain region examined and other factors related to specific preparations (see Janowsky and Sulser, 1987; Nakamichi et al., 1987). Part of the problem may also reside in the ability of clonidine to increase intracellular pH (see Marx, 1987). T h e change in pH has been related to clonidine’s ability to enhance Na+ / H exchange as demonstrated in platelets following stimulation of a,-adrenergic receptors. The increase in intracellular pH may trigger the decrease in cAMP often observed following a,-adrenergic receptor simulation in these cells. +

B. CLONIDINE AS A NEUROMODULATOR Despite this diversity of action, clonidine has been employed clinically for several years quite successfully. Although newer related drugs have purported to be associated with less severe side effects than clonidine, in fact, the drug is well tolerated by a large proportion of patients. Perhaps this selectivity is related to the drug’s marked potency for central autonomic pathways. This action is important for its antihypertensive and perhaps its antiwithdrawal properties, the current main clinical applications. In this respect, it is interesting that clonidine is a better antihypertensive agent than it is a hypotensive agent. Thus, in the presence of disease the drug’s actions are more apparent. The inhibitory action of clonidine on neurotransmitter systems is generally modulatory, its effectiveness in inhibiting transmitter release frequency dependent. Unlike direct receptor-blocking agents, clonidine’s modulatory ability could allow for a more subtle degree of regulation. Even in high doses, for example, clonidine does not usually alter the steady-state levels of neurotransmitter and does not completely inhibit the release or synthesis process. Finally, part of clonidine’s selectivity may be related to its singular effectiveness and potency in inhibiting central and peripheral cholinergic muscarinic activity. If this possibility has merit, then

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the role of central cholinergic neurons in mediating many of clonidine’s phartnacological properties as well as in the disease process itself deserves further investigation.

VII. Future Directions

There is no doubt that clonidine will continue to be employed as the experimental drug of choice for investigations examining the effect of central a,-adrenergic receptor stimulation. Some of the clinical indications for the drug will disappear, possibly its use as an antipsychotic or antianxiety agent. However, the use of the drug in withdrawal syndromes will continue to be examined. Clonidine’s analgesic actions will continue to be exploited and may actually surpass the use of opiates in the production of localized spinal analgesia and as a supplement to general anesthetics, before, during, and after surgical procedures. T h e advantage of clonidine and related drugs in this regard is the reduced capacity for producing central respiratory depression and an almost nonexistent abuse liability. Although the use of clonidine as a first-line antihypertensive agent has been supplanted by newer agents targeting peripheral nerves or blood vessels, a reexamination of clonidine’s property as a central sympatholytic agent may be in order. It is generally appreciated that lowering of blood pressure in hypertensive disease per se does always result in protection from secondary cardiovascular complications such as coronary artery disease, left ventricular hypertrophy, vascular damage to the eyes, kidney, and brain, and the production of ventricular arrhythmias (Rosenman, 1989). This kind of toxicity has been ascribed to excessive catecholamine excretion subsequent to enhanced sympathetic activity often associated with essential hypertension. In fact, the use of certain classes of peripherally acting antihypertensive agents may actually enhance sympatho-adrenal outflow though activation of cardiovascular reflex activity o r through plasma sodium and fluid loss (1220, 1989). To date there have been no large or multicenter studies regarding the incidence of cardiovascular-related morbidity or lethality following longterm treatment with a central versus peripheral antihypertensive treatment regimen. If such a study confirms the cardiovascular protective action of clonidine and related drugs, a resurgence in the utilization of this class of antihypertensive agent would be expected. It might be point-

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ed out that clonidine has been demonstrated to provide benefit in chronic heart failure and ischemic heart disease (Giles et al., 1985) and that in hypertensive rats, clonidine significantly reduced blood pressure and heart rate fluctuations, reflecting an enhanced baroreceptor control (Grichois et al., 1990). Finally, clonidine and related drugs may continue to be examined for potential antidementia effects. Despite the disappointing initial results in Alzheimer’s patients mentioned above, clonidine may exhibit beneficial actions in certain categories of human dementia. It might also be borne in mind that central cholinergic agonists such as physostigmine have produced limited benefit in Alzheimer’s patients. The possibility that combined treatment with clonidine and physostigmine may produce an added benefit fits with the observation of multiple neurotransmitter alterations in Alzheimer’s disease. Several studies (discussed above) indicate that clonidine can inhibit many of the pharmacological effects produced by cholinesterase inhibitors, but this does not necessarily imply incompatibility for their use in dementia. The reverse may actually be the case. Although clonidine does inhibit central cholinergic function, studies in our laboratory have demonstrated that this action is selective for certain brain regions. For example, the striatum and hippocampus, which exhibit a high degree of cholinergic innervation, are essentially spared from the inhibitory action of clonidine on their cholinergic neurons (Buccafusco and Spector, 1980a; Buccafusco, 1984a). One hypothesis that is under current investigation in this laboratory is that combined treatment with clonidine and physostigmine results in a greater than additive effect in a delayed matching paradigm in young and aged monkeys. In this situation, it is envisioned that clonidine may have some palliative action of its own on memory and/or learning processes, but may also obviate some of the autonomic side effects associated with physostigmine administration. This selective protective action of clonidine may be due to its inability to interfere with cholinergic function in the hippocampus, a crucial site for memory formation. Along these lines, it has been demonstrated that in rats having lesions of the ascending noradrenergic bundle, the memory-enhancing effect of physostigmine was blocked. A combination of physostigmine and clonidine was required to restore the beneficial effects of central cholinergic stimulation in the lesioned animals (Haroutunian et al., 1990). Thus, clonidine may widen the therapeutic window for physostigmine’s beneficial actions by reducing interfering side effects and by contributing to the relief of noradrenergic deficits associated with aging or the disease process.

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DEVELOPMENT OF THE LEECH NERVOUS SYSTEM Gunther S. Stent,* William 6. Kristan, Jr.,t Steven A. Terrence: Kathleen A. French,t and David A. Weisblat* *Department of Molecular and Cell Biology University of California, Berkeley, Berkeley, California 94720 tDepartment of Biology University of California, San Diego La Jolla, California 92093

I. Introduction to the Leech A. Historical Background B. Taxonomy C. Gross Anatomy D. The Leech Nervous System 11. Morphological Development and Staging 111. Behavioral Development IV. Developmental Cell Lineage A. Cell Lineage Tracing B. Genealogical Origins of the Segmental Neurons C. Origin of the Supraesophageal Ganglion D. Transfating V. Myogenesis and Neurogenesis A. Myogenesis B. Gangliogenesis C. Neurochemical Differentiation D. Electrophysiological Differentiation E. Morphological Differentiation F. Interactions between Neurons and Their Peripheral Targets G. Neuron-Neuron Interactions: The Origins of Unpaired Neurons VI. Conclusions References

1. Introduction to the leech

The nervous system presents two of the most challenging questions of contemporary biology: How do networks of neurons generate animal behavior? And how do the neurons and their specific connections arise during the development of an animal from the fertilized egg? The second question cannot be considered independently of the first, because 109 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 33

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the anatomy and function of the adult nervous system represent the endpoint of neural development. That is to say, detailed anatomical and functional knowledge is needed even to ask, let alone answer, wellfocused questions in developmental neurobiology. For gaining such knowledge, the leech with its relatively simple and electrophysiologically highly accessible nervous system is particularly suitable.

A. HISTORICAL BACKGROUND

'The tfierapeutic use of leeches, which had reached the peak of its popularity in the mid-nineteenth century, stimulated basic research on their reproduction, development, and anatomy. For example, leeches were the working material of one of the nineteenth century pioneers of modern experimental embryology, Charles 0. Whitman. In the 1880s, Whitman presented the first analysis of developmental cell lineage, describing the successive cleavages leading from the fertilized leech egg to the early embryo and the subsequent morphogenetic cell movements leading to the juvenile leech. On the basis of his studies, Whitman (1878, 1887) put forward the idea, then quite novel, that each identified cell of the early embryo, and the clone of its descendant cells, play a specific role in development. Despite these highly promising beginnings, embryological interest in leeches declined after the turn of the century. Similarly, the nervous system of the leech was studied by nineteenth century pioneers of modern neuroanatomy, such as Santiago Ramon y Cajal (1904) and Gustav M. Retzius (1891). Neurobiological interest in leeches also declined after the turn of the century, not to be rekindled until the 1960s, when Stephen Kuffler and David Potter first applied modern electrophysiological techniques to study glial cells in the leech nervous system (Kuffler and Potter, 1964). Their work was continued by John G. Nicholls and his students, who ascertained the modalities, receptive fields, and response characteristics of leech sensory neurons (Nicholls and Baylor, 1968), the fields of peripheral action of effector neurons (Stuart, 1970), and the integrative role of connections from the sensory neurons to the motor neurons (Nicholls and Purves, 1970, 1972). These findings, in turn, allowed the identification of specific cells and their synaptic connections responsible for generating not only some simple reflexes, but also some moderately complex integrated motor acts (Stent and Kristan, 1981). Acquiring such detailed knowledge of functional elements within the leech nervous system made it possible to ask specific, focused questions regarding the system's development. For that reason, and in view of the

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classical body of knowledge regarding its embryogenesis (Schleip, 1936; Dawydoff, 1959; Anderson, 1973), the leech seemed to offer considerable promise as an experimental object for developmental neurobiology. Thus studies of leech neurodevelopment began anew in the mid-1970s. This article presents an overview and synthesis of these recent studies; more detailed reviews of specific features of leech development are available '(Weisblat, 1981; Kristan et al., 1984; Weisblat & Shankland, 1985; Stent, 1985; Shankland and Stent, 1986; Weisblat and Astrow, 1989; Levine and Macagno, 1990; French and Kristan, 1991).

B. TAXONOMY Leeches form the class Hirudinea in the phylum Annelida. They are so closely related to earthworms (class Oligochaeta) that, together, leeches and earthworms are assigned to the superclass Clitellata (Anderson, 1973; Sawyer, 1972, 1986). An important difference between leeches and earthworms, however, is the number of body segments. In earthworms that number is variable, owing to continual addition of new segments from a posterior growth zone throughout life, whereas the number of body segments in leeches is constant after early embryogenesis (Harant and GrassC, 1959; Mann, 1962). The constant segment number in leeches may result from their possession of a caudal sucker, a structure that is developmentally incompatible with the type of posterior growth zone found in earthworms (Sawyer, 1981). Constancy of segment number allows a higher degree of specialization in different body regions than is found in earthworms, and leeches differ from earthworms, both anatomically and behaviorally, in ways that make them favorable experimental preparations for studying neurodevelopment. For instance, the earthworm nervous system consists of a diffuse distribution of metameric neurons along the ventral midline, whereas the leech CNS is organized into a chain of discrete segmental ganglia (Bullock and Horridge, 1965). T h e class Hirudinea comprises three orders (Pharyngobdellida, Gnathobdellida, and Rhynchobdellida), of which the latter two have been the object of neurobiological studies. Both of these orders feed by bloodsucking, but gnathobdellids bite the host with toothed, rasping jaws, whereas rhynchobdellids insert a muscular proboscis into the host. Most studies of the adult leech nervous system have been on species in the gnathobdellid order, especially on members of the family Hirudinidae, such as Hirudo medicinalis. In contrast, most neurodevelopmental studies have been on the embryologically more favorable

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GL'NTHER S. STENT t t a1 POSTERIOR

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FIG. 1. Body plan of the medicinal leech, Hirudo mtdicinalis. (A) Drawing of the dorsal aspect of an adult leech, indicating the location of the ventral nerve cord and its segmental ganglia. The external surface is divided by a series of circumferential grooves; the space between adjacent grooves is an "annulus." The large dots indicate the central annulus of each of the 21 midbody segments, numbered in rostrocaudal order. Most midbody segments contain five annuli, as indicated by the arrows bracketing segment 8, and each central annulus has several sensilla, indicated by small dots. The anterior brain includes a single pair of supraesophageal ganglia and four fused subesophageal ganglia; the posterior brain is composed of seven fused segmental ganglia. There are five pairs of eyes, one pair in each of the segments innervated by the anterior brain and one pair in the first midbody segment. The anterior sucker, containing the mouth, is on the ventral surface, just behind the anterior brain. (B) Drawing o f a transverse cut through the midbody, indicating the locations of the major muscle layers and blood vessels, as well as the nerve cord and the gut. The lateral blood sinuses serve as heart tubes because, unlike the dorsal and ventral blood sinuses, they have muscles in their walls and contract to move blood in the closed circulatory system. Two nerve roots-anterior and posterior-emerge from each ganglion and enter the body wall. The anterior root splits into an anterior and medial

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rhynchobdellid order, especially species in the family Glossiphoniidae: Helobdella, triserialis, Theromyzon rude, and Haementeria ghilianii. Helobdella triserialis is native to North America and feeds on aquatic snails. It reaches an adult length of 1-2 cm and propagates with an egg-to-egg generation time of about 9 weeks (Sawyer, 1972). Theromyzon rude is larger (about 2-4 cm in length) and feeds on the blood of aquatic birds, especially ducks; T. rude has not been successfully cultivated in the laboratory, but can be readily collected from ponds; its generation time in the wild is thought to be 1 year, Haementeria ghiliunii is native to South America and feeds on the blood of mammals. It reaches an adult length of up to 50 cm and has an egg-to-egg generation time of about 10 months in laboratory cultivation (Sawyer et al., 1981). The short generation time, simplicity of cultivation, and hardy embryo of Helobdella make it favorable for developmental studies, but its small size renders it less favorable for neurophysiology. In contrast, the enormous size of Haementeria makes even its embryonic nervous system accessible to recording and injection techniques that require penetrations of single cells, but the long generation time, demanding breeding conditions, and the more fragile embryos present drawbacks in comparison with Helobdella. Theromyzon embryos are as hardy as those of Helobdella and yet are large enough to provide some of the advantages of Haementeria. Fortunately, despite differences in size and habit, the three species are similar in adult body plan and embryonic development so that, for many purposes, the results obtained with one are applicable to the other two.

C. GROSSANATOMY

The tubular body of the leech consists of 32 segments, plus a nonsegmental prostomium (Fig. 1A). The anteriormost four (“head”)segments are fused, forming specialized cephalic structures, including pairs of eyes dorsally (0-9 pairs in different species) and a mouth surrounded by the anterior sucker ventrally. The posteriormost seven (“tail”)segments are also fused, forming the large caudal sucker. Between fused head and tail segments lie 21 unfused midbody segments, designated in rostrocaudal order as M1 to M21. (These ganglia have also been called nerve, and the posterior root splits into a dorsal and posterior nerve. All the major nerves are mixed, containing both sensory and motor axons. Axons run between ganglia via the connectives, consisting of a pair of large connectives and a single smaller connective between adjacent ganglia. (These drawings are variations of ones from Nicholls and Van Essen, 1974.)

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“abdominal” and “segmental” ganglia.) Along the entire length of the body, the skin is subdivided into circumferential rings, or annuli, of which there is a constant, species-specific number per midbody segment. This num’ber is 5 in the case of H i d o , whereas it is 5 on the ventral and 3 on the dorsal aspect of Haementeriu. In all species the number of annuli per segment is reduced in the head and tail segments, as well as in some of the anteriormost and posteriormost midbody segments. T h e central annulus of each segment contains a set of circumferentially distributed sensory organs, or sensilla, which comprise photo- as well as mechanoreceptors (Kretz et al., 1976; Derosa and Friesen, 1981).T h e excretory system of the leech consists of paired, metameric nephridia distributed in a species-specific mode over most of the midbody segments. Each individual nephridium excretes urine via a nephridiopore located on the ventral aspect (Hardnt and GrassC, 1959; Mann, 1962). Segments M 5 and M6 are the reproductive segments. T h e male pore, penis, and vas deferens lie in M5; sperm are produced and stored in metameric testes distributed over several more posterior segments. The paired ovaries lie in M6; eggs are fertilized internally and are laid through the female pore of M6. Below the epidermis lie three layers of muscle fibers (Fig. 1B). The outer layer consists of circumferential “circular” muscle fibers and the inner layFr of longitudinal fibers. T h e intermediate layer is formed by two thin sheets of crossed oblique muscle fibers: the fibers of one sheet lie at an angle of +45” and those of the other sheet lie at an angle of -45” to the longitudinal axis. T h e body of the leech is traversed by a fourth, dorsoventral set of muscle fibers, which insert into the dorsal body wall at one end and into the ventral body wall at the other. The length of individual longitudinal fibers is variable, ranging from about two-thirds of a segment to two segments (Cline, 1986); the lengths of the other muscle fibers in adult animals are not known. All four types of muscle fibers are arranged in discrete parallel fascicles. Each segment contains a fixed number of identifiable fascicles, with a fixed number of each type of muscle fascicle per segment. Contraction of each type of muscle works against the hydrostatic skeleton provided by the fluid-filled leech body tube to effect a characteristic change in body shape: contraction of the circular fibers causes constriction and lengthening, contraction of the longitudinal fibers causes shortening, and contraction of the dorsoventral fibers causes flattening and lengthening. T h e effect of contraction of the oblique fibers depends upon which other types of fibers happen to be contracting. During longitudinal fiber contraction (i.e., in a shortened animal), oblique fiber contraction produces elongation; during circular fiber contraction (i.e., in a fully extended animal), oblique

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fiber contraction produces shortening; when no other fibers are contracted, contraction of the oblique fibers stiffens the body wall at an intermediate body length. A fifth set of muscles, the annulus erectors (Stuart, 1970), is composed of short longitudinal fibers that traverse a single annulus just below the epidermis. Contraction of the erectors raises the annuli, forming a series of sharp ridges that make the epidermis resemble a washboard’s surface. D. THELEECHNERVOUS SYSTEM

1. Ventral Neme Cord T h e leech nervous system reflects the segmental body plan (Fig. 1A). T h e CNS consists of a ventral chain of 32 segmentally iterated ganglia (Mann, 1962). The anteriormost four and posteriormost seven segmental ganglia are fused, constituting rostral and caudal ganglionic masses (or “brains”),respectively. The rostral ganglionic mass, or subesophageal ganglion,’is linked at its anterior end via two circumesophageal connectives to a dorsally situated supraesophageal ganglion. The supraesophageal ganglion is part of the prostomium, and hence, unlike all other ganglia, is not a segmental organ. T h e unfused segmental ganglia are linked via an unpaired, median connective, called “Faivre’s nerve,” and two paired, lateral connective nerves. The connective nerves contain, in addition to interganglionic axons, several longitudinal muscle fibers whose contraction or distension is coordinated with changes in body length caused by the body wall musculature. Each segmental ganglion contains about 200 bilateral pairs of neurons (Macagno, 1980), as well as a few unpaired neurons (Fig. 2). Their cell bodies form an outer cortex around the ventral and lateral aspects of the ganglion. The neurons are monopolar; their processes project initially into a central neuropil, where they make synaptic contacts. From there, the axons of some neurons project to other ganglia via the connective nerves. Sensory and effector neurons send processes to peripheral targets via segmental nerves, whose roots emerge from the lateral edges of the ganglion. From either side of the typical midbody ganglion (Fig. 1B) emerge four main segmental nerves: the anteroanterior (AA), the medioanterior (MA), the dorsoposterior (DP), and the posteroposterior (PP) nerves (Ort et al., 1974). The detailed anatomy of these nerves differs somewhat among various leech species. The segmental nerves arborize extensively in the body wall, but all preserve a main, circumferential nerve trunk. At the dorsal midline, right and left main nerve trunks anastomose to form circumferential nerve rings.

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FIG.2 . Disposition of neuronal cell bodies on the ventral and dorsal surfaces of a segniental ganglion of Hir-udo medicinah. Cell bodies are distributed in a single layer around the cortex of the ganglion. The continuous lines with the ganglion indicate cell packet margins. Filled outlines indicate the approximate position of identified neurons of different function: sensory, motor, modulatory, or interneurons. Also indicated are identified neurons of unknown function, termed “partially characterized,” and as yet uncharacterized neurons. Most of the identified neurons are found in all ganglion, although some are found in specialized regions of the body. [Details of the identity of most of these neurons, a s well as references to their original identification, can be obtained from Muller et al. (1981).]

In each ganglion, the neuronal cell bodies are distributed among six cell packets: a pair of anterolateral packets, a pair of posterolateral packets, and a pair of ventromedial packets. The latter pair lie anteriorly and posteriorly in all but the anteriormost ganglia, where they lie nearly side by side. Each neuronal packet is enveloped by one giant glial cell. In addition, two giant glial cells are associated with the ganglionic neuropil, and additional giant glial cells are present in the interganglionic connective nerves (Coggeshall and Fawcett, 1964; Weisblat et al., 1980b). T h e anatomy of the leech ganglion is sufficiently stereotyped, and its cell bodies are sufficiently accessible to electrophysiological and anatomical analysis, that a substantial fraction of its neurons have been identified (Fig. 2). After characterizing a particular neuron in a particular ganglion of a particular specimen according to morphological and physiological criteria, homologous neurons can usually be found on the other side of that same ganglion, in other ganglia of that same specimen, in the

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ganglia of other specimens of the same species, and even in other leech species, families, or orders (Muller et al., 1981). It is likely that all neurons of the segmental ganglia are identifiable in this sense. Despite this high degree of neural stereotypy, some systematic variations in the number of cells do occur among different segmental ganglia within the same nerve cord and among corresponding ganglia in the nerve cords of different leech species. For instance, in H. medicinal&, the ganglia in the two reproductive body segments M5 and M6 contain nearly twice as many cells as do the other midbody ganglia; in H . ghilianii the corresponding ganglia contain only about 5% more cells than do ganglia in nonreproductive segments (Macagno, 1980).Moreover, some slight variations in the exact number of neurons per ganglion has been found among corresponding ganglia from different individuals of the same species. This “developmental noise” amounts to a variance of about 1% in the total number of neurons per corresponding ganglion, thus placing a conceptual limit on the idealized picture of the segmental ganglion as a fixed set of uniquely identifiable neurons. In fact, in at least one specimen of H . medicinalis, a set of supernumerary neurons corresponding to identified cell types was observed (Kuffler and Muller, 1974).

2 . Identijied Cells About one-quarter of the neurons in the segmental ganglia of H. medicinalis have been identified according to various criteria, including function (Fig. 2). Thus, many cells have been classified as sensory, effector, or interneurons, and their connectivity has been elucidated (Nicholls and Baylor, 1968; Baylor and Nicholls, 1969; Stuart, 1970; Nicholls and Purves, 1972; Lent, 1973; Ort et al., 1974; Thompson and Stent, 1976a,b,c; Friesen et al., 1978; Muller, 1979, 1981; Friesen, 1985; Nusbaum and Kristan, 1986; Lockery and Kristan, 1990b). These surveys have culminated in the description of sensory pathways and of neuronal networks controlling various behaviors, such as body shortening, heartbeat, and swimming (Stent et al., 1978, 1979; Kristan et al., 1988; Friesen, 1989). Despite their considerable phyletic distance from the hirudinid Hirudo, the glossiphoniid species share with Hirudo not only the same general structure of the CNS, but even many of the identified neurons (Kramer and Goldman, 1981). The identified sensory neurons include three types of mechanosensory cells, designated as T (for touch), P (for pressure), and N (for nociception) (Nicholls and Baylor, 1968).Each of these neurons projects its axons from the ganglion to a particular territory of the segmental skin, where its endings form specialized mechanoreceptors that respond specifically to slight (T),moderate (P), or intense (N) deformation of the

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GUNTHER S. STENT el al.

skin (Yau, 1976; Blackshaw and Nicholls, 1979; Blackshaw, 1981a,b). In the typical midbody ganglion there are pairs of T , TD,T,*,P , and P, cells, for which the subscripts v, D, and L designate that the ipsilateral ventral, dorsal, o r lateral skin, respectively, is the principal territory of innervation. T h e Hirudo ganglion has two pairs of N cells, whereas the Haementerza ganglion has only one pair; in both species, each N cell appears to innervate the entire hemilateral skin (Blackshaw, 1981c; Kramer and Goldman, 1981). Ganglia in specialized segments, e.g., the reproductive segments, contain a somewhat different complement of mechanosensory neurons, a pattern that varies from species to species (Johansen et ul., 1984). ‘The identified effector neurons include an ensemble of about two dozen paired excitatory and inhibitory motor neurons, each of which innervates a particular type of muscle in a particular territory within the contralateral segmental body wall (Stuart, 1970; Ort et ul., 1974; Norris and Calabrese, 1990). In addition, there are neurons that have modulatory effects on muscles and other neurons. For instance, the largest neurons in most midbody segments are the pair of Retzius neurons, which, by releasing serotonin, cause mucus release onto the skin surface (Lent, 1973), increase the rate of muscle contraction and relaxation (Mason et ul., 1979; Mason and Kristan, 1982), increase the probability of both swimming (Willard, 1981) and feeding (Lent and Dickinson, 1989), and increase the magnitude of both local bending and shortening movements (Lockery and Kristan, 1991; Wittenberg, 1991). Another pair of neurons, the Leydig cells, large opalescent somata at the posterolateral marginal of each midbody ganglion, influence the strength of the heartbeat (Calabrese and Arbds, 1989) and of the local bending response (Lockery and Kristdn, 1991). T h e identified interneurons include some of the few unpaired cells, the best studied of which is intersegmental interneuron S, whose giant axon courses in the median connective nerve. T h e axons of the single S cell in each ganglion are linked via strong electrical junctions to the axons of the homologous S cells in both the next anterior and the next posterior ganglion, so that an action potential arising in one segmental S cell is rapidly propagated over the entire nerve cord. This chain of electrically linked giant axons forms a fast through-conducting system over the whole length of the leech CNS (Frank et ul., 1975; Magni and Pellegrino, 1978; Muller and Carbonetto, 1979). Mechanosensory neurons are linked to the S cell chain via “coupling interneurons,” a single pair of small neurons that are dye coupled to the S cell in each ganglion (Muller and Scott, 1981).In addition, interneurons have been identified that contribute to the generation of the heartbeat (Thompson and Stent,

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1976a,b,c; Stent et al., 1979; Calabrese et al., 1989), swimming (Friesen et al., 1978; Poon et al., 1978; Weeks, 1982a,b; Friesen, 1985, 1989; Nusbaum and Kristan, 1986; Brodfuehrer and Friesen, 1986), local bending (Lockery and Kristan, 1990b), and shortening (Wittenberg, 1991). Finally, a few neurons have been identified by their morphology, by their electrical properties, o r by their connections to other neurons (Muller et al., 1981; Wadepuhl, 1989), but their behavioral functions remain unknown. There are also identified neurons found only in particular regions of the leech-in the supraesophageal ganglia, for instance, or in the midbody segments devoted to reproduction. These neurons have not been indicated in Fig. 2, and their identifying characteristics will be given as they are discussed in later sections. 3. Neurotransmitters T h e characterization of leech neurons has been extended to the identification of actual or putative neurotransmitters by electrophysiological, pharmacological, autoradiographic, histochemical, and immunohistological techniques. For example, the identified excitatory motor neurons innervating the body wall muscles are cholinergic (Kuffler, 19’78),whereas the corresponding identified inhibitory motor neurons are y-aminobutyric acid (GABA)ergic (Cline, 1986). There may be additional cholinergic and GABAergic neurons in the segmental ganglia, because many other cells contain choline acetyltransferase activity (Sargent, 1977) and a specific cholinesterase activity (Wallace and Gillon, 1982), as do the muscle excitors; other neurons have a high-affinity uptake system for GABA (Cline, 1983, 1986), as do the muscle inhibitors. Furthermore, glutamic acid (the metabolic precursor to GABA), known to serve as a neurotransmitter in other nervous systems, is concentrated in a small number of neurons in the anterior portion of the leech CNS (Brodfuehrer and Cohen, 1990); the release of glutamate is thought to mediate the initiation of swimming behavior. Neurons containing monoamine neurotransmitters are present in the leech as well. Segmentally iterated serotonin-containing neurons, referred to here as “serotonin neurons” (Fig. 3) (Rude, 1969; Stuart et al., 1974), can be identified by several criteria: they emit yellow-green fluorescence after exposure to formaldehyde or glyoxylic acid (Lent, 1982; Stuart et al., 1987), they accumulate the dye neutral red (Stuart et al., 1974), they react with an antiserotonin antibody (Stuart et al., 1982; Jellies et al., 1987), and they selectively accumulate serotonin from the extracellular fluid (Glover and Stuart, 1983). Each segmental ganglion includes three pairs of serotonin neurons: the Retzius (R) cells and two

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Frc,. 3. Identification of monoamine-containing cells in a midbody segment of Haemenfe% g/zz/iattzi.This is a summary diagram of monoamine-containing cell types found in midbody segments. based on histofluorescence induced by treatment with glyoxylic acid. The yellow-green fluorescence characteristic of serotonin is seen in nine cell bodies: the giant Retzius (R) cell pairs, the dorsolateral (dls) and ventrolateral (vls) cell pairs, and the paired anteromedial (anis) and unpaired posteromedial (pms) cell pairs. The blue-green fluorescence characteristic of dopamine is seen in three pairs of peripherally located cell bodies ( L D I , LD2, and MD) and their extensive arborizations (not indicated) within the central neuropil. Anterior is up; ganglia in adult leeches are about 500 Frn in width. (Drawing provided by D. K. Stuart.)

other pairs of smaller cells designated as dorsolateral (dls, or cell 2 1) and ventrolateral (vls, or cell 61) neurons. Most ganglia also contain one unpaired posteromedial (pms) neuron (Blair, 1983; Lent et al., 1979; Nusbaum and Kristan, 1986; Leake, 1986; Stuart et al., 1987). In Haementerin and Helobdella, pms is present only in ganglia Ml-M7 (Stuart et ad., 1987), and there is also an anteromedial (ams) pair of serotonin neurons in ganglia Ml-M3. In addition to labeling serotonin neurons, glyoxylic acid treatment induces a blue-green fluorescence in segmentally iterated dopaminecontaining neurons, referred to here as “dopamine neurons” (Fig. 3). One pair, designated as the medial dopamine (MD) neurons, is located just beyond the margin of the ganglion in all midbody segments except M 1. There, and in the head and tail segments, the MD pairs are located within the ganglia (Wallace, 1981). In glossiphoniid species, there are two additional pairs of dopamine neurons, designated as lateral dopamine neurons, o r LD1 and LD2. They lie near the lateral edge of the body wall, within the trunks of the PP and AA segmental nerves, respectively (Blair, 1983; Stuart et al., 1987). T h e dopamine neurons project axons into the CNS, where they arborize profusely throughout the ganglionic neuropil.

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A third biogenic amine, octopamine, has also been identified in the leech nervous system (Webb and Orchard, 1980, 1981). Localization of octopamirie is more problematic than the other two amines because it is not fluorogenic when treated with formaldehyde or glyoxylate, and there is no specific antibody to it. Despite this, radioenzymatic assays have shown that the cell bodies of the Leydig cells account for most of the octopamine in the ganglion (BClanger and Orchard, 1986); octopamine is thought to play a neurohormonal role in modulating activity levels (BClanger and Orchard, 1988). In addition to low-molecular-weight neurotransmitters, several neuropeptide transmitter candidates have been found in the leech nervous system. Neuropeptides related to, or derived from, FMRFamide are probably the best studied of these substances found in the leech. Antibodies to FMRFaniide stain about 40 cells per midbody ganglion, including almost all of the excitatory motor neurons except the excitors to the circular muscles and to the lateral dorsoventral muscles (Kuhlman et al., 1985; Evans and Calabrese, €989; Norris and Calabrese, 1990). Other neurons stained by anti-FMRFamide antibody include a swim-initiating interneuron (cell 204) and an inhibitory motor neuron (cell 101). HPLC analysis of Hirudo nerve cord extracts has revealed the presence of four FMRFamide-like immunoreactive peptides, including FLRFamide, YMRFamide, and, YLRFamide, as well as FMRFamide itself (Evans et al., 1990). The FMRFamide family of peptides modulates neuromuscular activity in the leech, in part by gradually potentiating the response of muscles to acetylcholine (Norris and Calabrese, 1990). Some of the neurons staining with anti-FMRFamide antibody also stain with antibody against the small cardiac peptide (SCP) B (Shankland and Martindale, 1989; Evans and Calabrese, 1989), although chromatographic analyses of Hirudo nerve cord failed to demonstrate the presence of authentic SCP (B. Evans and R. Calabrese, personal communication). Evidence for other leech neuropeptides is limited to immunohistochemical data. These include a proctolin-like immunoreactivity in inhibitory motor neurons (Li and Calabrese, 1985); substance P-like immunoreactivity in a single pair of neurons at the base of the circumesophageal commissures of the head brain (S. Ranganathan, D. K. Stuart, and L. Gleizer, personal communication); red pigmentconcentrating hormone (RPCH)-like immunoreactivity in approximately a dozen neurons in each segmental ganglion, plus others in the head and tail brains, including the supraesophageal ganglion (M. Nusbaum, personal communication); and enkephalin-like immunoreactivity in a pair of neurons in each segmental ganglion (Zipser, 1980).

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4. Sensory a d Motor Fields T h e receptive field of a leech mechanosensory neuron consists of a major field in the ipsilateral skin of the segment in which the ganglion containing the cell body is located and minor fields in the ipsilateral skin of both adjacent segments (Nicholls and Baylor, 1968; Yau, 1976; Blackshaw, 1981a,b,c; Kramer and Goldman, 1981). T h e major field is innervated by an axon that exits from the ganglion containing the cell body via a segmental nerve, and the minor fields are innervated by axon branches that course in the connectives to the adjacent ganglia, where they exit via homologous nerves to the periphery (Fig. 4). In addition, the major and minor sensory fields are composed of distinct subfields, with each subfield being innervated by a separate axon branch coursing within one of the main branches of the segmental nerves. These receptive fields are sufficiently stereotyped to allow a description of their typical characteristics: overall size, major and minor fields, structure, and skin territories of innervation. However, there is significant variation from segment to segment and from specimen to specimen in the number of axonal branches growing out to the skin from homologous cells, and hence in the number of subfields and in the location of the subfield borders (Kramer et al., 1985). Even in cases in which the same number of subfields is present, there can be substantial variation in the size, shape, and position of the subfields making u p the whole field. Thus, although a given mechanosensory neuron seems destined to innervate a specific territory of skin, the actual innervation pattern of that territory is subject to considerable indeterminacy, or “epigenetic noise,” in the developmental program (Waddington, 1957). T h e motor neurons also innervate their targets according to a specific pattern (Stuart, 1970). At the first level of specificity, a particular motor neuron always innervates a single type of muscle fiber. For example, a motor neuron servicing longitudinal fibers does not innervate any circular fibers, either in the course of normal development or after its axon has been cut and directed to a different region of the body wall (Van Essen and Jansen, 1977). At the second level of specificity, a particular motor neuron normally innervates only a subset of the potentially available fibers of the appropriate type (Stuart, 1970). For instance, the excitor designated d innervates longitudinal fibers only near the dorsal midline of the body wall, whereas the excitors 1 and v innervate longitudinal fibers only in the lateral and ventral body wall, respectively. Two further excitors, dl and vl, innervate longitudinal fibers only in the dorsolateral or ventrolateral body wall quadrants, respectively. One excitor,

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C

123

C

DM

LE

VM

- t - -I--

I

t -1-+PP

I

FIG.4. Receptive field of Pv, a mechanoreceptive neuron responsive to pressure on the ventral surface of the skin of Haementeria ghilianii. The parts of the receptive field innervated by the four different branches of Pv are mapped separately on the schematic representation of three segments of skin on one side of the leech from ventral (VM) to dorsal (DM) midlines. LE marks the lateral edge of the body. Vertical lines represent the margins of the annuli. The central (C) annulus in each segment contains the sensilla (small circles in the central annuli). In this species, the ventral territory in most midbody segments has five annuli and the dorsal territory has three. The complete receptive field is outlined in bold lines, except for the portion that crosses the ventral midline, which has been left out. The horizontally shaded and stippled areas constitute the major subfield: the shaded area is the part innervated by branches of the largest peripheral axon, which leaves the ganglion in the medial (MA) nerve; the stippled area is the part innervated by branches of a smaller axon, which leaves the ganglion by way of the posterior (PP) nerve. The cross-hatched areas are the minor subfields, which are innervated by branches of Pv axons that course through the interganglionic connectives, through the neuropil, and exit via peripheral nerves of the adjacent ganglia. (From Kramer and Goldman, 1981.) The receptive field of the PD neuron in the same segment innervates the dorsal territory; its receptive field overlaps that of Pv across LE. The receptive fields of sensory neurons were originally determined for mechanoreceptors in Hirudo medicinalis (Nicholls and Baylor, 1968);they are very similar to those shown here for Haementeria.

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A. Shortening

B. Local bending

D. Swimming

FIG. 5. Four behaviors whose neuronal basis has been studied in Hirudo medicinalis. (A) In response to a moderate mechanical stimulus anywhere along the body, the animal shortens by contracting all the longitudinal muscles in several segments on either side of the location touched. In response to stronger mechanical stimuli, particularly at the front end, the animal will contract longitudinal muscles in all segments. (B) In response to light or moderate mechanical stimuli, the segment touched will bend away from the stimulus by contracting the longitudinal muscles on the side of the touch and relaxing the longitudinal muscles on the opposite side. This response will occur in the same segment stimulated even when adjacent segments are producing the shortening response. (From Lockery and Kristan, 1990a.) (C) Crawling locomotory behavior is accomplished by an inchwormlike series of movements, using the front (to the right) and rear (to the left) suckers as points of attachment. The extension phase of the step is produced by contraction of circular muscles with the posterior sucker attached to a firm substrate; the contraction phase, in which the body is pulled forward, is accomplished by contraction of the longitudinal muscles with the anterior sucker attached. (From Stern-Tomlinson el al., 1986.) (D) Swimming locomotion is accomplished by a dorsoventral undulation of the body in water with both suckers unattached. Shown are body outlines at about 50-msec intervals, showing a complete cycle of undulation in a leech moving forward from right to left. The body is held extended by a continuous contraction of all the dorsoventral muscles in the body. The crest of each body wave is produced by contraction of the ventral longitudinal muscles in a restricted body region, and the troughs are produced by contraction of the ventral longitudinal muscles

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L, innervates all longitudinal fibers on one side of the segmental body wall. 5. Behavior The behavioral repertoire of leeches ranges from simple reflexes, through locomotion and feeding, to complex mating routines (Gee, 1913; Sawyer, 1981). The simple reflexes elicited by mechanical stimulation of the skin include shortening (Fig. 5A), curling, writhing, and local bending (effected by contraction of the longitudinal muscles on one side of a single segment and distension of the longitudinal muscles on the other side) (Fig. 5B). Reflex implementation varies with the location and intensity of the stimulus (Kristan et al., 1982). Leeches carry out two types of locomotory movements: crawling and swimming. Whereas all known leech species crawl, only some species swim; some species swim as juveniles but stop swimming as they reach adulthood (Sawyer, 1981). Crawling consists of a sequence of stepwise movements (Fig. 5C). At the beginning of each crawling step, the posterior sucker is attached to the substrate. The leech then extends its body forward by contracting the circular muscles and relaxing the longitudinal muscles, while the head searches for a suitable attachment site for the anterior sucker. After the anterior sucker attaches to the substrate, the rear sucker releases its hold and the rear is brought forward by contraction of the longitudinal muscles and relaxation of the circular muscles. Finally, the posterior sucker attaches to the substrate at a more forward location and the anterior sucker is detached. At this point, one crawling step has been completed, and the next can be taken (Stern-Tomlinson et al., 1986). The leech swims by undulating its extended and flattened body in the dorsoventral plane, forming a single wave that travels along the body from head to tail (Fig. 5D) (Gray et al., 1938; Gray, 1968; Kristan et al., 1974a,b). Swimming movements are produced by two types of muscles (von Uexkull, 1905). First, the body flattens by tonically contracting the dorsoventral muscles. Second, rhythmic local contraction and relaxation of longitudinal muscles alternately shorten and lengthen the dorsal and ventral body wall, with the ventral wall relaxed while the dorsal is contracted and vice versa, to form the troughs and crests of the wave. Swimming subserves both food seeking and escape. Starved leeches regionally. Forward progress is made by a coordinated anterior-to-posterior progression of the contraction patterns, such that the crests and troughs move backward along the body; in this manner, the body waves push backward on the water and produce a forward thrust. (From Stent et al., 1978.)

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swim toward the source of water waves created by a host animal (Mann, 1962; Young et al., 1981), a response that appears to be mediated by ciliated mechanoreceptors in the sensilla (Friesen, 198 1 ; Phillips and Friesen, 1982). Moreover, in response to tactile stimulation of its caudal skin, a stationary leech will swim. This response is likely to be mediated by the T and P mechanoreceptor neurons, because electrical stimulation of the skin adequate to activate these neurons usually elicits swimming, particularly when delivered to the rear end of the animal (Kristan et al., 1982). The behavior of leeches depends on their nutritional condition: starved leeches spend much of their time making searching movements and respond to stimuli that sated, quiescent animals ignore (Mann, 1962). These differences in behavior appear to depend upon differences in blood levels of serotonin, because very active leeches have high, and quiescent leeches have low, levels of serotonin in their body fluids (Willard, 198 1). One important source of circulating serotonin is the pair of Retzius cells present in each segmental ganglion. At a high rate of impulse activity, the Retzius cells release enough serotonin into the blood to increase significantly the concentration of circulating serotonin, and hence the frequency and/or duration of swimming episodes. Impulse activity in the two smaller, paired serotonin neurons (cells 21 and 61) also stimulates swimming. In contrast to the neurohormonal release of serotonin into the general circulation by the Retzius cell, however, the smaller cells make direct synaptic contact with, and provide strong excitation to, one of the interneurons that form part of the segmental neuronal circuit that generates the swim motor pattern (Kristan and Nusbaum, 1983; Nusbaum and Kristan, 1986). Serotonin also appears to play a role in the control of feeding (Lent et al., 1988; Lent and Dickinson, 1989). For instance, activity in serotonin neurons of the subesophageal ganglia and the anterior midbody ganglia evokes such feeding-related activities as pharyngeal pumping and salivation. Serotonin also intensifies simple reflexes, such as local bending (Lockery and Kristan, 1991) and shortening (Wittenberg, 1991), so that it may serve as a general enhancer of leech motor routines. Leeches can modify their behavior as a result of environmental contingencies. For instance, the body-shortening reflex is subject to classical conditioning (Henderson and Strong, 1972; Sahley and Ready, 1988), habituation, and sensitization (Belardetti et al., 1982; Boulis and Sahley, 1988). Other behaviors, such as local bending (Lockery and Kristan, 1991), crawling (Sahley and Ready, 1988), swimming (Debski and Friesen, 1986), and food preference (Karrer and Sahley, 1988) also exhibit various forms of conditioning.

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6. Identijied Circuits The simplicity, stereotypy, and experimental accessibility of the leech nervous system offers the possibility that all of the neurons participating in the generation of any leech behavior can be identified and their synaptic and humoral interactions characterized. This promise is strengthened by the possibility of eliciting many motor acts from semiintact preparations in which the nervous system has been exposed (Gray et al., 1938; Kristan, 1982; Kristan et al., 1974a, 1988; Thompson and Stent, 1976a,b,c; Lockery and Kristan, 1990a,b; Baader and Kristan, 1990; Wittenberg, 1991). In some cases, the motor neuron activity pattern characteristic of a behavioral act can be evoked in the isolated nerve cord, or even in parts of it (Kristan and Calabrese, 1976; Thompson and Stent, 1976a; Lockery and Kristan, 1990a; Wittenberg, 1991).To various degrees of completeness, neuronal circuits have been identified for four behaviors; local bending (Kristan, 1982; Lockery et al., 1989; Lockery and Kristan, 1990a,b), body shortening (Wittenberg, 199l), swimming (Stent et al., 1978; Friesen and Stent, 1977; Stent and Kristan, 1981; Kristan and Weeks, 1983; Friesen, 1989), and heartbeat (Stent et al., 1979; Stent and Kristan, 1981; Calabrese and Peterson, 1983; Calabrese and Arbas, 1989; Calabrese et al., 1989). These characterizations of neuronal circuits provide the points of departure for ascertaining the physiological, morphological, and molecular mechanisms responsible for the development of behavior.

II. Morphological Development and Staging

In all leeches, fertilization is internal and embryonic development begins as soon as the eggs are laid. Helobdella, Theromyzon, and Haementeria lay yolk-rich eggs about 0.4,0.8, and 2 mm in diameter, respectively. The eggs are laid in clutches, enclosed in transparent, soft-walled, salinefilled cocoons, which remain attached to the ventral body wall of the brooding parent for most of embryonic development. However, for experimental purposes, the eggs can be removed from the cocoon at any stage of development and cultured to maturity in saline, the composition of which resembles that of the cocoon fluid. The egg yolk provides the energy and organic matter needed for development, which proceeds under conditions of nearly constant volume. Upon exhaustion of the yolk, by which time the volume of the embryo is still not much larger than that of the egg, the juvenile leech takes its first meal from a host

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A

B

Vent ra I midline

Stage 7 (early)

Germinal plate Germinal band

Stage 7 ( m i d d l e )

m

Stage 8 ( l a t e )

Stage 10

W

Stage 8 (early)

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animal. Subsequent postembryonic growth and maturation of the juvenile leech occur by way of increases in both cell size and cell number (Weisblat, 1981). The embryonic development of glossiphoniid leeches has been divided into l l stages, beginning with egg deposition and extending up to the point at which the juvenile leech is ready for its first meal 3 (Helobdella) to 6 (Theromyzon and Haementeria) weeks later (Fernandez, 1980; Weisblat et al., 1980b; Stent et al., 1982; Bissen and Weisblat, 1989); some stages have been further divided into substages. The definition of the stages is based on morphological criteria discernible in living embryos; the staging scheme and cell nomenclature presented here include modifications and additions that reflect an increasing knowledge of the details of development. Figure 6 shows a schematic representation of key stages in the development of Helobdella. The criteria for the beginning and end of each stage are given in Table I. These stages are equally applicable to Theromyzon and Haementem'a. When first laid, the glossiphoniid leech egg is filled throughout with colored yolk and enclosed in a clear vitelline membrane. T h e first embryonic axis is marked initially by the production of two polar bodies at the animal pole early in stage 1 (Fig. 6A). Later, as the egg approaches its first cleavage, this axis is reinforced by domains of yolk-free cytoplasm, or teloplasm, which form at the animal and vegetal poles. The first cleavage, yielding blastomeres AB and CD, is meridional and establishes the second embryonic axis (stage 2). The second cleavage is also merid-

FIG.6. Embryonic development in glossiphoniid leeches. (A) A lineage tree summarizing the cell divisions leading to formation of polar bodies (pb), macromeres (capital letters A-D and combinations), micromeres (lowercase letters with primes), teloblasts (capital letters M, N, 0, P, and Q), and blast cells (lowercase letters without primes). Each cell division is indicated by a horizontal line, and the spacing between horizontal lines is proportional to the approximate times between subsequent cell divisions. The fact that there are bilaterally symmetric lineages after the cleavages of cells DM" and DNOPQ"' is denoted by broken horizontal lines. Embryonic stages are indicated at left. (B) Schematic representation of embryonic stages, showing the arrangement of teloblasts and their primary blast cell bandlets within the germinal band and germinal plate in early stage 8 (left) and views of whole embryos at selected stages (right). In the early stage 7 embryo, teloblasts have begun to produce blast cells in contact with the micromere cap; by midstage 7, the bandlets have formed recognizable germinal bands. At early stage 8, the heart-shaped germinal bands have begun to coalesce along the future ventral midline; this process leads to the formation of the germinal plate, which is complete by the end of stage 8. By stage 10, segmental tissues, including the segmental ganglia of the ventral nerve cord (black), are well differentiated. Late stage 8 and stage 10 embryos are as viewed from the ventral aspect; all other stages are as viewed from the animal pole. (From Bissen and Weisblat, 1987.)

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TABLE I LEECHEMBRYOGENESIS

STAGES OF (;LOSSIPHONIID

Stage

Stage name

1

Uncleaved egg

2 3 4a

T w o cells

4b

Macromere quintet

4c

Mesoteloblast formation

5

Ectoteloblast precursor

6a

N teloblast formation

6b

Q teloblast formation

7 8

Germinal band formation Germinal band coalescence

9

Segmentation

10

Body closure

11

Yolk exhaustion

Juvenile

Four cells Micromere quartet

Beginning

Egg laying Onset of first cleavage Onset of second cleavage Onset micromere formation (cleavage of D to form D’ + d’) Onset of D’ macromere cleavage to form DM + DNOPQ Onset of cleavage of cell DM” to form left and right M teloblasts Onset of cleavage of cell DNOPQ“‘ to form left and right NOPQ proteloblasts Onset of cleavage of cell NOPQ“ to form N and OPQ Onset of cleavage of cell OPQ” to form OP and Q Completion of cleavage of cell OP Onset of coalescence of left and right germinal bands Completion of germinal band coalescence Appearance of coelomic space in the 32nd somite Completion of fusion of the lateral edges of the germinal plate along the dorsal midline Exhaustion of the yolk in the embryonic gut and first feeding

ional; it divides the egg into four blastomeres, A, B, C, and D (stage 3), and D receives most of the teloplasm. The third cleavage is highly unequal, producing four vegetal macromeres A‘, B’, C’, and D‘ and four micromeres a’, b’, c’, and d’ (stage 4a); D’ then cleaves equatorially to yield cells designated DNOPQ and DM, while cells A’, B’, and C’ undergo another round of micromere production, yielding macromeres A , B”, and C and micromeres a”, b”, and c’’ (stage 4b). At this stage, according to the classical germ layer interpretation, macromeres A , B”, and C” constitute presumptive endoderm, and DNOPQ and DM constitute presumptive ectoderm and mesoderm, respectively (Whitman, 1887). Subsequently (stages 5 and 6), quadrants A, B, and C each undergo one more round of micromere production, whereas the D quadrant cells undergo a series of cleavages to generate five bilateral pairs of large,

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yolky stem cells called M, N, O/P, O/P, and Q teloblasts, as well as additional micromeres. These cleavages are presented schematically in Fig. 6A. Each teloblast carries out a series of 40-100 highly unequal cleavages, producing a bandlet of small primary blast cells (stage 7). T h e bandlets produced by the five teloblasts on either side of the midline rise to the surface and merge to form two ridges of cells, the right and left germinal bands. Between the germinal bands lies a cluster of cells, the “micromere cap,” derived mainly from the micromeres. The blast cells and their bandlets that are produced by the M, N, and Q teloblasts are designated as m, n, and q, respectively. In the germinal bands, the bandlets produced by the two O/P sister teloblasts lie between the n and q bandlets, with the bandlet lying next to the n bandlet being designated as o (and its O/P teloblast of origin being designated as “generative 0 teloblast”) and the bandlet lying next to the q bandlet being designated as p (and its O/P teloblast of origin being designated as “generative P teloblast”) (Shankland and Weisblat, 1984). In Theromyzon, the identities of the generative 0 and P teloblasts are apparent from the positions of the teloblasts themselves, which are simply designated 0 and P (Keleher and Stent, 1990). At this point, the ectodermal bandlets lie in mediolateral order q, p, 0, and n, whereas the mesodermal m bandlet lies under the four ectodermal bandlets (Fig. 6B). As the result of morphogenetic processes that are only poorly understood at present, the left and right germ bands come to join one another at their distal (future anterior) ends at the site of the future head (Fernandez and Olea, 1982). The areas between the bands, and the bands themselves, are covered by a simple squamous epithelium derived from the micromere cap. With ongoing blast cell production, the midportions of the bands move apart over the surface of the embryo, and then gradually, during stage 8, meet and coalesce zipperlike, from the future head rearward along the ventral midline, forming a sheet of cells called the germinal plate (Fig. 7A). The circumferential movement of the germinal bands prior to their coalescence on the ventral midline reverses the mediolateral order of the bandlets, so that the left and right n bandlets come into apposition at the ventral midline. As the germinal bands move over the surface of the embryo, the micromere-derived epithelium expands with them, eventually covering the entire surface of the embryo. Over the part of the embryonic surface not covered by the germinal plate, a superficial, simple squamous epithelium and an underlying layer of circumferentially oriented muscles combine to form a two-layered tissue, the provisional epithelium, which does not contribute to the eventual juvenile leech.

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With the proliferation of cells of the germinal plate to form the body tissues, the plate gradually thickens and expands over the surface of the embryo into dorsal territory. Even before the germinal bands have started to coalesce, the left and right mesodermal bandlets become partitioned into a series of discrete blocks of cells corresponding to hemisomites. During stage 8 and continuing through stage 9, the coelom arises as a cavity within each somite, so that the germinal plate becomes partitioned along its length into a series of tissue blocks, each separated from the others by transverse septa (Fig. 7). Each block corresponds to a future body segment. Segmentation starts at the front and progresses rearward; by the time the ganglion of the hindmost segment has formed (at the end of stage 9), the expanding germinal plate covers about onethird of the ventral surface. The embryo hatches from the vitelline membrane during stage 9. In stage 10, right and left leading edges of the expanding germinal plate meet and coalesce on the dorsal midline, closing the leech body. During this expansion of the germinal plate, the provisional integument retracts before, o r is pushed back by, the leading edges of the plate. By the time that the leading edges of the plate have coalesced on the dorsal midline, the crumpled remainder of the provisional integument has disappeared from the embryonic surface. Meanwhile, formation of the gut is underway. It first appears as a cylinder (filled with yolk provided by macromeres A, B, and C and the remnants of the teloblasts) and then becomes segmented by annular constrictions, which probably correspond to the segmental septa. These constrictions give rise to paired gut lobes, o r caeca, in register with the midbody segments. Gut segmentation is completed at body closure (at the end of stage 10); the embryo now has the general shape of the adult leech. The final steps of morphological development, including maturation of the posterior sucker, occur during stage 11. When the yolk in the gut is exhausted (at the end of stage 1 l), the juvenile leech is ready for its first meal. This description of embryogenesis up to the formation of the germinal plate applies to glossiphoniid leeches in general, such as Tfwromyzon, Helobdellu, and Huementeria, but not to the hirudinids, such as Hzrudo. Hirudo eggs are much smaller (about 0.1 mm in diameter). They contain little yolk and are deposited in a sealed cocoon that contains an albuminous fluid. T h e initial stages of embryogenesis produce a sac, inappropriately called a “Iarva,” complete with a mouth that ingests the albumin, providing an exogenous source of organic matter and energy for subsequent development. T h e embryo forms on the surface of this sac and rapidly increases in size. As in glossiphoniid leeches, embryogenesis in Hirudo proceeds via five pairs of small teloblasts that

B

A Anterior

Mouu7 ReproductiveStructures

Protonephridia

Larval Sac

Caudal Swker

-1

mm

FIG. 7. Drawings of leech embryos at the stage when ganglia are formed and neurons are growing their processes. (A) Drawing of Haementeria ghzlianii embryo at midstage 9. (From Kuwada and Kramer, 1983.) (B) Drawing of H i d o medicinalis embryo at a comparable stage, E12. The protonephridia and larval sac are nonembryonic tissues that are likely not to be retained in the adult leech. The mouth is at the anterior end. (Drawing provided by J. Jellies.) Embryos vary in size at this stage over a twofold range, hence the calibration is approximate.

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produce bandlets of primary blast cells, germinal bands, and a germinal plate. In the hirudinid embryo, germinal bands form on the future ventral (rather than on the future dorsal) surface and coalesce directly, without undergoing the circumferential migration characteristic of the glossiphoniids (Schleip 1936). Subsequent development follows much the same course in the hirudinids (Fig. 7B) as in the glossiphoniids (Schleip, 1936; Fernandez and Stent, 1983). N o staging system has yet been devised for the embryonic development of the hirudinids such as Hirudo. Instead, the developmental progress of Hirudo embryos is reported in days of development, from egg deposition (EO) to completion of the posterior sucker and maturation of the juvenile leech about a month later (E30). Formation of the posteriormost ganglion of H i d o is complete at about E10, and body closure is complete at about E20, corresponding to the end of stages 9 and 10, respectively, in glossiphoniid embryos.

111. Behavioral Development

The highly regular morphological development of the leech embryo is accompanied by similarly regular behavioral development. T h e behavioral repertoire of a given species of leech emerges in a stereotyped sequence. By the end of embryogenesis (stage 1 1 in glossiphoniids and about E30 in hirudinids), most adult behavioral routines have appeared, except those pertaining to reproduction. A schematic summary of the behavioral development of Haementena is presented in Fig. 8. During the first seven stages, i.e., for many days after the fertilized Haementeria egg is laid, there are no movements, either spontaneous or evocable, in the developing embryo. But as the germinal plate nears completion, peristaltic movements begin. These movements consist of longitudinal waves of circumferential constrictions that usually start at one end of the embryo and take 5-10 sec to reach the other end. Although they arise spontaneously and at irregular intervals, they can also be initiated by gentle mechanical stimulation at any site along the embryo. T h e constrictions are produced by the circumferential muscle cells of the provisional integument. These cells (which are not precursors of the definitive circular muscle fibers) disappear by the time body closure is complete, when peristaltic movements have ceased as well (A. P. Kramer, unpublished observations). T h e peristaltic movement leads to hatching of the embryo from the vitelline membrane. In addition, peristalsis may also circulate fluid in the developing embryo. T h e peristaltic

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rhythm is almost certainly myogenic in origin, because peristalsis begins before the embryonic nervous system becomes functional, and it can persist even after surgical removal of the germinal plate and its developing nervous system. T h e body-shortening reflex appears when body closure is almost complete. At first this reflex is evoked only by mechanical stimuli applied to the very front end of the embryo, and shortening occurs only in the anterior third of the animal. Over the next few days, the receptive field for the stimulus enlarges to cover the anterior half of the animal, and the extent of the shortening response enlarges to encompass most of the body. Within a day after shortening appears, another reflex, namely local body bending (Kristan, 1982), emerges. A clutch of embryos remains enclosed within its cocoon until stage 10, when the embryos emerge and attach themselves directly to the venter of their brooding parent by means of a sticky exudate, localized at the anteroventral end of the embryo. Early in stage 1 1, first the posterior and then the anterior suckers develop and begin to function, allowing the nearly mature embryo to control its attachment to solid substrates. By midstage 11, the embryos are able to use the suckers for wellcoordinated crawling. Before this, during a precrawling phase, the embryos execute a progression of movements that appear to be increasingly complex elements of crawling behavior (A. P. Kramer, unpublished observations). Toward the end of stage 11, the Haementeria embryo acquires the ability to swim, having undergone a preswimming phase during which it produces progressively more swimminglike movements. Is this gradual improvement in swimming performance the result of an autonomous developmental process, or does it depend upon practice? To answer this question, all serotonin neurons of the developing nervous system were ablated during stage 10, i.e., long before the onset of swimming, by injecting the embryo with 5,7-dihydroxytryptamine,a toxic analog of serotonin, (Glover and Kramer, 1982). Because (as will be discussed in more detail later) there is no regulative restoration of ablated neurons in leech development, embryos thus treated develop into juveniles whose nervous system is devoid of serotonin neurons. Despite their lack of what might seem to be a vital neurologic component, the treated leeches are morphologically and behaviorally quite normal, although they are lethargic and do not swim. Such leeches, which have never previously executed a body wave, start to swim as soon as they are either injected with serotonin o r are simply put into water containing serotonin. I t appears, therefore, that the capacity to generate the basic swimming rhythm, and hence the underlying neural circuit, is as-

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sembled via an autonomous developmental process that does not require practice. Even after they are able to swim, Haementeria embryos remain attached to the venter of the parent. Finally, when their store of yolk is exhausted, the embryos detach from their parent in the presence of a potential host animal. If the host has a sufficiently thin skin, the embryo inserts its proboscis through the skin and, by means of pumping movements of its pharyngeal muscles, ingests as much as 15 times its own body weight of blood. With this first feeding, the embryo advances to the status of juvenile leech. T h e behavioral development of hirudinids, which spend their entire embryonic development bathed in an albuminous fluid within a cocoon, is on the whole quite similar to that of the glossiphoniids (Fernandez and Stent, 1983). However, in the hirudinids the order of maturation of the two locomotory behaviors is reversed: they swim before they crawl, and both behaviors appear in embryos at a time when they would normally still be within their cocoon. If the embryos are removed from the cocoon and dropped into water the day after body closure becomes complete (corresponding to early stage 1 1 in glossiphoniids), they make primitive swimming movements, which over several days mature into the adultlike swimming rhythm (Reynolds and Kristan, 1989). Crawling movements and the competent use of the suckers first appear about halfway between body closure and normal emergence from the cocoon. Interestingly, the order in which the two locomotory behaviors appear matches the needs of leech embryos: the rhynchobdellids must use their suckers to attach to the parental venter during the late stages of embryogenesis and need to swim only upon the completion of development, when they must get to a host for their first feeding; the gnathobdellids, by contrast, may swim in the cocoon fluid, and need to crawl only after they emerge, to aid in finding a site for taking blood from their first host.

IV. Developmental Cell Lineage

A. CELLLINEAGE TRACING

To begin to address the fundamental question of which differentiated properties of an adult cell depend on the cell’s line of descent from the fertilized egg and which depend on interactions with other cells, it is necessary to ascertain exact developmental pedigrees. Whitman ( 1 878,

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1887) carried out the pioneering studies on developmental cell lineage in leech embryogenesis over a century ago. His method for ascertaining cell lineages was to observe living embryos under a microscope and keep track of successive cleavages. Cell lineage analyses were later extended to embryos of other species, not only by direct observation, but also by use of such techniques as selective cell ablation, application of extracellular marker particles, and production of chimeric and genetic mosaics (Wilson, 1892; Sturtevant, 1929; Tarkowski, 1961; Mintz, 1965; Stern, 1968; Garcia-Bellido and Merriam, 1969; Le Douarin, 1973; Sulston and Horvitz, 1977, 1981; Deppe et al., 1978; Sulston et al., 1983). More recently, Whitman’s century-old cell lineage studies on leech embryos have been refined and extended by the introduction of microinjected cell lineage tracers (Weisblat et al., 1978, 1980a; Gimlich and Braun, 1985; Stuart al., 1989) to establish the lines of descent of identified cells in the leech nervous system. In this procedure, a histologically detectable tracer molecule is injected directly into an identified cell of an embryo early in development. At a later embryonic stage, the cellular distribution of the tracer is observed. Cells containing the tracer are inferred to have descended from the originally injected cell. For this method to be useful the tracer molecule has to meet three conditions: (1) it must permit embryonic development to continue normally after it is injected, (2) it must remain intact and not be diluted too much during increases in cell size and number within the developing embryo, and (3) it must not pass through junctions linking embryonic cells, so that it is confined exclusively to descendants of the injected cell. (Because early hirudinid embryos have very small cells, microinjection has been used only in glossiphoniid embryos.) Horseradish peroxidase (HRP) was the first molecule to be employed as an intracellular lineage tracer (Weisblat et al., 1978). Soon thereafter covalently linked composites of large carrier molecules and the fluorescent dyes rhodarnine o r fluorescein came into use (Weisblat et al., 1980a). Presently, the most widely used carrier molecules are dextrans; the rhodamine-dextran tracer is designated RDX, and the fluorescein-dextran tracer is designated FDX. To obtain a fluorescent lineage tracer that binds to tissues after histological fixation, dextrans with linked lysine residues are used as carrier molecules (Gimlich and Braun, 1985; Stuart et al., 1989). These fixable composites of rhodamine o r fluorescein and lysinated dextran are designated RDA or FDA. In addition to its utility for tracing cell lineages, a fluorescein-labeled tracer can serve as a specific photosensitizer (Shankland, 1984). Upon illuminating an FDX-labeled cell at a wavelength of about 490 nm, some of the excited fluorescein fluorophores are quenched by transferring the

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absorbed energy to oxygen molecules in solution, converting them to the highly reactive singlet state. These reactive oxygen molecules, in turn, cause a generalized oxidation of cell constituents, killing the illuminated labeled cell (Miller and Selverston, 1979; Braun and Stent, 1989b). Cells are largely transparent to light at 490 nm unless they contain fluorescein, so injecting an embryonic progenitor cell with FDX makes it possible to photoablate its progeny selectively, even if they have become intermingled with cells from other lines of descent. Moreover, because the photosensitizer is also a lineage tracer, it automatically allows direct visual identification of cells to be ablated, as well as direct visualization of their normal fates in unirradiated control embryos.

B. GENEALOGICAL ORIGINS

OF THE

SEGMENTAL NEURONS

Because the paired n blast cell bandlets straddle the ventral midline of the germinal plate, Whitman (1887) and his contemporaries (Bergh, 1885) inferred that the cells of the ventral nerve cord are derived from the N teloblasts. However, using cell lineage tracers has shown that the leech nerve cord is, in fact, derived from all five teloblasts (Weisblat et al., 1984; Kramer and Weisblat, 1985; Weisblat and Shankland, 1985; Torrence and Stuart, 1986), as Apathy (1889) had suggested. The tracerlabeled descendants of each injected teloblast form a distinct pattern, which is repeated from segment to segment and is the same in every embryo in which a particular teloblast has been injected. Figure 9 illustrates the cells that each of the five teloblasts contributes to a midbody segment. Note that the labeled structures lie on the same side as that of the injected teloblast, except for axons projected contralaterally by labeled neuronal cell bodies. Hence in the course of neurogenesis there is no appreciable migration of neuronal precursor cells across the ventral midline. Detailed scrutiny of lineage-tracer-labeled stage 10 embryos shows that each of the four ectodermal precursor teloblasts-N, 0, P, and (2contributes some cells to the CNS, some to the peripheral nervous system, and some to the epidermis. The mesodermal precursor teloblast, M, contributes three to four pairs of neurons to each segmental ganglion, but its main contribution is to mesenchyme, nephridia, and muscle fibers, tissues to which embryologists traditionally assign a mesodermal origin. Thus, each individual teloblast generates progeny that are found in several types of tissue. Descendants of the n blast cell bandlet are found exclusively within the segmental ganglia, except for two or three peripheral neurons

GANGLION

t

VENTRAL MIDLiNE

FIG.9. Contributions of the ectodernial teloblasts and bandlets to a midbody segment. Each panel illustrates the pattern of tracer-labeled cells observed in one segment of'a stage 10 embryo of 7%rromyzon rude following injection of a lineage tracer into one ectodernial teloblast at stage 6 or 7. The right edge of the figure corresponds to the lateral margin of the germinal plate, and thus to the future dorsal midline. Anterior is uppermost. Major labeled axonal tracts are shown, but no attempt has been made to render detailed neuronal projection patterns within the CNS. LDI and LD2 designate identified dopaminecontaining neurons, as in Fig. 3. Names of other neurons (e.g., 921) include a letter indicating the bandlet of origin, the letter z to symbolize the (largely unknown) intermediate cell lineage, and a number. Labeled squamous epidermal cells are indicated by light stippling; epidermal rell florets (CF I -6), are more densely stippled. Top ganglion: Contrihution of the q handlet; AV, anterovenrral cluster of central neurons; CG, connective glioblast; MA, cluster of peripheral neurons along the MA nerve. This cluster includes the dopamine-containing neuron MD. Second ganglion: Contribution of the p bandlet. Third ganglion: Contribution of the o handlet; AD, anterodorsal cluster of central neurons; PV, posteroventral cluster of central neurons. Fourth ganglion: Contribution of the n bandlet; fine, labeled processes fill most of the contralateral neuropil (marked by an asterisk and outlined by a dashed line). (From Torrence and Stuart, 1986.)

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(depending on the species) and a few ventral epidermal cells. Descendants of the 0, p, and q blast cell bandlets contribute substantially fewer cells to the segmental ganglia and correspondingly more cells to the peripheral nervous system and the epidermis, including epidermal specializations called cell florets (Fig. 9). The stereotyped patterns of peripheral neurons that are derived from each blast cell bandlet indicate that the peripheral nervous system also consists of individually identifiable neurons, whose unique identity can be determined based on their position in the body wall and their teloblast of origin. The topography of the contribution each blast cell makes to the segmental epidermis is generally consistent with its relative position within the germinal plate. Thus, dorsal epidermis derives from the q bandlet, ventral epidermis derives mainly from the mediolateral o and p bandlets, and a few epidermal cells on the ventral midline derive from the n bandlet (Weisblat et al., 1980b). The ganglion cells contributed by any blast cell bandlet form discrete and coherent cell domains; they are not randomly distributed or uniformly mixed with those contributed by other teloblasts (Fig. 10). For instance, the n bandlet contributes two transverse slabs of cells in the anterior and posterior regions of the ipsilateral hemiganglion and a longitudinal band of cells adjacent to the midline on the ventral aspect of the ganglion. The cell domains derived from the other bandlets display similarly compact and stereotyped topographies. In fact, these other bandlets contribute smaller numbers of neurons, allowing some of their progeny to be individually recognized. For example, some of the progeny of the o bandlet lie in distinct anterodorsal (AD) and posteroventral (PV) neuron clusters, and some p bandlet progeny form a transverse, wedge-shaped cluster of neurons in the middle of the ventral aspect of the ganglion, including a single neuron just beyond the tip of the wedge. These regular, segmentally iterated patterns of neuronal descent indicate that the developmental cell lineages derived from each blast cell bandlet correspond to four identifiable Kinship groups (Stent et al., 1982; Weisblat et al., 1980b; Weisblat and Shankland, 1985) designated M, N, 0, P, and Q, in accord with their teloblasts of origin. The stereotyped location of each set of teloblast descendants within the embryonic ganglia suggests that each identified neuron and glial cell normally arises from a particular blast cell bandlet. This supposition has been confirmed by surveying the kinship groups in studies combining fluorescent lineage tracers with electrophysiological, anatomical, and histochemical techniques used for identifying individual neurons (Blair, 1983; Kramer and Weisblat, 1985; Stuart et al., 1987). Some results of this survey are summarized in Fig. 10. Do the members of a given neuronal kinship group share a unique

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Dorsal

AA MA

P

AA MA

AA MA

P

P

W

W

Q Ventrol

mcG

AA MA

P

FIG. 10. Schematic representation of the five kinship groups in a typical midbody ganglion of HaPmentrria glrilZa7& The N and 0 kinship groups occupy both dorsal and ventral aspects of the ganglion; P and Q kinship groups are confined to the ventral aspect; the M kinship group is divided between the dorsal aspect of the interganglionic connective (in the case of the muscle cells) and the center of the hemiganglion, midway between dorsal and ventral aspects (in the case of the mz neurons). The connective tracts traverse the dorsal aspect of the ganglion. Boundaries of cell packets, each of which is associated with a packet glial cell, are indicated by dashed lines. The cells in each kinship group are indicated as follows: large cross-hatched regions in N and 0 are clusters of uncounted cells; cross-hatched circles in M,P, and Q are cell bodies of single, unidentified neurons; solid circles with labels are cell bodies of identified neurons; open circles enclosing small solid cirrles denote glial cells. The neuropilar glial cell body is at the ventral edge o f t h e neuropil. The clusters of N-derived cells in the dorsal anterior region of the ganglion are ventral to the dorsal anterior cluster of 0-derived cells. Cell abbreviations are as follows: MCM and LCM, medial and lateral connective muscle cells, respectively; NG, neuropil glia; ALG. anterior lateral giant; K, Retzius neuron; AE, annulus erector motor neuron; P h i l , posteroniedial neuron; AL2 and AL3, anterolateral neurons; LPG and MPG, lateral

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set of properties that set them apart from the members of the other kinship groups, such as functional category (glial cell or sensory neuron, motor neuron, or interneuron) or type of neurotransmitter? The provisional answer to this question must be “no,” because current data (Fig. 10) reveal no obvious kinship group-specific neuronal properties (except that all serotonin neurons belong to the N kinship group and that the 0 and P kinship groups contain some apparently homologous mechanosensory, glial, and dopamine cells). For example, each of the four ectodermal kinship groups includes one or more glial cells; the N, 0, and P kinship groups contain mechanosensory neurons; and the 0, P, and Q kinship groups contain dopamine neurons. How many primary blast cells derived from each teloblast contribute to founding one hemisegmental primordium? The answer to this question turned out to be somewhat complex, but it provided insights into the process of segmentation in leeches. An upper limit of three blast cells has been suggested, because the total number of blast cells produced per teloblast is less than three times the total number of segments (Fernandez and Stent, 1980). The actual number of founder blast cells contributed by each bandlet was estimated by two different methods using lineage tracers: an indirect method, termed the “label boundary method” (Fig. 11) (Weisblat et al., 1984; Weisblat and Shankland, 1985; Bissen and Weisblat, 1987), and a direct method, termed the “double-label method” (Zackson, 1984). Both methods led to the same answer, namely that a single primary blast cell in each of the m, 0, and p bandlets contributes the entire hemisegmental complement of its kinship group, whereas two successively born primary blast cells in the n and q bandlets each contribute a specific subset to the hemisegmental complements of their respective kinship groups. The first-born (i.e., more anterior) of these two blast cells in each hemisegment are called ns and q,, and the younger (i.e., more posterior) cells are called n, and qp A beginning has been made in extending this lineage analysis beyond identifying which primary blast cell gives rise to the roughly 50-100 definitive differentiated cells in each hemisegmental kinship group. First, the initial division patterns of all seven primary hemisegmental founder blast cells were ascertained by observing the caudorostral disposition of second-, third-, and higher-order blast cells in m, n, 0, p, or q and medial packet glia, respectively; PD and Pv, dorsal and ventral pressure-sensitive mechanosensory neurons; the neurons of the P teloblast (pz1-4) and q teloblast (921-3) lineages; TD, TL, and Tv, dorsal, lateral, and ventral touch-sensitive mechanosensory neurons, respectively; N, nocioceptive mechanosensory neuron. The major peripheral nerves (AA, MA, and P) are shown. Anterior is up. (From Kramer and Weisblat, 1985.)

m

a

k-JLA.2 144

FIG. 1 1. Distribution of progeny from individual primary o or p blast cells, ascertained by the labeled boundary method. Neurons labeled with tracer are shown as solid black; labeled epidermal cells are stippled. Segmental ganglia are illustrated as oval outlines, and the ventral midline is indicated by a dashed line through the ganglia. Anterior is up. (A) Three segments from a stage 10 embryo of Helobdelh triserialis in which the generative 0 teloblast was injected with lineage tracer after it had produced some unlabeled blast cells. In the anteriomost segment containing labeled cells (the top segment illustrated), all 0 pattern elements are present but only a subset are labeled. Thus, this segment was populated in part by progeny of the last unlabeled blast cell produced before the teloblast was injected and in part by progeny of the first labeled blast cell. Because the same subset of pattern elements was found to be labeled regardless of where the boundary fell along the longitudinal axis of the embryo, all o blast cells must produce the same complement of progeny. (B) Two segments in an embryo in which the generative P teloblast was injected. As in the 0 boundary segment, only a subset of pattern elements is labeled in the P boundary segment, and that subset is independent of the longitudinal position of the boundary. (C) Three segments in an embryo in which a single primary o blast cell was injected, showing its progeny, as inferred from a comparison of the partially labeled boundary segment to more posterior, fully labeled segments obtained when the P teloblast was labeled (as in panel A). (D) Distribution of the clone of cells labeled after injecting tracer into a single primary p blast cell, inferred from boundary segments as illustrated in panel B. Abbreviations: adc, anterodorsal cluster of central neurons; mpg, medial packet glia; pvc, posteroventral cluster of central neurons: nt, nephridial tubule; cf, cell floret. Other abbreviations as in Fig. 9. (From Weisblat and Shankland, 1985.)

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cell bandlets that had been labeled by injecting lineage tracer into one of the parent teloblasts (Zackson, 1982, 1984; Shankland and Weisblat, 1984; Shankland, 1987a,b; Bissen and Weisblat, 1989; Keleher and Stent, 1990). These studies revealed that each of the seven founder blast cells divides in a sequence that is idiosyncratic and stereotyped with respect to timing, orientation, asymmetry of cell division, and cell cycle composition. Furthermore, insofar as has been determined, each blast cell line has its own pattern of gene expression (Wedeen and Weisblat, 1991). Second, the fate ofsecond- and third-order m, 0, and p blast cells was ascertained by injecting lineage tracer directly into them (Shankland, l987a.b; L. Gleizer and G. Keleher, personal communications). Each higher-order blast cell was found to generate a stereotyped subset of the hemisegmental kinship group issuing from its parental primary blast cell. In the majority of cases, each subset still contributed to more than one tissue; e.g., higher-order o and p blast cells produce both neurons and epidermal cells, whereas m-derived blast cells produce both muscle fibers and nonmuscle cells. Division of higher-order blast cells d o not, therefore, lead to the typological segregation of cell fates in any simple manner. In this way, these divisions resemble the earlier blastomere divisions that generate the four ectodermal precursor teloblasts. One might conclude from the results just described that the mesodermal and ectodermal tissues of each hemisegment arise as clones founded by seven primary blast cells. In fact, the situation is more complicated, as was first inferred from results of label boundary experiments (Weisblat and Shankland, 1985) and then was demonstrated directly by injecting lineage tracer into individual primary blast cells (Shankland, 1987a,b; L. Gleizer and D. K. Stuart, personal communications). Each m, nt, o, p, or qr primary blast cell contributes a stereotyped subset of its progeny to each of several successive morphologically defined hemisegments. Progeny o f each nr, 0, p, or q, blast cell are spread over two successive hemisegments, whereas progeny of each m blast cell are in at least three successive hemisegments. Thus, each morphologically defined hemisegmerit contains the progeny of not one, but of two or three primary blast cell clones, which interdigitate across segmental borders. By contrast, the contributions of the n, and q, primary blast cells appear to be confined to single hemisegments and do not cross segmental borders.

C. ORIGIN OF

THE

SUPRAESOPHAGEAL GANGLION

Thie supraesophageal ganglion is the only component of the leech CNS that fails to be labeled after tracer is injected into any of the five

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teloblasts (Weisblat et al., 1980b). Hence, the anteriormost cells of the CNS must arise from a source other than the blast cells of the germinal bands. T h e most likely alternative source for this tissue appeared to be some or all of the 25 micromeres that arise during early cleavage: 3 each from the A, B, and C quadrants and 16 from the D quadrant (Sandig and Dohle, 1988; Bissen and Weisblat, 1989). Lineage tracer experiments confirmed this hypothesis and showed that micromeres a‘, b‘, c’, and d’ contribute their progeny to the supraesophageal ganglion (Weisblat et al., 1984). More specifically, the c’ and d’ micromeres generate bilaterally symmetric sets of supraesophageal neurons, as well as some nonneuronal cells that include the provisional epithelium of the stage 8 embryo, the prostomial epidermis, and the muscle cells of the proboscis (F. Ramirez, personal communication). Another set of micromeres, designated nopq” (Fig. 8) appears to contribute neurons in the anterior region of the otherwise teloblast-derived subesophageal ganglion (S. Ranganathan, D. K. Stuart, and L. Gleizer, personal communication). T h e observation that the supraesophageal ganglion and circumesophageal connectives of leeches are derived from micromeres, rather than from blast cells in the germinal bands, carries phyletic significance. In another major class of annelids, the polychaetes, the highly developed and complex supraesophageal ganglion is obviously a prostomial organ. It not only lies in a region that is clearly rostra1 to the metameric body segments, but it also arises developmentally as the neural tissue of a nonsegmented larva (itself derived from micromeres) to which the teloblast-derived, segmental ventral nerve cord is added later, during metamorphosis (Dawydoff, 1959). By contrast, the status of the much less elaborate supraesophageal ganglion of leeches, whose development lacks a free-living larval stage complete with its own nervous system, has long been the subject of controversy. Whitman (1887) originally conjectured that the leech’s supraesophageal ganglion is derived from the micromeres rather than from the germinal bands. Modern lineage tracers have confirmed this hypothesis, although Whitman abandoned it when he concluded that the anteriormost ganglion is a homologue of the segmental ganglia rather than a prostomial organ (Whitman, 1892). D. TRANSFATING The analyses of cell lineages described thus far indicate that leech neurogenesis is, on the whole, highly “determinate,”in the sense that the developmental fate of a particular embryonic cell in a normal embryo

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can be reliably predicted. One exception to this general finding is the indeterminacy of the fates of the O/P sister teloblasts in Helobdella. These teloblasts arise from an apparently symmetric division of their mother cell, OP, and they are morphologically as well as genealogically indistinguishable. One of the O/P sisters, designated the “generative 0 teloblast,” gives rise to the 0 kinship group, whereas the other, designated the “generative P teloblast,” gives rise to the P kinship group (Figs. 9 and 11). Whether a particular O / P teloblast takes on the role of the generative 0 o r generative P tebbidst depends on the relative position of its bandlet of daughter blast cells within the germinal band (Weisblat and Blair, 1984). T h e O/P sister teloblasts thus form an “equivalence group” (Sulston and White, 1980) of pluripotent embryonic cells. T h e following rule governs the fate taken on by the sister teloblasts: whichever O/P bandlet lies next to the q bandlet when it enters the germinal band will give rise to the elements of the P kinship group, whereas the O/P bandlet lying next to the n bandlet will give rise to the elements of the 0 kinship group. Once its blast cell bandlet is in place, the fate of either OIP teloblast is determined, in that its fate as 0 or P can be predicted reliably based on the relative position of its bandlet in the germinal band. Although under normal conditions the fate of an O/P teloblast and of its blast cell bandlet becomes determined when the bandlet enters the germinal band, the primary blast cells have not yet lost their ability to switch fates at that time (Weisblatand Blair, 1984). This fact came to light when an 0 / P teloblast, identified as the generative P teloblast by the position of its bandlet containing the first dozen or so blast cells at the time, was ablated by intracellular injection of DNase, aborting further production of blast cells. At stage 10, in the posterior regions of the embryo that had been deprived of one of the p bandlets, the ipsiiateral primary o blast cells had changed their developmental fate and had given rise to cells of the P kinship group instead (Fig. 12). Such an experimentally induced change in developmental fate-of 0 bandlet cells to a P fate-has been designated “transfating.” By contrast, an equivalent ablation of the generative 0 teloblast was found to have little effect on the fate of the primary p blast cells, which continue to follow their normal P fate. These findings have been interpreted in several ways: that (1) the blast cell descendants of an O/P teloblast are developmentally pluripotent, i.e., capable of giving rise to either 0 or P fates; (2) these blast cells will take on one o r the other fate depending upon whether they lie next to the n o r q bandlet within the germinal band; and (3) the t w o fates form a developmental hierarchy, because if there is only one O/P

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xv

-

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BODY WALL

....

....””

’...., .....:

xv I XVll XVlll XIX

xx XXI FIG. 12. 0-to-P transfating. Camera lucida tracing of seven segments of a stage 10 embryo of Helobdella triserialir. Lineage tracer was injected into the generative 0 teloblast at stage 7; some hours later, after a number of labeled o blast cells had been produced, the generative P teloblast was killed. Labeled epidermal cells are stippled; other labeled cells are black. The nerve cord is drawn separately; its true position relative to the body wall is indicated by a dotted outline. The upper segments (XV-XVII) are populated by a full complement of cells, including progeny of p blast cells, that were born before the P teloblast was killed. Here, the labeled progeny of the 0 teloblast form a typical 0 pattern. Because of the ablation, the lower segments (XIX-XXI) lack progeny of the P teloblast. Here, progeny of the 0 teloblast form a typical P pattern; np, nephridiopore complex; other abbreviations as in Fig. 9. The segment labeled XV is M11 by the enumeration scheme used elsewhere in this paper. (From Shankland and Weisblat, 1984.)

teloblast-derived bandlet present, that bandlet will assume the P fate (said to be the “primary” fate of the equivalence group) (Weisblat and Blair, 1984). Hence, in a normal embryo, the o bandlet is diverted from the primary P fate to the “secondary”0 fate by some form of interaction with the p bandlet that lies beside it. Interactions with the n and q bandlets appear to have no role in this transfating; despite ablation of the N and/or Q teloblasts, the o bandlet cells still take on the 0 fate as long as the p bandlet is present, and they are transfated to the P fate if

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the p bandlet is ablated (Weisblat and Blair, 1984; Zackson, 1984). On the other hand, the epithelial cells overlying the germinal bands may play a role in the fate-determining interactions of the o and p bandlets, because producing localized photodynamic lesions in that epithelium at stage 7 produces partial transfating of o blast cells to the P fate, even in the presence of the adjacent p bandlet (Ho and Weisblat, 1987). Transfating experiments in Theromyzon have provided additional insights into the O-to-P transfating process. Unlike in Helobdella, when the generative P teloblast is ablated in Theromyzon, transfating of the o bandlet occurs in some, but not all, segments deprived of the p bandlet (Keleher and Stent, 1990). When these embryos were observed at stage 7 or 8, in some segments that lacked the p bandlet the o bandlet had moved over into the position of the absent p bandlet, and in others it had not moved. Scoring the same embryos at stage 10 for the development of 0 or Y fates indicated that transfating had occurred only in the segments in which the o bandlet had moved into an abnormal position next to the q bandlet. There was no transfating in segments in which the o bandlet remained in its normal position next to the n bandlet. These results suggest that positional cues derived from cells other than those belonging to the n, 0, p, o r q blast cell bandlets play a role in determining the fate of the pluripotent o and p blast cells. T h e underlying m bandlet cells may provide these signals. Using the photosensitizing lineage tracer FDX, it is possible to show that the o blast cells ultimately do become committed to the 0 fate and no longer respond to ablation of their apposed p bandlet by transfating (Shankland and Weisblat, 1984). In this experiment, the generative P teloblast was injected with FDX and the generative 0 teloblast was injected with a nonphotosensitizing tracer, such as RDX or HRP. At progressively later developmental stages, germinal bands containing these photosensitized blast cell bandlets were illuminated, ablating the p bandlet cells and their descendants. By the end of stage 10, many labeled o blast cell clones in these embryos had become committed to the 0 fate. However, this commitment occurred in a sequence of three successive steps, rather than in a single event affecting the fate of the entire descendant clone of each o bandlet cell. In each of the three steps, members of the o blast cell clone become committed only to a particular portion of the entire 0 fate, concomitantly losing their potency to transfate into a particular subset of the elements of the P fate (Shankland and Weisblat, 1984).T h e three commitment steps to the 0 fate, and the corresponding losses of pluripotency, appear to be associated with successive divisions of the o blast cell clone. In each of these divisions, one of the two daughter cells becomes committed to produce a subset of 0 fate ele-

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ments, while the other daughter cell remains pluripotent for the remainder of the, as yet uncommitted, elements of the 0 and P fate (Shankland and Stent, 1986). This orderly, stepwise sequence of partial commitments accompanying asymmetric cell divisions must reflect important aspects, as yet undetermined, about the molecular nature and organization of the determinants that commit embryonic cells to their eventual fates.

V. Myogenesis and Neurogenesis

A. MYOGENESIS In all leech species studied, the musculature of the leech body wall develops mainly during growth and expansion of the germinal plate. T h e outer layer of circular muscle fibers (Fig. 1B) and the inner layer of longitudinal fibers are first to appear; shortly thereafter, the intermediate layer of oblique muscle fibers appears between the circular and longitudinal layers. Observing the development of the musculature has been aided greatly by the isolation of a monoclonal antibody that binds selectively to leech muscle fibers (Zipser and McKay, 1981). T h e circular, longitudinal, and oblique muscle layers all arise according to the same morphogenetic scheme: scaffold cells lay down the basic architecture of the layer and myoblasts then collect around the scaffold cells to establish the definitive pattern of fascicles. The scaffold for the circular and longitudinal muscle layers of the body wall is provided by a segmentally iterated gridwork of individually identifiable founder fibers (Stuart et al., 1982; Torrence and Stuart, 1986). The first circular founder fiber in each hemisegment, the primary circularfiber (Fig. 13), appears late in stage 8 or early in stage 9 and spans the entire distance from the ventral midline to the lateral margin of the germinal plate. In accord with the rostrocaudal gradient that characterizes development of the germinal plate, this fiber is first seen in anterior segments (at late stage 8) and only later in more posterior segments (at early stage 9). Each primary circular founder fiber lies along the posterior margin of the intersegmental septum, and by convention it defines the anterior edge of a segment. T h e second circular founder fiber appears soon after the primary circular fiber. This fiber is designated the septalfiber, because it lies along the anterior margin of' the intersegmental septum. Additional circular muscle founder fibers (Fig. 13) appear progressively throughout the remainder of stage 9 and into stage 10.

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FIG. 13. Myogenesis and neuroblast migration in 14 segments of a stage 9 embryo of Theromyzon nrde. The entire width of the germinal plate is shown in this tracing from a fluorescence photomicrograph. Immunolabeled muscle fibers are stippled; lineage-tracerlabeled cells of the q bandlet (on one side only) are solid black. The longitudinal extent of one segment is indicated by the bracket labeled s, at the left. The increasing numbers of circular (horizontal) and longitudinal (vertical) muscle fibers, from the posterior segments at the bottom toward the older, more anterior segments at the top, reflect the orderly appearance of new fibers over developmental time. In the posteriormost segments, the labeled q bandlet is still coherent. In more anterior segments, major (M) and minor (m) groups of labeled cells leave the q bandlet and migrate into the future ganglia (go, dashed outline), which are outlined by the primary circular (pcf) and deep longitudinal (dlf) muscle fibers; scf, septa1 circular muscle fiber. Scale bar = 50 pm. (Drawing by S. A. Torrence.)

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Longitudinal muscle founder fibers begin appearing along the lateral margins of the germinal plate very soon after the consegmental primary circular fiber (Fig. 13). At about the time that the septa1 circular fiber first appears, a prominent band of longitudinal founder fibers also appears that lies deeper in the germinal plate (i.e., further from the epidermis) than do other longitudinal fibers of the body wall musculature. These are referred to as the deep longitudinalfibers, although by stage 11 they come to lie at the same depth as the rest of the longitudinal fibers. Additional longitudinal muscle founder fibers appear progressively throughout the remainder of stage 9 and into stage 10. The architecture of the oblique muscle layer of the hemisegment is provided by a single scaffold cell, the C-cell (Jellies and Kristan, 1988a; Jellies, 1990). In Hirudo, this cell grows about 70 parallel processes, which lie at an angle of 45" to the long axis of the embryo (Fig. 14A). These processes grow out between the layers of longitudinal and circular muscle precursors and they serve as the scaffold on which myoblasts gather and orient themselves to form individual oblique muscles (Fig. 14B).The spacing between the processes appears to depend on interac-

C-cells

FIG. 14. Morphology of the C-cells, precursors of the oblique muscles, in Hirudo medicinal&. (A) Tracings of HRP-filled C-cells in one segment of E12. The ventral midline of the germinal plate runs down the ganglion (indicated by dashed lines), and the nephridiopores are about two-thirds of the way to the lateral edge of the germinal plate. (Tracings provided by D. .M. Kopp.) (B) Diagram showing how myocytes line up along the processes of C-cells to form the oblique muscles in two midbody segments at about E13. (Drawing provided by J. Jellies.)

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tions among growth cones at the ends of the processes. T h e processes of C-cells in adjacent segments are transiently linked by gap junctions at the intersegmental border. Interactions via these junctions appear to establish the extent and spacing of the oblique muscles in successive segments, thus producing a uniform distribution of oblique muscles along the whole length of the body. Myoblasts begin aggregating around their scaffolds during stage 11 of glossiphoniid development. The myoblasts then elongate and differentiate into definitive muscle fibers. The orthogonal grid of founder fibers thus serves as a template for the development of the definitive muscle fascicles of the adult. It is not known whether the founder fibers for the circular and longitudinal muscles persist into the adult, but it appears that the C-cells die after completing their task of organizing the oblique muscle layer (Jellies and Kristan, 1988a). The use of scaffold cells for establishing muscle layers is a developmental strategy also encountered in the development of insects (Ho et d., 1983) and crustaceans Uellies, 1990).

B. GANGLIOGENESIS

1. Ganghonu: Rluliments The process of ganglion formation is similar in hirudinid (Fernandez and Stent, 1983) and glossiphoniid (Fernandez, 1980; Kuwada and Kramer, 1983; Torrence and Stuart, 1986; Stewart et al., 1987) leeches. The following detailed description is based primarily on observations of glossiphoniid embryos. In stage 8 and early stage 9, when the germinal plate of a glossiphoniid embryo has just been formed by the coalescence of the right and left germinal bands, its constituent bandlets are still coherent columns of cells. T h e four bilateral pairs of ectodermal bandlets lie in a single layer, and the single bilateral pair of mesodermal bandlets lies just below the ectodermal bandlets (Fig. 6). At this stage, before there are morphologically recognizable ganglionic rudiments, the primary circular and deep longitudinal muscle fibers described in the previous section first become detectable. When ganglionic rudiments appear slightly later in stage 9, each occupies the rectangular territory bounded by two successive primary circular fibers and by the right and left deep longitudinal fibers (Fig. 13). Thus, these muscle fibers outline the presumptive ganglionic territory in each segment. At the beginning of gangliogenesis, cells begin accumulating within

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the presumptive ganglionic territory by proliferation of cells locally and by migration from more lateral parts of the germinal plate. In the lateral regions of the presumptive ganglionic territories, bilaterally paired aggregations of cells form; they are the earliest visible morphological sign of ganglion formation (Fig. 7A). In accord with the rostrocaudal developmental gradient in the germinal plate, these cell aggregations appear first in the anteriormost segments and progressively later in ever more posterior segments. The longitudinal muscle fibers that will extend along the interganglionic connective nerves appear at about this stage as flat cells, stainable with the antimuscle antibody, on the deep (future dorsal) aspect of the nascent ganglia. Where it overlies the primary circular muscle fibers, the presumptive neural tissue is thinner, forming narrow clefts that demarcate the margins of individual ganglia. Adjacent ganglia remain in contact with one another throughout most of stage 9, during which time longitudinal nerve fiber tracts are established between them. Thus, when the midbody ganglia begin to move apart from one another late in stage 9, they are already linked via connective nerves. As the embryo elongates during later stages, the midbody ganglia move increasingly far apart and the connective nerves lengthen accordingly. Rather than separating, the anteriormost four and posteriormost seven ganglia fuse to form the subesophageal and caudal ganglia (Fernandez, 1980; Kuwada and Kramer, 1983; Torrence and Stuart, 1986). Ganglionic neurons are parceled into their six glial packets in stage 10 (Kuwada and Kramer, 1983). As in the vertebrate nervous system (Oppenheim, 1991), neuronal cell death helps to shape the developing nervous system. Studies of neuronal cell number during development of the hirudinid Huemopis mumnoruta (Stewart et ul., 1986) have shown that by E10, newly formed midbody ganglia contain approximately 14% more neuronal cell bodies than does an adult ganglion. Between El0 and E20, this number declines to the adult value. The presence of pycnotic and fragmenting cells during this decline indicates that cell death is probably responsible for the decrease in cell number. Cell death may perform three functions in nervous system development (Truman, 1984): ( 1 ) to regulate the size of neuronal populations, serving to match cell numbers between two regions of the CNS o r between the CNS and the periphery; (2) to remove unneeded cells whose production is a by-product of fixed cell lineages; and (3) to remove cells whose function is transient, after they are no longer needed. It is likely that cell death plays all of these roles in the development of the leech nervous system (Stewart et al., 1986), as illustrated by the following examples. First, cell death regulates cell number by removing some neu-

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rons that arise as pairs but are unpaired in adult ganglia. Second, an immunochemically identified pair of putative neurons that arise in each segmental ganglion of Hirudo appear to be transiently functional. These cells grow processes, persist for about 2 days, and then die (Stewart et al., 1987). Third, it seems likely that the stereotyped cell lineages of the leech generate the same complement of neural precursor cells in each segment, and that segment-specific patterns of cell death play some role in producing distinct segmental identities among midbody ganglia. The genesis of extra neurons also contributes to unique, segmentspecific characteristics of some ganglia. As mentioned above, ganglia in the reproductive segments, M 5 and M6, of adult leeches contain significantly more neurons than d o other midbody ganglia (Macagno, 1980). The origin of the increased cell number in ganglia of reproductive segments M5 and M 6 has been studied in embryos of the hirudinid Hnemopt-s. Here the ganglia in reproductive segments contain the same number of neurons as other midbody ganglia until E20 (Stewart et al., 1986). Thereafter, neuronal cell number gradually increases in ganglia of segments M 5 and M 6 through late embryonic and postembryonic development, while the cell number remains constant in other midbody ganglia. Immunohistochemical studies of ganglionic DNA replication indicate that the additional cells are generated by proliferation of neuroblasts within ganglia of the reproductive segments (Baptista et al., 1990). 2 . Neuroblmt Migration Migrations of neuroblasts and immature neurons have long been recognized as playing crucial roles in the development of vertebrate nervous systems (Rakic, 1972; Le Douarin, 1984), but they have been much less prominent in accounts of invertebrate neurogenesis. Attention was first drawn to the importance of cell migration in the neural development of glossiphoniid leeches when it was observed that all blast cell bandlets contribute cells to the CNS, although the p and q bandlets initially lie completely outside the presumptive ganglionic territory. Hence the progeny of p and q bandlet cells that are destined for the CNS must migrate to enter the ganglionic rudiments (Weisblat et a/., 1984). Studies analyzing the migrations of lineage-tracer-labeled cells have revealed that in each segment, small groups of cells follow stereotyped migration pathways from the site of their birth to their characteristic final positions. Cells of the q bandlet must migrate farther to reach the CNS than do any other ectodermal neural precursor cells and their migrations have been described in the greatest detail (Weisblat et al., 1984; Torrence and

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Stuart, 1986; Torrence, 1991) (Fig. 13). The description of the fates of these migrating cells reemphasizes the diversity of cell fates that are assumed by the progeny of the ectodermal bandlet cells. In every hemisegment, two small groups of cells leave the q bandlet to migrate toward the presumptive CNS. These cells divide several times during and after their migration. T h e major group migrates toward the ventral midline along the anterior margin of its segment, just posterior to the primary circular muscle fiber. Most of the cells of this group enter the ganglionic rudiment, where they give rise to particular glia and neurons (cf. Fig. 9). A few cells remain outside the ganglionic rudiment and they contribute to a cluster of peripheral neurons in the segmental MA nerve. The remainder of this peripheral cluster is derived from the smaller, and later-departing, minor group of migratory cells that also divide during their migration. These cells migrate medially along a midsegmental path, posterior to the path followed by the major group. After the peripheral cluster has formed, one cell leaves it to enter the ganglion. The q bandlet cells that d o not migrate give rise to the dorsal epidermis and peripheral neurons. A single group of cells in each segment migrates from the p bandlet toward the presumptive CNS, starting out at about the same time as the migratory q bandlet cells. Like the minor group of q bandlet cells, migratory cells from the p bandlet follow a midsegmental path, approximately halfway between successive primary circular muscle fibers. These cells will form cell floret 1 in the periphery, as well as central neurons and a packet glial cell (cf. Fig. 10) (Torrence and Stuart, 1986; Braun and Stent, 1989a). Most cells of the n and o bandlets arise within the presumptive ganglionic territory and are incorporated directly into the ganglionic rudiments. Nevertheless, they also undergo characteristic rearrangements and short migrations within the ganglionic rudiment to reach their definitive positions (Stuart et al., 1987; Braun and Stent, 1989a). For example, in the adult midbody ganglion, the serotonin neuron pms is positioned at the posterior margin of the segmental ganglion, although it arises from a primary blast cell clone that initially populates the anterior ganglionic quadrant (Stuart et al., 1987; Bissen and Weisblat, 1987). Thus, pms o r its precursor must migrate posteriorly a distance of at least one-half segment to reach its definitive position. A few central neurons turn out to be of mesodermal provenance (Fig. 10) (Weisblat et al., 1984; Kramer and Weisblat, 1985). They arise as a group of five morphologically immature, m bandlet progeny that migrate into each nascent hemiganglion to take up their specific positions (Shankland and Martindale, 1989). Some prospective peripheral neu-

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rons also must migrate to reach their characteristic positions. For example, peripheral neurons nzl, nz2, and nz3 (collectively referred to as nz neurons) (Fig. 9) arise within the presumptive ganglionic territory and migrate laterally to reach the periphery. Furthermore, several o or p bandlet-derived peripheral neurons (pz6, pzl0, and LD2) arise near the lateral margin of the presumptive ganglionic territory and migrate laterally to reach their definitive positions (Weisblat et al., 1978; Braun and Stent, 1989a). Precursors of nonneural ectodermal cells have also been shown to undergo similar migrations (Martindale and Shankland, 1988; Braun and Stent, 1989a). In summary, the determinate patterns of cell division that generate the neurons and glial cells of the leech nervous system do not place most cells directly into their characteristic final positions. Instead, the definitive pattern of neuronal positions is produced by extensive and stereotyped cell rearrangements and migrations. 3 . Tissue Interactions

To investigate the role of tissue interactions in controlling the migrations that generate the neuronal pattern of the leech nervous system, the development of lineage-tracer-labeled cells that are descended from the n and q bandlets has been studied in embryos selectively deprived of other bandlets. T h e results showed that many neurons differentiate even when deprived of their normal intractions. For example, identified neurons derived from the n bandlet, including the peripheral nz neurons and the central serotonin neurons (Figs. 3 and 10; also, see later, Fig. 19), found their characteristic positions, grew axons, and (in the case of the serotonin neurons) neurochemically differentiated in segments deprived of the o and p bandlets or of the q bandlet (Blair and Weisblat, 1982; Blair, 1983; Stuart et al., 1987, 1989). Similarly, cells derived from the q bandlet followed their normal migration pathways, found their normal final positions, and (in the case of the peripheral dopamine neuron MD) neurochemically differentiated in segments deprived of n, 0, or p bandlets (Blair, 1983; Torrence, 1991). Thus, interactions among cells of different ectodermal lineages are not required for the differentiation of at least some aspects of neuronal phenotype. In contrast, two types of teloblast ablations did have dramatic effects on n bandlet progeny. Although the mirror-symmetrical patterns formed by the left and right n bandlets are normally restricted to their respective sides of the embryos (Figs. 10 and 15A) (Weisblat et al., 1984; Kramer and Weisblat, 1985; Weisblat and Shankland, 1985; Torrence and Stuart, 1986; Stuart et al., 1989),elimination of either one n bandlet (Fig. 15B, left panel) (Blair and Weisblat, 1982; Blair, 1983; Stuart et al.,

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1987, 1989) o r one m bandlet (Fig. 15C, left panel) (Blair, 1982; Torrence et al., 1989) induces an abnormal cross-over of n bandlet cells to the opposite side. After n bandlet ablation, one hemiganglionic complement of several identified cell types was missing, including neuropil glial cells (Blair and Weisblat, 1982), serotonin neurons (Blair, 1983; Stuart et al., 1987, 1989), and the peripheral neurons nzl, nz2, and nz3 (Stuart et al., 1989), showing that descendents of the absent n bandlet were not regulatively restored from any other source. Furthermore, the single surviving n bandlet generated its normal segmental complement of descendant cells, but they were abnormally distributed, with some cells on one side of the ganglionic midline and some cells on the other. When peripheral nz neurons and central serotonin neurons abnormally crossed the ventral midline into hemisegments that were deprived of an n bandlet, they nearly always occupied the normal positions of their missing homologues (Fig. 15B, right panel). This observation might suggest that pluripotent n bandlet cells differentiate into a particular neuron because they occupy a particular location. However, this hypothesis predicts at least some bilateral duplications of identified neurons when the descendants of a single n bandlet are spread over both sides of the nervous system, because two pluripotent cells should sometimes happen to occupy homologous positions on the opposite sides of the same segment. Such duplications were never observed. Therefore, cells that cross over into contralateral territory must be committed to finding the characteristic position of absent homologous neurons. [Neural precursor cells differ in this regard from epidermal precursors, which are not committed to any particular position within the epithelium (Blair and Weisblat, 1984).] T h e results described so far do not exclude the possibility that a neuronal precursor cell initially is instructed only about its destination following migration, and it receives further instructions regarding the remainder of its phenotype once it has completed its migration. This possibility has been eliminated, however, by the behavior of n bandletderived neuronal precursor cells that have been deprived of their underlying m bandlet (Fig. 15C, left panel) (Blair, 1982; Torrence et al., 1989). Stage 10 or 11 embryos that were prevented from forming most of one m bandlet contained a zone of segments lacking mesodermal tissues on one side (Fig. 15C, right panel). Just as in the case of hemilateral n bandlet ablations, when one m bandlet is missing, some precursors cross the midline and the resulting immigrant neurons occupy their characteristic sites and develop their characteristic phenotypes. In this case, however, on the side containing the m bandlet there are duplicate n bandlet-derived neurons, one native and the other immigrant. In these

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embryos, some of the n bandlet-derived cells remain on the mesodermdeprived side, where they are scattered along the nerve cord and are not organized into recognizable patterns. Nevertheless, many of the stay-athome neurons differentiated as serotonin neurons (Fig. 15C, right panel) (Torrence et al., 1989). Several general conclusions can be drawn from these experiments. First, a precursor cell does not need to occupy its normal position nor does it need contact with a normal complement of mesoderm, to differentiate neurochemically. Second, the fact that migration of neuronal precursor cells from either side is normal on the side with mesoderm and disorganized on the side lacking mesoderm implies that the posiFIG. 15. Summary of experiments analyzing the effects of unilateral N (panel B) or M (panel C ) ablation on the development of identified neurons. In each panel, the protocol is diagrammed on the left and the results are illustrated on the right in four segments from a stage 11 embryo. Round black dots within the diagrammatic ganglia represent immunoreactive serotonin neurons (cf. Fig. 3); black ovals next to the ganglia represent peripheral nz neurons (cf. Fig. 9). Both cell types are derived from the n bandlets. Hatching and stippling represent differently colored lineage tracers. Anterior is up. (A) Normal fate mapping. Protocol: the left and right n bandlets were labeled with differently colored lineage tracers [red-fluorescing rhodamine dextran amine (RDA) and the yellow-fluorescing fluorescein dextran amine (FDA)]by injecting them into their parental teloblasts (circlesat the bottom of each bandlet) at stage 6 or 7. Result: cells descended from either n bandlet were restricted to their respective sides of the nerve cord. (B) Effects of unilateral n bandlet deprivation. Protocol: the left and right n bandlets were labeled as in A. Later, after a few labeled n blast cells had been formed, further formation of the right n bandlet was aborted by killing the right N teloblast by injecting it with DNase. Result: anterior control segments (top ganglion) contained progeny of both n bandlets, born before the teloblast was killed, and were completely normal. More posterior segments (bottom three ganglia) lacked progeny of the right n bandlet, and all serotonin and nz neurons were derived from the left n bandlet. In such segments, the left n bandlet gave rise to its normal complement of identified neurons. These neurons were abnormally distributed across both sides of the nervous system, but on either side they occupied positions appropriate for their cell types. ( C ) Effects of unilateral m bandlet deprivation. Protocol: the left n and m bandlets were labeled with differently colored lineage tracers, as in A. Later, after a few labeled blast cells had been formed, further formation of the left m bandlet was aborted by killing the left M teloblast with DNase. Result: anterior control segments (top ganglion) contained progeny of a complete Complement of blast cells, born before the teloblast was killed, and were completely normal. More posterior segments lacked progeny of the left m bandlet. Here, both n bandlets gave rise to serotonin and nz neurons. Some progeny of the mesodermdeprived left n bandlet crossed into nondeprived right hemiganglia, where they intermingled with their normally contralateral homologues and found appropriate, cell typespecific positions adjacent to those homologues (middle two ganglia). Other progeny of the left n bandlet remained in the mesoderm-deprived region. Such cells were not organized into recognizable patterns. In the extreme posterior segments (bottom ganglion) progeny of both n bandlets were disorganized. (A and B from Stuart et al., 1989; C from Torrence et al., 1989.)

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tional information used by migrating neuronal precursor cells is provided by mesodermal tissue. Third, the observation that n-derived neurons can cross the ventral midline, but d o not normally do so, implies that under normal conditions the apposition of the left and right n bandlets prevents the cells of both n bandlets from crossing the midline. Fourth, because immigrant n bandlet cells from a side lacking mesoderm intermingle freely with the resident n bandlet cells, the ventral midline of a leech embryo does not represent a line of clonal restriction of the same kind as compartment boundaries in insect embryos (Garcia-Bellido et al., 1973; Crick and Lawrence, 1975). T h e migration of cells derived from the q bandlet cells, following ablation of specific tissues, suggested a further role for mesoderm in guiding cell migrations. After the mesoderm is ablated on one side of an embryo, q bandlet-derived cells begin to migrate toward the ventral midline at the normal time, but they fail to follow normal migration pathways or to find their normal definitive positions. Thus, commitment of particular q bandlet-derived cells to migrate does not depend on the presence of mesoderm, but their ability to follow normal pathways and recognize their normal destinations does (Torrence, 199 1). T h e results of these ablation experiments strongly suggest that neuronal precursor cells attend to local cues to find their normal positions, and that at least some of the cues are of mesodermal origin (Stuart et ul., 1989; Torrence et al., 1989; Torrence, 1991). Because neurons are normally arranged with bilateral symmetry in a segmentally iterated array, it is likely that the mesodermal positional cues are similarly arranged. These characteristics accord well with the developmental origin of the mesoderm from the bilaterally symmetrical m bandlets, each of which products segmentally iterated complements of progeny cells from its longitudinal series of primary blast cells. C. NEUROCHEMICAL DIFFERENTIATION

Neurochemical differentiation in leech embryos has been followed by diverse methods. To determine when neurons could first synthesize acetylcholine (ACh) or serotonin, embryonic nerve cords that had been isolated at various developmental stages were exposed to [3H]choline o r to [3H]hydroxytryptophan and their rate of ["H]ACh or [3H]serotonin synthesis was assayed. In Hirudo, detectable conversion of 3H-labeled precursors into ACh or serotonin appeared during E7-E8, when ganglia are first forming, and then rose rapidly from E9 onward (Wallace, 198 1). Similarly, Huementeriu embryos synthesize very small amounts of

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ACh and serotonin early in stage 9 (Fig. 16) (Glover et al., 1987). The amount of transmitter synthesized rose slowly in middle or late stage 9, and then rapidly duriqg stage 10. By the beginning of stage 11, transmitter synthesis was 20-fold higher than at stage 9. Staining the embryonic nervous system for the presence and distribution of acetylcholinesterase (Fitzpatrick-McElligott and Stent, 1981) indicated that the capacity to hydrolyze ACh develops in parallel with the capacity to synthesize it. Also, there is a concomitant rise in the levels of ACh and serotonin, as measured by radioimmunoassay (Fig. 16) (Glover et al., 1987). The appearance and accumulation of various transmitters in identified neurons have also been followed using specific histochemical methods. In glossiphoniids, neuronal staining with an antiserotonin antibody is first detectable at midstage 9. The number of immunoreactive serotonin neurons in the nerve cord increases rapidly until early stage 10 and approaches that of the adult complement by early stage 11 (Fig. 16C) (Glover et al., 1987). This progressive increase in the number of immunoreactive serotonin neurons has two components. One is a progressive increase in the number of ganglia containing a given type of immunoreactive serotonin neuron; in accord with the rostrocaudal development gradient, serotonin neurons of a given type appear first in the anteriormost ganglia, and neurons of the same type are added successively in more posterior ganglia. The other component is a progressive addition of cell types within any given ganglion, with the Retzius neuron appearing first and the other serotonin neurons later. Retzius neurons are labeled by an antiserotonin antibody very early during axonogenesis, whereas the other serotonin neurons cannot be labeled until their axonogenesis is well under way (Glover and Mason, 1986; Glover et al., 1987; D. K. Stuart, unpublished observation). The characteristic blue-green fluorescence induced in dopamine neurons by glyoxylic acid treatment has been used to study the accumulation of dopamine in the peripheral neurons MD and LDl (Figs. 3 and 10). In Haementeria (Glover et al., 1987),stainable MD neurons first appear in anterior segments early in stage 10, at which time they have already projected axons into the CNS. During stage 10 and early stage 11, central arbors of MD neurons become more elaborate, and additional MD neurons appear in progressively more posterior segments. LD 1 neurons cannot be labeled before stage 11, In Hirudo, MD neurons become stainable in anterior segments by about E9-El0, shortly after completion of gangliogenesis. As in Haementm.u, the MD axons in Hirudo project into the CNS by the time they contain enough dopamine to be labeled (Dietzel and Gottmann, 1988). Interestingly, in ganglia of H i d o embryos that have been preincubated in dopamine between E7 and El 1,

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FIG. 16. Development of serotonin metabolism in Haementrrin gliilianii during stages 911. Each interval on the ordinate corresponds to 1 day of-development, with the first time point being taken early in stage 9 (approximately 10 days after egg deposition for this species) and the last early in stage 11. (A) Serotonin content of nerve cord (circles) and body wall (squares), i.e., germinal plate from which the nerve cord had been dissected. The asterisk represents the value obtained for a total germinal plate, from which the nerve cord was not dissected, at the earliest time point, showing that serotonin was undetectable at that stage. Error bars indicate standard deviation (SD) of the mean; if the SD was smaller than the height of the symbol. no error bar is shown. (B) Specific serotonin content, in picomoles of serotonin per microgram of protein; same data as in A. (C) The number of serotonin-irnmunoreactive neurons in the nerve cord. The dashed horizontal line indicates the adult complement of serotonin-containing neurons. (D) Total capacity for synthesis and accumulation of serotonin by the nerve cord, as measured in an electrophoretic assay following a 3-hr incubation in tritiated serotonin. (E) Specific capacity for synthesis and accuniulation of serotonin by the nerve cord per microgram of protein. (From Glover et al., 1987.)

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serotonin neurons have the blue-green fluorescence characteristic of dopamine following glyoxylic acid treatment (Dietzel and Gottmann, 1988). After E l 1, serotonin neurons gradually cease to stain for dopamine after dopamine preincubation. Thus, it appears that the developing serotonin neurons first acquire, and then lose, the ability to take up dopamine. GABAergic neurons, including the inhibitory motor neurons innervating the longitudinal muscles (Cline, 1983, 1986), possess high-affinity GABA uptake systems that provide rapid resorption of the transmitter from the synaptic cleft (Schousboe, 1981). The development of this uptake system in Huementeria has been studied by autoradiography of embryonic ganglia from various stages after incubation of the nerve cord in [3H]GABA. A pair of cell bodies that takes up [3H]GABA first appears in anterior ganglia at early stage 9. In a pattern similar to serotonin neurons, at progressively later times homologous GABAergic cells appear in increasingly more posterior ganglia, while within a given segmental ganglion, additional GABAergic cells appear in a stereotyped order until early in stage 11. By that time, the full adult complement of about 30 neurons that take up 13H]GABA is present in each ganglion (Glover et ul., 1987). Another set of adult neurons is labeled by an antibody directed against the molluscan small cardiac peptide B (Shankland and Martindale, 1989; Evans and Calabrese, 1989). In the following discussion, these neurons will be referred to as “SCP neurons,” although the leech neuropeptide that is recognized by this antibody is probably not identical with molluscan SCP (Evans and Calabrese, 1989). Many SCP neurons are also labeled by an antibody against the neuropeptide FMRFamide. In Theromyzon and Helobdellu, the neurons labeled by anti-SCP or antiFMRFamide antibody first appear late in stage 10 in anterior segments and near the beginning of stage 11 in posterior segments. They appear, therefore, considerably later than most serotonin neurons. The axons of SCP neurons in the interganglionic connective become immunoreactive at the same time as their cell bodies, suggesting that, unlike Retzius neurons, SCP neurons project axons before accumulating detectable amounts of neurotransmitter. Among the earliest SCP neurons to stain are the phenotypically asymmetrical rostra1 alternating SCP (RAS) (in anterior segments) and caudal alternating SCP (CAS) (in posterior segments) neurons, whose development will be discussed in more detail below. T h e number of immunoreactive cell bodies increases as development proceeds, and some cells appear that are labeled by the antiFMRFamide antibody, but not by the anti-SCP antibody (Shankland and Martindale, 1989).

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FIG 17. Development of electrogenesis and peripheral processes in the mechanoreceptive P cells of H a m t i t e r i n ghilianii. (A) Development of electrogenesis. The adult form of the action potential is shown in the top trace. N o active electrical responses could be elicited before the last day of stage 9, when a small voltage-dependent response is present on the largest depolarization shown in the leftmost trace. Early in stage 10 (middle trace), a response resembling an action potential can be elicited, which becomes progressively faster, larger in amplitude, and with an after-potential during stage 1 I (rightmost trace). All responses were elicited by passing current into the neurons during the time between the downward deflections near the beginnings of the traces and the upward deflections near the end of the traces. The calibrations apply to all four traces. (B) Development of the‘peripheral processes of Pv from late stage 9 to early stage 11. The outlines of the ganglia are shown in stages 9 and 10,but are left out of the stage 1 1 drawing in order to show the processes of PV,which pass under the ganglion. (C) The corresponding develop-

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In summary, neurochemical differentiation begins soon after the ganglion have formed in stage 9, but proceeds mainly during the expansion of the germinal plate and elaboration of the ganglia in stage 10. Each neuronal cell type undergoes neurochemical differentiation according to a characteristic schedule-some types begin to differentiate very soon after ganglion formation and others wait for another week or so. The early appearance of some aspects of neurotransmitter metabolism in leech embryos, coinciding with-or even preceding-the earliest stages of process outgrowth (cf. Section V , D ) , has led to the suggestion that neurotransmitters may participate in the regulation of neuronal or even of general development, in addition to their subsequent role in synaptic transmission (Fitzpatrick-McElligottand Stent, 1981; Glover et al., 1987). Such regulation has, in fact, been suggested in other organisms (e.g, Buznikov et al., 1968; Kusano et al., 1977; Lauder and Krebs, 1978; Haydon et al., 1984).

D.

ELECTROPHYSIOLOGICAL DIFFERENTIATION

To follow the electrophysiological differentiation of glossiphoniid leech neurons, intracellular recordings were made from embryonic neurons, glial cells, and muscle fibers in a germinal plate preparation viewed with transillumination under differential interference contrast (Nomarski) or fluorescence optics (Kuwada and Kramer, 1983; Kramer and Stent, 1985). By early stage 10,junction potentials can be recorded from the longitudinal muscle fibers, indicating that motor neurons have innervated their peripheral targets. The development of electrogenic properties has been followed for the mechanosensory P neurons (Kuwada and Kramer, 1983) (Fig. 17A).At midstage 9, the embryonic P neurons are electrophysiologically passive and their resting potentials and input resistances are -40 to -60 mV and 200 to 500 MR, respectively, as compared to values of -35 to -45 mV and 100 MR in adult neurons. By late stage 9, the mechanosensory P neurons begin to respond actively to passage of depolarizing current into the cell body, first with delayed rectification and then with an all-or-none depolarizing ment of PD. The images in B and C are tracings made from representative Lucifer Yellow fills of P cells; the size calibration applies to all the tracings. The dorsal (DM), lateral (LM), and ventral (VM) midlines are indicated in the drawings of the cells at stages 10 and 1 1 . The size calibration applies to all drawings in B and C. (A and B from Kuwada and Kramer, 1983; C based on data from Kuwada and Kramer, 1983, and Kramer and Kuwada, 1983.)

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transient. Early in stage 11, by which time process outgrowth is well under way, P neurons respond to depolarization with an overshooting action potential that resembles the characteristic action potential of the adult cells. However, the action potential lasts much longer in the embryonic than in the adult cells and can be extended even further by bathing the preparation in high-concentration Ca2+ saline. T h e P neuron action potential takes on the fully adult properties (Nicholls and Baylor, 1968) only during postembryonic growth of the juvenile leech. Spontaneous postsynaptic potentials, excitatory as well as inhibitory, can be recorded in the P cells from early stage 10, after their neuropilar processes have started to develop. Thus, formation of functional synapses in the CNS begins at about the same time as formation of neuromuscular junctions in the periphery. A similar time course of electrophysiological development has been found also for the excitatory L motor neuron of the longitudinal muscles (Kuwada, 1984). E. MORPHOLOGICAL DIFFERENTIATION To follow the morphological differentiation of leech neurons, especially the pattern of axonal outgrowth, horseradish peroxidase (Muiler and McMahan, 1976) o r Lucifer Yellow dye (Stewart, 1978) has been injected intracellularly via microelectrodes at various stages to reveal the anatomy of individual embryonic neurons (Kramer et al., 1985; jellies and Kristan, 1988a,b).In this way, it has been found that many cell bodies in the nascent segmental ganglia of glossiphoniids have not yet grown any processes late in stage 8. However, as judged by the intercellular passage of injected dye (Stewart, 1981), at this stage sets of cell bodies are dye coupled, implying that they are connected by gap junctions. By early stage 9, exuberant growth of fine processes is under way, and the dye coupling has largely disappeared. [Early dye coupling of cell bodies, followed later by its loss, occurs also among embryonic grasshopper neurons (Goodman and Spitzer, 1979).] T h e initial fine processes disappear by midstage 9, having been replaced by a single process that, for most cells, is directed toward the midline of the ganglion, traversing the region of the future neuropil. By late stage 9, the processes of particular cells have established the trajectories of the future segmental nerves and the connectives (Kuwada, 1982; Kuwada and Kramer, 1983). 1. Mechnosensory Neurons

The characteristic projections and receptive fields of mechanosensory neurons could arise during development in either of two ways. On the

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one hand, each neuron might first project axonal processes indiscriminately into all four major segmental nerves-AA, MA, DP, and PP-followed by later pruning of inappropriate processes through retraction or degeneration. On the other hand, each neuron might, from the very beginning, project only appropriate axonal processes (i.e., those that are retained into adulthood). The first possibility implies that specific axonal projection patterns arise by selective survival of haphazard axonal outgrowth, a process that might be mediated by interaction between axons and their appropriate targets. The second possibility suggests that specificity arises by directed outgrowth toward targets, a process mediated by recognition of landmarks on the growth substratum. In order to elucidate which of these mechanisms shapes the receptive fields of the mechanosensory neurons, the axonal arborization patterns of neurons P, and P, were examined at progressively later embryological stages (Fig. 17B and C) (Kuwada, 1982; Kuwada and Kramer, 1983; Kramer and Kuwada, 1983; Kramer and Stent, 1985). Like other leech neurons, these two neurons arise as morphologically undifferentiated neuroblasts in the ganglionic primordium. They can be identified in midbody ganglia of stage 9 embryos by their relatively large size and characteristic location. The first process emerges from the P, or P, cell body at its medial pole and grows toward the midline of the future ganglion neuropil. Just before reaching the midline, the process forms a T-junction, with one arm of the T growing toward the next anterior and the other arm growing toward the next posterior ganglion, via the connective nerve. In the case of cell P,, several additional, peripherally directed processes emerge concurrently from the lateral pole of the cell body and grow into the future ventral germinal plate bordering the ganglion. By late stage 9, one peripheral process of the P, neuron has grown far enough laterally to reach the future dorsal germinal plate. This process extends few branches before reaching dorsal territory, even though it is growing across uninnervated territory. Apparently the axon fails to branch because it grows along the surface of a broad, flat cell that extends from the edge of the ganglion to the future lateral edge of the embryo (Kuwada, 1982). This process will become the main adult axon of the P, neuron, which runs in the DP nerve. The other peripheral processes of the P, neuron, which have remained in the future ventral territory, disappear. Some of the ventral skin is eventually innervated by the P, neuron via an axon in the MA nerve, a process that forms as a branch of the central axon. By contrast, the P, neuron grows only one peripheral process that normally emerges from the lateral pole of the cell body and grows into the future ventral germinal plate. This process

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will become the main adult axon of the P, neuron, running in the MA nerve. The minor receptive fields of these neurons are formed as branches of the central P, and P, axons, which extend in the connectives to the adjacent ganglia and exit from nerves in these ganglia. These processes in adjacent segments form innervation fields similar to the major fields that are being formed simultaneously by the local P, and P, neurons. Meanwhile, the central processes also begin to proliferate neuropilar processes within the ganglia, to provide for the eventual network of synaptic connections. It should be noted that although both P neurons arise as bipolar neurons, with one (central) process emerging from the medial pole of the cell body and another (peripheral) process emerging from the lateral pole, they become monopolar by late stage 10. T h e conversion from bipolar to monopolar morphology of embryonic leech neurons is the result of peripheral and central axons merging at a Tjunction, where they remain attached to the cell body via the single axon that forms the stem of the T. A similar bipolar-to-monopolar conversion occurs in embryonic dorsal root ganglion cells of the vertebrate spinal cord (Tennyson, 1965). Like axons of many other embryonic neurons (Ramon y Cajal, 1929), the developing central and peripheral P cell processes carry numerous filopodia, both at their growth cone tip and on their sides. These filopodia are much more abundant on the peripheral processes that grow out first, directly from the cell bodies, than on the peripheral processes that grow out later from the central processes, either in the ganglion of origin o r in the adjacent ganglia. T h e large number of filopodia on the initial peripheral P cell process is consistent with their putative role in pioneering the peripheral pathways, because numerous filopodia are typical of growth cones at pathway choice points (Tosney and Landmesser, 1985; Caudy and Bentley, 1986). At the time the P, axons first contact skin, the germinal plate is still expanding circumferentially and large areas of the body wall have yet to form. The axon that grows peripherally from the cell body along the future MA nerve and that establishes the primary subfield appears to follow a predesignated path, because its initial peripheral branching pattern is highly stereotyped. Later, a branch of the central (neuropil) process of the P, cell may leave the ganglion via the PP nerve and establish a secondary sensory subfield. Upon circumferential expansion of the germinal plate, the peripheral axons grow and sprout additional branches to innervate new territories in the developing body wall. At no time during the outgrowth of the P, axon do any “tentative” processes enter the AA or DP nerve branches, and by the end of stage 10

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the fraction of cases in which P, processes extend into the PP branch is only slightly greater than in the adult. Hence it would appear that the adult projection pattern of this mechanosensory neuron results not from an initially haphazard projection that is selectively pruned, but rather from the specific and directed outgrowth of processes. These observations indicate, moreover, that the relationships among various components of the adult mechanosensory receptive fields are determined before the peripheral axons leave the ganglion. For example, in the major field of cell P , the primary subfield, which is the largest subfield and is invariably present, is the first to be established, and it arises by direct axonal outgrowth along the MA nerve. The secondary subfield, which is smaller and sometimes absent altogether, is established only later, by an axonal branch of neuropil processes, a branch that exits the ganglion via the PP nerve, if it forms at all. Similarly, the much smaller minor fields of cell P , within the body wall of the adjacent segments, are established by intersegmental axon branches that exit via the MA nerve of the adjacent ganglia. These branches grow out only after the primary axon that establishes the major field of the serially homologous P, neuron has already begun to innervate its own segmental body wall. This pattern suggests that some mechanism of territorial competition helps to establish the mechanosensory receptive fields, in which the earliest process to arrive has the advantage.

2. Growth Guidance by Muscle Fibers Stereotyped axonal outgrowth and mechanosensory receptive fields suggest that developing processes grow along prespecified pathways and form branches at prespecified points. The orthogonal grid of embryonic circular, longitudinal, and dorsoventral muscle fibers seems a likely source of this information, because the fibers are already in place in the germinal plate at the time axonogenesis begins. For instance, by late stage 9, some axons have formed a T-junction on reaching the midline of the ganglionic neuropil and have begun to grow longitudinally, anteriorly, and/or posteriorly out of the home ganglion and into the next ganglion. These longitudinal axons follow two pairs of the most medial longitudinal muscle fibers-founders of the future longitudinal muscle fascicles in the connective nerves. At this stage, these muscle fibers are the only overt structures connecting the ganglia (Kramer and Stuart, 1982; Stuart et al., 1982). Some interganglionic axons follow the more lateral of these muscle fibers, and thereby pioneer the paired lateral connective nerve tracts. Other axons course between the more medial muscle fiber pair and thus pioneer the unpaired connective nerve tract, or Faivre's nerve. Later, during stage 10, processes of sensory

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and effector neurons grow out of the lateral margin of the ganglion into the peripheral germinal plate, following circular muscle fibers. The axon of the P, neuron follows the septa1 circular muscle founder fiber, apparently thereby pioneering the MA segmental nerve trunk. At intersections between the circular founder fiber and particular longitudinal fibers, the outgrowing P, axon sprouts a longitudinal branch, apparently thereby pioneering the characteristic longitudinal branching pattern of the adult segmental nerve. All the higher-order axon branches of the P, neuron are also aligned with either a circular or a longitudinal muscle fiber. T h e axon of the P, neuron grows out along the most medial dorsoventral muscle precursor to the future dorsal territory of the germinal plate, among the first axons to appear in the nascent DP nerve (Kuwada, 1982).

3. Interneurons The axonal projection of the intersegmental interneuron S is of a particularly simple form, greatly facilitating observation of its development (McGlade-McCulloh and Muller, 1989). In the adult Hirudo nervous system, there is a single, unpaired S cell per midbody ganglion (Muller et ul., 1981). That cell sends two large axons into the median connective nerve, one anteriorly and the other posteriorly; these axons extend about halfway to the next anterior or posterior ganglion. The anterior axon makes an electrical synapse with the posterior axons of the next anterior S cell at this midway point, so that the ensemble of S cells forms a functionally syncytial chain along the ventral nerve cord (Muller and Carbonetto, 1979). T h e S cells in the anteriormost ganglia of Hirudo begin axonogenesis during E9-El0, as do other Hirudo neurons. By E12-El3, S cell axons from adjacent ganglia have met midway in the interganglionic connective nerves, and within a day of this meeting have become dye coupled. T h e embryonic S cell axons continue to elongate in the connective nerve for several days after this initial contact and coupling, until they overlap for as much as 76% of their length. Thus, the coupling of embryonic S cells does not inhibit their continued axonal extension (McGlade-McCulloch and Muller, 1989). By contrast, in adult specimens of H i n d u , S cell axons damaged by a crush of the connective nerve will regrow toward each other to reestablish their connection (Muller and Carbonetto, 1979), but axonal extension ceases as soon as dye coupling between anterior and posterior S cell axons has been reestablished (Scott and Muller, 1980).

4. Motor Neurons Most motor neurons project their axons across the ganglionic midline into the contralateral segmental nerve roots, on their way to the

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peripheral musculature. Unlike interneuron S or the mechanosensory neurons, adult motor neurons project no axons into anterior or posterior ganglia via the interganglionic connectives. Study of the morphogenesis of different types of motor neurons has revealed some significant differences in their developmental strategy. In Haementeria, motor neuron L (which forms extensive peripheral arborizations innervating the entire longitudinal musculature of the contralateral segmental body wall) begins axonogenesis early in stage 9, at about the same time as the other neurons of the ganglion (Kuwada, 1984). The L neuron projects a single axon, which grows across the midline into the contralateral posterior segmental nerve root, entering that root only after the axon of the mechanosensory P, neuron has done so. Growth cones of L axons are small, unlike the large and complex growth cones of P, and P, axons, which suggests that the growing L axons fasciculate with and follow the sensory axons in their peripheral outgrowth. Although each L neuron projects a short, anteriorly directed process into the contralateral neuropil of its ganglion, it produces no supernumerary axons that are later pruned or reabsorbed. Throughout development, the pattern of L axons is consistent with the adult pattern, as is the case for the axonal outgrowth pattern of vertebrate motor neurons (Lance-Jones and Landmesser, 1981; Tosney and Landmesser, 1985; Eisen et al., 1986). Further insights into the mechanism of L axon outgrowth were obtained from developmentally abnormal Haementeriu embryos in which the two halves of the germinal plate had failed to fuse, producing two separate haif-leeches with half-ganglia (Kuwada, 1984). In such specimens, the L axons could not follow their normal pathway across the ganglionic midline. Under these abnormal conditions, the L neuron produced several supernumerary axons that followed a diversity of abnormal pathways, such as into the ipsilateral segmental nerve roots and into the interganglionic connectives. This finding implies that, in response to abnormal developmental cues provided by the bisected germinal plate, the L motor neuron can generate more than a single axon and can send the supernumeraries along pathways not normally taken by its processes. L axons are, therefore, not absolutely constrained to follow the normal L pathways. In contrast to the single, definitive axon normally projected from the start by the L motor neuron of Haementeriu, the heart accessory (HA), annulus erector (AE), and “anterior pagoda” (AP) neurons of Himdo all generate supernumerary axons during their early development, axons that are lost in later development (Gao and Macagno, 1987a,b, 1988). At the outset of axonogenesis, all three types of neurons send axons across the ganglionic midline into both contralateral segmental nerve roots, as

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.. urn FIG. 18. HA neuron projection in Hirudo medzcinulis. Ganglionic outlines are indicated by dotted lines. (A) Normal projection pattern of mature HA neurons, consisting of an arborization within the neuropil and two major axons in the roots contralateral to the soma. Note that H A neurons normally lack axons in the interganglionic connective nerves. (B) Projection of H A neurons following the ablation of one embryonic H A neuron in MX. Central axons are maintained by the HA neurons that are ipsilateral to the ablated neuron in adjacent ganglia both anterior and posterior to the ganglion lacking one HA. Extra axons from M7 and M9 enter the periphery by way of the anterior and posterior nerve roots, in MX. The normal position of the ablated HA neuron is indicated by a circle. M7, M8, and M9 indicate ganglia in midbody segments 7, 8, and 9. (Data from Gao and Macagno, 1987b.)

well as into the anterior and posterior contralateral connective nerves. Later in development, the axons in the connectives regress, so that in adults the HA, AE, and AP neurons innervate only the periphery of their o w n segment and lack intersegmental projections (Fig. 18A). To determine whether interactions between ipsilateral intersegmental homologues play a role in this developmental loss of central axons, various neurons were ablated and the axonal projection patterns of neurons in adjacent segments were examined at later stages. Ablation of HA, AE, or AP neurons early in development enhanced the persistence of the central axons of their homologues in adjacent segments long after

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these axons normally would have disappeared (Fig. 18B) (Gao and Macagno, 1987a,b). This result is consistent with the hypothesis that transsegmental interactions among central axons of segmentally homologous neurons can lead to the elimination of interganglionic processes. However, severing the connection of AP and AE motor neurons to their peripheral muscle targets also enhanced the persistence of their central axons, suggesting that contact with their peripheral targets sensitizes these neurons to the central intersegmental interaction that promotes the elimination of their central processes (Gao and Macagno, 1988). 5. Other Effector Neurons Pruning of initially exuberant axonal outgrowth occurs also in the case of some Retzius neurons in Hirudo embryogenesis (Glover and Mason, 1986; Jellies et ul., 1987). These large, serotonergic neurons innervate muscles in the periphery and arborize extensively in the ganglionic neuropil. They are thought to have a modulatory effect on their targets (Lent, 1977; Mason and Kristan, 1982; Leake, 1986; Lent and Dickinson, 1989). In most segments, Retzius neurons project axons into the periphery by way of both ipsilateral segmental nerve roots and into the adjacent anterior and posterior ganglia by way of the ipsilateral connective nerves (Fig. 19A). The peripheral axons branch extensively within the muscles of the body wall. In the two reproductive segments, M5 and M6, however, the peripheral Retzius axons branch in the walls of the reproductive ducts peculiar to these segments, but do not extend into the muscle layers of the body wall. In the two reproductive segments, moreover, the Retzius neurons lack interganglionic axons (Fig. 19B). During the early phases of axonogenesis, all Retzius neurons in midbody ganglia extend axons into the periphery and toward adjacent ganglia via the interganglionic connectives. It is only at later developmental stages that the intersegmental processes extended by the Retzius neurons of segments M5 and M6 [labeled Rz(5) and Rz(6) in Fig. 201 stop growing and are eventually retracted. This loss of central processes is temporally correlated with the initiation of contact between the axons of Rz(5) and Rz(6) and their eventual peripheral targets, the reproductive ducts (Glover and Mason, 1986; Jellies et al., 1987). If interaction of Rz(5) and Rz(6) with their presumptive targets is prevented by ablating the embryonic reproductive ducts at about the time contact begins, the central axons of these neurons persist (Loer et ul., 1987). Thus, persistence of central processes of Rz(5) and Rz(6) neurons appears to depend upon the presence or absence of specific peripheral target tissue, as does the axonal pattern of motor neurons AE and AP. Unlike the case of the AE and AP neurons, however, intersegmental interactions among

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250pm FIG.19, Peripheral projection of Retzius neurons in standard and reproductive segments of H i r u h The outlines of ganglia are indicated by dotted lines. (A) Primary and secondary projection fields of Rz(9) in an embryo on E20. The projection reaches from the ventral midline (VM,dashed line) to the dorsal midline (DM, approximate position indicated by arrow) in three segments and consists largely of branches in an orthogonal pattern. (B) Projection of Kz(5) in an embryo on E20. Rz(5) lacks axons in the interganglionic connectives and branches densely around the male reproductive ducts, whose outline is indicated by dashed lines. (Unpublished data, K. A. French and W. B. Kristan, J r . )

central axons of segmental homologues appear to be unimportant in controlling the central morphology of Retzius neurons (Loer and Kristan, 1989~).

PERIPHERAL TARGETS F. INTERACTIONS BETWEEN NEURONSAND THEIR As in the developing vertebrate nervous system (Smith and Frank, 1987; Schotzinger and Landis, 1988), interactions between neurons and their peripheral targets play a role in leech neurogenesis (French and Kristan, 1992).These interactions have been examined most extensively

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in the reproductive segments M5 and M6 of Hirudo. The developmental interactions of Rz(5) and Rz(6) neurons with their target organs, the male and female reproductive ducts, affect not only the axonal projection pattern of these neurons, but also several other of their properties: soma size, complexity of neuropil arborization, synaptic inputs, and sign of cell membrane polarization in response to ACh (Fig. 20) (Jellies et al., 1987; Kristan and French, 1988; Loer and Kristan, 1989a; Wittenberg et al., 1990). For instance, during the early stages of axonogenesis, the morphology of Retzius neurons is indistinguishable in all midbody ganglia. But Rz(5) and Rz(6) begin to look different from their segmental homologues after their peripheral axons have approached the embryonic reproductive ducts (Glover and Mason, 1986; Jellies et al., 1987). If development is allowed to proceed normally, Rz(5) and Rz(6) develop smaller somata and sparser dendritic arborizations within the neuropil of their home ganglion, and they lack central intersegmental axons. Furthermore, stimulation of either a P mechanosensory neuron or of the neural circuit controlling swimming fails to evoke excitation of Rz(5) or Rz(6), whereas either stimulus is clearly excitatory for Retzius neurons in all other midbody ganglia. Finally, application of ACh to their somata hyperpolarizes Rz(5) and Rz(6), whereas all other Retzius neurons depolarize in response to applied ACh. Ablation of the embryonic reproductive ducts at the time when the peripheral Retzius axons would normally approach them causes Rz(5) and Rz(6) to develop a morphology (Loer et al., 1987; Loer and Kristan, 1989b), synaptic input patterns (Loer and Kristan, 1989a), and a response to ACh application (W. B. Kristan, Jr. and K. A. French, unpublished results) similar to that of standard Retzius neurons. The contact between embryonic reproductive ducts and growing axons of Rz(5) and Rz(6) generates immediate as well as long-term changes in their appearance. At the time Retzius axons first leave the ganglion, their growth cones are small and simple. Most Retzius growth cones remain simple, but within hours of reaching the reproductive ducts the growth cones of Rz(5) and Rz(6) expand, becoming both larger and more complex (French et al., 1992). When reproductive duct tissue is transplanted into nonreproductive segments, axons of the resident Retzius neurons approach the tissue and their growth cones enlarge, as they do in segments M5 and M6 (French et al., 1992). Although this early response to axonal contact with ectopic reproductive ducts in nonreproductive segments is similar to that normally seen in reproductive segments, it is not sufficient to confer the other morphological features of the Rz(5) and Rz(6) phenotype on Retzius neurons in segments other than M5 and M6 (Loer and Kristan, 1989~).

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The reproductive ducts affect the development of M5 and M6 in other ways, as well. In adults, M5 and M6 contain many more neurons than other midbody ganglia (Macagno, 1980), and most of these extra neurons are generated only near the end of embryogenesis and in postembryonic life (Stewart et al., 1986). However, in Hirudo there is at least one extra pair of neurons present solely in ganglion M6, the rostra1 penile evertor (RPE) cells (Zipser, 1979).These paired neurons are born and begin axonogenesis at about the same time as most other neurons, i.e., on E9 or E10. The presence of reproductive ducts has been implicated in both the generation of the extra cells in ganglia M5 and M6 and in shaping the final axonal pattern of the RPE neuron. If the reproductive ducts are ablated before E 16, no extra cells are subsequently added to the ganglia of M5 or M6. If the ducts are ablated on E 16 or thereafter, the extra cells appear on schedule (Baptista and Macagno, 1988a).Thus, the reproductive ducts influence segment-specific neurogenesis in these two ganglia (Baptista et al., 1990).This induction of extra neurons depends not only on a signal provided by the ducts, however, but also upon the competence of the neural precursor cells of ganglia M5 and M6 to respond to the signal; reproductive ducts transplanted ectopically into segments other than M5 and M6 fail to induce the production of extra neurons. Adult RPE neurons project axons to the contralateral periphery

FIG. 20. Features distinguishing Rz(5,6) from Rz(X) in Hirudo medicinalis. (A) Soma size and density of arborization within the neuropil (expressed as a percentage of the total neuropilar area containing processes of labeled neurons, as seen in camera lucida tracings). The soma size and the density of arborization increase much more rapidly between E9-11 and E20-24 in standard segments than they do in reproductive segments, causing Rz(5,6) to have distinctly smaller somata and smaller central arborizations by the end of embryogenesis. (Data from Jellies et al., 1987.) (B) Synaptic inputs onto Rz(X) lead to excitation when the tail is pinched (at times indicated by arrows in the left traces), when the swimcontrolling neural circuit is active (indicated by the horizontal bars in the middle traces, after tail pinches indicated by arrows), or when a pressure-sensitive mechanosensory neuron (P cell) in the same ganglion is stimulated by an intracellular electrode (at times indicated by the horizontal bars in the right traces). Rz(5,6) lack these inputs. The horizontal voltage calibration bar represents 2 mV for the third Rz(X) trace and for the second and third Rz(6) traces, 5 mV for the first Rz(6) trace, 10 mV for the first and second Rz(X) traces, and 20 mV for the P cell traces. The vertical time calibration bar indicates 1 sec for the first and second Rz(X) traces and for the first Rz(6) trace, 2 sec for the second Rz(6) trace, and 20 msec for the third traces for both Rz(X) and Rz(6). (Data from Loer and Kristan, 1989a.) (C) Response of Rz neurons to acetylcholine (ACh) ejected onto their soma in ganglia from which the glial sheath has been removed. Rz(X) neurons depolarize in response to ACh, whereas Rz(5,6) hyperpolarize. (Unpublished data, K. A. French and W. B. Kristan, Jr.)

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through the anterior segmental nerve root of ganglion M6, as well as via an interganglionic collateral process through the posterior segmental nerve root of ganglion M5 (Baptista and Macagno, 1988b). The reproductive ducts of segments M5 and M 6 are among the peripheral targets of the RPE axons. Early in axonogenesis, however, the RPE neurons send axons through both the anterior and posterior segmental nerve roots of ganglion M6 and both anteriorly and posteriorly into the interganglionic connective nerves. The supernumerary axons are pruned slowly in late embryonic life and possibly persist into postembryonic life. If contact between the RPE axons and the reproductive ducts is averted or disrupted, either by ablating the ducts or by severing the segmental nerves containing the RPE axons, the supernumerary axons persist (Baptista and Macagno, 1988b). T h e reproductive ducts thus seem to play an important role in shaping the definitive axonal projection pattern of RPE neurons. Thus, interactions between the embryonic reproductive ducts and neurons in segments M5 and M6 modify several disparate aspects of neuronal development, including neurogenesis, axonal growth and maintenance, synaptogenesis, and the production of neurotransmitter receptors. In some instances (e.g., in the differentiation of the Retzius, AE, and HA neurons) the interaction requires contact between peripheral processes and their targets, whereas in other cases (e.g., in the genesis of extra neurons) a diffusible factor might mediate the interaction.

G. NEURON-NEURON INTERACTIONS: THEORIGINS OF UNPAIRED NEURONS

Initial overproduction of neurons that subsequently compete with one another for survival is a prominent feature of vertebrate neurogenesis, wherein it serves to match neuronal populations to the available targets (Oppenheim, 1991; Truman, 1984). Although such overproduction is less prominent in the highly determinate development of the leech nervous system, the production of a dozen or so unpaired interneurons, in a typical segmental ganglion, from the initially symmetrical embryonic cell lineages of the leech nervous system offers several examples of competitive interactions that control neuronal survival or differentiation. Attention was first drawn to neuron-neuron competition in the leech when lineage tracers were used to determine the lineage of the unpaired, posteromedial serotonin neuron pms (Fig. 3). By double labeling

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embryos with lineage tracer and an antiserotonin antibody, it was shown that the pms neurons are derived from the N teloblast, like all other serotonin neurons. In some ganglia, pms arises from the right N teloblast and in other ganglia pms arises from the left (Blair and Stuart, 1982; Stuart et al., 1987), with the side of origin in a given segment varying randomly from specimen to specimen (Blair and Stuart, 1982; Stuart et al., 1987; Macagno and Stewart, 1987). Thus, each N teloblast gives rise to approximately half of the pms neurons in a normal embryo, but which half is indeterminate, so it was surprising when ablation of one N teloblast caused no deficit of pms neurons (Stuart et al., 1987). This result was explained by the observation that two pms neurons arise in each ganglion, one descended from each N teloblast. In a normal embryo, one o r the other pms dies during stage 10, but in an embryo deprived of one N teloblast, all of the pms neurons descended from the remaining N teloblast survive. Evidently, competition between pms cells derived from the two N teloblasts determines which pms neuron in a given ganglion survives and which dies (Stuart et al., 1987; Macagno and Stewart, 1987). T h e developmental mechanisms for producing two other unpaired neurons seem to be similar. Lineage-tracing studies indicate that the unpaired neurons pz4 (Figs. 9 and 10) and mz4 (Fig. lo), which descend from the P and M teloblasts, respectively, also arise from bilaterally paired precursors. One precursor in each segment dies near the end of stage 9 (Kramer and Weisblat, 1985; Shankland and Martindale, 1989; D. K. Stuart, S. M. Shankland, and S. A. Torrence, unpublished observations). Two other unpaired neurons represent a variation on this theme. These cells are the large rostra1 and caudal alternating SCP neurons (RAS and CAS; collectively called AS neurons) (Fig. 21A) (Blair et al., 1990). AS neurons, which stain intensely with an antibody against small cardiac peptide, are found on only the left or right side of any ganglion. Their development has been studied in glossiphoniid leeches. RAS neurons, which were shown to descend from the N teloblasts, are found only in ganglia Ml-M3 and in the fourth subesophageal ganglion. CAS neurons, which descend from the M teloblasts, are found only in ganglia M 18-M2 1 and in caudal ganglia. Each asymmetrically placed immunoreactive AS neuron seen in an adult nerve cord arises as one of a bilateral pair of cells that begin to stain with the anti-SCP antibody in late stage 10 o r early stage 1 1 . Subsequently, the cell that becomes the AS neuron maintains its immunoreactivity, while the other ceases to stain. T h e development of AS neurons differs from that of pms, pz4, or mz4 in that no evidence of cell death has been found, and it seems likely that

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FIG. 21. Phenotypic specification of neurons by interactions within a segment and between segments. (A) Segmental distribution of SCP-immunoreactive KAS and GAS neurons (black dots) in the nerve cord of Helobdella triserialir. (B) Effect of unilateral RAS or CAS precursor ablations on the asymmetry of homologous AS neuron differentiation in adjacent segments. Ablation of a single AS neuron precursor (at a site indicated by X) caused the contralateral AS precursor (stippled) to express the mature AS phenotype of SCP-like immunoreactivity. The numbers in the left and right sides of the adjacent ganglia are the numbers of experimental embryos in which an unpaired, immunoreactive AS neuron was observed in those locations. There was a strong tendency for AS neurons in adjacent ganglia to alternate sides with the AS neuron in the lesioned ganglion. The percentage of alternation is indicated in italics beside the ganglia. (From Blair el al., 1990.)

the AS homologue survives but expresses an alternative neurochemical phenotype (Shankland and Martindale, 1989).Like the survival of pms, the neurochemical phenotype of AS neurons is controlled by competition between contralateral homologues, because ablation of AS precursors on one side of appropriate segments causes the precursors on the other side invariably to take the AS (Le., SCP+) phenotype. By varying

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the timing of these ablations it was shown that the competitive interaction occurs after the terminal mitosis of the AS precursors (Martindale and Shankland, 1990). A striking feature of the distributions of many unpaired leech neurons is that the side of origin of a given neuron tends to alternate from one ganglion to the next along the nerve cord (Fig. 21A). This has been shown in Hirudo for the pms neurons (Macagno and Stewart, 1987) and six types of unpaired SCP neurons, including the homologue to CAS (Evans and Calabrese, 1989), and in Theromyzon and Helobdella for mz4 and the As neurons (Shankland and Martindale, 1989). Such segmentto-segment alternation suggests that the outcome of competition between bilaterally homologous cells within a given segment is biased by interactions with cells in adjacent segments. This proposal was directly tested for the AS neurons by ablating, in a single hemiganglion, a small cluster of neurons that included the precursor of an AS neuron (Fig. 21B) (Blair et al., 1990). This manipulation caused the contralateral homologue of the ablated precursor to take the AS phenotype, confirming the intraganglionic interaction proposed above and imposing a particular sidedness to the AS symmetry in the operated segment. T h e AS neurons in most segments adjacent to an operated segment were found on the same side as the lesion-they alternated sides with the AS neuron in the operated segment. Thus, consistent with the proposed intersegmental interaction, experimentally imposed AS sidedness in one ganglion biased the outcomes of AS competitions in the adjacent ganglia. Many of the unpaired neurons whose side of origin tends to alternate from one segment to the next project axons to one or both adjacent ganglia via the interganglionic connective nerves. These axonal projections are proposed to mediate the interganglionic interaction (Macagno and Stewart, 1987; Shankland and Martindale, 1989; Martindale and Shankland, 1990; Blair et al., 1990). To test this proposal, the connective nerves between CAS-containing ganglia were transected in late stage 10 o r early stage 11 Theromyzon embryos. As predicted, such transection reduced the frequency of CAS alternation across the cut (Martindale and Shankland, 1990).

VI. Conclusions

As shown by the research summarized in this review, embryogenesis of the leech nervous system is highly determinate in the sense that during normal development the genealogical origin of each identified neu-

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ron can be traced, via a sequence of stereotyped cleavages, to the zygote. This determinacy might suggest that the developmental fate of a given cell is somehow governed entirely by its particular line of descent. However, cellular interactions strongly affect the fate taken on by at least some neural precursor cells, as, for example, is the case for the O/P kinship group. Thus, although the characteristic phenotype of each identified neuron stems from a series of increasingly restrictive developmental commitments made by its ancestors, and although for many neurons these developmental choices would seem to rely strongly on cell lineage, interactions with other cells cannot be ignored as a source of significant developmental information. I t seems to be generally thought that cell fates become assigned when the developmental possibilities of pluripotent cell lines are limited by a set of sequential commitment steps that end in differentiation into the final set of phenotypes emanating from each pluripotent cell line. It has been thought that such a process depends on a stepwise, typologically hierarchic sequence of commitments (Slack, 1983). For instance, a cholinergic (excitatory) motor neuron would arise in a leech along the following pathway: ( 1 ) from a cell clone committed to expressing characters that distinguish ectoderm from mesoderm, to a neuroectoderm subclone committed to expressing characters that distinguish nervous tissue from epidermis; (2) from the neuroectoderm subclone to a neuronal subclone committed to expressing characters that distinguish neurons from glia; (3) from the neuronal subclone to a motor neuron subclone committed to expressing characters that distinguish motor neurons from sensory neurons; and finally, (4)from the motor neuron subclone to a cholinergic subclone committed to expressing the gene that encodes cholineacetyltransferase (typical of excitatory motor neurons), rather than expressing the gene for glutamic acid decarboxylase (typical of inhibitory motor neurons). Contrary to this hypothetical pattern, during the development of the leech nervous system the sequence of commitment steps appears to be typologically arbitrary, rather than hierarchic. In fact, the phenotype of particular neurons shows little correlation with membership in any particular kinship group (cf. Fig. 10). Indeed, even after a primary blast cell has undergone one o r two divisions in the generation of its segmental founder clone, one of its daughter blast cells may still give rise to a mixed clone of neural and epidermal cells (Shankland and Stent, 1986). Hence in the leech, instead of the proposed pattern, the neurodevelopmental pathways seem to be mainly topographically hierarchic, rather than typologzcally hierarchic. In other words, it is the position of two cells, rather than their phenotype, that is usually correlated with their geneological closeness.

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Admittedly, the serotonin neurons, all being derived from the N teloblast, are genealogically more closely related to one another than they are to the dopamine neurons, which are derived from the OPQ blastomere. This relationship might suggest that the NOPQ blastomere cleaves in a typologically hierarchic fashion, according to which the N teloblast daughter is committed to generate serotonin neurons and the OPQ blastomere daughter is committed to generate dopamine neurons (cf. Figs. 3, 9, and 10). However, these two types of monoamine neurons account for only a small fraction of all the diverse progeny of the N and OPQ cells, so it appears more reasonable to invoke a topographic interpretation of the cleavage pattern: nearly all of the N kinship group cells (including the serotonin neurons) are destined for the CNS, whereas the bulk of the OPQ-derived cells (including the dopamine neurons) are destined for the body wall (Weisblat et aZ., 1984). The topographic rationale can be extended further to the cleavage pattern of the OPQ blastomere, whose Q and OP daughter cells primarily generate descendants in the dorsal and ventral body wall, respectively. Nevertheless, the topographic hierarchy in the cleavage pattern is far from absolute. Some descendants of the Q and of the OP blastomeres are destined for the central nervous system on the ventral midline, whither they migrate from the lateral germinal plate, where the cleavage pattern initially places them. Thus, topography based on lineage pattern may exert a strong influence upon the choice of developmental fates among leech neurons, but many neurons are clearly not restricted to their original location. Two kinds of agents are commonly thought to contribute to developmental commitment under either the typologically or the topographically hierarchic mode. One of these agents consists of a set of intracellular determinants, which would account for the differential commitment of sister cells in terms of unequal partition of cellular elements in successive cell divisions. For instance, a pluripotent cell might possess two determinants, a and 6, that are necessary for producing cell types A and B respectively. Commitment of a daughter cell to fate A (and loss of pluripotency) would occur at an asymmetric cell division in which the daughter cell received only a and not b. Under this mechanism, cell lineage would play a governing role in cell commitment by consigning particular subsets of intracellular determinants to particular cells. The other commonly considered agent leading to commitment consists of a set of intercellular inducers whose elements are anisotropically distributed through the volume of the embryo. In this construction, a pluripotent cell would be one that was capable of responding to either of two inducers, a or 6, that were necessary for producing cell types A and B, respectively. Commitment of a cell to fate A (and the loss of pluripotency)

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would occur when the cell responded to inducer a at some crucial stage of development and lost the ability to respond to 6. Under this mechanism, cell lineage could play a crucial role in cell commitment by placing particular cells at particular sites within the inductive field, hence governing the pattern of their exposure to inducers. (Note that this idea leaves the source of the competence to respond to a and b open to conjecture.) Developmental studies carried out with nematodes (Laufer et al., 1982) and ascidians (Whittaker et al., 1977) have shown that cell lineage can play a governing role in cell commitment by bringing about the orderly, unequal partitioning of intracellular determinants among daughter cells in successive cell divisions. T h e mitotic partition of teloplasm during the very earliest leech embryonic cleavages appears, similarly, to exert a strong influence on the subsequent development of leech blastomeres A, B, C, and D (Astrow et al., 1987; B. Nelson, personal communication). It is also clear, however, that in the determinate development of the leech, the orderly topographic placement of cells relative to anisotropically distributed intercellular inducers must play a major role. This conclusion follows from the numerous cases of developmentally critical cellular interactions that are summarized in this review. Such interactions are important during early development, as in the potential transfating of primary O/P blast cells, and later when neuronal phenotype depends on contacting other neurons or nonneuronal tissue. Either of these two classes of agents, or perhaps a combination of the two, may determine the identity of particular leech neurons. However, the stereotyped patterns of migration and of specific process outgrowth that characterize many identified neurons suggest that the ability to recognize and to respond appropriately to particular signals in the environment plays an important role in shaping the details of connectivity within the leech nervous system. Migration and process outgrowth must depend upon intercellular signals, because manipulations that disrupt normal interactions but leave precursors of the identified neurons intact disrupt the precision of these patterns. The competence to respond to such external cues might originate either from particular intracellular agents or from a previous commitment response to intercellular agents. For example, the commitment to follow a particular migratory path apparently arises before central neurons derived from the OPQ clones begin their journey, and it could, thus, be the product of either type of agent. In contrast, fine tuning the peripheral and central processes of many neurons occurs long after the cells become postmitotic and depends upon the specific interactions to determine a final phenotype. 'These developmental events must rely upon intercellular agents, be-

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cause in the absence of the specific targets the final phenotype of the neuron is abnormal. Intracellular agents are insufficient, on their own, to generate the entire ensemble of normal characteristics in these cells. As described in this review, studies carried out over the past decade have produced a reasonably good understanding of the genesis of the leech nervous system, in many of its structural and some of its functional aspects, and at both the cellular and intercellular levels. There has recently been an explosive development of novel techniques for analyzing systems at the molecular biological level, and it seems likely that these techniques offer an excellent opportunity to understand the development of leeches-and hence the development of other organisms, as well-at a more detailed level than has heretofore been possible. Identified cells in leech embryos are readily accessible to various kinds of biochemical manipulations, exemplified by the injection of macromolecules, and leech embryos offer the unusual opportunity to follow animal development at the cellular level from uncleaved egg to mature tissue in embryos large enough to allow molecular and surgical manipulations. Thus, it is likely that identifying the molecular nature of both intracellular determinants and intercellular inducers will be one of the main items on the agenda for future research concerning the development of the leech nervous system. A second item on that agenda is likely to focus on the behavioral effects of the leech embryo’s increasingly complex nervous system. A major function of nervous systems is to generate behavior appropriate to conditions within the environment, and the complexity and precision with which elements in the system connect to one another are thought to determine the complexity and precision of the behavioral responses that are possible. Embryonic leeches begin behaving early, and this behavior has been found to differ, in several ways that are described in this review, from that in the adult. Correlating the increasing complexity and interconnected nature of the nervous system in embryonic leeches with their increasingly complex and precise behavior should teach us more about how the nervous system acts as the biological substrate for behavior.

References

Anderson, D. T. (1973). “Embryology and Phylogeny in Annelids and Arthropods.” Pergarnon, Oxford. Apathy, S. (1889). Biol. Centralbl. 9, 600-608. Astrow, S., Holton, B., and Weisblat, D. (1987).Dev.Biol. 120, 270-283.

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Baader, A,, and Kristan, W. B., Jr. (1990). In “Brain, Perception and Recognition” (N. Elsner and G. Roth, eds.), p. 60. Thierne, New York. Baptista, C. A , , and Macagno, E. R. (1988a).J. Neurobiol. 19, 707-726. Baptista, C. A., and Macagno, E. R, (1988b). Neuron 1, 949-962. Baptista, 5 pM) added to the extracellular solution. These data indicate that tetanization effects in the CAI hippocampal subfield are contingent upon activation of NMDA receptors: washout of D-APV and further tetani produce the usually observed tetanization-induced changes of synaptic potentials and receptor sensitivity, although the progression of alterations after washout of D-APV and further tetanization is somewhat faster compared with slices that had not been exposed to tetani in the presence of APV. The notion that the changes of synaptic inhibition occur through NMDA receptor action is further supported by the observation that the NMDA receptor sensitivity-as assessed by various parameters (rise time, amplitude, and duration of the response to iontophoretically applied NMDA)-is greatly enhanced by tetanization (cf. Fig. 5 ) . These data indicate that the efficacy of GABAergic inhibition is impaired postsynaptically through a reduction of GABA receptor sen-

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F i c . 3. Tetanic stimulation changes GABA and NMDA receptor sensitivity. (A) The biphasic hyperpolarizing-depolarizing responses to iontophoretically applied GABA as well as both IPSP components decline following tetanization. The responses to iontophoretically applied NMDA increase in amplitude and duration following tetanization.

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sitivity. To address the question of possible presynaptic modifications of GABAergic transmission, a series of experiments was performed to study effects of tetanization in interneurons located in or close to the CA 1 pyramidal cell layer. These interneurons, most likely inhibitory basket cells, have been characterized by electrophysiological and anatomical experiments (cf. Schwartzkroin and Mathers, 1978). Cells were chosen based upon membrane responses to depolarizing and hyperpolarizing current injection (cf. Fig. 6, control inset): brief spikes, short time constants, small input resistance, lack of inward rectification during hyperpolarizing current pulses, large afterhyperpolarization. The late IPSP upon orthodromic stimulation of the Schaffer collaterals was less or was not expressed in interneuron recordings (cf. Figs. 6 and 7). Tetanization produced similar changes of the orthodromic EPSPIPSP sequence as observed in CAl pyramidal cells following tetanization of the Schaffer collaterals (Figs. 6 and 7). The reduction of the IPSP and increase of the EPSP in interneuron cells occurred during the same time course as in CA1 pyramidal cells. Iontophoretic GABA application produced a biphasic hyperpolarizing-depolarizing response similar to the responses in CA1 pyramidal cells. In nonpyramidal cells, however, tetanic stimulation leads to an increase of the depolarizing iontophoretic GABA component (Fig. 6). The increase of the depolarizing response to iontophoretic GABA application resulted in burstlike events as soon as post tetanum one (Fig. 6). The orthodromically evoked IPSP following tetanization was reduced but remained hyperpolarizing after the first and second tetanus and did not match the large depolarizing response to exogenously applied GABA. The depolarizing GABA response in interneurons was accompanied by a large increase in action potential firing (Fig. 6). In this respect, depolarizing GABA actions resemble the burst responses in interneurons in the presence of 4-AP (cf. Muller and Misgeld, 1990; cf. Section IV,B). Depolarizing spontaneous fast IPSPs in interneurons are similarly reduced following tetanization (Fig. 7). However, large spontaneous depolarizing potentials develop, usually between the third and fourth tetani (Fig. 7). Spontaneous depolarizing potentials grow in amplitude upon further tetanization and eventually develop into large PDS-like events with bursts of action potentials riding on them (Fig. 7). At this stage of excitability, orthodromic stimulation (stratum radiatum fibers) (B) In the presence of D-APV,tetanization does not produce alterations of G A B A responses or orthodromically evoked synaptic potentials. Washout of APV and further tetanization result in changes of G A B A receptor sensitivity and inhibitory synaptic potentials commonly observed after tetanization (from Stelzer et al., 1987).

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FIG. 6. Tetanization of stratum radiatum fibers increases the depolarizing component of the biphasic hyperpolarizing-depolarizing control response to iontophoretically applied GABA in nonpyramidal cells. Orthodromically evoked synaptic potentials (upper row) are measured at the cell's resting potential (-67 mV); recordings of GABA responses were performed at the more depolarized potential of -61 mV (obtained by depolarizing DC current injection). The inset (upper row,left) shows the cell's IV characteristics, which are profoundly different from those of CAI pyramidal cells (cf. Fig. 3). The growth of the depolarizing GABA response was accompanied by high-frequency action potential discharges (bottom, post tet. 1 and 2) (note: frequency of discharges supersedes the chart's ability to monitor full lengths of action potentials).

produced burstlike depolarizing potentials in interneurons (Fig. 7, top). Spontaneous giant depolarizations and bursts occur infrequently, are not rhythmic, and neither the frequency nor the probability of occurrence is dependent on the membrane potential. Several lines of experimental evidence indicate that the depolarizing giant events are GABA, receptor mediated, despite the fact that amplitude and frequency of small spontaneous depolarizing IPSPs progressively diminish-most likely due to a loss of GABA, receptor sensitivity (see below). Giant potentials are not observed when bicuculline is applied at these later stages of tetanization. In addition, giant depolarizing potentials occur in slices in which the CA2/CA3 pacemaker region for synchronized activity in CA 1 is removed by dissection. Excitatory synchronized activity, including epileptiform activity, is not spontaneously generated in CA 1 (Brown et al., 1979; Alger and Nicoll, 1980; Ropert et at., 1990) due to the specific anatomical and functional properties of the circuitry, in particular the lack of recurrent excitatory connections in this area (Wong

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FIG. 7. Spontaneous giant IPSPs and bursts in nonpyramidal cells. Repeated tetanization in a nonpyramidal CAI neuron (IV characteristics as in cell of Fig. 6 are not depicted) produces spontaneous giant depolarizing potentials and bursts, which can be blocked by GABAA antagonists (not shown). Giant IPSPs and bursts occur despite the progressive fading of the amplitude and frequency of fast, spontaneous control IPSPs. The upper row depicts stimulation-evoked potentials, which resemble bursts at increased stages of excitability following the third and fourth tetanus.

and Traub, 1983; Schwartzkroin and Prince, 1978). The question as to whether giant IPSPs and GABAergic bursts occur as highly synchronized discharges of a large number of cells in the presence of the remainder of (reduced) GABA, sensitivity (after three or four tetani) or whether the synchronized GABAergic events are mediated by a different kind of receptor, the sensitivity of which is actually enhanced following tetanization (cf. Fig. 6), remains to be elucidated. The fact that tetanization-induced spontaneous giant IPSPs occur exclusively in nonpyramidal neurons in which the depolarizing iontophoretic GABA response is actually enhanced following tetanization (Fig. 6) strongly favors the latter notion. The infrequency of giant IPSPs after tetanization may be due to a lesser expression of synchronization of inhibitory interneurons compared with that in 4-AP. Another factor may be the isolation of the CA 1 hippocampal subfield in which inhibitory bursting activity can be generated (as inferred from 4-AP experiments; cf. Section IV,B), but to a lesser extent compared with other hippocampal

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subfields, in particular the hilar region (Michelson and Wong, 1991; Muller and Misgeld, 1990). I n summary, tetanic stimulation of Schaffer collaterals results in the following changes in CA 1-located interneurons: 1 . Orthodromically evoked PSPs shift toward increases in the EPSP and a reduction in IPSPs similar to CA1 pyramidal cells. 2. T h e depolarizing component of the biphasic hyperpolarizingdepolarizing control response to exogenous GABA is largely increased and the hyperpolarizing component is decreased. A functional evaluation of tetanization-induced changes in interneurons has to take into consideration the complex circuitry and connectivity of interneurons. However, the increased excitability of feedforward interneurons following tetanization of afferent pathways (Buzsaki and Eidelberg, 1982) (cf. Figs. 6 and 7) is indicative of an increase of inhibitory efficacy at presynaptic sites (with respect to CA 1 pyramidal cells). With respect to interneurons, however, underlying mechanisms of tetanization-induced interneuron hyperexcitability-in analogy to CA 1 principal cells-are most likely postsynaptic. Taken together, the changes produced by tetanization of afferents on the pre- and postsynaptic inhibitory efficacy in the hippocampal CA1 region may be opposite. Mechanisms as t o how alterations of the inhibitory circuit may result in a net increase of excitability are discussed below (cf. Sections V,B and V,D). SynchroniLed giant IPSPs and burst potentials, after repeated tetanization (Fig. 7), are reminiscent of 4-AP-induced changes of the inhibitory circuitry. Underlying mechanisms remain to be elucidated. T h e tetanization-induced shift of interneuron GABA sensitivity toward depolarization and a possible role of GABA acting as excitatory transmitter may, however, play a major role in the generation of synchronized inhibitory bursts (cf. Sections IV and VI). Another property of CA1 neurons, which may play a role in cellular synchronization, is characteristically altered following Schaf€er collateral tetanization: rhythmical membrane potential oscillations (MPOs) (Fujita and Sato, 1964) occur in hippocarnpal neurons in CA1 and CA3 (Leung and Yim, 1988). MPOs are subthreshold oscillations of the membrane potential of hippocampal neurons at frequencies between 3 and 11 Hz (Leung and Yim, 1988). Each cycle of an MPO probably consists of a depolarizing phase caused by N a + and Ca'+ currents that are below the spiking threshold, and a repolariation phase by K + currents (Leung and Yim, 1988). Spikes and hippocampal membrane oscillations are not linked, although membrane oscillations occur at membrane potentials close to the spiking threshold

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and most oscillations are accompanied by action potential firing. MPOs can be triggered by depolarizing current injection (Leung and Yim, 1988). The threshold of membrane potential oscillations is significantly reduced after tetanic stimulation (Fig. 8, bottom right). The lowering of the threshold for MPOs is a progressive development following tetanization: after four or more tetani, some cells exhibited large MPOs close to the resting potential. The frequency of spontaneous discharges in cells was largely increased despite the fact that the firing threshold remained unaltered following tetanization. MPOs occur in principal cells and interneurons under control conditions. The smaller gap between resting potential and action potential threshold in interneurons may have a more significant impact for interneuron oscillations after tetanization. Block of synaptic transmission by omission of Ca*+ attenuates MPOs (Leung and Yim, 1988). MPOs are sensitive to GABA shunting and are

CONTROL

STIM.

L POST TET.l

400 ms

FIG.8. Threshold for membrane potential oscillations (MPOs) is lowered by tetanization. Orthodromically evoked synaptic potentials (top row) are recorded at resting membrane potential (-73 mV); GABA responses are recorded at a depolarized membrane potential of -68 mV (by DC current injection). Before tetanization, the MPO threshold was about -63 mV (not shown). Action potential threshold of about -60 mV remained unchanged by tetanization. The cell's IV characteristics are typical of a CAI pyramidal cell. Similar observations are made in nonpyramidal interneurons. During the responses to iontophoretically applied GABA, MPOs were completely abolished (recording at bottom, right).

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completely abolished during the biphasic response to iontophoretically applied GABA (Fig. 8). In summary, two spontaneous events in CAI interneurons are more pronounced after tetanization of stratum radiatum fibers. T h e differentiating features of depolarizing (giant) IPSPs and MPOs are severalfold. T h e major difference lies in the fact that MPOs occur in principal cells and interneurons whereas giant IPSPs occur exclusively in cells with interneuron properties. T h e regularity and frequency of MPOs increase with the level of depolarization. Giant IPSPs (in CA 1) are solitary, infrequent events, the frequency of which is not dependent on membrane potential changes. Tetanization lowers the threshold for the generation of MPOs that are observed in controls at membrane potentials close to the firing threshold and are reliably triggered by depolarizing current injection. Giant IPSPs are not observed until several tetani are administered (usually after three to four tetani). Synaptic currents (GABA-mediated chloride currents) drive giant IPSPs and intrinsic currents (Na+, Ca2+, K + ) drive MPOs, but blockade of synaptic transmission attenuates the occurrence of MPOs (Leung and Yim, 1988). 3. Time Course of Effects of Tetanimtion

The tetanus-induced changes in excitability discussed herein, including the reduction of the GABA,-mediated early IPSP and the sensitivity of GABA,, and NMDA receptors, usually develop around I5 min after the application of a given tetanus and are fully expressed after about 30 min following individual tetani: if no further tetani are applied, the tetanus-induced effects are long-lasting and remain until the end of the experinient-up to 4 hr as assessed intracellular and u p to 8 hr measured extracellularly. Further tetanization (in 30-min intervals) resulted in a progressive development of the changes described. T h e time course and duration of the tetanization-induced effects indicate the involvement of intracellular regulation processes [second-messenger-mediated phosphoryiation processes o r activation of early genes (cf. Cole et al., 1989)]. Tetanization evokes several short-term changes (within 15 min following the tetanus) that affect GABA, and GABA, receptor sensitivity. Shifts of the ionic equilibria of ions mediating GABAergic inhibition (Cl- and K + ) contribute to these short-term changes, which reverse within a few minutes (Wong and Watkins, 1982; Thompson and Gahwiler, 1989a,b,c).N o change of the ionic equilibria and reversal potential of either phase of the orthodromic IPSPs is observed 20 min after individual high-frequency trains (Stelzer el al., 1987).

GABAA RECEPTOR C O N T R O L

23 1

B. LONG-TERM POTENTIATION Learning and memory processes are likely to involve long-lasting, use-dependent alterations in the efficiency of synaptic communication. Such alterations may result from changes at existing synapses (Hebb, 1949; Eccles, 1953) or from alterations in the number of functional synaptic connections (Tsukahara, 1981). Groups of synaptically associated cortical cells might represent a basis for information storage and might be formed if collateral synapses between them are strengthened (Lorente de NO, 1949; Hebb, 1949; Marr, 1971; Gardner-Medvin, 1976; Abeles, 1982; Miles and Wong, 1987b). A valuable model for investigating long-lasting synaptic alterations is long-term potentiation. LTP can reliably be induced by a short, highfrequency stimulus to afferent pathways in many areas of the mammalian CNS, most notably in the hippocampus (Bliss and Loemo, 1973). Several features of LTP support the notion of LTP as a possible physiological substrate for learning and memory: its long-lasting duration (up to days and weeks following tetanization in vivo) (Bliss and Loemo, 1973) and its relative input specificity (Dunwiddie and Lynch, 1978; Andersen et al., 1980b), cooperativity (McNaughton et al., 1978; Lee, 1983), and associativity (Levy and Steward, 1979; Sastry et al., 1986). A number of reviews cover various aspects of LTP (cf. Collingridge and Bliss, 1987; Malenka et al., 1989; Kennedy, 1989). The role of synaptic inhibition in LTP, in particular the role of GABA, receptors, will be the focus of the following discussion. Synaptic inhibition has been ruled out as a major factor in the expression of LTP by earlier reports (Haas and Rose, 1982; Wigstrom and Gustafsson, 1985; Abraham et al., 1987; Taube and Schwartzkroin, 1987). This conclusion may be premature in the light of the marked reduction of the inhibitory tone following high-frequency stimulation of afferent fibers and the prominent role of the inhibitory circuit in the control of the excitability of hippocampal population activity. 1. Physiological Stimulus LTP can reliably be obtained by a standard tetanus (duration between 200 psec and about 2 sec, frequencies between 50 and 100 Hz). However, a wide variety of stimuli of afferent fiber pathways produce LTP-like synaptic plasticity (cf. Gustafsson and Wigstrom, 1988). The survey of the different stimulation patterns that are capable of inducing LTP leads to the conclusion that depolarization of the postsynaptic membrane to a certain degree represents a necessary requirement for

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the induction of LTP (Douglas et al., 1982; Wigstrom and Gustafsson, 1985; Sastry et al., 1986). Recent studies have examined stimulation patterns similar to those produced in vivo. Short, high-frequency bursts (four-pulse bursts, 100 Hz) applied to the Schaffer commissural projections to the CA1 subfield elicited LTP in a repetition window between 0.1 and 2 sec: burst intervals of 2 sec and longer were not very effective whereas 200-msec repetition intervals were most effective in eliciting LTP (Larson et al., 1986). ‘These stimulation patterns resemble spike discharge patterns of hippocampal neurons in animals exposed to learning situations. Moreover, when bursts were applied to two different dendritic inputs to CAI pyramidal cells, the second bursts elicited robust LTP at an appropriate interval (200 msec) even when the t w o input sites innervated completely different regions of the postsynaptic cells (Larson and Lynch, 1986). LTP did not occur when the inputs were stimulated simultaneously o r when the second burst was delayed by 2 sec (Larson and Lynch, 1986). The data indicate that a single burst produces a transient, spatially diffuse priming effect that modifies subsequent bursts to produce synaptic plasticity in a spatially confined area. Similar stimulation and timing patterns (bursts consisting of about four spikes) occur physiologically in the hippocam pal formation: CA3 pyramidal cells generate periodic, spontaneous burst discharges (Wong and Prince, 1979) very similar to those applied in the patterned stimulation experiments by Larson et al. (1986). As discussed in Section VI,A (cf. Traub et al., 1989b), partially synchronized burst firing constitutes physiological brain activity, both in uiuo and in vitro. The functional state of GABA, receptors is a critical factor in the generation of synchronized activity (cf. Miles and Wong, 1987a; cf. Fig. 18) and determines the number of axonal fibers synchronously activated. Taken together, it is conceivable that synchronous burst discharges in the disinhibited CA3 hippocampal subfield may provide the cooperative activation of patterned afferent fiber stimulation required to elicit longlasting synaptic plasticity in CAI. Furthermore, the similarity of the temporal parameters of the priming effect [maximal LTP effect at frequencies of about 5 Hz (Larson and Lynch, 1986)l and the theta rhythm (4-7 Hz) that occurs in the hippocampus during learning episodes supports the notion of such periodic, synchronous bursting as a correlate of the LTP-inducing input. T h e output pattern controlled by the state of synchronized activity of the (entire) CA3 population or specific clusters of CA3 cells may represent a crucial factor in generating the physiological stimulus producing synaptic plasticity effects in CA1.

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2. Induction of LTP Induction of LTP in the CA1 hippocampal subfield is contingent upon the activation of NMDA receptors (Collinridge et al., 1983; Harris et al., 1984; Wigstrom and Gustafsson, 1985). NMDA receptor activation requires a sufficient depolarization of the cell membrane (Douglas et al., 1982; Wigstrom and Gustafsson, 1985; Malinow and Miller, 1986; Sastry et al., 1986) to alleviate the Mg2 block of NMDA channels (Mayer et al., 1984). This notion is supported by intracellular studies demonstrating that the EPSPs of single pulses or weak tetanic stimuli that are not sufficient to trigger LTP alone produce LTP when combined with intracellular depolarizing current application (Kelso et al., 1986; Sastry et al., 1986; Wigstrom et al., 1986). Tetanization fails to elicit LTP when the neuron is kept hyperpolarized (Malinow and Miller, 1986). A reduction of the inhibitory input during the depolarizing, LTP-inducing stimulation phase greatly facilitates the induction of LTP in both hippocampus (Wigstrom and Gustafsson, 1983) and neocortex (Artola and Singer, 1987). In addition, properly timed inhibitory inputs reduce or prevent the occurrence of LTP (Douglas et al., 1982). In neocortex, the block of GABAA-mediated inhibition represents a necessary condition for the induction of LTP by tetanic stimulation (Artola and Singer, 1987). These findings suggest that GABA, receptors in the feedforward circuitry control the amount of postsynaptic depolarization necessary for the activation of NMDA receptors in the induction phase of LTP. Two possible mechanisms may contribute to the disinhibition of the feedforward inhibitory pathway: first, a decrease in the efficacy of synaptic inhibition (either pre- or postsynaptic), and second, functional synaptic connections between inhibitory interneurons (Misgeld and Frotscher, 1986; Lacaille et al., 1987; Freund and Antal, 1988; Lacaille and Schwartzkroin, 1988b). +

3. Maintenance oJ: LTP LTP is commonly measured by extracellular or intracellular recordings. In extracellular recordings in the pyramidal cell layer, orthodromic stimulation evokes a characteristic sequence of field potentials consisting of an early antidromic spike (more pronounced in in vivo recordings) and a later negative-going orthodromic population spike, superimposed upon a positive synaptic wave, representing dendritic excitatory postsynaptic potentials. High-frequency stimulation increases largely the negative-going orthodromic population spike and the positive-going EPSP, leaving the negative-going antidromic spike almost unchanged

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(Bliss and Loemo, 1973). T h e population spike amplitude reflects the number of cells firing upon orthodromic conditioning stimulation. Dendritic excitatory potentials can be more directly assessed through extracellular recordings of field potentials in the dendritic area (usually recorded in stratum radiatum of CA1 o r CA3). In intracellular recordings, orthodromic stimulation of afferent fibers evokes a sequence of EPSPsIPSPs in pyramidal cell somata (cf. Section 111,A). T h e most common parameter measured in LTP experiments is the increase in the EPSP amplitude (with fast inhibition often blocked) or the EPSP slope. Recently, intradendritic (Taube and Schwartzkroin, 1988) and whole cell patch recordings in the slice (cf. Malinow and Tsien, 1990) have been performed to elucidate LTP mechanisms. Most studies focus on modifications of excitatory amino acid-mediated transmission properties underlying the expression of long-term potentiation: persistent modifications of synaptic transmission may occur presynaptically (Dolphin et al., 1982; Bekkers and Stevens, 1990; Malinow and Tsien, 1990) or postsynaptically. Possible postsynaptic modification sites following tetanization include a-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA) receptors, which mediate fast EPSPs (Muller et al., 1988; Kauer et al., 1988; cf. Kennedy, 1989), and NMDA receptors, which mediate slow components of excitatory postsynaptic potentials. Activation of NMDA receptors is a prerequisite (at least in some areas, such as the hippocampal CAI subfield) for the induction of LTP (Collingridge et al., 1983). A recent study indicates that NMDA receptor-mediated postsynaptic potentials may also be persistently enhanced following tetanization (Bashir et al., 1991). a. IPSP Reductionfollowing Tetanization. Following tetanic stimulation of afferent fibers, orthodromically evoked IPSPs were found to be unchanged, increased, or decreased using the same stimulation protocol (Yamamoto and Chujo, 1978; Misgeld et al., 1979; Abraham et al., 1987; Haas and Rose, 1982; Taube and Schwartzkroin, 1988). A typical sample of evoked IPSP alterations following tetanization was reported by Abraham et al. (1987), who found IPSP increases in 8 of 19 CA1 pyramidal cells, a decrease in 3 cells, and no IPSP changes in 8 cells. Similar inconsistent changes of evoked IPSPs were also obtained in intradendritic recordings (Taube and Schwartzkroin, 1988). A decrease of stimulationevoked IPSPs after tetanic stimulation was measured in CA3 (Yamamoto and Chujo, 1978; Misgeld et al., 1979; Misgeld and Klee, 1984; Miles and Wong, 1987b) and CA1 pyramidal cells (Haas and Rose, 1982; Larson and Lynch, 1986, Abraham et al., 1987; Stelzer et al., 1987; Taube and Schwartzkroin, 1987, 1988). IPSP reductions following tetanization were most commonly observed following repeated long and strong tetaniza-

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235

tions (Abraham et al., 1987; Misgeld and Klee, 1984). However, marked reductions of orthodromically evoked IPSPs were observed following prime pulses, which are relatively weak stimuli but resemble in viuo stimulation patterns (Larson and Lynch, 1976). IPSP increases following tetanization in CA3 were found to be pathway specific; decreases were consistently heterosynaptic in nature (Misgeld et al., 1979). Haas and Rose (1982) attributed the observed decrease in the stimulation-evoked IPSPs to the fact that the increase in the fast EPSP produced the concomitant reduction of the overlapping early IPSP. T h e conclusion of most studies was that tetanization-induced changes of IPSPs are not significant and inhibition does not play a major role in the maintenance of LTP (cf. Haas and Rose, 1982; Taube and Schwartzkroin, 1987; Abraham et al., 1987). As discussed in Section V,A, tetanization produces opposite pre- and postsynaptic changes of the inhibitory efficacy: an increase in interneuron excitability and a reduction of postsynaptic GABA sensitivity. Opposite pre- and postsynaptic changes of the inhibitory efficacy could account for inconsistent changes of orthodromically evoked IPSPs in LTP studies. If so, to what extent and under what conditions are preand postsynaptic alterations of inhibitory synaptic efficacy expressed after tetanization? b. GABA Sensitivity. As discussed in Section V,A, GABA receptor sensitivity in CA 1 is characteristically altered after tetanization of the Schaffer collaterals: in CAI pyramidal cells, both the hyper- and depolarizing components of the response to iontophoretically applied GABA are progressively reduced after application of repeated tetani. T h e control GABA response is most likely composed of several GABA receptor components, including GABA, and GABA, components. T h e impairment of the GABA response together with both phases of the orthodromically evoked IPSPs (Figs. 5 and 8) indicates that the loss of synaptic inhibition is due to a reduction of the sensitivity of GABA receptors, both GABA, and GABA,. I n nonpyramidal cells with characteristics similar to those recorded in CAI basket cells (Schwartzkroin and Mathers, 1978), iontophoretic GABA application produced a biphasic hyperpolarizing-depolarizing response similar to the responses in CA1 pyramidal cells. In these cells, however, tetanic stimulation produced an increase of the depolarizing iontophoretic GABA component (Fig. 6).The reduction of IPSPs in these cells occurred at the same rate as in other neurons in which the biphasic GABA response progressively faded. The increase of the depolarizing iontophoretic GABA response, however, occurred somewhat faster and iontophoretic GABA application after the second tetanus produced

burstlike events that outlasted the duration of GABA application considerably (Fig. 6). These data demonstrate specific alterations of the GABA sensitivity in different cell types of the CAI area following tetanization. In a previous study, GABA sensitivity was reported to be unaltered in slices following LTP (Scharfman and Sarvey, 1985). A possible explanation for these data may be found in opposite alterations of GABA sensitivity in CA 1 cells. In pyramidal cells, which outnumber interneurons t o a large extent (Cassell and Brown, 1977), both components of the hyperpolarizing-depolarizing control responses are progressively reduced following tetanization. In at least one class of nonpyramidal cells located close to the pyramidal cell layer, the depolarizing GABA response is highly increased. Extracellular measurements of GABA responses may not reveal such specific alterations and may not be the adequate approach in the light of opposite changes of GABA sensitivity following tetanization. c. Interneuron LTP. T h e results of recordings from interneurons demonstrate increased basket cell activity following tetanic stimulation of afferent fibers: LTP in CA1 interneurons was reported in in vivo (Buzsaki and Eidelberg, 1982; Kairiss et al., 1987) and in aztro (Taube and Schwartzkroin, 1987; Abraham et al., 1987) studies. The degree of interneuron LTP seems to be higher in in TWO preparations than in the slice (Abraham et al., 1987). It was hypothesized that increases in excitability following tetanic stimulation of afferent fibers occur when the degree of potentiation of the feedforward inhibitory system is smaller than the increase of excitatory transmission properties (Wilson et al., 1981; Abraham et al., 1987). Although the exact degree of tetanization-induced potentiation of basket cell activity remains to be determined, it is widely accepted that basket cell activity (in CA1) is not decreased following tetatiizatiori and that the potentiation of excitability following high-frequency stimulation cannot be explained by the observed changes in interneuron activity (Taube and Schwartzkroin, 1988). Additional factors that may contribute to a presynaptic strengthening of the inhibitory system include the activation of an increased number of inhibitory interneurons after the tetanus. Enhanced efficacy of inhibition may he further achieved by the synchronization of the inhibitory circuit (Fig. 7) and by the enhanced sensitivity of the depolarizing GABA response in interneurons (Fig. 6). In summary, tetanization results in increased interneuron activity (Buzsaki and Eidelberg, 1982; Kairiss et al., 1987; Taube and Schwartzkroin, 1987; Abraham et al., 1987) and an increased number of recurrent interneurons activated by the control stimulus. The observation of orthodromically evoked “unchanged” IPSPs measured in somatic intracellular recordings after a single tetanus implies, therefore, an actual reduction of the postsynaptic GABAergic

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efficacy in the presence of increased interneuron excitability. Mechanisms that strengthen synaptic inhibition presynaptically (activation of a larger number of interneurons, interneuron LTP) are likely pathway specific and may mask the actual reduction of the reduced postsynaptic GABA sensitivity. Repeated or stronger high-frequency stimulation may result in an overall reduction of the inhibitory efficacy by reducing (postsynaptic)GABA sensitivity to a larger degree than enhancing (presynaptic) interneuron activity. At this stage of excitability, orthodromically evoked IPSPs are generally reduced (for a more elaborate discussion, see Section V,D). d. E-S Potentiation. A number of studies demonstrate that potentiation of somatic excitability (measured by the population spike amplitude) exceeds by far the increase in the population EPSP (reflecting excitation at the dendritic site). This enhanced EPSP-spike relationship, termed E-S potentiation (Andersen et al., 1980b),has been observed in both dentate gyrus (Bliss and Loemo, 1973; Wilson etal., 1981)and CA1 (Andersen et al., 1980b; Reymann et al., 1987;Abraham et al., 1987; Taube and Schwartzkroin, 1987, 1988). Intradendritic recordings in CAI pyramidal cells confirm the poor correlation between dendritic and somatic LTP: population spike amplitudes were often potentiated without concomitant changes in the intradendritically recorded EPSP (Taube and Schwarukroin, 1988). A dendritic EPSP increase is always accompanied by population EPSP increases and vice versa (Taube and Schwartzkroin, 1988). These data demonstrate that alterations in the EPSP (measured intra- or extracellular) are not a prerequisite for potentiation of the population spike (Taube and Schwartzkroin, 1988). In a study to test a hypothesis put forward by Wilson et al. (1981) that tetanization induces greater LTP of the excitatory pathway than of the feedforward inhibitory pathway in the same area, Abraham et al. (1987) examined the contribution of synaptic inhibition in the E-S potentiation. It was postulated that alteration of the EPSP is not a prerequisite for potentiation of the population spike and that E-S potentiation is largely due to modification of inhibition (Abraham et al., 1987). The possibility of changes in the strength of the inhibitory circuit underlying this overproportional enhancement of the dendritic EPSP-spike relationship led to the suggestion that the term “ L T P should be confined to the changes of the dendritic EPSP (cf. Gustafsson and Wigstrom, 1988). As discussed in Section V,A, tetanization of afferent fibers produces opposite changes of the efficacy of synaptic inhibition: postsynaptically, a reduction of GABA receptor sensitivity; presynaptically, enhanced efficacy of GABAergic transmission through an increase of the number of discharging inhibitory interneurons upon orthodromic stimulation at a given stimulation intensity. Figure 10a demonstrates that a partial block-

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ade of GABA, receptors shortly after the washin of bicuculline results in an increase of all synaptic components including the GABA,-mediated IPSP. Longer exposure to bicuculline (and a more complete block of GABA, receptors) then leads to a progressive reduction of the GABAAmediated IPSP and progressive increasesof EPSP and late IPSP (cf. Fig. 1). These data show that the extent of GABA, receptor blockade determines whether the early IPSP upon orthodromic stimulation of the Schaffer collaterals is enhanced, unchanged, o r decreased. T h e extent of the increase of the EPSP amplitude (and duration) is determined by the extent of GABA, suppression; however, a marked “increase of the E P S P is expressed at any stage (including early stages) of GABA, receptor blockade (cf. Section 111,A). As inferred from the pharmacological blockade of GABA, receptors, the two opposite changes in the inhibitory efficacy after tetanization-suppression of GABA sensitivity and activation of the inhibitory circuit-may not be independent processes: activation of the inhibitory circuit is rather contingent upon reduction of GABA, sensitivity. As for the late, GABA,-mediated IPSP, tetanization results also in a decreased sensitivity of GABA, receptors, thus counteracting the effects of a reduction of GABA, receptor sensitivity following tetanization. When the stimulation intensity of the orthodromic conditioning pulse is adjusted after the tetanus such that the population spike amplitude measured in the CA1 pyramidal cell layer matches pretetanus controls (Fig. 9), “EPSP increases” can still be observed at depolarized potentials including the cell’s resting membrane potential (in the cell depicted about -68 mV) (Fig. 9). This EPSP increase at RMP occurs despite a much smaller EPSP measured at the chloride reversal potential (in the cell depicted at about -78 mV) and at more hyperpolarized membrane potentials. These data suggest that under physiological conditions (cell at resting membrane potential, all excitatory and inhibitory synaptic components preserved) a persistent “enhancement” of synaptic transmission following tetanic stimulation of afferent fibers in the CA1 hippocampal subfield is expressed when the reduction of the efficacy of synaptic inhibition is greater than the reduction of the efficacy of excitatory transmission. An increase of the fast EPSP mediated by AMPA receptors is not a requirement for the manifestation of IXP. It is conceivable that the tetanization-induced increases of intradendritically measured EPSPs are due to the same E-S potentiating mechanisms, i.e., reduction of inhibition (only at a smaller scale at spatially confined areas in distant dendritic regions). Such a notion is supported by the fact that tetanization-induced increases of intradendritically measured EPSPs are small or nonexistent (‘Taubeand Schwartzkroin, 1988).A recent study shows that tetanization of the Schaffer collaterals produced a long-term enhancement of the

239

GABAA RECEPTOR CONTROL POST TET.1

CONTROL

mrac.

Stimul. 1.5 V

Stimul. 2.5 V

Y 40 mo

h

nV

Intrac.

-

MP: 88 m

-98rnV

v

v

LL '

400mo

' zomv

FIG.9. Extracellular field potentials in the CAI pyramidal cell layer (upper row) and intracellular recordings of a CAI pyramidal cell (below). The stimulation strength of the conditioning pulse posttetanum 1 was adjusted to produce a similar population spike amplitude before and after the tetanus. Both, high-frequency tetanic stimuli and the conditioning pulse were administered to fibers in stratum radiatum. Intracellular recordings were performed at the cell's resting membrane potential (-68 mV), at the chloride reversal potential (-78 mV), and at a more hyperpolarized potential at which the GABAAmediated chloride component was reversed and depolarizing (here -98 mV).

compound EPSP in given CA1 pyramidal neurons but no increase in the unitary EPSP elicited by depolarizing current pulses of monosynaptically coupled CA3 neurons (Friedlander et al., 1990). The most probable explanation for these observations (although not taken into consideration by the authors of the study) is that the reduction of the feedforward inhibitory pathway, which involves both GABAA- and GABA,-mediated IPSPs, may have produced the increased dendritic compound EPSP. T h e (unitary) EPSP, however, is not enhanced due to the lack of activation of intercalated inhibitory interneurons in the monosynaptically coupled CA3-CAI neuronal connection. At resting membrane potential, very small increases of subthreshold EPSPs result in cell discharges following tetanization (Figs. 2 and 8). The main physiological mechanisms through which GABA, receptors control the excitability of neuronal populations are large conductance increases in spatially confined subcellular areas (shunting inhibition; cf. Section 111,A). GABAA-mediated potentials elicit only relatively small hyperpolarizing membrane changes at the cell's resting membrane po-

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tential due to the closeness of the chloride reversal potential. The combination of large conductance increases and small membrane potential changes restrict the propagation of GABA,-mediated IPSPs. Tetanization-induced reductions of GABA,-mediated IPSPs generated at far dendritic sites may not be recorded by intracellular recordings in the cell soma, whereas EPSP increases (due to local disinhibition at far dendritic sites, at which a reduction of GABA, sensitivity is effective) can be globally measured (e.g., in the soma) as a potentiated EPSP. It is conceivable that following tetanization, increased EPSPs and “unchanged” fast

A

1,

”.\;

6 Control ............................

Ij 1,

L------

4

Bic(5min) ..............................

2

\

\

40 msec

PT5

FIG.. 10. (A) Orthodroniic potentials of a CAI pyramidal cell during washin of bicuciIlline-methiodide (50 pkf). In comparison to the control response, the early IPSP is initially enhanced ( 5 mill after the start of bicuculline bath application, Bic 5 min), about equal in amplitude (8 miri after bicuculline application, Bic 8 min), and finally completely suppressed (Bic 16 rnin). The EPSP and the late IPSP are potentiated at any point of time following bicuculline application. Cell holding potential -60 mV (by DC current injection). (U) Orthotlromic responses in a CA 1 pyramidal cell in the presence of bicuculline-methiodide (50 pbf) before and at various intervals after tetanization. Large increases in both EPSP and late IPSP occur shortly after application of high-frequency stiniulation to the Schaffer collaterals. Orthodi-oniic responses decrease gradually with time and reach pretetanus control values sonie 20 to 30 min after the tetanus. Recordings were obtained at cell R M P (-68 mV).

GABAA RECEPTOR CONTROL

24 1

IPSPs are recorded in the soma when GABA,-mediated inhibition is effectively suppressed at dendritic sites. The notion that a reduction of fast inhibition represents the major mechanism in the expression of LTP is further supported by the consistent observation that in disinhibited slices long-term enhancement of excitability is not observed (Fig. 10). Tetanic stimulation of afferent fibers after complete blockade of GABA, receptors produces large increases in both EPSP and late IPSP shortly after the tetanus (Fig. 1Ob). However, orthodromic responses decrease gradually with time and reach pretetanus control values about 20 to 30 min after the tetanus (Fig. lob). In summary, GABA, receptors may play a critical role in all phases of LTP: 1. Synchronized bursts of CA3 cells could represent a physiological stimulus capable of producing LTP-like plasticity in CA 1. 2. Impairment of GABA,-mediated inhibition may be involved in the induction of LTP. The resulting block of the feedforward inhibitory pathway may promote membrane depolarization of the CA1 pyramidal required to activate NMDA receptors. 3. Reduction of GABA,-mediated synaptic inhibition due to an impairment of GABA, receptor sensitivity is conceivably the major component of potentiated synaptic transmission following tetanization.

C. INTRACELLULAR REGULATION OF GABA, RECEPTOR FUNCTION A number of recent studies have provided evidence for the concept of neuromodulation via intracellular second messenger-induced phosphorylation of ligand- and voltage-gated ion channels. It is now widely accepted that protein phosphorylation and dephosphorylation of receptor-channel complexes represent the primary mechanisms of controlling the efficacy of a number of voltage- and ligand-gated ion channels in the central nervous system (for review, see Nestler and Greengard, 1984; Schwartz and Greenberg, 1987). Historically, much more attention has been paid to the study of upregulation processes of phosphorylation, which have been considered more highly regulated and more relevant for the control of cellular processes. Yet protein phosphorylation functions as a reversible signaling system and equally effective dephosphorylation of protein kinase target enzymes is required to terminate the responses and maintain responsive phosphorylation systems. The state of phosphorylation of a target protein is determined by competition between a phosphorylating protein kinase and a dephosphorylating phosphatase (for review, see, for exam-

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ple. Krebs and Beavo, 1979; Cohen, 1980, 1989; Klee et al., 1988). Several brain phosphatases have recently been described and dephosphorylation as a signal transduction mechanism has been established in the regulation of many cellular processes in the CNS (Nestler and Greengard, 1984; Klee et al., 1988; Armstrong, 1989). Recent studies indicate that phosphatases are regulated by second messengers and protein modulators: activation is either direct, as in the case of phosphatase-2B, which is activated by calmodulin (Klee et al., 1979; Stewart et al., 1982; Klee and Cohen, 1988), o r indirect, through regulation of other phosphatase inhibitors (e.g., CAMP-dependent DARPP-32), as in the case of phosphatase type 1 (Hemmings et al., 1984; Halpain et al., 1990). 1. PhosphoqdutionlDephosphorylation Regulates GABA, Receptor Function Despite the huge body of information about the extracellular modifiability of the GABA,, receptor function (cf. Olsen and Venter, 1986), little was known about possible intracellular regulatory sites until very recently. The advent of the patch-clamp recording technique, in particular the whole cell-clamp approach, has provided a tool to study intracellular regulation processes in a variety of preparations (cultured neurons, acutely dissociated neurons, brain slices) (cf. Hamill et al., 1981; Edwards et al., 1989). The use of low-resistance electrodes (1-3 M a ) allows a rapid dialysis (within 1 to 2 min) of small diffusible molecules and ions and a proportionally slower exchange of larger molecules and small cell organelles (Marty and Neher, 1983). This property becomes even more prominent in recordings from acutely isolated cells in which the widespread deridritic arborization is largely cut (Kay and Wong, 1986). In acutely isolated CA 1 hippocampal neurons of adult guinea pigs. GABA-mediated chloride currents measured in the whole cell-clamp configuration diminish progressively when the intracellular contents of these neurons are perfused with a “minimal” intracellular solution (130 mM trismethanesulfonate, 10 mM HEPES, 10 mM BAPTA, and 0.1 mM leupeptin, pH 7.3) (Stelzer et al., 1988) (Fig. 11A). The decline of the

FIG.1 1. Rundown of GABA, currents in Mg-ATP-free intracellular perfusate resulting from the loss of GABAA conductance. (A) Whole cell recordings of GABA,, outward currents at I , 3 , and 5. inin after cell penetration (top to bottom). (B) Normalized GABA, currents ( A ) and GABA, conductances (m) decreased with time; cell input resistance ( 0 ) and GABAA reversal potential (+) remained unchanged. (C) rundown o f GABAA currents (recordings at 1, .5, and 10 niin following cell penetration at - 10 mV) and stability of glutamate responses (recordings at 30 sec following respective GABAA currents at -60 mV) (from Stelzer t t al., 1988).

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GABA.,-mediated current is accompanied by a proportionate reduction of the GABA, conductance, whereas the GABA reversal potential and the general condition of the recorded neurons are unaltered (as assessed by parallel measurements of cell input resistance and voltage-gated sodium and glutamate-activated inward currents) (Fig. 11B and C). A similar decline of GABA, currents was observed in hippocampal cultured neurons (Vicini et al., 1986; StelLer et al., 1988) and chick sensory neurons (Gyenes et al., 1988). T h e decline of the GABA,-activated conductance is significantly reduced by adding M g 2 + ions and adenosine triphosphate to the intracellular medium: in the presence of 4 mM intracellular MgCl, and 2 mM ATP, GABA, whole cell currents are maintained to about 95% of the control value after 10 min of cell penetration, whereas in the absence of these "stabilizing" factors, GABA, whole cell currents are suppressed to less than 10% of control values at the same point in time (Stelzer et al., 1988; Chen et al., 1990). T h e intracellular presence of Mg-ATP is necessary but not sufficient to maintain GABA, receptor function in hippocampal pyramidal cells. A loss of [Ca2+jichelators during dialysis (cf. Byerly and Moody, 1984) results in elevations of [Cay+],.In the absence of chemical [Ca2+],buffer in the dialysate, GABA, currents arid conductance diminish over a time course similar t o that in the absence of intracellular Mg-ATP (Fig. 12).

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FIG. 12. Elevated [(;a2+ ]i suppresses GABAA-mediated currents. (A) GABA currents recorded in the presence of high [Gas+], and Mg-ATP. Averaged recordings of four cells; the inset depicts recordings of one cell obtained at 0 . 5 , and 10 inin following cell penetration. (B) Decreasing GABA responses in intracellular solution containing Mg-ATP and high [Ca'+], are reversed upon introduction of low [Ca2+Ii.GABA currents below the graph are recorded at the labeled points of time (from Chen el al., 1990).

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The inclusion of 10 mM BAPTA into the recording pipette is a further requirement to stabilize the GABAA response (Stelzer et al., 1988; Chen et al., 1990). There are at least two possible causes for the time-dependent decrease of the GABA, conductance in the absence of [Ca2+Ii buffer. First, GABA channels could be irreversibly destroyed by proteolysis during whole cell recording. The irreversible process of proteolysis is largely eliminated by providing leupeptin, an inhibitor of [Ca2 Ii-dependent proteases. Most notably, the rundown caused by elevation of [Ca2+Ii (1 mM BAPTA, 1 mM Ca2+, yielding about 1 pA4 free calcium in the dialysate) can be reversed by buffering [Ca2+Iito low levels during the whole cell recording (Fig. 12B). The reversibility of GABAA rundown argues against a mechanism via proteolysis of the GABA, receptorfchannel protein. Rundown caused by intracellular perfusion of a Mg-ATP-free solution can similarly be reversed by reintroducing phosphorylation factors (Mg-ATP) (Chen et al., 1990). T h e requirement for Mg-ATP and low [Ca2+Ii suggests that the GABA, receptor or a closely associated regulatory protein needs to be phosphorylated for the receptor to respond to GABA: in the absence of phosphorylation factors (Mg-ATP) or physiological or chemical [Ca2+Ii buffer, dephosphorylation becomes dominant and causes rundown (Stelzer et al., 1988). T h e results of a series of recent experiments support the notion of a (reversible) phosphorylation-dephosphorylation cycle regulating GABA, receptor function. GABA, current rundown due to prevailing dephosphorylation is demonstrated by intracellular application of the unspecifically dephosphorylating enzyme alkaline phosphatase in the presence of phosphorylating factors (Mg-ATP) (Chen et al., 1990) (Fig. 13). Alkaline phosphatase are nonspecific enzymes that hydrolyze phosphorus-containing compounds (McComb et al., 1979). When intracellular ATP is replaced by its more hydrolysisresistant analog adenosine 5'-0-(3-thiotriphosphate)(ATP-y-S), the alkaline phosphatase-induced decline of GABA currents is significantly retarded (Chen et al., 1990) (Fig. 13).The ATP analog ATP-y-S serves as a substrate for protein kinases and the resulting thiophosphate is far more resistant to hydrolysis by phosphatases than is the corresponding phosphate group (Eckstein, 1985). Similarly,the loss of the GABAA conductance caused by high [Ca2 Ii (cf. Fig. 12A) is significantly slower in the presence of ATP-y-S (cf. Chen et al., 1990). T h e latter observation also militates against the notion that the main intracellular effect of Mg-ATP is to maintain low levels of [Ca2+Ii at the inner surface of the plasma membrane (cf. Byerly and Yazejian, 1986): if the primary effect of Mg-ATP in maintaining GABA, receptor function were to lower [Ca2+Ii,one might expect ATP-y-S to +

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FIG. 13. ATP-7-S antagonizes the effect of alkaline phosphatase of reducing GABAAmediated currents. Alkaline phosphatase was introduced into cells that contained equivalent aniounts of ATP and ATP-7-S, respectively. Control responses are averages of four cells containing Mg-ATP (front Chen el nl., 1990).

enhance rundown of GABA, currents, as it is a poor substitute for ATP in active transport processes. T h e conspicuously slower rate of [Ca2+Iiinduced rundown of GABA, currents in the presence of W-7, a calmodulin inhibitor (cf. Tanaka et al., 1982), indicates that [Ca2+]],-activated GABA, rundown is mediated by calmodulin (Chen et al., 1990) (Fig. 14). Calmodulin-dependent phosphatase, calcineurin (phosphatase-BB), has been isolated from many tissues (Klee et al., 1979, 1988; Stewart et al., 1982; Ymg et al., 1982; Krinks et al., 1988; Farber et al., 1987; for review, see Tallant and Cheung, 1986). Calcineurin is-in contrast to phosphatase- 1, -2A, or -2C-not involved in dephosphorylation of metabolism-controlling proteins, indicating a relative restricted substrate specificity (cf. Ingebritsen and Cohen, 1983; Klee and Cohen, 1988). Immunohistochernical studies demonstrate a highly specific regional and subcellular distribution (cf. Wood et al., 1980; Goto et al., 1986): although calcineurin is present in neurons throughout the brain, a marked regional variation with higher levels in the hippocampal formation, the caudatoputamen, and substantia nigra is observed. Calcineurin is specific for neurons and is not detected in glial cells. Like CaM, calcineurin is enriched in dendrites and the somata. T h e abundance of

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FIG. 14. W-7, a calmodulin inhibitor, retards the high [Ca2+]i-induced rundown of GABA currents. Averages of peak amplitudes of GABA currents are depicted for control (low [Ca*+];),high [Ca*+]i (A),and high [Ca2+]i plus W-7. All intracellular solutions contained Mg-ATP (from Chen et al., 1990).

calcineurin in the CNS, its heterogeneous distribution, and its narrow substrate specificity suggest an important contribution of calcineurin to brain function. Intracellular perfusion with the catalytic subunit of calcineurin results in a rapid rundown of GABA, responses that is retarded by additional inclusion of ATP-y-S or W-7 (Q. X. Chen, A. Stelzer, A. R. Kay, and R. K. S. Wong, unpublished observations). In summary, the results discussed in this section indicate that GABA, receptor function is regulated by a phosphorylation/dephosphorylation cycle. The GABA, receptor or a closely associated regulatory protein can exist in a phosphorylated or a dephosphorylated state. With the provision of Mg-ATP, a protein kinase phosphorylates the molecule and maintains the receptor in a functional form. Phosphorylation competes with a Ca2 /calmodulin/phosphatase-dependentdephosphorylation process that renders the GABA, receptor nonfunctional. A similar phosphorylation-dephosphorylation cycle critical for channel function has been proposed for voltage-activated [Ca2 Ii channels (Doroshenko et al., 1984; Eckert and Chad, 1984; Chad and Eckert, 1986; Hosey et al., 1986; Kameyama et al., 1987; Hescheler et al., 1987; Chad et al., 1987; Byerly and Hagiwara, 1988; Armstrong, 1989). Similar to GABA, receptors, [Ca2+]i/caimodulin-dependent calcineurin renders voltage-activated Ca2 channels nonfunctional (cf. Armstrong, 1989). +

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2. Regulation of GABA, Receptor Function b~ [Ca2+],

Effective intracellular [Cay li buffering systems control the homeostasis of [Ca2+Ii, maintaining low levels of [Ca' +Ii and transmembrane Ca' gradients over four orders of magnitude (for review, see McBurney and ISeering, 1987). Both low levels of [Ca2+J i and high transmembrane