Journal of Physiology (1991), 441, pp. 755-778 With 11 figures Printed in Great Britain

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EFFECT OF CONDUCTION BLOCK AT AXON BIFURCATIONS ON SYNAPTIC TRANSMISSION TO DIFFERENT POSTSYNAPTIC NEURONES IN THE LEECH

BY XIAONAN GU* From the Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, FL 33101, USA

(Received 16 May 1990) SUMMARY

1. The cutaneous receptive field of the medial pressure (mP) sensory neurone in the leech has been examined. The cell has one major receptive field and an anterior and a posterior minor receptive field, principally on lateral and dorsal skin. The two minor receptive fields are contiguous with the major receptive field and are innervated by fine anterior and posterior axons, but there is no overlap between major and minor receptive fields. 2. At low frequencies of stimulation of the minor receptive fields, conduction block takes place in the mP cell at the central branch point within the leech ganglion. 3. The mP cell synapses directly with many other cells in the leech ganglion, including the anterior pagoda (AP) cell, longitudinal (L) motoneurone and the annulus erector (AE) motoneurone, which were studied as a group of postsynaptic neurones. Conduction block in the mP cell affects its synaptic transmission to all three postsynaptic neurones, but the effect can be different in different postsynaptic neurones. Block at the central branch point for an impulse travelling along the anterior axon reduces transmission to the AE cell much more than to the AP or L cells, while block at the central branch for an impulse travelling along the posterior axon has the reverse effect. 4. The distribution of functional connections of the branches of the mP cell with each postsynaptic cell was studied. For this analysis, branches of the mP cell were selectively silenced either during conduction block or by laser microsurgery. Generally, nearly all of the functional connections with the L and AP cell were made by anterior branches of the mP cell while the connection with the AE cell was primarily made by posterior branches of the mP cell. 5. The possible sites of contact between the mP cell and postsynaptic cells were determined by injecting separate markers into the mP cell and a postsynaptic cell. In confirmation of physiology, the mP cell's posterior branches had few, if any, contacts with the AP cell, while anterior branches had few, if any, contacts upon the AE cell. 6. Conduction block can thus act as a switch in the central nervous system (CNS), altering the mP cell's pattern of synaptic transmission to different postsynaptic *

Present address: Department of Biology, B-022, UCSD, La Jolla, CA 92093, USA.

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neurones depending upon the region of a single sensory neurone's receptive field that is stimulated. This effect, dependent upon inputs to a single neurone, may be expected to influence the performance of the system and its outputs. INTRODUCTION

While much signal processing in the nervous system occurs at the synapse, additional switching may occur within the presynaptic neurone owing to conduction block at branch points before signals reach the synapse. Can conduction block at central branch points in a single sensory neurone route signals to different postsynaptic neurones depending upon the spatial or temporal pattern of sensory stimulation ? Previous studies have shown that conduction block can occur at branch points of axons both in the peripheral and central nervous system (PNS and CNS) for reasons of neuronal geometry (Barron & Matthews, 1935; Grossman, Spira & Parnas, 1973; Hatt & Smith, 1976; Yau, 1976; Liischer, Ruenzel & Henneman, 1983; Dyball, Grossmann, Leng & Shibuki, 1987). This is especially likely in situations in which thin axons join thick axons and action potentials originate within the thinner processes, but can occur with a suitable load beyond the branch point (Rall, 1959; Joyner, Westerfield, Moore & Stockbridge, 1978; Parnas & Segev, 1979; Stockbridge, 1989). When block occurs, propagating action potentials may fail to reach synaptic terminals beyond the site of block. Therefore, one consequence of conduction block in the presynaptic neurone can be to silence some presynaptic terminals (Parnas, 1972; Muller & Scott, 1981; Macagno, Muller & Pitman, 1987). Since axons in diverse nervous systems appear to branch in a manner consistent with producing conduction block, conduction block might widely affect processing of signals and thus play a significant role in nervous system functioning. Although conduction block has been reported in both the PNS and CNS in some animals, little is known about conduction block and its effects in the CNS. Most of this is owing to the complexity of the CNS and the small size of most neurones. The experimental accessibility of mechanosensory neurones in the leech CNS, which are presynaptic to other identified neurones including motoneurones and other sensory cells, has made them useful to study the effect of conduction block on synaptic transmission. Conduction block occurs at central branch points in all three types of mechanosensory neurones in the leech ganglion - touch (T), pressure (P), and nociceptive (N) neurones (Van Essen, 1973; Yau, 1976; Macagno et al. 1987). Block in these cells is due to the particular geometry of the branch point, where the thin axon in the connective nerve meets a thick axon of the segmental nerves (the roots). Previous studies showed that block in these sensory neurones could reduce transmission at the electrical synapses that T sensory neurones make with certain other neurones and at the chemical synapses the lateral P (lP) sensory neurone makes with the longitudinal (L) motoneurone (Muller & Scott, 1981; Macagno et al. 1987). While these studies have demonstrated that conduction block in a presynaptic neurone can reduce its synaptic transmission to one of its postsynaptic targets, it is unknown whether conduction block might differentially affect synaptic transmission to separate postsynaptic neurones and what function it might play in a whole nervous network.

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The present experiments tested the effect of conduction block in the medial pressure (mP) sensory neurone on synaptic transmission to three separate postsynaptic neurones - the L motoneurone, the annulus erector (AE) motoneurone and the anterior pagoda (AP) cell. From the published morphology of the cells (Nicholls & Purves; 1970, Muller & McMahan, 1976; Sunderland, 1980), it seems likely that conduction block occurring while stimulating different parts of the mP cell's receptive field in the skin would selectively activate different postsynaptic neurones. The present experiment was designed to test this hypothesis. Thus, it was of interest to learn (1) whether regions of apparent contact between neurones, as viewed in the light microscope, are indeed regions in which there are functional connections and (2) whether conduction block in the presynaptic sensory neurones affects different targets differentially. Thus, does segregation of synapses with different cells enable block at the branch point to act as a switch, so that one set of postsynaptic neurones may be excited while others are not? A similar switch has been seen at the neuromuscular junction (Parnas, 1972; Grossman et al. 1973), but not reported for central synapses. METHODS

Animals and preparation The preparation used in this study was a chain of mid-body segmental ganglia from the nerve cord of the leech Hirudo medicinalis. Specimens were from the same stocks as those in the companion paper (Gu, Muller & Young, 1991). Typically, chains of three ganglia with dorsolateral skin attached on one side, including the minor receptor fields of an mP cell in the centre ganglion, were pinned ventral side up in Sylgard-coated dishes. Solutions During electrophysiological recording preparations were bathed in leech saline as described (Gu et al. 1991), except that for testing monosynaptic connections between the mP cell and its three postsynaptic cells, saline containing 15 mM-Ca2l and 18 mM-Mg2+, both replacing Na+ mole for mole, was used. In this high [Ca2+]-high [Mg2"] saline, synaptic potentials mediated by an excitable interneurone should be eliminated (Nicholls & Purves, 1970). This is because the divalent cations increase the threshold of all neurones with little change in presynaptic transmitter release, reducing the likelihood of an interneurone firing. Preparations could be maintained for 1-2 days pinned in a modified Leibowitz-15 (L-15) medium used by Ready & Nicholls (1979).

Electrophysiological recording Standard electrophysiological methods were used for intracellular and extracellular recording and stimulating (Muller, Nicholls & Stent, 1981; Macagno et al. 1987). Intracellular microelectrodes of thin-walled glass (Haer 30-30-0, Frederick Haer and Co., Brunswick, Maine) were filled with 4 Mpotassium acetate and had resistances of 25-35 MQ measured in the bath. Electrodes were placed into the soma of the mP cell and into each postsynaptic cell in turn, recording pairwise. Suction electrodes with tip diameters of approximately 150 /sm were used for stimulating cutaneous receptive fields of the mP cell. Signals were amplified by a Grass P-15 preamplifier and displayed on an oscilloscope. At the same time signals were digitized at a sampling rate of 2-5 kHz by a Tecmar A/D converter and stored by an IBM XT-compatible computer for later analysis. A Haer window discriminator triggered the computer sampling. Intracellular staining Three different intracellularly injected markers, horseradish peroxidase (HRP), Lucifer Yellow (LY), and 5,6-carboxyfluorescein (CF), were used to stain cells either individually or in pairs as described (Gu et al. 1991). Typically, makers were injected under pressure into the cell soma (Muller

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& McMahan, 1976) through bevelled microelectrodes constructed of thick-walled glass (Hilgenberg Glaswarenfabrik, Malsfeld, Germany) having resistances in the range of 50-105 MQ7 in physiological saline. Alternatively, LY was injected using 0 5 s current pulses of -1 to -5 nA at 1 Hz. Laser microsurgery Laser microsurgery was performed basically as described by Gu, Macagno & Muller (1989), although bath [Ca2+] was varied as described below. Axotomized neurones usually recovered their resting potential and excitability within about two hours. Intracellular recording showed that lesioned cells maintained those physiological properties that were measured, including firing action potentials, releasing transmitter from presynaptic terminals and producing synaptic potentials within postsynaptic targets. The input resistance of lesioned cells was usually similar to that before cutting if lesions were more than about 200-400 mm from the soma and possibly higher if cut close to the soma. The strength of transmission after injury was more variable. In control experiments, the laser cut an mP cell axon near the connectives without removing presynaptic terminals in the ganglion. Of a total of twenty-one presynaptic cells cut in control experiments, synaptic potentials increased in more than 40%, remained the same in about 30%, and diminished slightly in 30% of preparations. This variability in controls made it difficult quantitatively to compare synaptic potentials before and after cutting. Many laser-lesioned cells remained healthy, but others did not survive. Irreversible damage appeared to be associated with excessive illumination with blue light used for viewing or excessive laser irradiation, especially when the lesioning spots were close to the soma. Both the amount of Ca2+ in the saline bath and the intensity of illumination affected the extent of degeneration and degree of survival after lesioning. Indeed, too much light even at a distance from the soma could kill the irradiated cell. At lower but effective intensities for lesioning, the amount of degeneration proximal to the lesion increased as [Ca2+] in the bath was increased from 1-4 to 10 mm in different preparations. Yet, higher [Ca2+]. might have also helped lesioned cells to survive. For example, some neurones survived although only the soma remained after cutting in solutions containing elevated [Ca2+]. Thus, survival of lesioned cells depended both upon laser intensity and bath [Ca2+]. In one experiment to test the effect of Ca2 , twelve ganglia from the same animal were placed into two groups, A and B, with six in each. One AE cell in each of the twelve ganglia was axotomized with the laser beam, using the same illumination for both groups. In group A, the bathing solution contained 1-8 mM-Ca2 , while in group B it contained 7-5 mr Ca2t Group A cells seemed to have less degeneration and all retained a length of main axon with some branches attached. Cells in group B all looked quite different. They had little or no main axon and no branches remaining. Additional experiments showed that there was no effect of elevated Ca2+ if it was present before or after cutting, but not during illumination. The degeneration caused by Ca2+ was greater for branches and thinner axons than for thicker axons. This was advantageous for accurate removal of branches or thinner axons with the laser by allowing irradiation at a distance from the intended site of lesion. For example, to destroy one of the two thin anterior and posterior connective axons beginning at its connection with the thick axon, the thin axon was irradiated at a distance from the branch point in an elevated Ca2+ concentration. By irradiating at a distance from the soma, damage was confined to the intended part of the cell, and that part degenerated completely. Protease injection to kill single neurones Protease was used selectively to destroy some neurones to remove them as potential sources of synaptic input (Bowling, Nicholls & Parnas, 1978). Protease solution was made fresh for each experiment by dissolving 2 mg protease (Sigma type VIII) in 3 ml stock solution containing 0-4% Fast Green (FCF; Fisher) and 0-2 M-KCl. Electrodes filled with protease solution were bevelled to 60-90 MQ measured in the bath. Protease was injected under pressure into the somata of ipsilateral mP cells in the anterior and posterior ganglia and the injected tissue was maintained in L-15 medium overnight to permit protease to kill the injected cells. Protease was also used to eliminate electrically coupled contralateral homologues from the circuit (Bowling et al. 1978). The P cells are not electrically coupled in the same ganglion or between ganglia. However, the AE cell is weakly electrically coupled to its contralateral homologue in the same ganglion, and the AP and the L cells are more strongly coupled. It was previously shown that the L cell connection from sensory neurones is direct rather than through the coupled homologue (Bowling et al. 1978). None the less, in the present experiments to ensure that direct connections were examined, the contralateral AP

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and L homologues were injected with protease and AE cells were treated as in the companion paper (Gu et al. 1991). RESULTS

Receptive fields of the mP cell and conduction block The mP cell on each side of leech segmental ganglia resembles the T (Yau, 1976), N (Blackshaw, Nicholls & Parnas, 1982) and lP (Macagno et al. 1987) cells in innervating the skin of its own segment with one or two large axons (one axon for the mP cell) and contiguous regions of skin anteriorly and posteriorly by means of thinner axons. The thinner axons extend into the ipsilateral connectives and through adjacent ganglia, as shown in Fig. 1. This was characteristic of the six cells whose receptive fields were mapped in detail. The mP and lP cells can be distinguished by the locations of their somata in the ganglion as well as their dorsal and ventral receptive fields, respectively (Nicholls & Baylor, 1968). Moreover, study of thirtythree HRP-injected mP cells showed that the cell extends only one rather than two thicker axons out of the ipsilateral segmental nerves (roots) of its own ganglion, and that axon enters the dorsal segmental nerve. Within the mP cell's own ganglion, the two thin axons and one thick axon join at a pair of asymmetrical forks. Many secondary branches emerge from thin axons within the ganglion and extend into the neuropile where they form synaptic contacts with other neurones (Muller & McMahan, 1976). As shown in Fig. 1, one or more of these secondary branches may extend into the contralateral neuropile and form synapses there (Macagno et al. 1987; Gu et al. 1991). These contralateral branches can emerge from either the anterior or posterior axon. Among the postsynaptic cells of the mP cell are the ipsilateral L and AE motoneurones and the AP cell, whose function is unknown. Figure 2 shows the morphology of the mP cell and its postsynaptic cells stained with HRP. Figure 1 shows the receptive fields of an mP cell mapped with a suction electrode focally stimulating the skin. The receptive field of each mP cell extends ipsilaterally over three segments and consists of a major receptive field covering the cell's own segment, innervated by the thick axon, and two minor receptive fields in both adjacent segments, each innervated by a thin axon. The three subfields are contiguous, with no overlap, as can be demonstrated by mapping before and after selective axotomy. Although the threshold for stimulation of all three subfields was similar, action potentials generated in the anterior and posterior minor receptive fields typically failed to invade the cell soma either from the onset of recording or, in other preparations, during a train of 1 Hz or even lower. By analogy with other leech sensory neurones (Yau, 1976; Macagno et al. 1987), the geometry suggests that impulses arising in a minor receptive field may block at the central fork, indicated by filled arrows in Fig. 1, and can therefore reach only part of the cell, while impulses generated in the major field should propagate through the neurone. Block of action potentials which arise in the anterior minor field and travel in the anterior axon towards the soma is referred to as anterior block, while block of action potentials travelling in the posterior axon towards the soma is called posterior block. Only the anterior axon and its associated secondary branches should be excited during anterior block, while only the posterior axon and its associated branches should be excited during posterior block. Conduction block in the mP cell occurs because of (1) the lower safety factor at the

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Fig. 1. Cutaneous receptive fields of the medial pressure (mP) sensory cell. Here and in all following figures, the open arrow denotes anterior. The mP cell has three cutaneous axons innervating dorsal skin, an anterior and a posterior axon innervating minor receptive fields (cross-hatched area) and a thick middle axon innervating the major field (hatched area) in the neurone's own segment. The three axons converge at forks within the cell's ganglion. Secondary branches emerge from anterior and posterior axons in the neuropile where they synapse with other neurones. Most branches are ipsilateral but some extend to the contralateral neuropile. Typically, impulses originating in either the anterior or posterior axon will block at a central fork, the anterior or posterior central branch point (filled arrows). Usually only impulses arising in the major field propagate throughout the cell. DM, dorsal midline; VM, ventral midline.

branch point, where a thin axon joins a thick axon (Yau, 1976; Grossman, Parnas & Spira, 1979a), and (2) the hyperpolarization (Van Essen, 1973) and resultant increasing threshold (Bennett, Hille & Obara, 1970) of the mP cell during activity, which activates both a Ca2+-dependent K+ conductance and the Na+-K+-ATPase (Jansen & Nicholls, 1973). Previous studies of conduction block in sensory cells in the

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leech CNS have indicated indirectly that block probably occurs at the central fork between thick and thin axons. In the present study two lines of evidence support this assumption. First, as has been seen for other sensory neurones, action potentials generated in the minor fields can propagate throughout the cell, but during block

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Fig. 2. Structure of pre- and postsynaptic cells. The presynaptic neurone, the medial pressure sensory neurone (mP), was filled with HRP and reacted with diaminobenzidine to reveal its arborization within the leech segmental ganglion. i and c represent ipsilateral branches and contralateral branches of the mP cell, respectively. M at triangle marks ganglion mid-line. The postsynaptic neurones were the anterior pagoda (AP) cell, the longitudinal (L) motoneurone, and the annulus erector (AE) motoneurone, also injected with HRP and stained. The soma and most ipsilateral branches of the AP cell are located anteriorly in the ganglion, those of the L cell centrally, and those of the AE cell posteriorly in the ganglion. All panels are to same scale.

action potentials cannot be recorded in the soma or the root. Therefore, this indicated that during conduction block action potentials generated in either minor field did not pass the central branch point. Second, the size of the failed action potential recorded in the soma strongly depends on the distance to the presumed site

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of block. In each of seventeen experiments, the amplitude of the blocked impulse recorded in the soma consistently correlated with the locations of the branch points, which were invariably bifurcations rather than a true trifurcation. Their arrangement varied, so that in those five cells whose axon in the anterior connective branched A

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Fig. 3. Impulses that fail at branch points closer to the soma are larger. Anterior and posterior axons join with the middle axon at the anterior and posterior branch point, respectively. These two branch points are rarely at exactly the same place, so one is closer to the soma than the other. When the mP cell is stimulated electrically (see Methods) in its anterior minor receptive field (top row of traces) and in its posterior minor receptive field (bottom row of traces) as shown in Fig. 1, the impulses generated typically block at a central branch point before reaching the soma. The size of the blocked action potential at the soma strongly depends on the distance between the particular branch point and the soma. When the anterior branch point is closer to the soma, the signal in the soma is larger during anterior block than during posterior block (A). When the posterior branch point is closer to the soma the situation is reversed (B). In fewer than 10% of mP cells, the anterior and posterior axons join before linking with the main axon, which leaves the ganglion along the posterior root (C) and in these cells the blocked action potentials are identical. When these cells were hyperpolarized further, at a certain level the attenuated action potential abruptly became smaller, thus independently confirming the presence of block at the more distal branch point. Sizes of blocked action potentials were consistent with the distance from branch to soma, indicating that conduction block in the mP cell occurred at these branch points.

from the main axon proximal to the branch point for the posterior axons, the blocked anterior impulse was larger (Fig. 3A). In ten others the situation was reversed with the anterior axon's bifurcation located distally to that of the posterior, and the posterior blocked impulse was larger (Fig. 3B). In the remaining 12 % of preparations, (two cells) there was a single bifurcation of the thicker axon to produce

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a thin branch, and the thinner axon bifurcated again to produce anterior- and posterior-going axons in the connectives. Blocked impulses originating in the two minor fields were equal in amplitude when recorded in the soma (Fig. 3C), as one might predict. This was equal to the fraction of HRP-injected mP cells overall (4/33, or 12%) that had a single branch point.

Synaptic transmission from the mP cell to separate types of postsynaptic cells The L and AE motoneurones and the AP cell used in this study are ipsilateral and postsynaptic to the mP cell in the same ganglion. The morphologies of these cells are shown in Fig. 2. Cells in the separate ganglia were injected with HRP, stained with diaminobenzidine and H202, and observed in wholemounts under the microscope. As shown in Fig. 2, the three postsynaptic cells have distinctive arbors with some shared features. Each cell extends its axon from its soma across the ganglion to bifurcate and leave the ganglion through both contralateral roots. Most secondary branches of these cells emerge from the main axon before it bifurcates and they ramify within the neuropile at the centre of the ganglion, where synaptic contacts are made upon then. Ipsilateral branches of the L and AP cells are principally in the anterior and middle regions of the ganglion and intermingle with branches of the mP cell in these regions. In contrast, ipsilateral secondary branches of the AE motoneurone principally stay in the posterior region of the ganglion and should overlap with the P cell's branches there. The mP cell elicits monosynaptic EPSPs in all three cells. Those in the L and AE motoneurones were previously characterized (Nicholls & Purves, 1970, 1972; Muller & Nicholls, 1974). Chemical transmission predominates from the mP to the L and AE cells, with a very weak component due to a rectifying electrical synapse. In the present study, synaptic transmission between the mP cell and AP cell persisted in saline containing 15 mM-Ca2l and 18 mM-Mg2+, consistent with a monosynaptic connection (Nicholls & Purves, 1970). In addition, a small synaptic potential was recorded in 20 mM-Mg2" solution, suggesting the presence of a weak electrical synaptic potential. By passing current pulses into each of the two cells in the same high [Mg2"] solution, it was found that positive currents flowed from the mP cell to the AP cell: the two cells were coupled by a weak rectifying electrical junction. Thus, the synapse with the AP cell resembles the monosynaptic connections reported between the mP cell and the L and AE motoneurones. Before study of the effect of conduction block, in each preparation a collision test was done to confirm that no other cell produced synaptic potentials in the postsynaptic cells when the skin receptive field was stimulated, as is shown in Fig. 4. It is of technical interest that the somata of leech mechanosensory neurones are electrically excitable, in contrast with motoneurones, as described in the companion paper (Gu et al. 1991). In the test, an action potential initiated in cutaneous axon terminals and an action potential initiated in the soma of the mP cell collided, preventing the cutaneous action potential from reaching the middle ganglion. The postsynaptic response was eliminated by the collision (Fig. 4C), showing that the response was caused only by action potentials in the mP cell.

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Effect of conduction block in the mP cell on synaptic transmission Transmission from the mP cell to all three postsynaptic cells - AP, L and AE - in the same ganglion was examined during anterior conduction block, during posterior block, and with no block. In about half the preparations (6/13), recordings

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Fig. 4. Cutaneous synaptic potentials in the AE motoneurone can be produced solely by the mP cell. Before studying the effect of conduction block in the mP cell on its synaptic transmission, collision experiments were done to confirm that (1) the small signals recorded at the soma did represent blocked action potentials and (2) that synaptic potentials recorded in each postsynaptic cell were produced by the mP cell. Intracellular recordings (A, B and C) were from the soma of the mP cell (upper traces) and AE cell (lower traces). A, the control shows that an action potential in the mP cell initiated (arrow) at the soma produced an EPSP in the AE cell. B, the posterior minor field of the mP cell was stimulated (arrow), generating an action potential at the time marked by a recording artifact. The propagating action potential blocked before reaching the soma but produced a similar size EPSP in the AE cell. C, the soma was stimulated (arrow) before the posterior minor field, producing a collision of two action potentials in the posterior periphery. The peripherally generated action potential would have produced a small blocked potential and synaptic potential at the time marked by arrowheads had it not collided with the outgoing somatic action potential. This showed that neither the blocked action potential or synaptic potential were caused by impulses in another neurone that synapsed upon them.

were similar to those shown in Fig. 5. In these ganglia, during anterior block in the mP cell, which excited only its anterior axon and branches, EPSPs recorded from the L and AP cells (middle traces in Fig. 5B) persisted (compare Fig. 5A), but little or no EPSP was recorded from the AE cell (lower trace in Fig. 5B). In contrast, during posterior block in the mP cell, which excited only its posterior axon and branches, no EPSPs could be recorded from either the AP or L cells, while EPSPs recorded from the AE cell were nearly unchanged during block (lower trace in Fig. 5C). Thus in these ganglia, during anterior block the mP cell transmitted as usual to the AP and L cells but stopped transmitting to the AE cell. Conversely, during posterior block, the mP cell transmitted fully to the AE cell but not to the AP and L cells. Furthermore, the results indicated that only anterior branches of the mP cell synapsed with the AP and L cells, while only posterior branches synapsed with the AE cell in preparations such as those in Fig. 5.

DIFFERENTIAL TRANSMISSION AND CONDUCTION BLOCK 765 In the remaining preparations (7/13), the effect of conduction block on synaptic transmission to these three postsynaptic neurones in a ganglion was somewhat different from that just described in Fig. 5. In some of those ganglia (3/7), during anterior block in the mP cell instead of almost no change in EPSPs, the EPSPs A

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Fig. 5. Conduction block differentially eliminates transmission to separate neurones. Impulses in the mP cell, shown in top traces, produced synaptic potentials that were recorded in AP, L and AE cells in a single ganglion (lower traces). A, full size synaptic potentials produced without block; their amplitudes are indicated by horizontal lines in B and C. B, blocked action potential generated in the anterior minor field and recorded in the mP cell body, and the postsynaptic potentials it produced. C, the effects of a blocked action potential arising in the posterior minor receptive field. Arrows indicate timing of peak of unblocked synaptic potentials. Traces are computed averages of 20-40 sweeps. The AP and L cells, whose responses were similar, evidently received input chiefly from the anterior axon, while the AE cell's input was from the posterior axon of the mP cell. This situation was seen in six of thirteen (46%) experimental ganglia.

recorded from both the AP and L cells were distinctly reduced in amplitude and in these cells small EPSPs were recorded during posterior block. A record from an AP cell is shown in Fig. 6A. The responses in the AE cell were as in Fig. 5, above. In other ganglia (3/7), EPSPs recorded from the AE cells showed a slight reduction in amplitude during posterior block in the P cells, and small EPSPs were recorded from these AE cells during anterior block, as shown in Fig. 6B. For these ganglia the responses in the L and AP cells were as in Fig. 5, above. In one ganglion the EPSPs were reduced in this fashion in all three cells - AP, L and AE. Considering the relative strengths of transmission by anterior and posterior branches of the mP cell to each postsynaptic cell, recordings from these ganglia indicated that the mP cell's anterior branches contributed most of the transmission to the AP and L cells and little to the AE cell, while the mP cell's posterior branches did just the opposite. Concerning the effect of conduction block on transmission to each postsynaptic

X. GU neurone, data from thirteen preparations show that in about 70 % (9/13) of ganglia, anterior block in the mP cell produced no discernible effect on transmission to the AP and L cells, while posterior block completely eliminated transmission. In the remaining 30 % (4/13) of ganglia anterior block measurably reduced transmission to 766

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Fig. 6. Conduction block in the mP cell may differentially reduce but not eliminate transmission to separate neurones. A, full size synaptic potentials produced by the mP cells without block in an AP cell and an AE cell in two separate ganglia; amplitudes of EPSPs are indicated by horizontal lines in B and C. B and C show transmission to the AP and AE cells during anterior block and posterior block, respectively. Partial reductions were seen in seven of thirteen experimental ganglia. In four ganglia synaptic potentials in the AP cell were reduced during anterior block and a residual response persisted during posterior block. In four ganglia, synaptic potentials in the AE cells were reduced during posterior block and a residual response persisted during anterior block. (In one of the seven ganglia synaptic potentials in both the AP and AE cells had partial reduction.) Results of recording from the L cells (not shown) were consistent with those seen in the AP cells.

the AP and L cells, while during posterior block some transmission remained. The opposite results were obtained for the AE cell: posterior block in the mP cell produced no effect on the transmission in about 70% (9/13) of ganglia and reduced the transmission in another 30 % (4/13) of ganglia, which were largely different from those four of thirteen that had residual transmission to the AP cells during posterior block. Since blocked action potentials do passively spread beyond the site of block and can be recorded from the soma of the P cell, the question arises whether this

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electrotonic potential can cause branches beyond the site of block to release transmitter. A previous study by Muller & Scott (1981) found that blocked action potentials in the T cell did not transmit detectably at electrical synapses beyond the site of block. Can blocked action potentials transmit at chemical synapses beyond the B

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Fig. 7. The anterior and posterior components of the EPSPs in the AP cell (A) and AE cell (B) sum (middle traces) to equal the unblocked (full) EPSPs (top traces). Averages are of thirty episodes. Top and middle traces are superimposed at bottom. This indicates that the effect of the presynaptic impulse does not spread beyond the central branch point to release transmitter. The anterior and posterior synapses are spatially distinct and generate small voltage changes, apparently permitting the potentials recorded in the cell body to sum linearly (see text). Presumably, linear summation also occurs at the site of impulse initiation at the primary axon bifurcation (see below).

site of block? In the mP cell, during anterior block and posterior block action potentials evidently invade separate parts of the mP cell, the anterior and the posterior portions, respectively. If blocked action potentials do not cause transmitter release from branches beyond the site of block, then each of the two parts of the cell should transmit independently during anterior and posterior block and transmit together when conduction is not blocked. Therefore, the full EPSP recorded in a postsynaptic cell without presynaptic block should equal the sum of two fractions of EPSPs recorded during anterior and posterior block. If, instead, the passively spread components of the action potentials were to release transmitter during conduction block, then the sum of the anterior and posterior blocked EPSPs would be larger than the EPSPs recorded when there was no presynaptic conduction block. The EPSPs recorded in an AP cell during anterior and posterior block (shown in Fig. 6A) were summed by computer, as seen in Fig. 7A. Similar sums of EPSPs recorded during anterior and posterior conduction block were computed for the AE cell, as shown in Fig. 7B. The summed synaptic potentials in each cell were almost equal at their peaks to the full synaptic potentials recorded in the same cells without block. This result suggests that the passively spread components of blocked action potentials might be too weak to produce detectable transmission. In three cases the sum of the two blocked EPSPs was smaller than the unblocked EPSP; when these mP cells were examined under a microscope, they were seen to have sizeable branches that emerged

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directly from the thick axon, which differed from the usual configuration for mP cells. Those branches would presumably be excited only when there was no block but not during either anterior or posterior block. In this analysis it was valid to add voltages rather than current or conductances when comparing normal transmission with transmission during presynaptic

m~ ~ ~~~

Fig. 8. Diagram of functional connections from the mP to three postsynaptic cells, based on conduction block results such as those in Figs 5 and 6. Anterior branches of the mP cell principally excite the AP and L cells with little excitation of AE cells, while its posterior branches excite the AE cell more strongly than the AP and L cells. Weaker connections are represented by thinner lines. Synapses are on somata for diagrammatic purposes; they actually occur within the neuropile. Ganglion is outlined.

conduction block. Voltages normally do not sum linearly, but the synaptic potentials in our study were less than a few mV and with a resting potential of -50 mV the correction for non-linear summation would be less than 5 % (Martin, 1955, 1966). The membrane conductance at rest was ohmic. Thus the results were consistent with the independence of the anterior and posterior arbors of the mP cells. There was little spread of effective current beyond the P cell's branch point, and there was a nearly linear summation of the postsynaptic potentials at low amplitudes.

Distribution offunctional connections compared with apparent contacts between neurone terminals The above results from conduction block, which can activate different regions of the mP cell separately, indicate that the mP cell makes its principal functional contacts with the L and AP cells with branches from its anterior axon, while contacts with the AE cell are from its posterior axon. Based on the physiological study of conduction block, a pattern of connections between the P cell and three postsynaptic cells in a ganglion is proposed in Fig. 8. To determine more precisely the morphological basis of this pattern of connections of the mP cell with different postsynaptic neurones, physiology and morphology were

DIFFERENTIAL TRANSMISSION AND CONDUCTION BLOCK 769 combined. Varicosities of the P cells are presynaptic sites (Muller & McMahan, 1976), and in a detailed study of the connection between the IP cell and L motoneurone using electron microscopy it was found that apparent contacts as detected in the light microscope were always sites of synapses (Macagno et al. 1987). This makes it likely that apparent contacts between the P cell and those postsynaptic neurones described here are sites of synapses, but electron microscopy would be required to prove it and this has not been done. After detailed study of synaptic transmission with and without conduction block, preparations were double-stained by labelling one postsynaptic cell at a time with HRP and the mP cell with LY (see Methods). Apparent contacts between branches of the mP cell and postsynaptic cells (see arrows in Fig. 9B) could be observed under the microscope and those branches of the P cell that made those contacts could be traced to identify their origins, either from the anterior axon or the posterior axon or, in a few cases, from the thick axon. These apparent contacts were counted under the microscope and compared with physiological measurements in Table 1, which shows the total number of putative contacts for each cell pair. From Table 1 the average strength of each contact made by mP cells can be calculated and is 72 ,uV for AE cells and 40 ,sV in AP cells. Also in Table 1 are given the fractions of contacts made by secondary branches of the anterior or posterior axon branch, respectively. These values are compared with the fraction of total synaptic potential associated with each of those branches (represented by the numbers in parentheses) as determined from conduction block. The physiological data correlated well with the morphological data in all pairs of pre- and postsynaptic cells, as shown in Table 1. For the last mP-AP cell pair in the table, the sum both of apparent contacts and of partial EPSPs is less than 1. This is because for that mP cell some branches which made apparent contacts emerged from the thick axon of the root rather than the anterior or posterior axon and these branches would not have been expected to be excited during either anterior or posterior conduction block. There are several lines of indirect evidence which all indicate that it is likely that the weak connections of the mP cell's anterior axon with the AE cell and of its posterior axon with the AP cell and L cell were by means of its contralateral branches (Table 1). As mentioned before, most mP cells have some contralateral branches that appear to emerge either from the anterior or from the posterior axon. If these branches form functional connections with postsynaptic neurones, as it appears (Gu et al. 1991) they would account for the persistence of transmission to the AE cells during anterior block when they emerge from the anterior axons or, when from the posterior axons, persistent transmission to the AP and L cells during posterior block. Indeed, of those mP cells with residual transmission, three were stained after physiological recording and found to have such contralateral branches, although the postsynaptic cells they contacted were not stained. Further, such contralateral branches were seen emerging from the anterior axon in the two cases when there was a residual transmission to the AE cell during anterior block and from posterior axon in that case in which there was residual transmission to the AP and L cells during posterior block. In addition, morphological study of double-labelled pre- and postsynaptic cells showed such contralateral branches of the mP cell did form apparent contact with branches of postsynaptic cells. Laser axotomy experiments

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Fig. 9. Pre- and postsynaptic neurones filled with LY and HRP respectively. Apparent contacts between the mP cell and postsvnaptic cells are charted by injecting separate markers. A. the mP cell was injected with LY; the AE cell was injected with HRP and stained in the living ganglion. The brightly fluorescent mP cell soma was removed after

DIFFERENTIAL TRANSMISSION AND CONDUCTION BLOCK

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also provided some indirect evidence, as mentioned below. Thus, contralateral branches of the mP cell emerged from its anterior axon when there was a residual EPSP in the AE cell, while they emerged from the posterior axon when the L cell and/or AP cell revealed residual EPSPs.

Laser axotomy can isolate either the anterior or the posterior axon of the mP cell Conduction block apparently reduces or eliminates synaptic transmission because of a failure to excite a portion of the sensory cell beyond the axon fork. As a further TABLE 1. Number of apparent contacts vs. strength of transmission Total Fraction of contacts contacts (Fraction of total EPSP) (Full Posterior EPSP in mV) Anterior mP-AE 24 (1-7) 10 (10) 00 (00) 01 (00) 27 (2) 09 (10) (n = 2) 65 (1-2) 05 (05) 05 (05) mP-AP 01 (00) 40 (1-2) 0-9 (10) 52 (30) 06 (07) 04 (03) (n = 4) 42 (2 3) 0-6 (0-6) 0 1 (0 1) In the table, the total number of putative contacts and amplitude of unblocked EPSP is given for each cell pair, followed by the fraction made by secondary branches of the anterior or posterior axon branch, respectively. For comparison, the numbers in parentheses represent the fraction of total synaptic potential associated with each of those branches. For the last mP-AP cell pair the sums are less than 1 but are in agreement. In that mP cell some branches emerged from the main axon rather than the anterior or posterior axon. Contralateral branches emerged from the posterior axon of the first, third and fifth cells, and from the main axon of the sixth. Those branches in the third and fifth cell pairs can account for their residual synaptic potentials.

test of this hypothesis, an alternative means of activating only a part of the neurone would be by selectively cutting or destroying another part of the cell and stimulating the remainder. Microsurgery with a laser beam was used to cut either the anterior axon or posterior axon of the mP cell at its fork. In preparations in which posterior axons of the mP cells were selectively cut, action potentials in the cells activated only the anterior axon, a situation resembling anterior block. In other preparations in which anterior axons were cut from the mP cells, only the posterior axon could be activated, which resembled the situation occurring during posterior block. Transmission from the lesioned mP cell to two different postsynaptic cells, the AP and AE cells, was compared during conduction block and following axotomy. Typically, when the posterior axon of the mP cell was cut and only the anterior axon remained, the mP cell excited only the AP cell and not the AE cell (Fig. lOB). The fixation to aid in viewing fine branches near it. M mark ganglion mid-line. B, apparent contacts (arrow-heads) were viewed and counted in fixed tissue at higher magnification (400 x ) with a water immersion objective (numerical aperture = 0 75). Results from such counts are given in Table 1.

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reverse was true after cutting the mP cell's anterior axon (Fig. lOD). Of thirteen mP cells that had their posterior axons cut and destroyed, as confirmed by intracellular marking and recording, all thirteen cells continued to transmit to the AP cells but only three mP cells transmitted to the AE cells. All those three still transmitting to

B

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Fig. 10. Laser axotomy mimics anterior and posterior conduction block. A, micrograph shows an mP cell that was filled with CF dye, irradiated and cut at its posterior axon, as marked by the arrow, with the microbeam of an argon laser. After being cut, the mP cell was filled with HRP and stained. B, the AP cell but not the AE cell was excited by the action potential in the cut mP cell. Averages of twenty to forty sweeps. Rapid deflections in postsynaptic traces are electrical artifacts. C, the anterior axon of the mP cell was severed with the laser, destroying it at the point indicated by the arrow. D, the posterior branches, which remained, excited the AE motoneurone but not the AP cell. Data shown here confirm the results in Fig. 5.

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: @;.~AE

DIFFERENTIAL TRANSMISSION AND CONDUCTION BLOCK

773

the AE cells had contralateral branches that emerged from the anterior axons but the remaining ten did not (Fig. 1 lB). This is indirect evidence that the mP cells' active synapses with the AE cells might be contralateral, as demonstrated directly in Gu et al. (1991). Twelve mP cells had their anterior axons cut completely with the laser

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Fig. 11. Contralateral branches can account for residual synaptic potentials in the AE cell following laser axotomy. The mP cells were cut as in Fig. 10, with the posterior axon cut (arrow) in A and the anterior axon cut (arrow) in C. The cut cells were filled with HRP and stained. Each cell had a fine branch (arrowheads) that crossed the ganglion and arborized in the contralateral neuropile. Close examination revealed that the contralateral axon emerged from the anterior and posterior axons, respectively, and not the root axon. B, with a contralateral branch present the mP excited the AE cell, albeit weakly, in addition to exciting the AP cell. D, with a contralateral branch present the mP cell excited the AP cell, weakly, in addition to the AE cell. Averages of twenty to forty sweeps. Rapid deflections in postsynaptic records are electrical artifacts.

microbeam. All twelve cells continued to excite the AE cells, but only three mP cells still transmitted, weakly, to the AP cells (Fig. 1 lD). Again all three mP cells had contralateral branches that emerged from posterior axons, while the remaining nine

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did not. Evidently it was those contralateral branches, emerging from the posterior axons, that contacted the AP cells. Since for three of thirteen cells a contralateral branch emerged from the anterior axons and for three of twelve one emerged from the posterior axons, and it is rare for contralateral branches to arise from both the anterior and posterior axons, then about half the mP cells might be expected to have contralateral branches. In summary, these results of laser axotomy experiments independently confirmed results of the conduction block experiment (Figs 5 and 6), and they support the hypothesis that contralateral branches might mediate the weak connections from the anterior axon to the AE cells or from the posterior axon to the AP and L cells in about half the preparations. DISCUSSION

Receptive field of the mP cell and conduction block in the cell This study has shown that the receptive field of a single mP cell is innervated by three cutaneous axons separately and is thus divided into three sub-fields, a major receptive field and two minor receptive fields, in adjacent segments which are contiguous with the major field and do not overlap it. The receptive field innervation of the mP cell in the leech Hirudo medicinalis is similar to that reported for its homologue in another leech, Haementeria ghilianii (Kramer & Kuwada, 1983). For the mP cell, impulses from axons innervating the minor fields were often blocked centrally from the onset of the experiments, but otherwise block was more likely to occur as the frequency of impulses increased to no more than 1 Hz. This is greater susceptibility to block than in some other sensory cells in the leech CNS, such as T cells. Without block, impulses initiated from minor fields travel through the central ganglion and out toward the periphery along the thick axon. If the outgoing impulses were to collide with impulses coming from the major field, information from that field would be lost. The probability of collision increases as the frequency of impulses increases. Conduction block prevents this collision, thereby permitting signals to arrive in the ganglion from the major field. It is difficult to identify the exact location of block in most types of neurones, although theoretical calculations (Rall, 1959; Parnas, Hochstein & Parnas, 1976; Parnas & Segev, 1979; Stockbridge, 1989) and a few key experiments (Yau, 1976; Grossman et al. 1979a; Grossman, Parnas & Spira, 1979b; Stockbridge & Stockbridge, 1988) have shown that block occurs at the branch point. The present experiments also show that block occurs at the central branch point of axons, based in part on the consistent correlation between amplitude of the blocked impulse and branch-point location and on the effects of laser axotomy.

Effect of conduction block on signalling What function might conduction block play in the nervous system? Conduction block in leech sensory cells substantially reduced synaptic transmission to individual neurones, as confirmed in the present experiments. Moreover, it has now been shown that the reduction is different and distinctive for different postsynaptic neurones. This possibility had been predicted from the morphology and distribution of possible contacts of another sensory neurone with morphology like that of the P cell, the T

DIFFERENTIAL TRANSMISSION AND CONDUCTION BLOCK

775

cell and two of the postsynaptic cells studied here, the L and AE motoneurones (DeRiemer & Macagno, 1981), but the prediction was untested. Thus for the mP cell, conduction block can allow it to selectively excite certain postsynaptic cells but not others, temporarily changing the pattern of functional connections with postsynaptic targets. In this way, stimulating a single sensory cell at different locations in the skin might produce different responses. These results appear to be the first demonstration that conduction block can function as a switch between separate targets in a nervous system, resembling a similar phenomenon at the neuromuscular junction (Bittner, 1968; Parnas, 1972; Grossman et al. 1973; Smith, 1983). One question arising in the study of conduction block is whether blocked action potentials can passively spread within the neurone beyond the point of block to activate presynaptic terminals. Experiments with leech touch sensory neurones have shown that impulses do not transmit at electrical synapses beyond the point of block (Muller & Scott, 1981; Gu et al. 1989). The present study extends this result to chemical synapses. One consequence of conduction block is that the receptive fields of sensory neurones shrink, and thus become sharper. This study confirms that the sharpening, which is evident while recording from the soma (Yau, 1976), is transmitted to postsynaptic cells (Muller & Scott, 1981; Macagno et al. 1987). For leech mechanosensory neurones (Yau, 1975), this is similar to the sharpening of retinal ganglion cell receptive fields during light adaptation in mammals (Kuffler, 1953), which operates by a different mechanism involving lateral interactions in a group of cells. In the leech this is done by a single sensory neurone with conduction block. Such efficiency might be useful for an animal which has only about 400 neurones in each ganglion. Conduction block has been reported in mechanosensory afferents in the mammalian spinal cord, where it affects transmission from Ia afferent fibres to amotoneurones (Henneman, Liuscher & Mathis, 1984). It can also occur in excitable dendrites of the cerebellar Purkinje cell (Nicholson & Llina's, 1971), affecting integration of synaptic inputs received by different dendrites. Thus, conduction block can affect signalling in pre- and in postsynaptic neurones.

Are apparent contacts sites of synapses? Experiments in which the number of apparent contacts was compared with the physiologically measured strength of transmission showed a good correlation between the two (Table 1). Although electron microscopy would be required to demonstrate that apparent contacts (sometimes referred to simply as contacts) are definitely functional synaptic sites, an earlier study of synapses made by the lP cell with the L motoneurone showed in every case in which the two appeared to be making contact when viewed in the light microscope, examination of that apparent contact in the electron microscope revealed a synapse (Macagno et al. 1987). In that study a strength per contact was determined by dividing the amplitude of the synaptic potential by the number of apparent contacts between the cells, and a range from 20 to 100 ,tV/contact, with typical values of 30-50,aV, was reported. In Table 1 for the present study the strength per contact for the AE and AP cells is calculated to be 72 and 39 ,IV/contact, respectively. These values agree with those in the same

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range obtained in the study on the IP cell synapse with the L cell, as might be expected if individual contacts between different cells are equally effective and are sites of synapses. The focus of the present study was on the synapses that the mP cell makes with ipsilateral L, AP and AE cells, but synapses are also made with the cells' contralateral homologue. Since the AP and L neurones are coupled to their contralateral homologues, the experiments on these cells were conducted following selective destruction of the contralateral homologue with a protease. This was not necessary for the weakly coupled AE cells (see Gu et al. 1991). The results confirmed earlier studies of sensory synapses upon the L cell (Bowling et al. 1978; Macagno et al. 1987) showing that the measured synaptic potentials are direct. Further investigation is required to determine the role of pairs of postsynaptic neurones in sensory-motor integration during conduction block.

Segregation of the mP cell synapses with separate targets The mP cell's synapses are distributed within the ganglion like those of other leech sensory neurones (Muller & McMahan, 1976; Muller & Scott, 1981; Macagno et al. 1987; Gu et al. 1989). Yet the synapses from the mP cell to different postsynaptic cells can be restricted to separate portions of the ganglion. This might account for the differences previously seen in facilitation and depression of the P cell's synaptic transmission to the L and AE cells (Muller & Nicholls, 1974), for these changes in transmission are probably presynaptic (Del Castillo & Katz, 1954). Does the segregation of synapses reflect their selective formation by different parts of the cell, so that the anterior branch, for example, synapses preferentially with the AP and L cells; or is there simply no opportunity to synapse with the AE cell because it has few branches within the anterior region of the neuropile ? The AE cell is paired and the mP cell makes excitatory synapses with both AE cells. Branches of the contralateral AE cell project both anteriorly and posteriorly in the ganglion and should encounter and be available to receive synapses from the anterior axon of the mP cell. However, both conduction block and laser axotomy experiments similar to those reported here (X. Gu & K. J. Muller, unpublished) show that it is principally the posterior branches of the mP cell that form synapses with the contralateral AE cell. Thus, proximity of branches does not guarantee that pre- and postsynaptic cells will form synapses. It is unclear whether the underlying mechanism for such segregation is a competition for targets or a selective matching or some other mechanism possibly related to function of postsynaptic neurones. I thank K. Muller for contributions throughout the course of this work, A. Morrissey and R. Harris for technical assistance, D. McColloh for writing the data acquisition and analysis programs, and E. McGlade-McCulloh and S. Young for useful discussions. This work was performed in partial fulfilment of requirements for a Ph.D. Supported in part by USPHS Grant RO1-NS 20607 to K. J. Muller.

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REFERENCES

BARRON, D. H. & MATTHEWS, H. C. (1935). Intermittent conduction in the spinal cord. Journal of Physiology 85, 73-103. BENNETT, M. V. L., HILLE, B. & OBARA, S. (1970). Voltage threshold in excitable cells depends on stimulus form. Journal of Neurophysiology 33, 585-594. BITTNER, G. D. (1968). Differentiation of nerve terminals in the crayfish opener muscle and its functional significance. Journal of General Physiology 51, 731-758. BLACKSHAW, S. E., NICHOLLS, J. G. & PARNAS, I. (1982). Physiological responses, receptive fields and terminal arborizations of nociceptive cells in the leech. Journal of Physiology 326, 251-260. BOWLING, D., NICHOLLS, J. & PARNAS, I. (1978). Destruction of a single cell in the central nervous system of the leech as a means of analysing its connexions and functional role. Journal of Physiology 282, 169-180. DEL CASTILLO, J. & KATZ, B. (1954). Statistical factors involved in neuromuscular facilitation and depression. Journal of Physiology 124, 574-585. DERIEMER, S. A. & MACAGNO, E. R. (1981). Light microscopic analysis of contacts between pairs of identified leech neurons with combined use of horseradish peroxidase and lucifer yellow. Journal of Neuroscience 1, 650-657. DYBALL, R. E. J., GROSSMANN, R., LENG, G. & SHIBUKI, K. (1987). Action potential conduction failure in the rat neurohypophysis. Journal of Physiology 388, 13P. GROSSMAN, Y., PARNAS, I. & SPIRA, M. E. (1979a). Differential conduction block in branches of a bifurcating axon. Journal of Physiology 295, 283-305. GROSSMAN, Y., PARNAS, I. & SPIRA, M. E. (1979b). Ionic mechanisms involved in differential conduction of action potentials at high frequency in a branching axon. Journal of Physiology 295, 307-322. GROSSMAN, Y., SPIRA, M. E. & PARNAS, I. (1973). Differential flow of information into branches of a single axon. Brain Research 64, 379-386. Gu, X., MACAGNO, E. R. & MULLER, K. J. (1989). Laser microbeam axotomy and conduction block show that electrical transmission at a central synapse is distributed at multiple contacts. Journal of Neurobiology 20, 422-434. Gu, X., MULLER, K. J. & YOUNG, S. R. (1991). Synaptic integration at a sensory-motor reflex in the leech. Journal of Physiology 441, 733-754. HATT, H. & SMITH, D. 0. (1976). Synaptic depression related to presynaptic axon conduction block. Journal of Physiology 259, 367-393. HENNEMAN, E., LUSCHER, H. R. & MATHIS, J. (1984). Simultaneously active and inactive synapses of single Ia fibres on cat spinal motoneurones. Journal of Physiology 352, 147-161. JANSEN, J. K. S. & NICHOLLS, J. G. (1973). Conductance changes, an electrogenic pump and the hyperpolarization of leech neurones following impulses. Journal of Physiology 229, 635-665. JOYNER, R. W., WESTERFIELD, M. MOORE, J. W. & STOCKBRIDGE, N. (1978). A numerical method of model excitable cells. Biophysical Journal 22, 155-170. KRAMER, A. P. & KUWADA, J. Y. (1983). Formation of the receptive fields of leech mechanosensory neurons during embryonic development. Journal of Neuroscienre 3, 2474-2486. KUFFLER, S. W. (1953). Discharge patterns and functional organization of mammalian retina. Journal of Neurophysiology 16, 37-68. LUSCHER, H. R., RUENZEL, P. & HENNEMAN, E. (1983). Composite EPSPs in motoneurones of different sizes before and during PTP: implications for transmission failure and its relief in I a projections. Journal of Neurophysiology 49, 269-289. MACAGNO, E. R., MULLER, K. J. & PITMAN, R. M. (1987). Conduction block silences parts of a chemical synapse in the leech central nervous system. Journal of Physiology 387, 649-664. MARTIN, A. R. (1955). A further study of the statistical composition of the end-plate potential.

Journal of Physiology 130, 114-122.

MARTIN, A. R. (1966). Quantal nature of synaptic transmission. Physiological Reviews 46, 51-66. MULLER, K. J. & MCMAHAN, U. J. (1976). The shapes of sensory and motor neurones and the distribution of their synapses in ganglia of the leech: A study using intracellular injection of horseradish peroxidase. Proceedings of the Royal Society B 194, 481-499. MULLER, K. J. & NICHOLLS, J. G. (1974). Different properties of synapses between a single sensory neurone and two different motor cells in the leech C.N.S. Journal of Physiology 238, 357-369.

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X. GU

MULLER, K. J., NICHOLLS, J. G. & STENT, G. S. (1981). Neurobiology oftheLeech. Cold Spring Harbor Laboratory, New York. MULLER, K. J. & SCOTT, S. A. (1981). Transmission at a 'direct' electrical connexion mediated by an interneurone in the leech. Journal of Physiology 311, 565-583. NICHOLLS, J. G. & BAYLOR, D. A. (1968). Specific modalities and receptive fields of sensory neurons in the CNS of the leech. Journal of Neurophysiology 31, 740-756. NICHOLLS, J. G. & PURVES. D. (1970). Monosynaptic chemical and electrical connexions between sensory and motor cells in the central nervous system of the leech. Journal of Physiology 209, 647-667. NIcHOLLS, J. G. & PURVES, D. (1972). A comparison of the chemical and electrical synaptic transmission between single sensory cells and a motoneurone in the central nervous system of the leech. Journal of Physiology 225, 637-656. NICHOLSON, C. & LLINA(S, R. (1971). Electrophysiological properties of dendrites and somata in alligator Purkinje cells. Journal of Neurophysiology 34, 532-551. PARNAS, I. (1972). Differential block at high frequency of branches of a single axon innervating two muscles. Journal of Neurophysiology 35, 903-914. PARNAS, I., HOCHSTEIN, S. & PARNAS, H. (1976). Theoretical analysis of parameters leading to frequency modulation along an inhomogeneous axon. Journal of Neurophysiology 39, 909-923. PARNAS, I. & SEGEV, I. (1979). A mathematical model for conduction of action potentials along bifurcating axons. Journal of Physiology 295, 323-343. RALL, W. (1959). Branching dendritic tree and motoneuron membrane resistivity. Experimental Neurology 1, 491-527. READY, D. F. & NICHOLLS, J. (1979). Identified neurones isolated from leech CNS make selective connections in culture. Nature 281, 67-69. SMITII, D. 0. (1983). Axon conduction failure under in vivo conditions in crayfish. Journal of Physiology 344, 327-333. STOCKBRIDGE, N. (1989).Theoretical response of a bifurcating axon with a locally altered axial resistivity. Journal of Theoretical Biology 137, 339-354. STOCKBRIDGE, N. & STOCKBRIDGE, L. L. (1988). Differential conduction at axonal bifurcations. I. Effect of electrotonic length. Journal of Neurophysiology 59, 1277-1285. SUNDERLAND, A. J. (1980). A hitherto undocumented pair of neurons in the segmental ganglion of the leech which receive synaptic input from mechanosensory cells. Comparative Biochemistry and Physiology 67a, 299-302. VAN ESSEN, D. C. (1973). The contribution of membrane hyperpolarization to adaptation and conduction block in sensory neurones of the leech. Journal of Physiology 230, 509-534. YAU, K.-W. (1975). Receptive Fields, Geometry and Conduction Block of Sensory Cells in the Leech Central Nervous System. Ph.D. Thesis, Harvard University, Cambridge, MA, USA. YAU, K.-W. (1976). Receptive fields, geometry and conduction block of sensory neurones in the CNS of the leech. Journal of Physiology 263, 513-538.