Identified serotonergic neurons LCB1 and RCB1 in the cerebral ganglia of Aplysia produce presynaptic facilitation of siphon sensory neurons

<jats:p>Several lines of evidence suggest that 5-HT plays a significant role in presynaptic facilitation of the siphon sensory cells contributing to dishabituation and sensitization of the gill- and siphon-withdrawal reflex in Aplysia. Most recently, Glanzman et al. (1989) found that treatment with the 5-HT neurotoxin, 5,7-DHT markedly reduced both synaptic facilitation and behavioral dishabituation. To provide more direct evidence for a role of 5-HT, we have attempted to identify individual serotonergic facilitator neurons. Hawkins (1989) used histological techniques to locate several serotonergic neurons in the ring ganglia that send axons to the abdominal ganglion and are therefore possible serotonergic facilitators. These include one neuron in the B cluster of each cerebral ganglion, which we have identified electrophysiologically and named the CB1 cells. Both glyoxylic acid histofluorescence and 5-HT immunofluorescence indicate that the CB1 neurons are serotonergic. In a semiintact preparation, the CB1 neurons respond to cutaneous stimulation which produces dishabituation and sensitization (such as tail shock) with an increase in firing, which may outlast the stimulation by 15 min. Intracellular stimulation of a CB1 neuron in a manner similar to its response to tail shock produces facilitation of the EPSPs from siphon sensory neurons to motor neurons, as well as broadening of the action potential in the sensory neurons in tetraethylammonium solution. These results strongly suggest that the identified serotonergic CB1 neurons participate in mediating presynaptic facilitation contributing to dishabituation and sensitization of the gill- and siphon-withdrawal reflex in Aplysia.</jats:p>

section_content: Steven L. Mackey, ' Eric Ft. Kandel, 'Center for Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, *New York State Psychiatric Institute, and 3Howard Hughes Medical Institute, New York, New York 10032 Several lines of evidence suggest that 5-HT plays a significant role in presynaptic facilitation of the siphon sensory cells contributing to dishabituation and sensitization of the gill-and siphon-withdrawal reflex in Aplysia. Most recently, Glanzman et al. (1999) found that treatment with the 5-HT neurotoxin, 5,7-DHT markedly reduced both synaptic facilitation and behavioral dishabituation. To provide more direct evidence for a role of 5-HT, we have attempted to identify individual serotonergic facilitator neurons. Hawkins (1999) used histological techniques to locate several serotonergic neurons in the ring ganglia that send axons to the abdominal ganglion and are therefore possible serotonergic facilitators. These include one neuron in the B cluster of each cerebral ganglion, which we have identified electrophysiologically and named the CBl cells. Both glyoxylic acid histofluorescence and 5-HT immunofluorescence indicate that the CBl neurons are serotonergic. In a semiintact preparation, the CBl neurons respond to cutaneous stimulation which produces dishabituation and sensitization (such as tail shock) with an increase in firing, which may outlast the stimulation by 15 min. Intracellular stimulation of a CBl -neuron in a manner similar to its response to tail shock produces facilitation of the EPSPs from siphon sensory neurons to motor neurons, as well as broadening of the action potential in the sensory neurons in tetraethylammonium solution. These results strongly suggest that the identified serotonergic CBl neurons participate in mediating presynaptic facilitation contributing to dishabituation and sensitization of the gill-and siphon-withdrawal reflex in Aplysia. 

As reviewed in the 2 preceding papers (Glanzman et al., 1989; Hawkins, 1989), several lines of evidence suggest that 5-HT plays a significant role in presynaptic facilitation of the siphon sensory cells contributing to dishabituation and sensitization of the gill-and siphon-withdrawal reflex in Aplysia. Application of serotonin mimics the effects of cutaneous or nerve stimulation in producing facilitation of the EPSPs from sensory cells to follower cells, as well as broadening of action potentials and elevation of CAMP in the sensory neurons (Brunelli et al., 1976; Klein and Kandel, 1978; Bemier et al., 1982). Furthermore, 5-HT is detectable in the abdominal ganglion (the location of the sensory cells) by several methods, and 5-HT immunoreactive processes come in close contact with the sensory cells (Kistler et al., 1985). Most recently, we have found that treatment with the 5-HT neurotoxin, 5,7-DHT, significantly reduced both facilitation of the sensory cells and dishabituation of the reflex (Glanzman et al., 1989). 

However, since this evidence is all indirect, and since at least 2 other transmitters (the small cardioactive peptide SCP and the unknown transmitter of the L29 cells) can produce facilitation of the sensory cells (Hawkins et al., 198 1; Abrams et al., 1984), we sought more direct evidence for a role of 5-HT in facilitation, dishabituation, and sensitization. The preceding paper (Hawkins, 1989) describes experiments combining glyoxylic histofluoresccnce with retrograde fluorescent labeling, which located approximately 10 serotonergic neurons in the ring ganglia that send axons to the abdominal ganglion and are therefore possible serotonergic facilitator neurons. These include 4 neurons in each pedal ganglion and 1 neuron in the B-cell cluster of each cerebral ganglion. Since the cerebral neurons showed the most consistent double labeling, we have studied them first. In this paper, we show that these identified serotonergic cells are excited by cutaneous stimuli that produce dishabituation and sensitization, and that firing them produces presynaptic facilitation of the siphon sensory cells.
section_name: Identified Serotonergic Neurons LCBI and RCBI in the Cerebral Ganglia of Aplysia Produce Presynaptic Facilitation of Siphon Sensory Neurons

section_content: EIectrophysiology. Aplysia californica weighing 40-250 gm were obtained from Pacific Biomarine (Venice, CA) or Marinus (Long Beach, CA). Animals were anesthetized by injection of isotonic MgCIZ (25-40% body weight) and were dissected in a 50% isotonic MgCl,, 50% artificial seawater solution. When studying neural responses to cutaneous stimulation, we used a modified "split-foot" preparation (Hening et al., 1979). The viscera, buceal mass, opaline gland, and purple gland were excised, and the anterior half of the foot and back were bisected longitudinally, revealing the nervous system and blood vessels. The preparation was then pinned dorsal side up to the wax floor of a lucite recording chamber filled with circulating, aerated artificial seawater. The cut end of the cephalic artery was cannulated and perfused with artificial seawater (in mM: 460 NaCl, 10 KCl, 55 MgCl,, 11 C&l,, 10 HEPES, pH 7. 6 ) at room temperature. The cerebral ganglia were pinned on a Sylgard stage and partially desheathed, and the pleural-abdominal connectives were wrapped around stimulation posts to facilitate identification of neurons. Neurons were impaled with single-barreled glass microelectrodes filled with 2. 5 M KCl for recording and stimulation through a Wheatstone bridge circuit. Cells were examined for their response to cutaneous tactile stimulation using a glass probe or electrical stimulation (75 mA a. c., 1. 0 set duration) using capillary electrodes. The cells were then filled with rhodamine and processed for glyoxylic histofluorescence as described in the preceding paper (Hawkins, 1989). 

When examining facilitation of siphon sensory neuron-motor neuron synapses, we discarded the body except for the tail, which was left connected to the ring ganglia via the posterior pedal nerves (P9). The ring and abdominal ganglia were dipped in 0. 5% glutaraldehyde for 45 set, pinned to the Sylgard floor of the recording chamber, and partially desheathed. The preparation was then perfused with artificial seawater at room temperture. Candidate cerebral cells, siphon sensory neurons (LE cells), and siphon or gill motor neurons (usually LFS or L7) were impaled. 'The motor net& was hyperpolarized 30 mV below. spike threshold and tested for receivina an EPSP from LE cells. EPSPs were considered monosynaptic if the; latency was less than 10 msec and constant. If the PSPs were complex, the amplitude was measured at the first peak or inflection point. 

When examining spike broadening in the siphon sensory cells, we used the isolated ring and abdominal ganglion preparation. Cerebral cells and LE sensory cells were impaled and the perfusion was changed to 100 mM tetraethylammonium chloride (TEA) in artificial seawater. Five-millisecond depolarizing current pulses were injected into the sensory cells causing them to fire single action potentials. The duration of the action potential was measured at the midpoint of its vertical excursion. 

Immunohistochemistty. In some of the electrophysiology experiments, cerebral cells were filled with Lucifer yellow and processed for serotonin immunoreactivity, using a modification of the whole-mount technique of Longley and Longley ( 1986). At the end of the experiment, the cells were repenetrated with electrodes filled with 5% Lucifer yellow in distilled water and filled iontophoretically (500 msec, l-2 nA hyperpolarizing pulses at 1 Hz for 15-30 min). After iontophoresis, the ring ganglia were fixed using 4% paraformaldehyde in 0. 1 M Na phosnhate (DH 7. 4) containina 30% sucrose at 4°C for 24 hr. The tissue was ihen p&meabhzed usingTriton X-100 in PBS (0. 1 M Na phosphate, 0. 17% Na azide, 2% Triton X-100) for 24 hr. Afterward, the tissue was incubated for 24 hr in nolvclonal rabbit anti-5-HT antibodv (Immuno-Nuclear Corporation, Stillwater, WI) diluted 1:200 in T&on X-100 PBS. Next, the tissue was washed in Triton PBS for another 24 hr and then incubated for 24 hr in rhodamine-labeled goat anti-rabbit antiserum (Cappel Laboratories, Cochranville, PA) diluted 1:50 in Triton PBS. After another 24-hr wash in Triton PBS, the tissue was rinsed in 0. 03 M Na phosphate and mounted in glycerol. The slides were viewed on a Leitz epifluorescence microscope, using the N2 and D or I2 filter packs for rhodamine and Lucifer yellow, respectively, and photographed with Kodak Ektachrome 400 film. 

Results Hawkins (1989) found that several cells in the B cluster of each cerebral ganglion show 5-HT histofluorescence and several show retrograde fluorescent labeling from the abdominal ganglion, but only one cell in each ganglion shows both. That cell is therefore a potential serotonergic facilitator for the siphon sensory cells. In a series of experiments before we knew how to identify that cerebral cell electrophysiologically (Mackey et al., 1986), we tested whether any cerebral cells that had axons going to the abdominal ganglion (as evidenced by receiving antidromic action potentials from stimulation of the pleural-abdominal connectives) (1) produced facilitation of the EPSP from a siphon sensory neuron to a motor neuron, and (2) showed 5-HT histofluorescence. In those experiments, cerebral cells were stimulated at a relatively high frequency ( 10 Hz) for 25-50 sec. Ten of thirty-one cerebral B cells with axons in the pleural-abdominal connectives produced facilitation of the sensory neuronmotor neuron EPSP. Only 1 cell in each B cluster produced facilitation; in 2 experiments, such a cell was found in both the left and right clusters of the same animal. At the end of the experiments, some of the cells that produced facilitation were filled with rhodamine and processed for glyoxylic histofluorescence as described in the preceding paper (Hawkins, 1989). Three of three cells that produced facilitation in 3 different preparations showed positive 5-HT histofluorescence. The results of these experiments were consistent with the idea that one of the cells in the B cluster of each cerebral ganglion is a serotonergic facilitator. However, to be more confident of that conclusion, it was necessary to identify that cell electrophysiologically. In this paper, we report on identification of an individual neuron in each B cluster (the CB 1 cells) and address the following questions: (1) Are the CB 1 cells serotonergic? (2) Are they excited by cutaneous stimuli known to produce dishabituation and sensitization? (3) Does intracellular stimulation of CB 1 cells, in a manner similar to their response to cutaneous stimulation, produce facilitation of EPSPs from LE cells to follower cells? (4) Is this effect direct? (5) Is this effect presynaptic? Electrophysiological ident$cation of CBI neurons We routinely found up to 3 cells in each B cluster that received an antidromic action potential from stimulation of either one or both pleural-abdominal connectives. However, we found that only 1 of those cells also receives excitatory synaptic input from the tail or posterior pedal nerve (P9); the other cells are inhibited. That cell, which we have designated CBl, is pale yellow, approximately 75-100 pm in diameter, and is located in the anterior region of the B cluster (Fig. 1 ). It has a low rate of spontaneous firing, receives spontaneous EPSPs and IPSPs, and receives an antidromic action potential from stimulation of either pleural-abdominal connective.
section_name: Methods

section_content: To test whether the CB 1 cells are serotonergic, we injected them with lissamine rhodamine and processed the ganglion for glyoxylic acid histofluorescence as described in the preceding paper (Hawkins, 1989). Figure 2A shows an identified CB 1 cell injected with rhodamine and viewed with rhodamine filters. Figure 2B shows. the same cell viewed with histofluorescence filters, revealing positive 5-HT histofluorescence. As an independent test of whether the CBl cells are serotonergic, we also examined whether those cells show 5-HT immunoreactivity. Cerebral cells were selected as described above and filled with Lucifer yellow. The cells were then processed for wholemount immunohistochemistry using a polyclonal anti-S-HT antibody. Identified CB 1 cells showed positive 5-HT immunoreactivity (Fig. 3A ). These experiments also revealed the axonal branching pattern of CBl: its axon bifurcates and sends one branch to the ipsilateral cerebral-pleural connective, while the other branch crosses the cerebral commissure and enters the contralateral cerebralpleural connective (Fig. 3B ). The positions of the axons in the cerebral-pleural connectives are similar to those of the serotonergic processes ("Sl" and "S2") described by Longley and Longley (1986), who showed that those axons project to the abdominal ganglion and branch extensively in the neuropil (Fig. 4 ). No processes of CB 1 were noted in peripheral cerebral nerves.
section_name: CBl neurons show 5-HT histofluorescence and immunoreactivity

section_content: We used a semiintact preparation as illustrated in Figure 5A to assess the response of cerebral cells to cutaneous stimulation. An example of the response of CB 1 neurons is shown in Figure 5B and C. CBl neurons, which show irregular spiking at rest, respond with a phasic increase in firing to moderate tactile stimulation anywhere on the body surface (Fig. 5B ). A noxious stimulus such as tail shock, however, causes a prolonged increase in firing rate, which in some cases lasts over 15 min (Fig. SC ). This response was observed in CBl cells in several different preparations and could be produced repeatedly in the same preparation. The prolonged response is not due to continued stimulation from pulling against the restraints, since stimulation of the posterior pedal nerve (P9) also produced a similar response

in the isolated nervous system. The cellular basis of this prolonged increase in firing rate has not yet been investigated.
section_name: CBI neurons are excited by cutaneous stimuli that produce dishabituation and sensitization

section_content: We next addressed the question of whether stimulating the CBl cells in a manner similar to their response to tail shock produces reliable facilitation of the EPSP from LE sensory cells to follower cells. CBl neurons were identified as described above. An LE sensory cell and a follower neuron (usually an LFS motor neuron) were then impaled. Follower neurons were hyperpolarized by 30 mV to prevent spiking. The LE cell was stimulated intracellularly with a brief current pulse causing it to 6re an action potential once every 30 sec. After at least 3 pretest stimuli, the CBl cell was fired intracellularly at a rate of l-3 Hz for 180-200 sec. The EPSP from the LE cell to the follower cell was tested for 6 trials during CB 1 stimulation and 3 trials poststimulation. 

An example of the results is shown in Figure 6A. The average results from 10 experiments are plotted in Figure 6B. The amplitude of the EPSP has been normalized to the last pretest (-1). As can be noted, the EPSP decreased in amplitude slightly from pretest -3 to -1. After the start of CB 1 stimulation, the amplitude of the EPSP increased on average to a peak of 142 f 17% of pretest by trial 3, and facilitation was maintained throughout CB 1 stimulation. After the end of CB 1 stimulation, the EPSP declined. At posttest 1, the EPSP was on average 128 f 10% of pretest; by posttest 3, the EPSP was 107 f 8% of pretest. The average amplitude ofthe EPSP during and following CBl stimulation was significantly greater than pretest (F,,* = 45. 6, p < O. Ol), and planned comparisons at the individual time points show significant facilitation on trials 3, 4, and 6 during CBl stimulation and posttest 1 (p < 0. 01 in each case). The average magnitude of facilitation, although modest, is similar to that produced by tail stimulation (Mackey et al., 1987). Although the duration of facilitation is substantially less than that seen after tail shock, CBl was also stimulated for a shorter interval than it fires after tail shock (cf. Fig. 6A and Fig. 5C' ). This suggests that at least part of the time course of facilitation following tail shock may be due to continued firing of the CBl neurons and continued release of 5-HT. 

These results support the idea that the CB 1 cells mediate some of the facilitatory effects of tail shock. Tail shock is still capable of producing facilitation when the cell body of an individual CBl neuron is hyperpolarized to prevent it from liring, suggesting that a single CBl cell does not mediate all of the facilitatory effects of the shock (although this procedure may not prevent firing in distant processes of the cell). We have not yet performed enough to these experiments to know whether there is a quantitative reduction in the facilitation, nor have we hyperpolarized both CBl cells simultaneously. 

In 5 of the 10 experiments illustrated in Figure 6B, EPSP facilitation during and following CB 1 stimulation was tested in an artificial seawater solution with elevated divalent cations (in c CEREBRAL G. ABDOMINAL G. Figure 4. Proposed axonal projections of the CBl neurons, based on retrograde labeling from the abdominal ganglion (Hawkins, 1989), antidromic stimulation by shocking the pleural-abdominal conneetives, intracellular dye tilling (Pig. 3), and S-HT immunofluorescence (Fig. 3 and Langley and Langley, 1986). The diagram is modified from Langley and Langley (1986). mM: 260 NaCl, 10 KCl, 60 CaCl,, 140 MgClJ in order to raise action potential threshold and thereby reduce intemeuron firing. This treatment produced no decrease in the duration or magnitude of facilitation. While these results by no means exclude the possibility that the facilitation observed during CBl stimulation may be via other intemeurons, the results are consistent with the idea that CBl produces facilitation directly. 

The facilitation produced by CBl stimulation could be either pre-or postsynaptic. Possible postsynaptic mechanisms include hyperpolarization of the membrane potential (increasing the driving force) and increased input impedance. LFS cells showed either no change in membrane potential or occasionally a slight depolarization following CBl stimulation. In order to test whether CB 1 stimulation produces changes in LFS input impedance, at the end of 6 out of 10 EPSP hcilitation experiments, the LFS cell was injected with hyperpolarizing pulses lasting 600-800 msec. The current was adjusted to produce a membrane change similar in magnitude to that of the EPSP. Using the same test protocol as in the PSP experiments, hyperpolarizing pulses were injected at 30-set intervals before, during, and
section_name: Stimulation of CBI produces facilitation of EPSPs from LE cells to follower cells

section_content: The results of the above experiments show that postsynaptic changes in membrane potential or input resistance (as measured in the cell body) cannot account for the EPSP facilitation during and following CB 1 stimulation. Although we have not excluded other postsynaptic mechanisms (for example, changes in receptor sensitivity), our results are consistent with the possibility that EPSP facilitation is mediated at least in part presynaptically. Application of 5-HT has previously been shown to cause broadening of the action potential in LE sensory neurons, contributing to presynaptic facilitation. This effect is more dramatic in seawater containing TEA, which blocks a non-5-HT-sensitive K+ current, making the action potential more sensitive to changes in the remaining currents (Klein and Kandel, 1978). We were interested to see whether stimulation of CBl also produces spike broadening in LE cells. Using an isolated ring and abdominal ganglion preparation, we identified CBl neurons as described above. Next, we impaled LE sensory cells and perfused the ganglia with seawater containing 100 mM TEA. Action potentials were produced in the LE cells by 5 msec, depolarizing pulses with a 20-set intertrial interval. After at least 3 pretests, the CBl cell was fired at l-3 Hz for 60-200 sec. LE cells were stimulated for 3-10 trials during CBl stimulation and 3 trials poststimulation. 

An example of the results is shown in Figure 7A, and the average of 10 experiments is plotted in Figure 7B. Spike width has been normalized to the last pretest (-1). On average there was a small decrease in spike width from trial -3 to -1. Following the start of CBl stimulation, the spike width increased to 133 + 19% of pretest by trial 3. After the end of CBl stimulation, the spike width declined and was 117 +-11% of pretest by posttest 3. The average spike width during and following CBl stimulation was significantly greater than pretest (F,,d, = 50. 16, p < 0. 0 l), and planned comparisons at the individual time points show significant facilitation on trial 3 during CBl stimulation and posttests 1 and 2 0, < 0. 01 in each case). These results are similar in time course and magnitude to the results of the PSP facilitation experiments. In addition, the magnitude of spike broadening, while modest, is similar to that seen following tail stimulation (Mackey et al., 1987).
section_name: Stimulation of CBI produces spike broadening in LE sensory neurons

section_name: Discussion

section_content: In summary, we have identified a pair of neurons in the cerebral ganglia (the left and right CBl cells) which show both 5-HT histofluorescence and immunoreactivity. Each CB 1 sends bilateral axons into the pleural-abdominal connectives, which are probably the 5-HT immunoreactive axons "S 1" and "S2" that Longley and Longley (1986) showed project to the abdominal ganglion. The CB 1 cells are excited by cutaneous stimuli known to produce dishabituation and sensitization of the gill-and siphon-withdrawal reflex and facilitation of the synaptic connections of the LE sensory neurons. Intracellular stimulation of the CBl cells at a frequency similar to their response to cutaneous stimuli produces facilitation of the EPSPs from the LE cells to follower cells. The facilitation is not reduced in a high-divalent solution that raises the firing threshold of interneurons, suggesting the effect is direct. The EPSP facilitation cannot be explained. by postsynaptic changes in membrane potential or conductance. Rather, intracellular stimulation of CBl produces broadening of the sensory neuron action potential in TEA, suggesting that the facilitation is at least in part presynaptic. 

Although the evidence that the CBl neurons are serotonergic is strong, it is not complete. 5-HT mimics the physiological effects of CB 1, and 2 independent histochemical techniques have shown that CBl contains 5-HT. However, it would be desirable to use biochemical techniques to test whether CBl also synthesizes and releases 5-HT. 

A second issue is whether the facilitatory effects of CBl are direct. Although suggestive, experiments conducted in high divalents by no means exclude the possibility that at least part of the effect may be mediated by interposed interneurons. Preliminary evidence indicates that CBl has no synaptic connections with the abdominal facilitatory neuron L29. We have not tested whether there are coMections with the facilitatory neurons L22 or L28. It will also be important to test whether at least some of the serotonergic varicosities observed in the region of the LE sensory neurons are contributed by CB 1. The facilitation produced by stimulating a single CBl, although reliable, is modest. We have not yet tested whether simultaneously stimulating both CB 1 neurons enhances the facilitatory effect. We doubt, however, that the 2 CBl cells can account for most of the facilitation produced by, tail shock. First, tail or nerve stimulation recruits not only CBl but also the abdominal interneurons L22, L28, and L29 (Hawkins et al., 198 1; Hawkins and Schacher, 1989). Each of these neurons has been shown to produce facilitation comparable to that produced by CBl (Hawkins et al., 198 1). Second, in experiments in which a single CBl has been hyperpolarized to prevent spiking, tail stimulation is still capable of producing facilitation. Further experiments will be needed to determine whether there are quantitative differences in either the magnitude or time course of facilitation under conditions in which CBl is prevented from spiking. Third, since cutaneous stimuli such as tail shock recruit both facilitation and inhibition of the siphon sensory neurons (Mackey et al., 1987), the actual facilitation elicited by tail shock is probably greater than the net facilitation observed. Therefore, comparing the facilitation produced by firing CBl to the net facilitation measured following tail shock probably overestimates the contribution of the CBl cells to total facilitation. For these reasons, we think that there may be additional facilitator neurons for the gill-and siphon-withdrawal reflex, some of which may be serotonergic (Glanzman et al., 1989). If so, it will be interesting to see whether their properties are similar to or different from those of CB 1. 

Comparison with other identified facilitator neurons Hawkins et al. (198 1) reported the identification of 3 groups of neurons on the ventral surface of the left abdominal ganglion which produce heterosynaptic facilitation of complex PSPs in the motor neuron L7: L22 (a cluster of about 3 cells), L28 (a single cell), and L29 (a cluster of about 5 weakly electrically coupled cells). All 3 types of cells are excitatory interneurons in the circuit for the gill-and siphon-withdrawal reflex. The L29 cells have been the most thoroughly studied. Brief, high-frequency intracellular stimulation of an L29 cell produces facilitation of monosynaptic EPSPs from siphon sensory cells to follower cells and broadening of action potentials in sensory cells in TEA solution (Hawkins et al., 1981; Hawkins, 1981). 

The facilitation produced by L22 and L28 has not been analyzed and may involve other mechanisms, such as PTP of the L22-L7 connection (Hawkins et al., 198 1). Although indirect evidence initially suggested that the L29 cells might be serotonergic (Bailey et al., 198 1, 1983), no serotonergic neurons have been seen on the left ventral surface of the abdominal ganglion with either histofluorescence or immunocytochemistry (Tritt et al., 1983; Goldstein et al., 1984; Ono and McCaman, 1984; Longley and Longley, 1986; Salimova et al., 1987). Moreover, identified L29 cells marked with fluorescent dye do not show 5-HT histofluorescence or immunofluorescence (Kistler et al., 1985; Hawkins, 1989). 

It is interesting to speculate why A&& has several different populations of facilitator neurons for the gill-and siphon-withdrawal reflex. A comparison of L29 and CBl may provide insights into different roles subserved by these interneurons. L29 and CBl show significant differences in several characteristics including (1) receptive field, (2) response to graded stimuli, and

(3) time course of facilitation. In terms of receptive field, L29 shows some degree of site specificity. It receives excitatory input from the posterior part of the body, but it is inhibited by stimuli directed to the anterior part of the body (Hawkins and Schacher, 1989). CBl, by contrast, is excited by tactile stimulation over all areas of the body including the tail, siphon, mantle, neck, rhinophores, and anterior tentacles. Stimulation of any of these areas can produce dishabituation and sensitization of the gilland siphon-withdrawal reflex (Pinsker et al., 1970; Carew et al., 1971 Carew et al.,, 1981;; Mackey et al., 1988; Marcus et al., 1988; V. F. Castellucci and R. D. Hawkins, unpublished observations). L29 thus appears to play a more local role in the gill-and siphonwithdrawal reflex, whereas CBl may have a more general role. For example, Hawkins et al. (198 1) have suggested that 1 function of L29 may be to modulate the time course of habituation of the withdrawal reflex to different intensity stimuli. The fact that L29 is an excitatory interneuron in the withdrawal circuit, with direct and indirect connections to motor neurons, may also explain why it has a limited receptive field. CB 1, on the other hand, does not appear to be an excitatory intemeuron and does not show site specificity. 

L29 and CBl also differ in terms of their response to graded tactile stimuli. L29 neurons are sensitive to light tactile stimuli, responding with a high-frequency burst of spikes. Noxious stimuli prolong the spiking to the duration of the stimuli, but not beyond (Hawkins and Schacher, 1989). CBl neurons respond to moderate tactile stimuli with a brief phasic response and respond to noxious stimuli with a tonic increase in firing lasting many minutes. These differences in response characteristics may be related to the differences in time course of facilitation produced by these neurons. Brief intracellular stimulation of L29 produces facilitation that peaks within 10 set and subsequently declines slowly with a time course parallel to the decrement seen prior to stimulation (Hawkins et al., 198 1). The facilitation produced by prolonged CB 1 stimulation peaks after 90 set and remains elevated for the duration of CBl firing. Thereafter, it returns to near baseline within 1 min. These results suggest that L29 and CBl might play different roles in dishabituation and sensitization ofthe withdrawal reflex. Marcus et al. (1988) showed that dishabituation can be produced by relatively weak stimulation and has a rapid onset, whereas sensitization requires stronger stimulation and may have a delayed onset (this distinction between dishabituation and sensitization is also consistent with the results of Pinsker et al., 1970; Carew et al., 197 1, 1981; Mackey et al., 1988; Castellucci and Hawkins, unpublished observations). The characteristics of L29 (low threshold and rapid facilitation) suggest that it may be preferentially involved in dishabituation, whereas the characteristics of CBl (higher threshold and slower facilitation) suggest that it may be preferentially involved in sensitization. However, there is considerable overlap, and both neurons could contribute to both effects.
section_name: Identified serotonergic facilitator neurons

section_content: There are now several identified serotonergic neurons in Aplysia, permitting a comparison of serotonergic function in different systems within the same organism. The MCCs modulate the feeding circuit at several sites, increasing the rhythmic activity of the feeding pattern generator, depolarizing buccal motor neurons, and enhancing buccal muscle contractions (Weiss et al., 1982). RB,, accelerates heart rate and thereby influences circulation (Mayeri et al., 1974; Liebeswar et al., 1975). CBl produces facilitation of LE sensory cells involved in defensive withdrawal. In all 3 cases the cellular responses are characterized by slow and predominantly modulatory effects which are mediated, at least in part, by CAMP (Brunelli et al., 1976; Mandelbaum et al., 1979; Bemier et al., 1982; Weiss et al., 1982). 

We have not yet examined whether CBl, like MCC, may have modulatory effects at multiple sites within the siphonwithdrawal circuit. Our data show no evidence of conventional synaptic input to either L7 or LFS motor neurons. It will be interesting to test whether firing CBl mimics the effects of 5-HT on neuronal elements in the circuit in other ways, such as inhibiting the L30 inhibitory interneurons or increasing the spontaneous firing rate of the LFS siphon motor neurons (Frost et al., 1988). We have also not yet tested whether CBl's role is limited to facilitation of the EPSP from siphon sensory neurons to gill and siphon motor neurons or generalizes to sensory neurons in other response systems. Preliminary evidence suggests that CBl has little or no effect on pleural sensory neurons that mediate the tail withdrawal reflex. Possible modulatory effects on cerebral sensory neurons have not yet been examined. Since some of the cerebral sensory neurons respond to 5-HT and nerve stimulation with spike broadening, and others respond with spike narrowing (Rosen et al., 1989), it will be interesting to see whether firing CB 1 can mimic either of these effects. The identification of the CB 1 neurons should allow us to begin to answer some of these questions and hopefully thereby to obtain new insights into the organization of serotonergic systems in Aplysia.
section_name: Serotonergic systems in Aplysia

section_content: This work was supported by grants from the National Institute of Health ( MH262 12 ) and the Howard Hughes Medical Institute. We thank D. Glanzman, L. Eliot, and I. Kupfermann for their comments, K. Hilten and L. Katz for preparing the figures, and H. Ayers and A. Krawetz for typing the manuscript.