Serotonin mimics tail shock in producing transient inhibition in the siphon withdrawal reflex of Aplysia

Tail shock-induced modulation of the siphon withdrawal reflex of Aplysia has recently been shown to have a transient inhibitory component, as well as a facilitatory component. This transient behavioral inhibition is also seen in a reduced preparation in which a cellular reflection of the inhibitory process, tail shock-induced inhibition of complex EPSPs in siphon motor neurons, is observed. The biogenic amine serotonin (5-HT) is known to play a role in the facilitatory aspects of sensitization in Aplysia. The aim of this article was to examine whether 5-HT might also contribute to the inhibitory effects of tail shock in the siphon withdrawal reflex. To examine this question, we carried out two kinds of experiments. First, in the isolated abdominal ganglion, we recorded intracellularly from siphon motor neurons and examined the effects of 5-HT on (1) complex (polysynaptic) EPSPs, produced by siphon nerve stimulation, and, simultaneously, (2) monosynaptic EPSPs from siphon sensory neurons. We found that, paralleling the effects of tail shock in the reduced preparation, 5-HT produced transient inhibition of the complex EPSP; the monosynaptic EPSP was facilitated by 5-HT. Second, we examined the behavioral effects of 5-HT on siphon withdrawal in a reduced preparation. We found that 5-HT again paralleled tail shock by producing transient inhibition of the siphon withdrawal reflex. Our results suggest that, in addition to its well-established facilitatory role in reflex modulation in Aplysia, 5-HT might play an important inhibitory role, as well.     

Depletion of serotonin in the nervous system of Aplysia reduces the behavioral enhancement of gill withdrawal as well as the heterosynaptic facilitation produced by tail shock

Noxious stimuli, such as electrical shocks to the animal's tail, enhance Aplysia's gill- and siphon-withdrawal reflex. Previous experimental work has indicated that this behavioral enhancement, known as dishabituation (if the reflex has been habituated) or sensitization (if it has not been habituated), might be mediated, at least in part, by the endogenous monoaminergic transmitter serotonin (5-HT). To assess 5-HT's role in dishabituation and sensitization of Aplysia withdrawal reflex, we treated Aplysia with the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). We found that 5,7-DHT treatment significantly reduced the dishabituation of the withdrawal reflex produced by tail shock. Treatment with the neurotoxin also blocked the heterosynaptic facilitation of monosynaptic connections between siphon sensory neurons and their follower cells, which contributes to the behavioral enhancement. Analysis by high-performance liquid chromatography indicated that 5,7-DHT treatment significantly reduced 5-HT levels in the Aplysia CNS. Moreover, the neurotoxic effects of 5,7-DHT appeared to be relatively specific for serotonergic pathways. Thus, 5,7-DHT treatment did not disrupt the ability of nonserotonergic facilitatory interneurons, the L29 cells, to facilitate the connections of siphon sensory neurons. Also, 5,7-DHT reduced 5-HT-dependent, but not dopamine-dependent, histofluorescence in Aplysia central ganglia. Finally, 5,7-DHT does not reduce the levels of the facilitatory peptides SCPA and SCPB within the Aplysia CNS. Our results, together with those of Mackey et al. (1989), indicate that 5-HT plays a major role in mediating dishabituation and sensitization of Aplysia's withdrawal reflex.     

A cellular analysis of inhibition in the siphon withdrawal reflex of Aplysia

Recent behavioral experiments examining the siphon withdrawal reflex of Aplysia have revealed inhibitory effects of strong tail shock, a stimulus commonly used as an unconditioned stimulus in studies of associative and nonassociative learning in Aplysia. We utilized a reduced preparation to perform a cellular analysis of tail shock-induced inhibition in the siphon withdrawal reflex. First, we carried out behavioral studies that showed that the reduced preparation exhibits a siphon withdrawal reflex to water jet stimuli, and that tail shock produces inhibitory behavioral effects comparable to those in the intact animal: (1) strong shock produces transient inhibition of nonhabituated responses, and (2) a habituated response is facilitated by weak shock, but not by strong shock, suggesting that increasing tail shock intensity recruits the inhibitory process that competes with facilitation of habituated reflexes. Next, we carried out cellular studies that showed that the amplitude of the complex EPSP in siphon motor neurons elicited by water jet stimuli to the siphon also exhibits the inhibitory patterns produced by tail shock: (1) the nondecremented complex EPSP (a neural correlate of a nonhabituated siphon withdrawal reflex) is significantly inhibited 90 sec after strong tail shock and recovers to preshock levels 10 min later, and (2) the decremented complex EPSP (a neural correlate of a habituated reflex) is significantly facilitated by weak shock, but is not facilitated by strong shock. In addition to the complex EPSP, we simultaneously examined the monosynaptic connection between siphon sensory neurons and siphon motor neurons. The monosynaptic EPSP does not show the pattern of inhibitory modulation by tail shock exhibited by the siphon withdrawal reflex and the complex EPSP: (1) the nondecremented monosynaptic EPSP is not inhibited 90 sec after strong shock, but tends to be above preshock levels; and (2) the decremented monosynaptic EPSP is facilitated by weak as well as strong tail shock. Our results suggest that an important component of the inhibitory process triggered by strong tail shock is mediated by neural elements presynaptic to the siphon motor neurons. Because modulation of the monosynaptic connection between identified siphon sensory and siphon motor neurons does not parallel the tail shock-induced inhibitory patterns observed in the siphon withdrawal reflex and in the complex EPSP, other synaptic connections are likely to play an important role in mediating tail shock-induced inhibition in the siphon withdrawal reflex.     

The use of elevated divalent cation solutions to isolate monosynaptic components of sensorimotor connections in Aplysia

A commonly used method to remove polysynaptic components of test PSPs is to elevate action potential threshold of interneurons with high extracellular concentrations of divalent cations ('Hi-Di'). Extrapolation to normal conditions requires that Hi-Di have negligible effects on synaptic transmission. We examined effects of Hi-Di on EPSPs from sensory neurons (SNs) onto motor neurons (MNs) of Aplysia in the pleural-pedal and abdominal ganglia, and in dissociated cell culture. In ganglia, standard Hi-Di solutions eliminated spontaneous input from interneurons as well as polysynaptic components of PSPs evoked by single action potentials in single SNs, but failed to block polysynaptic PSPs evoked by nerve stimulation. Hi-Di solutions had no effect on activity-dependent synaptic depression or posttetanic potentiation, or facilitation by serotonin (5-HT). Unexpectedly, standard Hi-Di solutions substantially reduced sensorimotor EPSPs in all preparations, whereas a solution containing 2.2x[Ca(2+)] and 2x[Mg(2+)] blocked the polysynaptic component of EPSPs without obvious changes to the monosynaptic component. In contrast to previous observations in Aplysia, and to predictions of the (J. Physiol. 193 (1967) 419) model, tripling the normal extracellular concentrations of Ca(2+) and Mg(2+) failed to increase sensorimotor EPSPs. Depression of EPSPs by these Hi-Di solutions may result from reduced spike invasion into presynaptic terminals.     

Modulation of calcium current and diverse K+ currents in identified Hermissenda neurons by small cardioactive peptide B

The molluscan neuropeptides, small cardioactive peptides A and B (SCPA,B), are known to modulate the responses of many molluscan central and peripheral target cells (see review by Lloyd, 1986), but their full range of physiological actions remains unknown. External application of SCPB (1-10 microM) modified diverse ionic conductances in a set of giant identifiable neurons in the brain of the marine mollusk Hermissenda crassicornis. SCPB caused a transient depolarization and increased input resistance that enhanced or promoted cell firing. Under voltage-clamp, SCPB reduced a "background" residual current (IR), reduced early transient K+ current (IA), reduced a delayed K+ current (IK(V], and enhanced ICa, IBa, and a Ca2+-activated K+ current, IK(Ca). In tetraethylammonium chloride (TEA) saline, SCPB enhanced the amplitude and duration and reduced the threshold of evoked Ca and Ba spikes. Immunocytochemical staining techniques have localized an endogenous SCPB-like peptide in numerous somata and their neurites in the nervous system of Hermissenda (Longley and Longley, 1985; Watson and Willows, 1986). These data are consistent with a role for SCPB as a neurotransmitter/neurohormone modulator of neuronal excitability in Hermissenda. A neurotransmitter role for endogenous SCPs has been proposed for a synaptic pair of cultured neurons in the Aplysia buccal ganglion (Lloyd et al., 1986). SCPB has been implicated in the control of feeding motor output in Aplysia (Sossin et al., 1986) and Tritonia (Willows and Watson, 1986), and in the presynaptic facilitation of sensory neurons mediating the gill and siphon defensive withdrawal reflex in Aplysia (Abrams et al., 1984).     

Identified facilitator neurons L29 and L28 are excited by cutaneous stimuli used in dishabituation, sensitization, and classical conditioning of Aplysia

Tactile or electrical stimulation of the skin can be used to produce dishabituation, sensitization, and classical conditioning of the gill- and siphon-withdrawal reflex in Aplysia. These behavioral effects are thought to involve presynaptic facilitation at the synapses from siphon sensory neurons to gill and siphon motor neurons. Facilitation of PSPs onto the motor neurons can also be produced by intracellular stimulation of single identified neurons in the abdominal ganglion, including L29 and L28. In this paper, we further characterize L29 and L28. First, we show that they are excited by cutaneous stimuli similar to those used to produce dishabituation, sensitization, and classical conditioning and may therefore participate in mediating those behavioral effects. The results are also consistent with a possible role of L29 and L28 in higher-order features of conditioning. Second, we show that 5-HT does not mimic some of the PSPs of L29, in agreement with previous evidence that L29 is not serotonergic. Third, we present 2 types of evidence that L29 acts directly to produce facilitation of the sensory cells: (1) L29 comes into close contact with sensory cells in fluorescent double-labeling experiments, and (2) L29 produces facilitation of sensory cells in dissociated cell culture. Together with the results of the preceding paper (Mackey et al., 1989), these results indicate that facilitation of sensory cell synapses contributing to behavioral enhancement of the reflex can be produced by identified neurons that use 2 different transmitters: 5-HT (the transmitter of CB1) and the unknown transmitter of L29.     

The contribution of facilitation of monosynaptic PSPs to dishabituation and sensitization of the Aplysia siphon withdrawal reflex.

To examine the relationship between synaptic plasticity and learning and memory as directly as possible, we have developed a new simplified preparation for studying the siphon-withdrawal reflex of Aplysia in which it is relatively easy to record synaptic connections between individual identified neurons during simple forms of learning. We estimated that monosynaptic EPSPs from LE siphon sensory neurons to LFS siphon motor neurons mediate approximately one-third of the reflex response measured in this preparation, which corresponds to siphon flaring in the intact animal. To investigate cellular mechanisms contributing to dishabituation and sensitization, we recorded evoked firing of LFS neurons, the siphon withdrawal produced by stimulation of an LFS neuron, the complex PSP in an LFS neuron, and the monosynaptic PSP from an "on-field" or "off-field" LE neuron to an LFS neuron during behavioral training. Unlike the simplified gill-withdrawal preparation (Cohen et al., 1997; Frost et al., 1997), in the siphon-withdrawal preparation we found no qualitative differences between the major cellular mechanisms contributing to dishabituation and sensitization, suggesting that dissociations that have been observed previously may be attributable to transient inhibition that does not occur for this component of the reflex. Furthermore, in the siphon-withdrawal preparation, all of the various cellular measures, including monosynaptic PSPs from either on-field or off-field LE neurons, changed approximately in parallel with changes in the behavior. These results provide the most direct evidence so far available that both dishabituation and sensitization involve multiple mechanisms, including heterosynaptic facilitation of sensory neuron-motor neuron PSPs.     

Respiratory pumping: Neuronal control of a centrally commanded behavior in aplysia

Respiratory pumping in Aplysia californica is a relatively stereotyped behavioral pattern with three components: (1) withdrawal of gill, siphon and mantle shelf; (2) closing of parapodia; (3) heart inhibition accompanied by a decrease in vasomotor tone. This phasic behavior is triggered by a central burst-generating network of interneurons in the abdominal ganglion. During respiratory pumping, motor neurons innervating the several effector organs receive a burst of either excitatory or inhibitory synaptic input which has previously been attributed to an unidentified central command cell called Interneuron II. Several of these motor cells are also concomitantly release from tonic synaptic input, which is opposite in sign to that which they receive from Interneuron II. This tonic input has been attributed to an unidentified cell called Interneuron XI. In this paper we identify and describe some of the neurons which contribute to the burst generating network; specifically, we focus on the neurons that produce the synaptic action attributed to Interneurons II and XI. The synaptic actions attributed to Interneuron XI are produced by a single, spontaneously active neuron, cell L24. This cell is a multi-action interneuron: it produces inhibitory synaptic potentials in some follower motor neurons, excitatory synaptic potentials in other follower cells, and a conjoint excitatory-inhibitory synaptic action onto gill motor neuron L7. At low frequency, L24 is excitatory to L7. With high frequency firing of L24, the synaptic potential produced in L7 converts from excitatory to inhibitory. In contrast to Interneuron XI, which is a single cell, the synaptic potentials previously attributed to Interneuron II are actually produced by a cluster of at least 3 respiratory command cells which we call L25, L26 and L27. Each of these cells accounts for only a limited portion of the synaptic input that drives the motor neurons during respiratory pumping. For most motor neurons innervated by both the respiratory command cells and Interneuron XI, the two synaptic inputs are opposite in sign. Mutually inhibitory connections between Interneuron XI and some of the central respiratory command cells ensure that the synaptic potentials from these two sources are constrained to occur at different times. Thus, centrally commanded synaptic inhibition or excitation of these motor neurons is made more effective by simultaneous disexcitation or disinhibition of Interneuron XI input. In addition to their role in generating respiratory pumping, L24 and L26 also contribute to the mediation of the defensive gill and siphon withdrawal reflex.     

Inhibition of lateral vestibular nucleus neurons by 5-hydroxytryptamine derived from the dorsal raphe nucleus

Electrophysiological studies were performed to elucidate the effect of 5-hydroxytryptamine (5-HT) originating in the dorsal raphe nucleus (DR) on neuronal activity in the lateral vestibular nucleus (LVN) neurons, using cats anesthetized with alpha-chloralose. LVN neurons were classified into monosynaptic and polysynaptic neurons according to their responses to vestibular nerve stimulation. Conditioning stimuli applied to the DR inhibited orthodromic spikes elicited by vestibular nerve stimulation predominantly in polysynaptic neurons of the LVN. The iontophoretic application of 5-HT also inhibited orthodromic spikes of the LVN neurons. A close correlation was observed between the effects of DR conditioning stimulation and iontophoretically applied 5-HT in the same neurons. These inhibitions with both treatments were antagonized during the application of methysergide, a 5-HT antagonist. In the majority of LVN polysynaptic neurons that responded to antidromic stimulation of the ipsilateral or contralateral abducens nucleus, orthodromic spikes elicited by vestibular nerve stimulation were inhibited by DR conditioning stimulation and the iontophoretic application of 5-HT. In contrast, LVN neurons that responded to antidromic stimulation of the vestibulospinal tract were rarely affected by these treatments. These results indicate that 5-HT derived from the DR inhibits the synaptic transmission of LVN polysynaptic neurons ascending to the abducens nucleus, and suggest that 5-HT derived from the DR is involved in the regulation of the vestibulo-ocular reflex.     

Pathway of the blink reflex in the brainstem of the cat: Interneurons between the trigeminal nuclei and the facial nucleus

Blink reflex responses evoked by electrical stimulation of the supraorbital nerve were examined using cats and the pathway of the blink reflex in the brainstem was elucidated. Both early response (ER) and late response (LR) were mediated by the main sensory trigeminal nucleus and the spinal trigeminal nucleus. However, a lesion of the main sensory trigeminal nucleus had less effect on the blink reflex than a lesion of the spinal trigeminal nucleus. The ER was mediated not only by the shorter disynaptic pathway of 3 neurons through the trigeminal nerve, the trigeminal nuclei and the facial nucleus but also by a polysynaptic pathway of 4 neurons. The interneurons were located between the trigeminal nuclei and the facial nucleus. Some of these interneurons participated in the production of both ER and LR. The area of the brainstem responsible for ER and LR of the blink reflex was the reticular formation from the rostral part of the medulla to the pons except the medial area around the median sulcus. The LR interneurons were distributed more widely than the ER interneurons.     

Participation of AMPA- and NMDA-type excitatory amino acid receptors in the spinal reflex transmission,in rat

Classical in vitro and in vivo models and electrophysiological techniques were used to investigate the role of AMPA- and NMDA-type glutamate receptors in various components of spinal segmental reflex potentials. In the rat hemisected spinal cord preparation, the AMPA antagonists NBQX and GYKI 52466 abolished the monosynaptic reflex (MSR) potential but caused only partial inhibition of the motoneuronal population EPSP. NMDA antagonists had no noticeable effect on the MSR in normal medium, but markedly depressed the late part of EPSP. However, an NMDA receptor antagonist sensitive monosynaptic response was recorded in magnesium-free medium at complete blockade of the AMPA receptors. In spinalized rats, the AMPA antagonists completely blocked all components of the dorsal root stimulation evoked potential. MK-801 (2mg/kg, i.v.) reduced monosynaptic responses in a frequency dependent way, with no effect at 0.03 Hz and 22% inhibition at 0.25 Hz. The reduction of the di- and polysynaptic reflex components was about 30% and did not depend on stimulation frequency. Long-latency reflex discharge responses, especially when evoked by train stimulation, were more sensitive to MK-801 than the polysynaptic reflex.These results suggest that glutamate activates MSR pathways through AMPA receptors. However, under certain conditions, NMDA receptors can modulate this transmission through plastic changes in the underlying neuronal circuits. AMPA and NMDA receptors play comparable roles in the mediation of longer latency reflex components.     

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

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.     

The Synapse Between LE Sensory Neurons and Gill Motoneurons Makes Only a Small Contribution to the Aplysia Gill-withdrawal Reflex

The monosynaptic connection between the mechano-sensory neurons in the LE cluster and gill motoneurons has been extensively studied and used as a model for the gill-withdrawal reflex and its behavioural plasticity. In an attempt to evaluate the contribution of this synapse to the behaviour, we used voltage-sensitive dye recording to determine the number of activated LE neurons and the number of spikes made by each neuron in response to a light touch. In five preparations, light touch activated a median of five sensory cells with a median of 1.6 spikes per cell. From a comparison of the sizes of the motoneuron synaptic potentials elicited by LE spikes and elicited by a light siphon touch, we estimate that the LE sensory neurons contribute ˜5% of the motoneuron synaptic potential in response to this touch. This result casts doubt on the validity of using this synaptic connection as a model for gill-withdrawal behaviour. Siphon nerve recordings reveal the existence of short-latency, low-threshold neurons that may provide much of the sensory input in response to a light touch.     

Segmental reflex pathways in spinal shock and spinal spasticity in man

Activity in three segmental pathways was compared in normal subjects, patients with spinal shock, and patients with established spinal spasticity. The Achilles tendon reflex (ATR) was used to estimate transmission in the Ia monosynaptic pathway. Evidence is produced implying that vibration activates motoneurones principally through a polysynaptic pathway. The tonic vibration reflex (TVR) was used to estimate transmission in this Ia polysynaptic pathway. The percentage of the motoneurone pool (M-response) that could be activated by these pathways was used as a measure of transmission. The H reflex (vibration)/H reflex (control) ratio was used as an estimate of the degree of presynaptic inhibition of the Ia monosynaptic pathway. The findings led to the following conclusions. (1) In spinal shock presynaptic inhibition is greater than normal, transmission in the Ia monosynaptic pathway is reduced, and in the Ia polysynaptic pathway virtually abolished. (2) In established spasticity presynaptic inhibition is impaired, transmission in the Ia monosynaptic pathway is increased, but transmission in the Ia polysynaptic pathway never recovers. (3) The failure of presynaptic inhibition associated with spasticity is a gradual process. A hypothesis to explain these findings is proposed.     

Synaptic interactions of identified nerve cells in the spinal cord of the sea lamprey

As part of a continuing study on the organization of the lamprey nervous system, two additional groups of interneurons have been identified by physiological and morphological criteria in the isolated spinal cord of Petromyzon marinus. These and other identified nerve cells were tested for synaptic interactions using separate intracellular microelectrodes for stimulation and recording.

• 1 Edge cells were identified by their unusual location in the lateral fiber tracts of the spinal cord. Their axons extended rostrally either on the ipsilateral or contralateral side of the cord.
• 2 Some edge cells showed polysynaptic EPSP's and IPSP's after stimulation of sensory dorsal cells, but interactions with other identified neurons were rare. A single cell was excited by a giant interneuron. One edge cell produced IPSP's in a contralateral edge cell, and another produced IPSP's in lateral cells on the opposite side of the spinal cord. Thus, some edge cells have an inhibitory function.
• 3 Lateral cells were distinguished from giant interneurons and edge cells by their cell bodies in the lateral grey of the spinal cord in the gill and trunk regions, their ipsilateral dendrites, and their long ipsilateral axons extending as far as the tail.
• 4 Lateral cells were excited and inhibited polysynaptically by sensory dorsal cells and, in turn, produced weak IPSP's in unidentified neurons. Stimulation of lateral cells produced neither visible movements peripherally nor synaptic potentials in other lateral cells, in giant interneurons, or in edge cells.
• 5 Giant interneurons were previously identified on the basis of their cell bodies in the caudal half of the spinal cord, their bilateral dendrites, and their long contralateral axons extending towards the brain. Giant interneurons exhibited unitary composite EPSP's when more caudal giant interneurons were stimulated. The two components of the EPSP were due to electrical and chemical transmission. Under the electron microscope a contact between a dendrite of one giant interneuron and the probable axon of another had separate junctions resembling chemical and electrical synapses.
• 6 Intracellular stimulation of sensory dorsal cells produced both monosynaptic and polysynaptic EPSP's in giant interneurons. Some dorsal cells produced unitary composite EPSP's Giant interneurons are part of a convergent, multispecific sensory system extending towards the brain.

     

SCP-containing R20 neurons modulate respiratory pumping in Aplysia

Respiratory pumping in Aplysia consists of transient, synchronous pumping actions of the gill, siphon, mantle shelf, and parapodia. This behavior has previously been shown to be driven by a network of coupled interneurons in the abdominal ganglion, the R25 and the L25 cells. We describe here a pair of electrically coupled cells, the R20 cells, which when active can initiate respiratory pumping or increase its spontaneous rate of occurrence. This action is mediated by a slow, long-lasting excitation of the endogenous burst mechanism of the cells in the R25/L25 network. The R20 cells, which are located in the abdominal ganglion, also make slow inhibitory connections to the RB cells and to the RG cells in that ganglion, and to the gill motoneurons in the branchial ganglion. The R20 cells are immunoreactive to SCPB, a molluscan neuropeptide. Biochemical purification studies demonstrate that each of the R20 cells synthesizes not only SCPB, but also SCPA, a closely related molecule known to be encoded by the same gene as SCPB. The R20 cells also synthesize in abundance several other low-molecular-weight, methionine-containing peptides. The excitatory actions of the R20 cells on the R25/L25 network are mimicked by SCPA and SCPB. However, the inhibitory actions of the R20 cells on the RB cells, the RG cells, and on the cells of the branchial ganglion are not mimicked by the SCPs. Thus, the data support the hypothesis that the R20 cells release SCPA and SCPB and at least one other unidentified transmitter.     

Localization of potential serotonergic facilitator neurons in Aplysia by glyoxylic acid histofluorescence combined with retrograde fluorescent labeling

A variety of evidence suggests that 5-HT participates 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) have shown that the 5-HT neurotoxin 5,7-DHT markedly reduces both the synaptic facilitation and behavioral dishabituation produced by tail shock. To provide more direct evidence for a role of 5-HT, I have used histological techniques to try to locate individual serotonergic facilitator neurons. I first used a modification of the glyoxylic acid histofluorescence technique to map serotonergic and dopaminergic neurons in the CNS of Aplysia. Intracellular fluorescent labeling combined with histofluorescence indicates that the previously identified L29 facilitator neurons are not serotonergic. Nerve transection experiments suggest that most of the perisomatic 5-HT histofluorescence in the abdominal ganglion (the location of the siphon sensory cells) comes from neurons whose cell bodies are located in the pedal or cerebral ganglia. As there are at least 500 serotonergic neurons in those ganglia, I combined retrograde fluorescent labeling with histofluorescence to identify a small subset of those neurons which send processes to the abdominal ganglion and are therefore potential serotonergic facilitators. In the following paper, Mackey et al. (1989) show that stimulation of 2 of those neurons in the cerebral ganglia (the CB1 cells) produces presynaptic facilitation of the siphon sensory cells contributing to dishabituation and sensitization of the withdrawal reflex.     

Heterogeneous synaptic inputs from the ventrolateral periaqueductal gray matter to neurons responding to somatosensory stimuli in the rostral ventromedial medulla of rats

The ventrolateral cell column of the midbrain periaqueductal gray matter (vl-PAG) plays a major role in the attenuation of pain behaviour. It is established that this effect is exerted via modulation of neuronal activities in the rostral ventromedial medulla (RVM). Until recently it has been generally accepted that the vl-PAG exerts its modulatory effects upon RVM neurons through a direct monosynaptic pathway. However, recent data suggest that an additional indirect, di- or polysynaptic pathway may also exist. Using in vivo intracellular recordings we tested this hypothesis, by studying synaptic responses of somatosensory receptive RVM neurons evoked by electric stimulation of the vl-PAG in rats. RVM neurons were regarded as somatosensory receptive if they responded to electrical stimulation of the sciatic nerve. Most of the recorded RVM cells were excited by vl-PAG stimulation. Some of them responded with a short onset latency (3.6+/-0.9 ms) corresponding to monosynaptic excitation. All of these neurons were also excited by sciatic nerve stimulation at nociceptive intensities. In contrast to this, another proportion of the recorded RVM neurons responded with a four times longer (14.8+/-3 ms) onset latency to the vl-PAG stimulation, corresponding to polysynaptic modulation. All of these neurons were inhibited by sciatic nerve stimulation. The findings show that RVM neurons receive heterogeneous monosynaptic and polysynaptic inputs from the vl-PAG. The results also suggest that the monosynaptic and polysynaptic pathways modulate the activity of functionally distinct groups of RVM neurons.     

Afferents to the trigeminal and facial motor nuclei in pigeon (Columba livia L.): Central connections of jaw motoneurons

Trigeminal and facial motor nuclei innervating the pigeon's jaw muscles were identified using a combination of microstimulation and EMG recording and HRP injections were made iontophoretically. The trigeminal motor nucleus receives an ipsilateral projection from sensory neurons in the trigeminal mesencephalic nucleus which forms the afferent limb of the monosynaptic stretch reflex of the jaw-closers. Both the trigeminal and facial motor nuclei receive bilateral projections from interneurons in the intertrigeminal area and the lateral (parvocellular) reticular formation of the pons and medulla. These neurons serve as premotor elements in the control of jaw movements, mediating ascending, descending and internuclear connections. The similarity of inputs to the trigeminal and facial nuclei may reflect their common function as jaw motoneurons in this species.     

Serotonin produces long-term changes in the excitability of Aplysia sensory neurons in culture that depend on new protein synthesis

When isolated and grown in cell culture, the sensory and motor neurons of the gill withdrawal reflex of Aplysia readily form synaptic connections. Repeated exposures to 5-HT cause facilitation of the synaptic connections between co-cultured sensory and motor neurons lasting at least 24 hr. As a first step toward understanding the locus and the mechanisms underlying this long-term synaptic facilitation, we have examined the membrane excitability of the isolated presynaptic sensory neurons grown alone in dissociated cell culture. Four repeated applications of 1 microM 5-HT caused a significant increase in the excitability of sensory neurons, lasting at least 24 hr. This resembles the short-term changes in excitability seen in response to a single application of 5-HT. Unlike the short-term effect, this long-lasting change was blocked by exposure of the cells during the 5-HT treatment to 10 microM anisomycin, an inhibitor of protein synthesis. Thus, like the synaptic facilitation, the long-term change in excitability of the isolated presynaptic neurons differs from the short-term in requiring the synthesis of new protein. This finding suggests that the sensory neuron uses gene products to modulate membrane currents in its long-term response to repeated external stimuli that are not required in its short-term response to a single stimulus.