Premotor neurons in the feeding system of Aplysia californica

Central pattern generator (CPG) circuits control cyclic motor output underlying rhythmic behaviors. Although there have been extensive behavioral and cellular studies of food-induced feeding arousal as well as satiation in Aplysia, very little is known about the neuronal circuits controlling rhythmic consummatory feeding behavior. However, recent studies have identified premotor neurons that initiate and maintain buccal motor programs underlying ingestion and egestion in Aplysia. Other newly identified neurons receive synaptic input from feeding CPGs and in turn synapse with and control the output of buccal motor neurons. Some of these neurons and their effects within the buccal system are modulated by endogenous neuropeptides. With this information we can begin to understand how neuronal networks control buccal motor output and how their activity is modulated to produce flexibility in observed feeding behavior.     

Functional role of serotonergic neuromodulation in Aplysia.

The serotonergic metacerebral cell (MCC) of the mollusk Aplysia produces slow synaptic potentials in motor neurons of the buccal muscle, and increases the rate of ongoing rhythmic burst output of the buccal ganglion. In addition, the MCC acts peripherally to enhance the strength of buccal muscle contractions that are produced by firing of motor neurons. The potentiation of contraction is not associated with any detectable changes of resting membrane potential of muscle cells. Although MCC activity produces a small enhancement of excitatory junctional potentials, several experiments clearly indicate that the MCC has a direct potentiating effect on excitation-contraction coupling. The data suggest that potentiation of contraction might be mediated by cAMP. For example, activity of the MCC enchances the rate of accumulation of cAMP in buccal muscle, application of phosphodiesterase resistant analogs of cAMP potentiates muscle contraction, and a phosphodiesterase inhibitor enhances the effect of MCC stimulation. Recordings from free-moving animals indicate that the MCC becomes activated by exposure of the animal to food stimuli, and that the activation parallels the presence of a food-arousal state. Food-arousal is characterized by enhanced strength and increased frequency of biting responses. Both these effects can result from activity of the MCC. Thus, in this system, modulatory synaptic actions function to provide the substrate for a type behavioral modulation.     

The octopamine-containing buccal neurons are a new group of feeding interneurons in the pond snail Lymnaea stagnalis

In the pond snail, Lymnaea stagnalis, the paired buccal ganglia contain 3 octopamine-immunoreactive neurons, which have previously been shown to be part of the feeding network. All 3 OC cells are electrically coupled together and interact with all the known buccal feeding motoneurons, as well as with all the modulatory and central pattern generating interneurons in the buccal ganglia. N1 (protraction) phase neurons: Motoneurons firing in this phase of the feeding cycle receive either single excitatory (depolarising) synaptic inputs (B1, B6 neurons) or a biphasic response (hyperpolarisation followed by depolarisation) (B5, B7 motoneurons). Protraction phase feeding interneurons (SO, N1L, NIM) also receive this biphasic synaptic input after OC stimulation. All of protraction phase interneurons inhibit the OC neurons. N2 (retraction) phase neurons: These motoneurons (B2, B3, B9, B10) and N2 interneurons are hyperpolarised by OC stimulation. N2 interneurons have a variable (probably polysynaptic) effect on the activity of the OC neurons. N3 (swallowing) phase: OC neurons are strongly electrically coupled to both N3 phase (B4, B4cluster, B8) motoneurons and to the N3p interneurons. In case of the interneuronal connection (OC<->N3) the electrical synapse is supplemented by reciprocal chemical inhibition. However, the synaptic connections formed by the OC neurons or N3p interneurons to the other members of the feeding network are not identical. CGC: The cerebral, serotonergic CGC neurons excite the OC cells, but the OC neurons have no effect on the CGC activity. In addition to direct synaptic effects, the OC neurons also evoke long-lasting changes in the activity of feeding neurons. In a silent preparation, OC stimulation may start the feeding pattern, but when fictive feeding is already occurring, OC stimulation decreases the rate of the fictive feeding. Our results suggest that the octopaminergic OC neurons form a sub-population of N3 phase feeding interneurons, different from the previously identified N3p and N3t interneurons. The long-lasting effects of OC neurons suggest that they straddle the boundary between central pattern generator and modulatory neurons.     

Regulation of afferent transmission in the feeding circuitry of Aplysia

Although feeding in Aplysia is mediated by a central pattern generator (CPG), the activity of this CPG is modified by afferent input. To determine how afferent activity produces the widespread changes in motor programs that are necessary if behavior is to be modified, we have studied two classes of feeding sensory neurons. We have shown that afferent-induced changes in activity are widespread because sensory neurons make a number of synaptic connections. For example, sensory neurons make monosynaptic excitatory connections with feeding motor neurons. Sensori-motor transmission is, however, regulated so that changes in the periphery do not disrupt ongoing activity. This results from the fact that sensory neurons are also electrically coupled to feeding interneurons. During motor programs sensory neurons are, therefore, rhythmically depolarized via central input. These changes in membrane potential profoundly affect sensori-motor transmission. For example, changes in membrane potential alter spike propagation in sensory neurons so that spikes are only actively transmitted to particular output regions when it is behaviorally appropriate. To summarize, afferent activity alters motor output because sensory neurons make direct contact with motor neurons. Sensori-motor transmission is, however, centrally regulated so that changes in the periphery alter motor programs in a phase-dependent manner.     

GABA regulates the buccal motor output of Helisoma by two pharmacologically distinct actions

GABA was tested for its effects on patterned motor activity (PMA) underlying feeding. Using buccal motoneuron B19 to monitor PMA through intracellular recordings, GABA was found to exert effects at two levels. First, GABA stimulated rhythmic patterned activity resembling fictive feeding, which is under the control of the buccal CPG. In addition, GABA produced a direct inhibition of neuron B19. Both effects were observed when the buccal ganglia were studied in isolation from the rest of the central nervous system, suggesting local interactions with GABA receptors of buccal neurons. Furthermore, these two actions of GABA were found to be pharmacologically distinguishable. The direct hyperpolarization of neuron B19 was mimicked by muscimol, but not baclofen, and involved an increased chloride conductance, which was blocked by picrotoxin.Baclofen duplicated CPG activation by GABA. Picrotoxin had no effect on GABA- or baclofen-induced PMA.These results demonstrate that the Helisoma buccal ganglia have two GABA receptor types which resemble, pharmacologically, mammalian GABA_A and GABA_B receptors, and that GABA plays a key role in feeding patterned motor activity in Helisoma.Abbreviations CPG central pattern generator - GABA gammaamino butyric acid - HPLC high performance liquid chromatography - IPSP inhibitory postsynaptic potential - PMA patterned motor activity - SLRT supralateral radular tensor muscle     

Physiological basis of feeding behavior in Tritonia diomedea. III. Role of depolarizing afterpotentials

The nature and role of the depolarizing afterpotentials (DAPs) of buccal motoneurons of Tritonia diomedea were examined. Neuron B5 exhibits a DAP whose ionic dependence and modifiability by TEA and 4-AP suggest a similarity to the DAP previously described in pleural pacemaker neurons. Reduction of the DAP severely reduces the ability of these neurons to generate bursts of action potentials. Certain other motoneurons (B1 and B6) are reexcited by a slow DAP (SDAP) which appears to be of synaptic origin. It is concluded that DAPs, which are dependent upon motoneuron activity, contribute to the synthesis of motor output by the buccal ganglion.     

Modulation of the feeding system by a radular mechanosensory neuron in the terrestrial slug,Incilaria fruhstorferi

We investigated the modulatory role of a radular mechanoreceptor (RM) in the feeding system of Incilaria. RM spiking induced by current injection evoked several cycles of rhythmic buccal motor activity in quiescent preparations, and this effect was also observed in preparations lacking the cerebral ganglia. The evoked rhythmic activity included sequential activation of the inframedian radular tensor, the supramedian radular tensor, and the buccal sphincter muscles in that order.In addition to the generation of rhythmic motor activity, RM spiking enhanced tonic activities in buccal nerve 1 as well as in the cerebrobuccal connective, showing a wide excitatory effect on buccal neurons. The excitatory effect was further examined in the supramedian radular tensor motoneuron. RM spiking evoked biphasic depolarization in the tensor motoneuron consisting of fast excitatory postsynaptic potentials and prolonged depolarization lasting after termination of RM spiking. These depolarizations also occurred in high divalent cation saline, suggesting that they were both monosynaptic.When RM spiking was evoked in the fictive rasp phase during food-induced buccal motor rhythm, the activity of the supramedian radular tensor muscle showed the greatest enhancement of the three muscles tested, while the rate of ongoing rhythmic motor activity showed no increase.Abbreviations CPG central pattern generator - EPSP excitatory postsynaptic potential - RBMA rhythmic buccal motor activity - RM radular mechanosensory neuron - SMT supramedian radular tensor neuron     

Octopamine: a new feeding modulator in Lymnaea

The role of octopamine (OA) in the feeding system of the pond snail, Lymnaea stagnalis, was studied by applying behavioural tests on intact animals, and a combination of electrophysiological analysis and morphological labelling in the isolated central nervous system. OA antagonists phentolamine, demethylchlordimeform (DCDM) and 2-chloro-4-methyl-2-(phenylimino)-imidazolidine (NC-7) were injected into intact snails and the sucrose-induced feeding response of animals was monitored. Snails that received 25 to 50 mg kg^-1 phentolamine did not start feeding in sucrose, and the same dose of NC-7 reduced the number of feeding animals by 80 to 90% 1 to 3 hours after injection. DCDM treatment reduced feeding by 20 to 60%. In addition, both phentolamine and NC-7 significantly decreased the feeding rate of those animals that still accepted food after 1 to 6 hours of injection. In the central nervous system a pair of buccal neurons was identified by electrophysiological and morphological criteria. After double labelling (intracellular staining with Lucifer yellow followed by OA-immunocytochemistry) these neurons were shown to be OA immunoreactive, and electrophysiological experiments confirmed that they are members of the buccal feeding system. Therefore the newly identified buccal neurons were called OC neurons (putative octopamine containing neurons or octopaminergic cells). Synchronous intracellular recordings demonstrated that the OC neurons share a common rhythm with feeding neurons either appearing spontaneously or evoked by intracellularly stimulated feeding interneurons. OC neurons also have synaptic connections with identified members of the feeding network: electrical coupling was demonstrated between OC neurons and members of the B4 cluster motoneurons, furthermore, chemically transmitted synaptic responses were recorded both on feeding motoneurons (B1, B2 cells) and the SO modulatory interneuron after the stimulation of OC neurons. However, elementary synaptic potentials could not be recorded on the follower cells of OC neurons. Prolonged (20 to 30 s) intracellular stimulation of OC cells activated the buccal feeding neurons leading to rhythmic activity pattern (fictive feeding) in a way similar to OA applied by perfusion onto isolated central nervous system (CNS) preparations. Our results suggest that OA acts as a modulatory substance in the feeding system of Lymnaea stagnalis and the newly identified pair of OC neurons belongs to the buccal feeding network.     

Ingestion motor programs of Aplysia are modulated by short-term synaptic enhancement in cerebral-buccal interneuron pathways

In the sea slug Aplysia, buccal synapses of cerebral-buccal interneurons (CBIs) CBI-2 and CBI-12 exhibit short-term synaptic enhancement (STE), including frequency-dependant facilitation and augmentation/post-tetanic potentiation (AUG/PTP). The STE that results from driving CBI-2 or CBI-12 is associated with significantly decreased latency to burst onset in buccal premotor neurons and motor neurons, increased cycle frequency of ingestion buccal motor programs (iBMPs) and increased intraburst firing frequency of buccal neurons during iBMPs. Tests of paired-pulse facilitation during AUG/PTP suggest that the locus for this plasticity is presynaptic. The AUG/PTP is not elicited by heterosynaptic pathways, indicating that its origin is homosynaptic. At low CBI-2 and CBI-12 firing frequencies, STE is likely to contribute to iBMP initiation, while at higher firing frequencies, STE is correlated with increased cycle frequency of iBMPs. Thus, STE is an important component of the mechanisms whereby cerebral neurons regulate cyclic feeding programs and likely contributes to observed variations in behavioral responses, including feeding arousal. Electronic Publication     

Cerebral-buccal pathways in Aplysia californica: synaptic connections, cooperative interneuronal effects and feedback during buccal motor programs

Ingestion of seaweed by Aplysia is in part mediated by cerebral-buccal interneurons that drive rhythmic motor output from the buccal ganglia and in some cases cerebral-buccal interneurons act as members of the feeding central pattern generator. Here we document cooperative interactions between cerebral-buccal interneuron 2 and cerebral-buccal interneuron 12, characterize synaptic input to cerebral-buccal interneuron 2 and cerebral-buccal interneuron 12 from buccal peripheral nerve 2,3, describe a synaptic connection between cerebral-buccal interneuron 1 and buccal neuron B34, further characterize connections made by cerebral-buccal interneurons 2 and -12 with B34 and B61/62, and describe a novel, inhibitory connection made by cerebral-buccal interneuron 2 with a buccal neuron. When cerebral-buccal interneurons 2 and 12 were driven synchronously at low frequencies, ingestion-like buccal motor programs were elicited, and if either was driven alone, indirect synaptic input was recruited in the other cerebral-buccal interneuron. Stimulation of BN2,3 recruited both ingestion and rejection-like motor programs without firing in cerebral-buccal interneurons 2 or 12. During motor programs elicited by cerebral-buccal interneurons 2 or 12, high-voltage stimulation of BN2,3 inhibited firing in both cerebral-buccal interneurons. Our results suggest that cerebral-buccal interneurons 2 and 12 use cooperative interactions to modulate buccal motor programs, yet firing in cerebral-buccal interneurons 2 or 12 is not necessary for recruiting motor programs by buccal peripheral nerve BN2,3, even in preparations with intact cerebral-buccal pathways.     

Models for unambiguousE-vector navigation in the bee

Summary The metacerebral giant (MCG) neurons of the molluskPleurobranchaea have been analyzed using a wide range of methods (cobalt staining, histochemical, biophysical and electrophysiological) on several types of preparations (isolated nervous systems, semi-intact preparations, and behaving whole-animal preparations). The MCG is serotonergic. The bilaterally-symmetrical neurons have somata in the anterior brain. Each MCG neuron sends an axon out the ipsilateral mouth nerve of the brain and also into the ipsilateral cerebrobuccal connective which descends to the buccal ganglion. The descending axon sends one or more branches out most buccal nerves.The MCG makes mono- and polysynaptic chemical excitatory and inhibitory connections with identified feeding motoneurons in the buccal ganglion. In quiescent preparations (isolated CNS or semi-intact), MCG stimulation caused coordinated eversion activity followed immediately by withdrawal activity. During an ongoing feeding rhythm (spontaneous output or induced by stimulation of the stomatogastric nerve), tonic stimulation of one or both MCG's at physiological discharge frequencies typically caused a significant increase in the frequency of the rhythm, and usually emphasized the eversion component at the expense of the withdrawal component. Phasic stimulation of one or both MCG's at physiological discharge frequencies and in normal discharge patterns (bursts; see below) accelerated and phaselocked the feeding rhythm.The MCG neurons receive synaptic feedback from identified neurons in the feeding network. Brain motoneurons are reciprocally coupled with the MCG by non-rectifying electrical synapses, while buccal ganglion neurons (the previously identified corollary discharge neurons) inhibit the MCG. Recordings from the MCG during cyclic feeding show that it discharges cyclically and that its membrane potential oscillates in phase with the feeding rhythm, presumably reflecting the above synaptic feedback. Two biophysical properties of the MCG membrane, namely anomalous rectification and postspike conductance increase, are presumed to contribute to the MCG's oscillatory activity.Chemosensory (food stimuli) and mechanosensory inputs from the oral veil excite the MCG's. In whole-animal preparations, these sensory inputs typically cause discharge in the MCG's and other descending neurons, accompanied by feeding motor output.The data collectively suggest that the MCG's ofPleurobranchaea are members of a population of neurons that normally function to command (i.e., arouse, initiate and sustain) the rhythmic feeding behavior. The demonstrated central feedback to the MCG is presumed to amplify these command functions.Supported by an NIH Postdoctoral Fellowship (1 F22 NS00511) to R.G. and NIH Research Grants NS 09050 and MH 23254 to W.J.D. We thank Kathryn H. Britton for histological assistance. We also thank Mark P. Kovac, who produced the records of Figures 8 and 18, for permission to reproduce them here.     

The timing of activity in motor neurons that produce radula movements distinguishes ingestion from rejection in Aplysia

1. We have studied the neural circuitry mediating ingestion and rejection in Aplysia using a reduced preparation that produces ingestion-like and rejection-like motor patterns in response to physiological stimuli.
2. We have characterized 3 buccal ganglion motor neurons that produce specific movements of the radula and buccal mass. B8a and B8b act to close the radula. B10 acts to close the jaws and retract the radula.
3. The patterns of activity in these neurons can be used to distinguish the ingestion-like and rejection-like motor patterns. B8a, B8b and B10 are active together during the ingestion-like pattern. Activity in B8a and B8b ends prior to the onset of activity in B10 during the rejection-like pattern.
4. Our data suggest that these neurons undergo similar patterns of activity in vivo. During both feeding-like patterns, the activity and peripheral actions of B8a, B8b, and B10 are consistent with radula movements observed during ingestion and rejection. In addition, the extracellular activity produced by these neurons is consistent with neural activity observed in vivo during ingestion and rejection.
5. Our data suggest that the different activity patterns observed in these motor neurons contribute to the different radula movements that distinguish ingestion from rejection.

     

Synaptic diversity and differentiation: Crustacean neuromuscular junctions

Crustacean motor neurons exhibit a wide range of synaptic responses. Tonically active neurons generally produce small excitatory postsynaptic potentials (EPSPs) at low impulse frequencies, and are able to release much more transmitter as the impulse frequency increases. Phasic neurons typically generate large EPSPs in their target cells, but have less capability for frequency facilitation, and undergo synaptic depression during maintained activity. These differences depend in part upon the neuron's ongoing levels of activity; phasic neurons acquire physiological and morphological features of tonic neurons when their activity level is altered. Molecules responsible for adaptation to activity can be sought in single identified phasic neurons with current techniques. The fact that both phasic and tonic neurons innervate the same target muscle fibers is evidence for presynaptic determination of synaptic properties, but there is also evidence for postsynaptic determination of specific properties of different endings of a single neuron. The occurrence of high- and low-output endings of the same tonic motor neurons on different muscle fibers suggests a target-specific influence on synaptic properties. Structural variation of synapses on individual terminal varicosities leads to the hypothesis that individual synapses have different probabilities for release of transmitter. We hypothesize that structurally complex synapses have a higher probability for release than the less complex synapses. This provides an explanation for the larger quantal contents of high-output terminals (where the proportion of complex synapses is higher), and also a mechanism for progressive recruitment of synapses during frequency facilitation.     

The molluscan neuropeptide, SCPB, increases the responsiveness of the feeding motor program of Limax maximus

Small cardioactive peptide B (SCPB) has an excitatory effect on both buccal neurons and musculature in numerous molluscan species. The present study reports the effects of SCPB on the activity of specified buccal neurons and the expression of the feeding motor program of the terrestrial slug, Limax maximus. Superfusion of an isolated CNS preparation with 10(-6)M SCPB results in a 3-4-fold increase in the burst frequency of the fast salivary burster neuron (FSB), while having no effect on the activity of another endogenous burster, the bilateral salivary neuron (BSN). The response of the FSB to SCPB is dose dependent, with a threshold concentration of 2 X 10(-8)M. The response of the FSB to SCPB showed no indication of desensitization, even after long-term exposure (20 min). The feeding motor program (FMP) in Limax is a discrete pattern of cyclical motor activity that can be initiated by lip nerve stimulation. In the presence of SCPB a previously subthreshold stimulus can initiate the full FMP. The pattern of the FMP, once initiated, appears unaffected by SCPB. Thus it is the responsiveness of the initiation process that is enhanced by SCPB. Histochemical studies revealed a number of buccal neuron somata and fibers that stain for SCPB-like immunoreactive material (SLIM).     

Patterning and organization of motor neuron dendrites in the Drosophila larva

Precise patterns of motor neuron connectivity depend on the proper establishment and positioning of the dendritic arbor. However, how different motor neurons orient their dendrites to selectively establish synaptic connectivity is not well understood. The Drosophila neuromuscular system provides a simple model to investigate the underlying organizational principles by which distinct subclasses of motor neurons orient their dendrites within the central neuropil. Here we used genetic mosaic techniques to characterize the diverse dendritic morphologies of individual motor neurons from five main nerve branches (ISN, ISNb, ISNd, SNa, and SNc) in the Drosophila larva. We found that motor neurons from different nerve branches project their dendrites to largely stereotyped mediolateral domains in the dorsal region of the neuropil providing full coverage of the receptive territory. Furthermore, dendrites from different motor neurons overlap extensively, regardless of subclass, suggesting that repulsive dendrite-dendrite interactions between motor neurons do not influence the mediolateral positioning of dendritic fields. The anatomical data in this study provide important information regarding how different subclasses of motor neurons organize their dendrites and establishes a foundation for the investigation of the mechanisms that control synaptic connectivity in the Drosophila motor circuit.     

Electrophysiological studies of the gill ganglion inAplysia californica

1. An electrophysiological analysis was made of gill ganglion neurons in Aplysia californica. 2. Gill ganglion neurons behave similarly to neurons in the abdominal ganglion (the central nervous systems; CNS) that are involved with gill withdrawal behaviors. 3. Some gill ganglion neurons are motor neurons much like those in the CNS. 4. Neurons in the gill ganglion are electronically and dye-coupled. In addition, they receive common chemical synaptic inputs from the Int-II network in the CNS. 5. Tactile stimulation of the gill or siphon evokes synaptic activity in gill ganglion neurons whether or not the CNS is present. 6. Pedal nerve stimulation results in synaptic activity in gill ganglion neurons and facilitates synaptic input evoked by tactile stimulation of the gill or siphon. 7. Antibody staining reveals serotonin-like fibers in the branchial nerve close to the gill ganglion but no cell bodies in the ganglion. 8. The gill ganglion may play a role in the mediation of adaptive gill reflex behaviors. It may be one of the loci where the CNS and peripheral nervous system (PNS) interact and form an integrated circuit to mediate gill withdrawal reflex (GWR) behaviors.     

Arginine vasotocin,an endogenous neuropeptide of Aplysia,suppresses the gill withdrawal reflex and reduces the evoked synaptic input to central gill motor neurons

The superfusion (15 min) of arginine vasotocin (AVT; 10^?9–10^?12M) over the abdominal ganglion of Aplysia californica suppressed the amplitude of the gill withdrawal reflex evoked by tactile stimulation of the siphon, increased the rate of gill reflex habituation, and decreased the evoked synaptic activity to central gill motor neurons. The suppressive effects of AVT on gill reflex behaviors were not due to toxic effects of the hormone since the effects were completely reversible following washout and 3 h rest. The results obtained with AVT were similar to those previously found using the mammalian neuropeptide arginine vasopressin. AVT may act by increasing the activity of central neurons which exert suppressive control over both gill reflex behaviors and evoked activity to central gill motor neurons.     

Transfer of habituation in Aplysia: contribution of heterosynaptic pathways in habituation of the gill-withdrawal reflex

Habituation of the Aplysia gill-withdrawal reflex (and siphon-withdrawal reflex) has been attributed to low-frequency homosynaptic depression at central sensory-motor synapses. The recent demonstration that transfer of habituation between stimulation sites occurs in this model system has prompted the hypothesis that heterosynaptic inhibitory pathways also play a role in the mediation of habituation behavior. To test this hypothesis, the sites and mechanisms of neural plasticity which underlie transfer of habituation in Aplysia were examined. Transfer of habituation is a reduction in the reflex evoked at one stimulation site (siphon) due to repeated presentation of a stimulus to a second site (gill). Centrally mediated transfer of habituation, measured in a preparation lacking the siphon-gill peripheral nervous system (PNS), was associated with a reduced excitatory response in central motor neurons. Repeated tactile stimulation of the gill did not attenuate the gill response evoked by electrical stimulation of the branchial nerve nor the mechanoreceptor response recorded in LE sensory neurons. In contrast, repeated stimulation of siphon or gill at a site which was "off" the sensory field of a specific mechanoreceptor led to a diminution in synaptic transmission between that sensory neuron and its followers (motor neurons and inter-neurons). These data demonstrate that centrally mediated transfer of habituation results from heterosynaptic modulation of synaptic transmission at the sensory-motor (and sensory-interneuron) synapses. Therefore, habituation behavior in Aplysia is mediated through the conjoint action of homosynaptic and heterosynaptic inhibitory processes.     

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal¹. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal^2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture^4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.Open in a separate windowClick here to view.^{(105M, flv)}