Neuropeptide localization in varicosities of Aplysia sensory neurons is regulated by target and neuromodulators evoking long-term synaptic plasticity

The synapses between the sensory neuron (SN) and motor neuron of Aplysia undergo long-term functional and structural modulation with appropriate behavioral training or with applications of specific neuromodulators. Expression of molecules within the presynaptic terminals may be regulated in parallel with the changes evoked by the neuromodulators. We examined with immunocytochemical methods whether the level of sensorin, the SN-specific neuropeptide, is modulated in SN varicosities by the location of interaction with the target motor cell L7 and by applications of either 5-HT that evoke long-term facilitation or FMRFamide that evoke long-term depression of Aplysia sensorimotor connections in vitro. A significantly higher proportion of SN varicosities are sensorin positive when they are in contact with the proximal axons of L7 compared to varicosities of the same SNs in contact with distal L7 neurites. Both 5-HT and FMRFamide evoked changes in the efficacy and structure of sensorimotor connections that are accompanied by changes in the frequency of sensorin-positive varicosities contacting the axons of L7. More preexisting SN varicosities are stained after 5-HT, and fewer preexisting SN varicosities are stained after FMRFamide. These results suggest that the postsynaptic target and the neuromodulators not only regulate overall structure but also regulate the level of SN neuropeptide at synaptic sites. © 1996 John Wiley & Sons, Inc.     

Inhibitors of protein and RNA synthesis block structural changes that accompany long-term heterosynaptic plasticity in Aplysia.

Synaptic connections between the sensory and motor neurons of Aplysia in culture undergo long-term facilitation in response to serotonin (5-HT) and long-term depression in response to FMRFamide. These long-term functional changes are dependent on the synthesis of macromolecules during the period in which the transmitter is applied and are accompanied by structural changes. There is an increase and a decrease, respectively, in the number of sensory neuron varicosities in response to 5-HT and FMRFamide. To determine whether macromolecular synthesis is also required for the structural changes, we examined in parallel the effects of inhibitors of protein (anisomycin) or RNA (actinomycin D) synthesis on the structural and functional changes. We have found that anisomycin and actinomycin D block both the enduring alterations in varicosity number and the long-lasting changes in synaptic potential. These results indicate that macromolecular synthesis is required for expression of the long-lasting structural changes in the sensory cells and that this synthesis is correlated with the long-term functional modulation of sensorimotor synapses.     

Postsynaptic regulation of the development and long-term plasticity of Aplysia sensorimotor synapses in cell culture

The monosynaptic component of the neuronal circuit that mediates the withdrawal reflex of Aplysia californica can be reconstituted in dissociated cell culture. Study of these in vitro monosynaptic connections has yielded insights into the basic cellular mechanisms of synaptogenesis and long-term synaptic plasticity. One such insight has been that the development of the presynaptic sensory neurons is strongly regulated by the postsynaptic motor neuron. Sensory neurons which have been cocultured with a target motor neuron have more elaborate structures—characterized by neurites with more branches and varicosities—than do sensory neurons grown alone in culture or sensory neurons that have been cocultured with an inappropriate target cell. Another way in which the motor neuron regulates the development of sensory neurons is apparent when sensorimotor cocultures with two presynaptic cells are examined. In such cocultures the outgrowth from the different presynaptic cells is obviously segregated on the processes of the postsynaptic cell. By contrast, when two sensory neurons are placed into cell culture without a motor neuron, thier processes readily grow together. In addition to regulating the in vitro development of sensory neurons, the motor neuron also regulates learning-related changes in the structure of sensory neurons. Application of the endogenous facilitatory trasmitter serotonin (5-HT) causes long-term facilitation of in vitro sensorimotor synapses due in part to growth of new presynatpic varicosities. But 5-HT applied to sensory neurons alone in cultuer does not produce structural changes in these cells. More recently it has been found that sensorimotor synapses in cell culture can exhibit long-term potentiation (LTP). Like LTP of some hippocampal synapses, LTP of in vitro Aplysia syanpses is regulated by the voltage of the postsynaptic cell. Pairing high-frequency stimulation of sensory neurons with strong hyperpolarization of the motor neuron blocks the induction of LTP. Moreover, LTP of sensorimotor synapses can be induced in Hebbian fashion by pairing weak presynaptic stimulation with strong postsynaptic depolarization. These findings implicate a Habbian mechanism in classical conditioning in Aplysia. They also indicate that Hebbian LTP is a phylogenetically ancient form of synaptic plasticity. 1994 John Wiley & Sons, Inc.     

Cell-specific changes in expression of mRNAs encoding splice variants of aplysia cell adhesion molecule accompany long-term synaptic plasticity

Aplysia neurons express several splice variants of apCAM, a member of the Ig superfamily of cell adhesion molecules. The major transmembrane isoform is endocytosed in sensory neurons (SNs) during the early phases of long-term facilitation (LTF) of SN synapses evoked by serotonin (5-HT) or in the motor neuron L7 during the early phases of long-term depression (LTD) of SN synapses evoked by Phe-Met-Arg-Phe-amide (FMRFa). We used single cell RT-PCR to evaluate whether expression of mRNAs encoding for different apCAM isoforms in SNs and L7 is regulated during LTF produced by 5-HT, and LTD produced by FMRFa. Single SNs and L7s express mRNAs encoding for all major isoforms, but the proportion of each isoform expressed differs for the two cells. SN expresses more mRNA encoding for GPI-linked isoforms, while L7 expresses more mRNA encoding for the major transmembrane isoform. The neuromodulators produced significant changes in the proportional levels of mRNAs encoding for specific apCAM isoforms during the first 4 h after treatments without affecting overall levels of apCAM mRNA. 5-HT evoked changes that exaggerated cell-specific differences in isoform expression. FMRFa evoked changes that reduced cell-specific differences in isoform expression. The effects of the neuromodulators on apCAM mRNA expression were not detected when cells were cultured alone or when SNs were cocultured with another motor cell that failed to induce synapse formation (L11). The results suggest that rapid cell-specific regulation of splice variant expression may contribute to different forms of long-term synaptic plasticity.     

Changes in functional glutamate receptors on a postsynaptic neuron accompany formation and maturation of an identified synapse

N-Methyl-D-aspartate (NMDA)-type glutamate receptors play important roles at developing synapses and in activity-dependent synaptic plasticity. Recent studies in Aplysia suggest that NMDA-like receptors may contribute to some forms of plasticity of sensorimotor synapses accompanying associative learning. We examined at various times after plating neurons in culture the contribution of NMDA- and alpha-amino-3 hydroxy-5 methyl-4 isoxazole proprionic acid (AMPA)-like glutamate receptors to responses evoked in motor cell L7 either by action potentials in sensory neurons (SNs) or by focal applications of glutamate. We found that (D,L)-2-amino-5-phosphopentoic acid-sensitive receptors contributed significantly to postsynaptic responses in 1-day cultures but contributed little in the same cultures on day 4. By contrast, postsynaptic responses on day 4 increased significantly in amplitude by the addition of functional 6-cyano-7 nitroquinoxaline-2,3-dione- or 1-(4-aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine hydrochloride-sensitive receptors. Receptors with NMDA-like properties are detected on day 1 only at sites on L7 apposed to SN varicosities, and are not detected on L7 cultured alone. The results indicate that changes in expression and distribution of functional receptors on L7 accompany the formation and maturation of SN synapses. Signals from the SN appear to trigger expression and clustering of functional NMDA-like receptors at sites contacted by presynaptic structures capable of transmitter release. With time, functional AMPA-like receptors are added to these sites enhancing synaptic efficacy. The results are consistent with the idea that the expression and sequential clustering of NMDA- and AMPA-type receptors may be essential for the formation and maturation of central synapses.     

Tetanic stimulation and cyclic adenosine monophosphate regulate segregation of presynaptic inputs on a common postsynaptic target neuron in vitro

Previous studies indicated that Aplysia sensory neurons (SNs) compete when reestablishing synapses with a motor cell target (1.7) in vitro. The competition is characterized by a cell number-dependent decrease in the efficacy of each connection, an increase in the elimination of SN varicosities, a reduction in the formation of new SN varicosities, and the segregation of varicosities of each SN to restricted portions of the target axons. The changes do not require spike activity, since both the SNs and L7 do not fire spontaneously. Here, we examined whether adding activity to SNs during the early stages of synapse formation with stimuli known to evoke facilitatory responses in stable SN-L7 connections—tetanic stimulation or increase in intracellular cyclic adenosine monophosphate (cAMP)—would modulate the intrinsic segregatory process. Tetanic stimulation to one SN increased synapse efficacy and the number of varicosities of the stimulated SNs while reducing the functional changes by the nonstimulated SNs in the same cultures. An increase in the stability of preexisting varicosities contributed to the overall increase in varicosities evoked by tetanus. The functional changes evoked by tetanus were not expressed when the same tetanic stimulation was also given to the other SN, or when L7 was hyperpolarized during the tetanus to the SN. Raising cAMP levels in one SN increased synapse efficacy and the rate of new varicosity formation by the injected SNs without affecting the development of the connections formed by the noninjected SNs. These results suggest that different forms of presynaptic and postsynaptic activities in neurons can regulate specific aspects of the competitive process associated with the fine-tuning of connections formed by converging presynaptic inputs. © 1996 John Wiley & Sons, Inc.     

Cell‐specific changes in expression of mRNAs encoding splice variants of Aplysia cell adhesion molecule accompany long‐term synaptic plasticity

Aplysia neurons express several splice variants of apCAM, a member of the Ig superfamily of cell adhesion molecules. The major transmembrane isoform is endocytosed in sensory neurons (SNs) during the early phases of long‐term facilitation (LTF) of SN synapses evoked by serotonin (5‐HT) or in the motor neuron L7 during the early phases of long‐term depression (LTD) of SN synapses evoked by Phe‐Met‐Arg‐Phe‐amide (FMRFa). We used single cell RT‐PCR to evaluate whether expression of mRNAs encoding for different apCAM isoforms in SNs and L7 is regulated during LTF produced by 5‐HT, and LTD produced by FMRFa. Single SNs and L7s express mRNAs encoding for all major isoforms, but the proportion of each isoform expressed differs for the two cells. SN expresses more mRNA encoding for GPI‐linked isoforms, while L7 expresses more mRNA encoding for the major transmembrane isoform. The neuromodulators produced significant changes in the proportional levels of mRNAs encoding for specific apCAM isoforms during the first 4 h after treatments without affecting overall levels of apCAM mRNA. 5‐HT evoked changes that exaggerated cell‐specific differences in isoform expression. FMRFa evoked changes that reduced cell‐specific differences in isoform expression. The effects of the neuromodulators on apCAM mRNA expression were not detected when cells were cultured alone or when SNs were cocultured with another motor cell that failed to induce synapse formation (L11). The results suggest that rapid cell‐specific regulation of splice variant expression may contribute to different forms of long‐term synaptic plasticity. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 152–161, 2000     

Structural plasticity at identified synapses during long-term memory in Aplysia

We have used the gill- and siphon-withdrawal reflex of Aplysia californica to determine the morphological basis of the prolonged changes in synaptic effectiveness that underlie long-term habituation and sensitization. We have found that clear structural changes accompany behavioral modification and have demonstrated that these can be detected at the level of identified sensory neuron synapses, a critical site of plasticity for the short-term forms of both types of learning. These alterations occur at two different levels of synaptic organization and include (1) changes in focal regions of synaptic membrane specialization--the number, size and vesicle complement of sensory neuron active zones are larger in sensitized animals and smaller in habituated animals compared with controls--and (2) a parallel but more dramatic and global trend involving modulation of the total number of presynaptic varicosities per sensory neuron. Quantitative analysis of the time course over which these structural alterations occur during sensitization has further demonstrated that changes in the number of varicosities and active zones persist in parallel with the behavioral retention of the memory. This increase in the number of sensory neuron synapses during long-term sensitization in Aplysia is similar to changes in the number of synapses in the mammalian brain following various forms of environmental manipulations and learning (Greenough, 1984). Therefore learning may involve a form of neuronal growth across a broad segment of the animal kingdom, thereby suggesting a role for structural synaptic plasticity during long-term behavioral modifications.     

Protein synthesis at synapse versus cell body: enhanced but transient expression of long-term facilitation at isolated synapses

Protein synthesis at synaptic terminals contributes to LTP in hippocampus and to the formation of new synaptic connections by sensory neurons (SNs) of Aplysia. Here we report that after removal of the SN cell body, isolated SN synapses of Aplysia in culture express protein-synthesis dependent long-term facilitation (LTF) produced by 5-HT that decays rapidly. Changes in expression of a SN-specific neuropeptide sensorin in isolated SN varicosities parallel the changes in synaptic efficacy. At 24 h after 5-HT the magnitude of LTF produced at isolated SN synapses was significantly greater than that produced when SN cell bodies were present. LTF was maintained at 48 h at connections with SN cell bodies, but not at isolated SN synapses. The increase in synaptic efficacy at isolated SN synapses at 24 h was blocked by the protein synthesis inhibitor anisomycin. LTF was accompanied by changes in expression of sensorin. The increase in sensorin level at isolated SN varicosities with 5-HT was blocked by anisomycin or was reversed 48 h after 5-HT treatment alone. The results suggest that, as is the case for initial synapse formation between SNs and L7, changes in protein synthesis at synaptic terminals may contribute directly to LTF of stable synapses. Changes in expression within the cell body provide additional contributions for long-term maintenance of the new level of synaptic efficacy that was initiated directly by local changes in protein synthesis at or near synaptic terminals.     

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)}     

Bidirectional plasticity gated by hyperpolarization controls the gain of postsynaptic firing responses at central vestibular nerve synapses

Linking synaptic plasticity with behavioral learning requires understanding how synaptic efficacy influences postsynaptic firing in neurons whose role in behavior is understood. Here, we examine plasticity at a candidate site of motor learning: vestibular nerve synapses onto neurons that mediate reflexive movements. Pairing nerve activity with changes in postsynaptic voltage induced bidirectional synaptic plasticity in vestibular nucleus projection neurons: long-term potentiation relied on calcium-permeable AMPA receptors and postsynaptic hyperpolarization, whereas long-term depression relied on NMDA receptors and postsynaptic depolarization. Remarkably, both forms of plasticity uniformly scaled synaptic currents evoked by pulse trains, and these changes in synaptic efficacy were translated into linear increases or decreases in postsynaptic firing responses. Synapses onto local inhibitory neurons were also plastic but expressed only long-term depression. Bidirectional, linear gain control of vestibular nerve synapses onto projection neurons provides a plausible mechanism for motor learning underlying adaptation of vestibular reflexes.     

En passant synaptic varicosities form directly from growth cones by transient cessation of growth cone advance but not of actin-based motility.

Formation of terminal synapses at sites such as the neuromuscular junction involves transformation of the motile growth cone into the nonmotile synaptic terminal. However, transformation does not need to be the mechanism when a neurite forms multiple widely spaced synaptic varicosities along a target in an en passant configuration. Synaptic varicosities could form here by specialization of the neurite after the growth cone has advanced past the site. We examined this issue by using cocultures of identified sensory (SN) and motor (L7) neurons from Aplysia. Living SNs were labeled with fluorescent dye and their neurites were observed at high resolution every few minutes growing along the axon of L7, allowing a fine-grained analysis of the behavior of the growth cone at the sites of synapse formation. All varicosities whose formation was observed indeed developed from the growth cone. Sensory varicosities were shown by electron microscopy to contain features characteristic of active zones for transmitter release within a day of their formation on the motor axon. Growth cone advance slowed or stopped transiently during varicosity formation, but the motile activity of the peripheral region of the growth cone (veils and filopodia) was maintained. These results suggest that target "stop signals" involved in the formation of synapses, at least of the en passant variety, may be of a different type from the growth inhibitory molecules, such as the collapsins, which guide axons to their targets.     

Rapid bidirectional modulation of mRNA expression and export accompany long-term facilitation and depression of Aplysia synapses

Serotonin (5-HT) and the neuropeptide Phe-Met-Arg-Phe-amide (FMRFa) modulate synaptic efficacy of sensory neurons (SNs) of Aplysia in opposite directions and for long duration. Both long-term responses require changes in mRNA and protein synthesis. The SN-specific neuropeptide, sensorin A, is a gene product that appears to be increased by 5-HT and decreased by FMRFa. We examined whether changes in sensorin A mRNA levels in the cell body and neurites of SNs accompany long-term facilitation and depression. Both 5-HT and FMRFa evoked rapid changes in sensorin A mRNA levels in the SN cell bodies: an increase with 5-HT and a decrease with FMRFa. Parallel changes in sensorin A mRNA levels in SN neurites were detected 2 h and 4 h later. These rapid changes in mRNA expression and net export required the presence of the appropriate target motor cell L7. The neuromodulators failed to produce changes in mRNA expression or export when SNs were cultured alone or with the inappropriate target cell L11. The changes in mRNA expression were transient because mRNA levels returned to control values 24 h after treatment, while synaptic efficacy remained altered by the respective treatments. These results indicate that two neuromodulators produce distinct, but transient, target-dependent effects on expression and export of a cell-specific mRNA that correlate with changes in synaptic plasticity.     

Protein synthesis at synapse versus cell body: Enhanced but transient expression of long‐term facilitation at isolated synapses

Protein synthesis at synaptic terminals contributes to LTP in hippocampus and to the formation of new synaptic connections by sensory neurons (SNs) of Aplysia. Here we report that after removal of the SN cell body, isolated SN synapses of Aplysia in culture express protein‐synthesis dependent long‐term facilitation (LTF) produced by 5‐HT that decays rapidly. Changes in expression of a SN‐specific neuropeptide sensorin in isolated SN varicosities parallel the changes in synaptic efficacy. At 24 h after 5‐HT the magnitude of LTF produced at isolated SN synapses was significantly greater than that produced when SN cell bodies were present. LTF was maintained at 48 h at connections with SN cell bodies, but not at isolated SN synapses. The increase in synaptic efficacy at isolated SN synapses at 24 h was blocked by the protein synthesis inhibitor anisomycin. LTF was accompanied by changes in expression of sensorin. The increase in sensorin level at isolated SN varicosities with 5‐HT was blocked by anisomycin or was reversed 48 h after 5‐HT treatment alone. The results suggest that, as is the case for initial synapse formation between SNs and L7, changes in protein synthesis at synaptic terminals may contribute directly to LTF of stable synapses. Changes in expression within the cell body provide additional contributions for long‐term maintenance of the new level of synaptic efficacy that was initiated directly by local changes in protein synthesis at or near synaptic terminals. © 2003 Wiley Periodicals, Inc. J Neurobiol 56: 275–286, 2003     

A neural network model for the intersensory coordination involved in goal-directed movements

A neural network model for a sensorimotor system, which was developed to simulate oriented movements in man, is presented. It is composed of a formal neural network comprising two layers: a sensory layer receiving and processing sensory inputs, and a motor layer driving a simulated arm. The sensory layer is an extension of the topological network previously proposed by Kohonen (1984). Two kinds of sensory modality, proprioceptive and exteroceptive, are used to define the arm position. Each sensory cell receives proprioceptive inputs provided by each arm-joint together with the exteroceptive inputs. This sensory layer is therefore a kind of associative layer which integrates two separate sensory signals relating to movement coding. It is connected to the motor layer by means of adaptive synapses which provide a physical link between a motor activity and its sensory consequences. After a learning period, the spatial map which emerges in the sensory layer clearly depends on the sensory inputs and an associative map of both the arm and the extra-personal space is built up if proprioceptive and exteroceptive signals are processed together. The sensorimotor transformations occuring in the junctions linking the sensory and motor layers are organized in such a manner that the simulated arm becomes able to reach towards and track a target in extra-personal space. Proprioception serves to determine the final arm posture adopted and to correct the ongoing movement in cases where changes in the target location occur. With a view to developing a sensorimotor control system with more realistic salient features, a robotic model was coupled with the formal neural network. This robotic implementation of our model shows the capacity of formal neural networks to control the displacement of mechanical devices.     

Contribution of cerebellar sensorimotor adaptation to hippocampal spatial memory

Complementing its primary role in motor control, cerebellar learning has also a bottom-up influence on cognitive functions, where high-level representations build up from elementary sensorimotor memories. In this paper we examine the cerebellar contribution to both procedural and declarative components of spatial cognition. To do so, we model a functional interplay between the cerebellum and the hippocampal formation during goal-oriented navigation. We reinterpret and complete existing genetic behavioural observations by means of quantitative accounts that cross-link synaptic plasticity mechanisms, single cell and population coding properties, and behavioural responses. In contrast to earlier hypotheses positing only a purely procedural impact of cerebellar adaptation deficits, our results suggest a cerebellar involvement in high-level aspects of behaviour. In particular, we propose that cerebellar learning mechanisms may influence hippocampal place fields, by contributing to the path integration process. Our simulations predict differences in place-cell discharge properties between normal mice and L7-PKCI mutant mice lacking long-term depression at cerebellar parallel fibre-Purkinje cell synapses. On the behavioural level, these results suggest that, by influencing the accuracy of hippocampal spatial codes, cerebellar deficits may impact the exploration-exploitation balance during spatial navigation.     

Developmentally restricted synaptic plasticity in a songbird nucleus required for song learning

We provide evidence here of long-term synaptic plasticity in a songbird forebrain area required for song learning, the lateral magnocellular nucleus of the anterior neostriatum (LMAN). Pairing postsynaptic bursts in LMAN principal neurons with stimulation of recurrent collateral synapses had two effects: spike timing- and NMDA receptor-dependent LTP of the recurrent synapses, and LTD of thalamic afferent synapses that were stimulated out of phase with the postsynaptic bursting. Both types of plasticity were restricted to the sensory critical period for song learning, consistent with a role for each in sensory learning. The properties of the observed plasticity are appropriate to establish recurrent circuitry within LMAN that reflects the spatiotemporal pattern of thalamic afferent activity evoked by tutor song. Such circuit organization could represent a tutor song memory suitable for reinforcing particular vocal sequences during sensorimotor learning.     

Serotonin-induced regulation of the actin network for learning-related synaptic growth requires Cdc42, N-WASP, and PAK in Aplysia sensory neurons

Application of Clostridium difficile toxin B, an inhibitor of the Rho family of GTPases, at the Aplysia sensory to motor neuron synapse blocks long-term facilitation and the associated growth of new sensory neuron varicosities induced by repeated pulses of serotonin (5-HT). We have isolated cDNAs encoding Aplysia Rho, Rac, and Cdc42 and found that Rho and Rac had no effect but that overexpression in sensory neurons of a dominant-negative mutant of ApCdc42 or the CRIB domains of its downstream effectors PAK and N-WASP selectively reduces the long-term changes in synaptic strength and structure. FRET analysis indicates that 5-HT activates ApCdc42 in a subset of varicosities contacting the postsynaptic motor neuron and that this activation is dependent on the PI3K and PLC signaling pathways. The 5-HT-induced activation of ApCdc42 initiates reorganization of the presynaptic actin network leading to the outgrowth of filopodia, some of which are morphological precursors for the learning-related formation of new sensory neuron varicosities.     

Sources of inputs to longitudinal muscle motor neurons and ascending interneurons in the guinea-pig small intestine

Light- and electron-microscopic studies were used to investigate connections between specific subgroups of neurons in the myenteric plexus of the guineapig small intestine. Inputs to two classes of calretinin-immunoreactive (IR) nerve cells, longitudinal muscle motor neurons and ascending interneurons, were examined. Inputs from calbindin-IR primary sensory neurons and from three classes of descending interneurons were studied. Electron-microscopic analysis showed that calbindin-IR axons formed two types of inputs, synapses and close contacts, on calretinin-IR neurons. About 40% of inputs to the longitudinal muscle motor neurons and 70% to ascending interneurons were calbindin-IR. Approximately 50% of longitudinal muscle motor neurons were surrounded by bombesin-IR dense pericellular baskets and 40% by closely apposed varicosities. At the electron-microscope level, the bombesin-IR varicosities were found to form synapses and close contacts with the motor neurons. Dense pericellular baskets with bombesin-IR surrounded 36% of all ascending interneurons, and a further 17% had closely apposed varicosities. Somatostatin-and 5-HT-IR descending interneurons provided no dense pericellular baskets to calretinin-IR nerve cells. Thus, calretinin-IR, longitudinal muscle motor neurons and ascending interneurons receive direct synaptic inputs from intrinsic primary sensory neurons and from non-cholinergic, bombesin-IR, descending interneurons.