Identified target motor neuron regulates neurite outgrowth and synapse formation of aplysia sensory neurons in vitro

To determine the influence that an appropriate target cell has on the axonal structure of a presynaptic neuron in vivo, we examined the morphologies of individual Aplysia sensory neurons in dissociated cell culture in the presence or absence of identified target motor neurons. We find that an appropriate target, the motor cell L7, regulates the morphological differentiation of the presynaptic sensory neurons in two ways: the target induces the axons of the sensory neurons to develop a more elaborate structure and to form active zones, and the target guides the outgrowth of the sensory neurons. The influence of the appropriate target, L7, on the morphological differentiation of sensory neurons appears to be related to the formation of chemical synaptic connections between the sensory neurons and L7, since sensory neurons co-cultured with an inappropriate target motor neuron do not exhibit a comparable elaboration of their axonal processes.     

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.     

Presynaptic modulation of sensory afferents in the invertebrate and vertebrate nervous system

• 1.1. Ultrastructural examination of the central terminals of sensory afferent neurons in both invertebrates and vertebrates demonstrates that the synapses that form the substrate for presynaptic inhibition and facilitation are almost universally present.
• 2.2. Presynaptic modulation of afferent input acts in many ways which tailor the inflow of sensory information to the behaviour of the animal, effectively providing a means of turning this on and off, or of combining information of the same or different modalities to refine responsiveness or clarify ambiguity.
• 3.3. Presynaptic modulation may act in several different roles on the same afferent.
• 4.4. A comparison of the mechanisms of presynaptic inhibition in different animals demonstrates the likelihood of a variety of common mechanisms,several of which may act simultaneously on the same terminal.These include changes in the conductance of the afferent membrane to Cl^-, K⁺and Ca²⁺ions, in addition to less well understood mechanisms that directly affect transmitter release.
• 5.5.A single transmitter can produce several effects on a terminal through the same or different receptors.
• 6.6. Ultrastructural studies of afferent terminals reveal that only a proportion of boutons on a given afferent may receive presynaptic input and that this may depend on the region of the nervous system in which these are found or on the identity of the postsynaptic neurons contacted.
• 7.7. The synaptic relationships of afferent terminals can be complex. In invertebrates different types of presynaptic neuron may interact synaptically,as may postsynaptic dendrites in vertebrates.
• 8.8. Axons presynaptic to afferent terminals in vertebrates frequently synapse also with dendrites postsynaptic to the afferents.
• 9.9. In both invertebrates and vertebrates reciprocal interactions between afferents and postsynaptic neurons are seen.
• 10.10. Ultrastructural immunocytochemistry reveals the likely dominance of GABA as an agent of presynaptic inhibition but also demonstrates the possible presence of other transmitters some of whose roles are less completely understood.

     

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.     

Postsynaptic target specificity of neurotrophin-induced presynaptic potentiation

The role of the target cell in neurotrophin-induced modifications of glutamatergic synaptic transmission was examined in cultured hippocampal neurons. Brain-derived neurotrophic factor (BDNF) induced rapid and persistent potentiation of evoked glutamate release when the postsynaptic neuron was glutamatergic, or excitatory (E-->E), but not when it was GABAergic, or inhibitory (E-->1). This target-specific action of BDNF was also found at divergent outputs of a single presynaptic neuron innervating both glutamatergic and GABAergic neurons, suggesting that individual terminals can be independently modified. Surprisingly, BDNF increased the frequency of miniature postsynaptic currents at both E-->E and E-->I, although it had no effect on evoked currents at E-->I. Finally, potentiation by neurotrophin-3 (NT-3) was also target specific. The selective effect at E-->E suggests that retrograde signaling by the postsynaptic target cell endows a localized presynaptic action of neurotrophins.     

The afferent synapse of cochlear hair cells

Mechanosensory hair cells of the cochlea must serve as both transducers and presynaptic terminals, precisely releasing neurotransmitter to encode acoustic signals for the postsynaptic afferent neuron. Remarkably, each inner hair cell serves as the sole input for 10-30 individual afferent neurons, which requires extraordinary precision and reliability from the synaptic ribbons that marshal vesicular release onto each afferent. Recent studies of hair cell membrane capacitance and postsynaptic currents suggest that the synaptic ribbon may operate by simultaneous multi-vesicular release. This mechanism could serve to ensure the accurate timing of transmission, and further challenges our understanding of this synaptic nano-machine.     

Interactions between entorhinal axons and target hippocampal neurons: a role for glutamate in the development of hippocampal circuitry

A coculture system consisting of input axons from entorhinal cortex explants and target hippocampal pyramidal neurons was used to demonstrate that glutamate, released spontaneously from afferent axons, can influence both dendritic geometry of target neurons and formation of presumptive synaptic sites. Dendritic outgrowth was reduced in hippocampal neurons growing on entorhinal axons when compared with neurons growing off the axons. Presumptive presynaptic sites were observed in association with hippocampal neuron dendrites and somas. HPLC analysis showed that glutamate was released from the explants in an activity- and Ca2(+)-dependent manner. The general glutamate receptor antagonist D-glutamylglycine significantly increased dendritic outgrowth in pyramidal neurons associated with entorhinal axons and reduced presumptive presynaptic sites. Tetrodotoxin and reduction of extracellular Ca2+ also promoted dendritic outgrowth and reduced the formation of presumptive synaptic sites. The results suggest that the neurotransmitter glutamate may play important roles in the development of hippocampal circuitry.     

Target cell contact suppresses neurite outgrowth from soma-soma paired Lymnaea neurons

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma-soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non-target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth-promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12-24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non-target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma-soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre- and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma-soma paired cells was transient, and neuronal sprouting began after a delay of 48-72 h. In contrast, when paired with VD1, both RPeD1 and this non-target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma-soma paired cells was target cell contact/synapse specific and Ca(2+) dependent. Specifically, soma-soma pairing in CM containing either lower external Ca(2+) concentration (50% of its control level) or Cd(2+) resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre- and postsynaptic neurons in a time-dependent and cell-specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca(2+) concentration.     

Modulation of sensory input to the spinal cord by presynaptic ionotropic glutamate receptors

Sensory input from peripheral nerves to the dorsal horn of the spinal cord is mediated by a variety of agents released by the central terminals of dorsal root ganglion (DRG) neurons. These include, but are not limited to, amino acids, especially glutamate, peptides and purines. The unraveling of the mechanisms of synaptic transmission by central terminals of DRG neurons has to take into account various ways in which the message from the periphery can be modulated at the level of the first central synapse. These include postsynaptic and presynaptic mechanisms. Homomeric and heteromeric complexes of receptor subunits for the different transmitters released by DRG neurons and interneurons, clustered at the postsynaptic site of central synapses, can be expressed in different combinations and their rate of insertion into the postsynaptic membrane is activity-regulated. Inhibitory mechanisms are an important part of central modulation, especially via presynaptic inhibition, currently believed to involve GABA released by inhibitory intrinsic neurons. Recent work has established the occurrence of another way by which sensory input can be modulated, i.e. the expression of presynaptic ionotropic and metabotropic receptors in central terminals of DRG neurons. Microscopic evidence for the expression, in these terminals, of various subunits of ionotropic glutamate receptors documents the selective expression of glutamate receptors in functionally different DRG afferents. Electrophysiological and pharmacological data suggest that activation of presynaptic ionotropic glutamate receptors in central terminals of DRG neurons may result in inhibition of release of glutamate by the same terminals. Glutamate activating presynaptic receptors may spill over from the same or adjacent synapses, or may be released by processes of astroglial cells surrounding synaptic terminals. The wide expression of presynaptic ionotropic glutamate receptors, especially in superficial laminae of the dorsal horn, where Adelta- and C fibers terminate, provides an additional or alternative mechanism, besides GABA-mediated presynaptic inhibition, for the modulation of glutamate release by these fibers. Since, however, presynaptic ionotropic glutamate receptors are also expressed in terminals of GABAergic intrinsic interneurons, a decrease of GABA release resulting from activation of these receptors in the same laminae, may also play a role in central sensitization and hyperalgesia.     

Formation of the neuromuscular junction: molecules and mechanisms

The vertebrate skeletal neuromuscular junction is the site at which motor neurons communicate with their target muscle fibers. At this synapse, as at synapses throughout the nervous system, efficient and appropriate communication requires the formation and precise alignment of specializations for transmitter release in the axon terminal with those for transmitter detection in the postsynaptic cell. Classical developmental studies demonstrate that synapse formation at the neuromuscular junction is a mutually inductive event; neurons induce postsynaptic differentiation in muscle cells and myofibers induce presynaptic differentiation in motor axon terminals. More recent experiments indicate that Schwann cells, which cap axon terminals, also play an active role in the formation and maintenance of the neuromuscular junction. Here, we review recent advances in the identification of molecules mediating such inductive interactions and the mechanisms by which they produce their effects. Although our discussion concerns events at developing neuromuscular junctions, it seems likely that similar molecules and mechanisms may act at neuron–neuron synapses in the peripheral as well as the central nervous system. BioEssays 20 :819–829, 1998. © 1998 John Wiley & Sons, Inc.     

Neurochemical aspects of interneuronal communication

It is suggested that the term neurotransmission, which is used to designate neuronal communication at synaptic level, be associated to the less restrictive term neuromodulation. These two types of intercellular communication seem in fact to be two basically different mechanisms, both of which contribute to neuronal integration. The integration of neuronal information at cellular level appears to be more complex than the simple addition of excitatory plus inhibitory influences eliciting postsynaptic responses. Evidence has been obtained that non synaptic transmission can alter the capacity of a given synapse to transfer neuronal information from the presynaptic element to the postsynaptic neuron. For instance, presynaptic mechanisms provide evidence for the functional independence of the nerve terminals, since the release of neuromediators by the latter is sometimes independent of the axonal firing rate. Similarly, the somato-dendritic part of some neurons exhibits intrinsic functions, such as a dendritic release of neuromediator, suggesting that the control of the axonal firing rate takes place partly at this somato-dendritic level and does not depend for the totality on afferent axonic information. The intercellular operations which organize individual neurons into neuronal networks will also occur either at somato-dendritic level or at the level of specific nerve terminals selected as the result of presynaptic interactions. This integration of neuronal information also seems to take place at postsynaptic level, where cooperative interactions have been shown to occur between various receptors. These mechanisms will function at the level of a single nerve terminal containing more than one neuromediator. Neuromodulation can therefore be said to involve very efficient adaptive processes, which help to account for the fact that such large behavioral responses are expressed by such a small number of neuronal elements.     

Anatomical Organization of Multiple Modulatory Inputs in a Rhythmic Motor System

In rhythmic motor systems, descending projection neuron inputs elicit distinct outputs from their target central pattern generator (CPG) circuits. Projection neuron activity is regulated by sensory inputs and inputs from other regions of the nervous system, relaying information about the current status of an organism. To gain insight into the organization of multiple inputs targeting a projection neuron, we used the identified neuron MCN1 in the stomatogastric nervous system of the crab, Cancer borealis. MCN1 originates in the commissural ganglion and projects to the stomatogastric ganglion (STG). MCN1 activity is differentially regulated by multiple inputs including neuroendocrine (POC) and proprioceptive (GPR) neurons, to elicit distinct outputs from CPG circuits in the STG. We asked whether these defined inputs are compact and spatially segregated or dispersed and overlapping relative to their target projection neuron. Immunocytochemical labeling, intracellular dye injection and three-dimensional (3D) confocal microscopy revealed overlap of MCN1 neurites and POC and GPR terminals. The POC neuron terminals form a defined neuroendocrine organ (anterior commissural organ: ACO) that utilizes peptidergic paracrine signaling to act on MCN1. The MCN1 arborization consistently coincided with the ACO structure, despite morphological variation between preparations. Contrary to a previous 2D study, our 3D analysis revealed that GPR axons did not terminate in a compact bundle, but arborized more extensively near MCN1, arguing against sparse connectivity of GPR onto MCN1. Consistent innervation patterns suggest that integration of the sensory GPR and peptidergic POC inputs occur through more distributed and more tightly constrained anatomical interactions with their common modulatory projection neuron target than anticipated.     

Different extrinsic trophic factors regulate neurite outgrowth and synapse formation between identified Lymnaea neurons

The requirement for trophic factors in neurite outgrowth is well established, though their role in synapse formation is yet to be determined. Moreover, the issue of whether the trophic factors mediating neurite outgrowth are also responsible for synapse specification has not yet been resolved. To test whether trophic factors mediating neurite outgrowth and synapse formation between identified neurons are conserved in two molluscan species and whether these developmental processes are differentially regulated by different trophic factors, we used soma-soma and neurite-neurite synapses between identified Lymnaea neurons. We demonstrate here that the trophic factors present in Aplysia hemolymph, although sufficient to induce neurite outgrowth from Lymnaea neurons, do not promote specific synapse formation between excitatory partners. Specifically, the identified presynaptic neuron visceral dorsal 4 (VD4) and postsynaptic neuron left pedal dorsal 1 (LPeD1) were either paired in a soma-soma configuration or plated individually to allow neuritic contacts. Cells were cultured in either Lymnaea brain-conditioned medium (CM) or on poly-L-lysine dishes that were pretreated with Aplysia hemolymph (ApHM), but contained only Lymnaea defined medium (DM; does not promote neurite outgrowth). In ApHM-coated dishes containing DM, Lymnaea neurons exhibited extensive neurite outgrowth, but appropriate excitatory synapses failed to develop between the cells. Instead, inappropriate reciprocal inhibitory synapses formed between VD4 and LPeD1. Similar inappropriate inhibitory synapses were observed in Aplysia hemolymph-pretreated dishes that contained dialyzed Aplysia hemolymph. These inhibitory synapses were novel and inappropriate, because they do not exist in vivo. A receptor tyrosine kinase inhibitor (Lavendustin A) blocked neurite outgrowth induced by both Lymnaea CM and ApHM. However, it did not affect inappropriate inhibitory synapse formation between the neurons. These data demonstrate that neurite outgrowth but not inappropriate inhibitory synapse formation involves receptor tyrosine kinases. Together, our data provide direct evidence that trophic factors required for neurite outgrowth are conserved among two different molluscan species, and that neurite extension and synapse specification between excitatory partners are likely mediated by different trophic factors.     

Independent development of sensory and motor innervation patterns in embryonic chick hindlimbs

Previous studies suggest that sensory axon outgrowth is guided by motoneurons, which are specified to innervate particular target muscles. Here we present evidence that questions this conclusion. We have used a new approach to assess the pathfinding abilities of bona fide sensory neurons, first by eliminating motoneurons after neural crest cells have coalesced into dorsal root ganglia (DRG) and second by challenging sensory neurons to innervate muscles in a novel environment created by shifting a limb bud rostrally. The resulting sensory innervation patterns mapped with the lipophilic dyes DiI and DiA showed that sensory axons projected robustly to muscles in the absence of motoneurons, if motoneurons were eliminated after DRG formation. Moreover, sensory neurons projected appropriately to their usual target muscles under these conditions. In contrast, following limb shifts, muscle sensory innervation was often derived from inappropriate segments. In this novel environment, sensory neurons tended to make more "mistakes" than motoneurons. Whereas motoneurons tended to innervate their embryologically correct muscles, sensory innervation was more widespread and was generally from more rostral segments than normal. Similar results were obtained when motoneurons were eliminated in embryos with limb shifts. These findings show that sensory neurons are capable of navigating through their usual terrain without guidance from motor axons. However, unlike motor axons, sensory axons do not appear to actively seek out appropriate target muscles when confronted with a novel terrain. These findings suggest that sensory neuron identity with regard to pathway and target choice may be unspecified or quite plastic at the time of initial axon outgrowth.     

Presynaptic modulation of transmitter release via alpha2-adrenoceptors: nonsynaptic interactions

It is generally accepted that neurochemical transmission occurring at the synapse is the primary way of sending messages from one neuron to another. Neurotransmitters released from axon terminal in a [Ca2+]0-dependent manner act transsynaptically on the postsynaptic site. The past 30 years have witnessed something of a revolution in the understanding of how neurons communicate with each other. It has been shown that the exocytotic release of transmitters from axon terminals is subject to presynaptic modulation via presynatic hetero- and auto-receptors. For example via stimulation of alpha2-adrenoceptors expressed on varicosities and coupled to G-protein the stimulation-evoked release of different transmitters can be inhibited. This review will focus on nonsynaptic interactions between axon terminals. The present data clearly show that transmitters released from axon terminals without synaptic contact play an important role in the fine tuning of communication between neurons within a neuronal circuit.     

Target cell contact suppresses neurite outgrowth from soma–soma paired Lymnaea neurons

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma–soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non‐target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth‐promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12–24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non–target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma–soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre‐ and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma–soma paired cells was transient, and neuronal sprouting began after a delay of 48–72 h. In contrast, when paired with VD1, both RPeD1 and this non‐target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma–soma paired cells was target cell contact/synapse specific and Ca²⁺ dependent. Specifically, soma–soma pairing in CM containing either lower external Ca²⁺ concentration (50% of its control level) or Cd²⁺ resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre‐ and postsynaptic neurons in a time‐dependent and cell‐specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca²⁺ concentration. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 357–369, 2000     

Molecular architecture of glycinergic synapses

Synapses can be considered chemical machines, which are optimized for fast and repeated exocytosis of neurotransmitters from presynaptic nerve terminals and the reliable electrical or chemical transduction of neurotransmitter binding to the appropriate receptors in the postsynaptic membrane. Therefore, synapses share a common repertoire of proteins like, e.g., the release machinery and certain cell adhesion molecules. This basic repertoire must be extended in order to generate specificity of neurotransmission and allow plastic changes, which are considered the basis of developmental and/or learning processes. Here, we focus on these complementary molecules located in the presynaptic terminal and postsynaptic membrane specializations of glycinergic synapses. Moreover, as specificity of neurotransmission in this system is established by the specific binding of the neurotransmitter to its receptor, we review the molecular properties of glycine receptor subunits and their assembly into functional glycine receptors with different functional characteristics. The past years have revealed that the molecular machinery underlying inhibitory and especially glycinergic postsynaptic membrane specializations is more complex and dynamic than previously anticipated from morphological studies. The emerging features include structural components as well as signaling modules, which could confer the plasticity required for the proper function of distinct motor and sensory functions.     

Endogenous cannabinoids mediate retrograde signals from depolarized postsynaptic neurons to presynaptic terminals

Endogenous cannabinoids are considered to function as diffusible and short-lived modulators that may transmit signals retrogradely from postsynaptic to presynaptic neurons. To evaluate this possibility, we have made a paired whole-cell recording from cultured hippocampal neurons with inhibitory synaptic connections. In about 60% of pairs, a cannabinoid agonist greatly reduced the release of the inhibitory neurotransmitter GABA from presynaptic terminals. In most of such pairs but not in those insensitive to the agonist, depolarization of postsynaptic neurons and the resultant elevation of intracellular Ca2+ concentration caused transient suppression of inhibitory synaptic currents, which is mainly due to reduction of GABA release. This depolarization-induced suppression was completely blocked by selective cannabinoid antagonists. Our results reveal that endogenous cannabinoids mediate retrograde signals from depolarized postsynaptic neurons to presynaptic terminals to cause the reduction of transmitter release.     

The SAD-1 kinase regulates presynaptic vesicle clustering and axon termination

During synapse formation, presynaptic axon outgrowth is terminated, presynaptic clusters of vesicles are associated with active zone proteins, and active zones are aligned with postsynaptic neurotransmitter receptors. We report here the identification of a novel serine/threonine kinase, SAD-1, that regulates several aspects of presynaptic differentiation in C. elegans. In sad-1 mutant animals presynaptic vesicle clusters in sensory neurons and motor neurons are diffuse and disorganized. Sensory axons fail to terminate in sad-1 mutants, whereas overexpression of SAD-1 causes sensory axons to terminate prematurely. SAD-1 protein is expressed in the nervous system and localizes to synapse-rich regions of the axons. SAD-1 is related to PAR-1, a kinase that regulates cell polarity during asymmetric cell division. Overexpression of SAD-1 causes mislocalization of vesicle proteins to dendrites, suggesting that sad-1 affects axonal-dendritic polarity as well as synaptic development.     

Effects of cholecystokinin-8s in the nucleus tractus solitarius of vagally deafferented rats

We have shown recently that cholecystokinin octapeptide (CCK-8s) increases glutamate release from nerve terminals onto neurons of the nucleus tractus solitarius pars centralis (cNTS). The effects of CCK on gastrointestinal-related functions have, however, been attributed almost exclusively to its paracrine action on vagal afferent fibers. Because it has been reported that systemic or perivagal capsaicin pretreatment abolishes the effects of CCK, the aim of the present work was to investigate the response of cNTS neurons to CCK-8s in vagally deafferented rats. In surgically deafferented rats, intraperitoneal administration of 1 or 3 mug/kg CCK-8s increased c-Fos expression in cNTS neurons (139 and 251% of control, respectively), suggesting that CCK-8s' effects are partially independent of vagal afferent fibers. Using whole cell patch-clamp techniques in thin brain stem slices, we observed that CCK-8s increased the frequency of spontaneous and miniature excitatory postsynaptic currents in 43% of the cNTS neurons via a presynaptic mechanism. In slices from deafferented rats, the percentage of cNTS neurons receiving glutamatergic inputs responding to CCK-8s decreased by approximately 50%, further suggesting that central terminals of vagal afferent fibers are not the sole site for the action of CCK-8s in the brain stem. Taken together, our data suggest that the sites of action of CCK-8s include the brain stem, and in cNTS, the actions of CCK-8s are not restricted to vagal central terminals but that nonvagal synapses are also involved.