Crustacean Neuromuscular Junctions Research Articles

Serotonin and Synaptic Transmission at Invertebrate Neuromuscular Junctions

The serotonergic system in vertebrates and invertebrates has been a focus for over 50 years and will likely continue in the future. Recently, genomic analysis and discovery of alternative splicing and differential expression in tissues have increased the knowledge of serotonin (5-HT) receptor types. Comparative studies can provide useful insights to the wide variety of mechanistic actions of 5-HT responsible for behaviors regulated or modified by 5-HT. To determine cellular responses and influences on neural systems as well as the efferent control of behaviors by the motor units, preparations amenable to detailed studies of synapses are beneficial as working models. The invertebrate neuromuscular junctions (NMJs) offer some unique advantages for such investigations; action of 5-HT at crustacean NMJs has been widely studied, and leech and Aplysia continue to be key organisms. However, there are few studies in insects likely due to the focus in modulation within the CNS and lack of evidence of substantial action of 5-HT at the Drosophila NMJs. There are only a few reports in gastropods and annelids as well as other invertebrates. In this review we highlight some of the key findings of 5-HT actions and receptor types associated at NMJs in a variety of invertebrate preparations in hopes that future studies will build on this knowledge base.

NMDA R1 receptor distribution in the cyprid of Balanus amphitrite (= Amphibalanus amphitrite) (Cirripedia, Crustacea)

The ontogenetic cycle of the barnacle Balanus amphitrite (= Amphibalanus amphitrite) (Darwin, 1854) includes a cyprid that binds submerged surfaces, metamorphosing into a sessile adult. γ-Aminobutyric acid (GABA) and GABA receptors have recently been located in its cyprid with a similar pattern to other crustaceans. Since NMDA R1 ionotropic glutamatergic receptors have been identified in crustacean neuromuscular junctions, we have investigated their presence in the B. amphitrite cyprid. The presence of NMDA R1 receptors might indicate a role for glutamate in neuromuscular control in B. amphitrite cyprids, therefore we studied the presence and distribution of the NMDA R1 by immunohistochemistry. Its distribution was observed in the peripheral nervous system and in non-neuronal elements. Actually, NMDA R1 immunoreactivity was detected in thoracic appendages, at the level of neuromuscular junctions, thus suggesting an involvement in motor control functions, as already demonstrated in other crustaceans. Immunoreactivity was also detected in ommatidia cells of the eye, in antennules, and in epidermal cells. The distribution pattern comparable to that of GABAergic molecules could indicate an interrelated agonistic/antagonistic role for these two systems, which could be considered as potential targets of combined antifouling strategies.

Proteomics of presynaptic differentiation

Event Abstract Back to Event Proteomics of presynaptic differentiation Lorelei Silverman1*, Moshe Praver1 and Milton Charlton1 1 University of Toronto, Department of Physiology, Faculty of Medicine, Canada The synapses of Crustacean neuromuscular junctions provide an extraordinary opportunity to study presynaptic differentiation. For instance, at individual fibres of the crayfish leg extensor muscle, the phasic motor neuron makes synapses that have a high probability of transmitter release and depress easily with low-frequency stimulation. On the same muscle fiber, the tonic motoneuron makes synapses that have a probability of transmitter release hundreds of times lower than that of the phasic synapses, but the probability increases dramatically with repetitive stimulation (Bradacs et al., 1997; Atwood and Karunanithi, 2002). Low frequency depression of phasic synapses was observed by recording from both leg extensor preparations and intact leg stimulated at 0.2 Hz. Our long term goal was to determine the molecular basis of this differentiation using a proteomic approach in both crayfish and Drosophila synapses at various locations as well as at cerebelar co-culture between granule cells and Purkinje cells (phasic) and inferior olivary-Purkinje cells (tonic). Since dephosphorylation of the cytoskeleton is involved in low frequency depression (Silverman-Gavrila and Charlton, 2009) we wanted first to determine if there are any differences in the cytoskeleton between the phasic and tonic terminals. Using double immunostaining and confocal imaging of actin and tubulin we found that tubulin is more abundant in tonic than phasic axons trunks, while actin seems to be more concentrated in phasic axons. We isolated and dissociated the phasic and the tonic terminals using enzymatic and mechanical manipulation. Once enough material is collected, proteins from the two terminals are extracted. Next the phasic proteins are stained with Cy3 dye and the tonic proteins with Cy5 dye, then submit them to 2D DIGE (two dimensional differential in gel electrophoresis) for separation. Next, the gel is simultaneously scanned and protein spots analyzed for differential expression. Protein spots whose expression is different for samples from phasic and tonic synapses are digested and then identified by mass spectrometry. Next these proteins will be further investigated for their specific function. The identification of proteins that are differentially expressed will advance the identification of molecular basis of synaptic plasticity. Acknowledgements: NSERC fellowship to LBSG and CIHR grant to Milton Charlton. Special thanks to Rene Prashad for his continuous technical assistance and helpful discussions. Conference: B.R.A.I.N. platform in Physiology poster day 2009, Toronto, ON, Canada, 16 Dec - 16 Dec, 2009. Presentation Type: Poster Presentation Topic: Poster presentations Citation: Silverman L, Praver M and Charlton M (2009). Proteomics of presynaptic differentiation. Front. Neurosci. Conference Abstract: B.R.A.I.N. platform in Physiology poster day 2009. doi: 10.3389/conf.neuro.03.2009.17.053 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 18 Dec 2009; Published Online: 18 Dec 2009. * Correspondence: Lorelei Silverman, University of Toronto, Department of Physiology, Faculty of Medicine, Toronto, Canada, lorelei.silverman.gavrila@utoronto.ca Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Lorelei Silverman Moshe Praver Milton Charlton Google Lorelei Silverman Moshe Praver Milton Charlton Google Scholar Lorelei Silverman Moshe Praver Milton Charlton PubMed Lorelei Silverman Moshe Praver Milton Charlton Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Calcium Sensitivity of Neurotransmitter Release Differs at Phasic and Tonic Synapses

The efficacy of synaptic transmission varies greatly among synaptic contacts. We have explored the origins of differences between phasic and tonic crustacean neuromuscular junctions. Synaptic boutons of a phasic motor neuron release three orders of magnitude more quanta to a single action potential and show strong depression to a train, whereas tonic synapses are nearly unresponsive to single action potentials and display an immense facilitation. Phasic and tonic synapses display a similar nonlinear dependence on extracellular [Ca2+]. We imposed similar spatially uniform intracellular [Ca2+] ([Ca2+]i) steps in phasic and tonic synapses by photolysis of presynaptic caged calcium. [Ca2+]i was measured fluorometrically while transmitter release was monitored electrophysiologically from single boutons in which the [Ca2+]i was elevated. Phasic synapses released the readily releasable pool (RRP) of vesicles at a much higher rate and with a shorter delay than did tonic synapses. Comparison of several kinetic models of molecular events showed that a difference in Ca2+-sensitive priming of vesicles in the RRP combined with a revision of the kinetic Ca2+-binding sequence to the secretory trigger produced the best fit to the markedly different responses to Ca2+ steps and action potentials and of the characteristic features of synaptic plasticity in phasic and tonic synapses. The results reveal processes underlying one aspect of synaptic diversity that may also regulate changes in synaptic strength during development and learning and memory formation.

Mechnisms underlying different facilitation forms at the lobster neuromuscular synapse

At the crustacean neuromuscular junction, facilitation elicited by a repetitive stimulation reaches a plateau level that is proportional to the stimulation frequency. In the present study we demonstrated that plateau facilitation (Fplateau) does not depend on Ca2+ manipulations. We manipulated Ca2+ concentration in the following ways: (1) applying cell permeable chelators BAPTA-AM or EGTA-AM; (2) decreasing Ca2+ concentration in the extracellular media; (3) enhancing Ca2+ influx by 4-aminipyridin. We found that neither Fplateau is decreased by lowering Ca2+ nor it is increased by enhancing Ca2+ influx. In contrast, facilitation elicited by a short train of stimuli (Fgrowth) was altered by Ca2+ manipulations. These results suggested that Fplateau does not result from accumulation of free intracellular Ca2+. We hypothesized that Fplateau results from the accumulation of synaptic vesicles properly activated for transmitter release, the readily releasable pool (RRP). To test this hypothesis, we measured the increase in RRP employing local applications of hypertonic solutions (HS). We found that the size of RRP was significantly increased after Fplateau was induced. Our results suggest that facilitation is mediated by two mechanisms: the increase in the residual Ca2+ and the increase in RRP. Frequency facilitation during continuous stimulation, Fplateau, is primarily controlled by the increase in RRP.

Active zones and receptor surfaces of freeze-fractured crayfish phasic and tonic motor synapses.

Deep and superficial flexor muscles in the crayfish abdomen are innervated respectively by small populations of physiologically distinct phasic and tonic motoneurons. Phasic motoneurons typically produce large EPSP's, releasing 100 to 1000 times more transmitter per synapse than their tonic counterparts, and exhibiting more rapid synaptic depression with maintained stimulation. Freeze-fracturing the abdominal flexor muscles yielded images of phasic and tonic synapse-bearing terminals. The two types of synapse are qualitatively similar in ultrastructure, displaying on the presynaptic membrane's P-face synaptic contacts recognized by relatively particle-free oval plaques which are often framed by the muscle fiber's E-face leaflet with its associated receptor particles. Situated within these presynaptic plaques are discrete clusters of large intramembrane particles, forming active zone (AZ) sites specialized for transmitter release. AZs of phasic and tonic synapses are similar: 80% had a range of 15-40 large particles distributed in either paired spherical clusters or in linear form, with a few depressions denoting sites of synaptic vesicle fusion or retrieval around their perimeters. The packing density of particles is similar for phasic and tonic AZs. The E-face of the muscle membrane displays oval-shaped receptor-containing sites made up of tightly packed intramembranous particles. Phasic and tonic receptor particles are packed at similar densities and the measured values resemble those of several other crustacean and insect neuromuscular junctions. Overall, the similarity between phasic and tonic synapses in the packing density of particles at their presynaptic AZs and postsynaptic receptor surfaces suggests similar regulatory mechanisms for channel insertion and spacing. Furthermore, the findings suggest that morphological differences in active zones or receptor surfaces cannot account for large differences in transmitter release per synapse.

Hyperosmolarity reduces facilitation by a Ca(2+)-independent mechanism at the lobster neuromuscular junction: possible depletion of the releasable pool.

1. At the crustacean neuromuscular junction, action potential-evoked neurosecretion increases in proportion to stimulation frequency, a process termed frequency facilitation. In the present study we examined how frequency facilitation is affected by osmotic pressure. 2. Hypertonic solution (HS) was applied by local superfusion of the synaptic area. Quantal release was monitored by focal extracellular recordings of postsynaptic potentials. Several stimulation frequencies (f) in the range from 1 to 10 Hz were employed, and quantal content (m) together with the number of releasable units (n) and release probability (p) was evaluated for each frequency. 3. Osmotic pressure enhanced quantal release at the lowest f tested (1 Hz) but suppressed neurosecretion at higher f (7-10 Hz). Thus, hyperosmolarity enhanced action potential-evoked release but suppressed frequency facilitation. 4. Chelation of intracellular calcium by BAPTA showed that the effect of HS was calcium independent. 5. Binomial analysis of quantal content revealed that HS suppressed the increase in the number of releasable units, which was very pronounced during facilitation under control conditions. Since HS also stimulated asynchronous quantal release, the observed effect of HS on facilitation can be explained by the depletion of the releasable pool of quanta caused by the asynchronous neurosecretion. 6. To test this hypothesis we increased the available pool of vesicles using serotonin and demonstrated that the suppressing effect of HS on facilitation was reversed. 7. The observed effects of HS on facilitated neurosecretion could be described quantitatively using our model for mobilization of vesicles into the releasable pool enhanced by action potentials.

Reduced facilitation and vesicular uptake in crustacean and mammalian neuromuscular junction by T-588, a neuroprotective compound.

Bath application of compound T-588, a neuroprotective agent, reduced paired-pulse and repetitive-pulse facilitation at mammalian and crustacean neuromuscular junctions. In addition, it reduced voltage-gated sodium and potassium currents in a use-dependent fashion, but had only a small effect on the presynaptic Ca(2+) conductance. By contrast, it blocked FM 1-43 vesicular uptake but not its release, in both species. Postsynaptically, T-588 reduced acetylcholine currents at the mammalian junction in a voltage-independent manner, but had no effect on the crayfish glutamate junction. All of these effects were rapidly reversible and were observed at concentrations close to the compound's acute protective level. We propose that this set of mechanisms, which reduces high-frequency synaptic transmission, is an important contributory factor in the neuroprotective action of T-588.

Presynaptic mitochondria and the temporal pattern of neurotransmitter release.

Mitochondria are critical for the function of nerve terminals as the cycling of synaptic vesicle membrane requires an efficient supply of ATP. In addition, the presynaptic mitochondria take part in functions such as Ca2+ buffering and neurotransmitter synthesis. To learn more about presynaptic mitochondria, we have examined their organization in two types of synapse in the lamprey, both of which are glutamatergic but are adapted to different temporal patterns of activity. The first is the giant lamprey reticulospinal synapse, which is specialized to transmit phasic signals (i.e. bursts of impulses). The second is the synapse established by sensory dorsal column axons, which is adapted to tonic activity. In both cases, the presynaptic axons were found to contain two distinct types of mitochondria; small 'synaptic' mitochondria, located near release sites, and larger mitochondria located in more central parts of the axon. The size of the synapse-associated mitochondria was similar in both types of synapse. However, their number differed considerably. Whereas the reticulospinal synapses contained only single mitochondria within 1 micron distance from the edge of the active zone (on average 1.2 per active zone, range of 1-3), the tonic dorsal column synapses were surrounded by clusters of mitochondria (4.5 per active zone, range of 3-6), with individual mitochondria sometimes apparently connected by intermitochondrial contacts. In conjunction with studies of crustacean neuromuscular junctions, these observations indicate that the temporal pattern of transmitter release is an important determinant of the organization of presynaptic mitochondria.

Depression of synaptic efficacy in high- and low-output Drosophila neuromuscular junctions by the molting hormone (20-HE).

The molt-related steroid hormone, 20-hydroxyecdysone (20-HE), was applied to muscles 6 and 7 of third instar larval of Drosophila melanogaster neuromuscular junction preparations to examine if rapid, nongenomic responses could be observed as was shown recently to occur in crustacean neuromuscular junctions. At a dose of 10 microM, the excitatory junction potentials were reduced in amplitude within minutes. To elucidate the site of action of the hormone, focal-macropatch recordings of synaptic currents were obtained over the neuromuscular junctions. The results showed that the high-output (Is) and the low-output (Ib) motor nerve terminals, which innervate muscles 6 and 7, released fewer synaptic vesicles for each stimulation while exposed to 20-HE. Because the size and shape of synaptic currents from spontaneous releases did not change, the effects of the 20-HE are presynaptic. The rapid effects of this hormone may account in part for the quiescent behavior associated with molts among insects and crustaceans.

Temporal dynamics of convergent modulation at a crustacean neuromuscular junction.

At least 10 different substances modulate the amplitude of nerve-evoked contractions of the gastric mill 4 (gm4) muscle of the crab, Cancer borealis. Serotonin, dopamine, octopamine, proctolin, red pigment concentrating hormone, crustacean cardioactive peptide, TNRNFLRFamide, and SDRNFLRFamide increased and -allatostatin-3 and histamine decreased the amplitude of nerve-evoked contractions. Modulator efficacy was frequency dependent; TNRNFLRFamide, proctolin, and allatostatin-3 were more effective when the motor neuron was stimulated at 10 Hz than at 40 Hz, whereas the reverse was true for dopamine and serotonin. The modulators that were most effective at high stimulus frequencies produced a significant decrease in muscle relaxation time; those that were most effective at low stimulus frequencies produced modest increases in relaxation time. Thus modulator actions that appear redundant when examined only at one stimulus frequency are differentiated when a range of stimulus dynamics is studied. The effects of TNRNFLRFamide, serotonin, proctolin, dopamine, and -allatostatin-3 on the amplitude and facilitation of nerve-evoked excitatory junctional potentials (EJPs) in the gm4 and gastric mill 6 (gm6) muscles were compared. The EJPs in gm4 have a large initial amplitude and show relatively little facilitation, whereas the EJPs in gm6 have a small initial amplitude and show considerable facilitation. Modulators that enhanced contractions also enhanced EJP amplitude; -allatostatin-3 reduced EJP amplitude. The effects of these modulators on EJP amplitude were modest and showed no significant frequency dependence. This suggests that the frequency dependence of modulator action on contraction results from effects on excitation-contraction coupling. The modulators affected facilitation at these junctions in a manner consistent with a change in release probability. They produced a change in facilitation that is inversely related to their action on EJP amplitude.

A pharmacological and HPLC analysis of the excitatory transmitter of the cardiac ganglion in the heart of the isopod crustacean Bathynomus doederleini

In crustacean neurogenic hearts, the myocardium contracts under the tight control of rhythmically active neurons of the cardiac ganglion located inside the heart. We demonstrated that the myocardium of Bathynomus doederleini was sensitive to glutamate, quisqualate, and kainate, and that the tension of myocardial cells developed in a dose-dependent manner. The threshold concentrations were 10-5 M for quisqualate, 10-4 M for glutamate, and 3 × 10-4 M for kainate. Concanavalin A, known to prevent desensitization of glutamate receptors at crustacean neuromuscular junctions, augmented excitatory junctional potentials evoked by the cardiac ganglionic neurons in myocardial cells. Using a high performance liquid chromatography (HPLC), we analyzed glutamate in extracts of the cardiac ganglion and myocardium. We obtained values for glutamate concentrations, 8741.2 ± 184.2 and 678.2 ± 10.7 pmol/mg, respectively. Although we attempted to measure monoamines in the extracts by HPLC, these were not detected at measurable (more than 1 fmol per 10-µL sample) levels. In conclusion, myocardial cells in isopod crustaceans were suggested to receive glutamatergic motor innervation.Key words: HPLC, glutamate, heart, crustacean, neurotransmitter.

A pharmacological and HPLC analysis of the excitatory transmitter of the cardiac ganglion in the heart of the isopod crustacean Bathynomus doederleini.

In crustacean neurogenic hearts, the myocardium contracts under the tight control of rhythmically active neurons of the cardiac ganglion located inside the heart. We demonstrated that the myocardium of Bathynomus doederleini was sensitive to glutamate, quisqualate, and kainate, and that the tension of myocardial cells developed in a dose-dependent manner. The threshold concentrations were 10(-5) M for quisqualate, 10(-4) M for glutamate, and 3 x 10(-4) M for kainate. Concanavalin A, known to prevent desensitization of glutamate receptors at crustacean neuromuscular junctions, augmented excitatory junctional potentials evoked by the cardiac ganglionic neurons in myocardial cells. Using a high performance liquid chromatography (HPLC), we analyzed glutamate in extracts of the cardiac ganglion and myocardium. We obtained values for glutamate concentrations, 8741.2 +/- 184.2 and 678.2 +/- 10.7 pmol/mg, respectively. Although we attempted to measure monoamines in the extracts by HPLC, these were not detected at measurable (more than 1 fmol per 10-microL sample) levels. In conclusion, myocardial cells in isopod crustaceans were suggested to receive glutamatergic motor innervation.

Decoding Synapses

The strength of many synapses is modified by various use and time-dependent processes, including facilitation and depression. A general description of synaptic transfer characteristics must account for the history-dependence of synaptic efficacy and should be able to predict the postsynaptic response to any temporal pattern of presynaptic activity. To generate such a description, we use an approach similar to the decoding method used to reconstruct a sensory input from a neuronal firing pattern. Specifically, a mathematical fit of the postsynaptic response to an isolated action potential is multiplied by an amplitude factor that depends on a time-dependent function summed over all previous presynaptic spikes. The amplitude factor is, in general, a nonlinear function of this sum. Approximate forms of the time-dependent function and the nonlinearity are extracted from the data, and then both functions are constructed more precisely by a learning algorithm. This approach, which should be applicable to a wide variety of synapses, is applied here to several crustacean neuromuscular junctions. After training on data from random spike sequences, the method predicts the postsynaptic response to an arbitrary train of presynaptic action potentials. Using a model synapse, we relate the functions used in the fit to underlying biophysical processes. Fitting different neuromuscular junctions allows us to compare their responses to sequences of action potentials and to contrast the time course and degree of facilitation or depression that they exhibit.

Synaptic diversity and differentiation: Crustacean neuromuscular junctions

Crustacean motor neurons exhibit a wide range of synaptic responses. Tonically active neurons gener- ally 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 dif- ferences depend in part upon the neuron's ongoing levels of activity; phasic neurons acquire physiological and morpho- logical 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 influ- ence 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.

Activity-induced changes in synaptic release sites at the crayfish neuromuscular junction

Crustacean motor axons provide a model in which activity-dependent changes in synaptic physiology and synaptic structure can be concurrently observed in single identifiable neurons. In response to a train of stimulation, crustacean neuromuscular junctions undergo pronounced facilitation of transmitter release. The effects of maintained high-frequency stimulation may persist for at least several hours ("long-term facilitation"). Electrophysiological studies suggest that the number of "active" synapses contributing transmitter quanta at low frequencies of stimulation increases during and after a train of high-frequency stimulation. However, at different terminal recording sites the effect of stimulation varies, and it was observed that not all released quanta produce a voltage change in the postsynaptic muscle cell. Electron microscopic examinations of serial sections from nerve terminals subjected to stimulation were made to determine whether changes in synaptic structure could be correlated with activity-induced long-lasting enhancement of transmission. A procedure was introduced for marking a recorded terminal with fluorescent polystyrene microspheres, which are visible in electron micrographs of the recording site. Crustacean nerve terminals possess a large number of discrete synapses, a small fraction of which have multiple presynaptic "active zones" (dense bodies with clustered synaptic vesicles, thought to represent sites of evoked transmitter release). In terminals previously stimulated, the proportion of synapses with multiple "active zones" is greater than in control unstimulated terminals. Total synaptic vesicle counts and readily releasable vesicles at synapses are not significantly different in previously stimulated terminals and controls. In terminals fixed during stimulation a few synapses show evidence of division in "active zones," and synaptic vesicle counts are lower than in controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular regulation of GABAA-receptor function.

γ-Aminobutyric acid (GABA) was reported to occur in brain tissue in 1950 in three studies (Awapara et al., 1950; Roberts and Frankel, 1950; Udenfriend, 1950). A long and often controversial discussion about the function of GABA (for review see Roberts, 1986) followed. Physiological effects of GABA were first studied at the crustacean neuromuscular junction (for review see Takeuchi, 1976 Kravitz et al., 1968). The role of GABA as inhibitory transmitter in the mammalian CNS was established in 1967 through electrophysiological studies comparing the properties of evoked inhibitory postsynaptic potentials with responses to iontophoretically applied GABA: GABA-induced hyperpolarization was accompanied by a large increase in membrane conductance (Krnjevic and Schwartz, 1967). It is now widely accepted that GABA is the main transmitter of synaptic inhibition in the mammalian CNS. The notion of a transmitter function of GABA has been corroborated by a variety of studies: the GABA-synthesizing enzyme glutamate decarboxylase (L-glutamate-1carboxylase, EC 4.1.1.15) is concentrated in nerve terminals of GABAergic neurons (Salganicoff and De Robertis, 1965; Fonnum, 1968; Fonnum and Walberg, 1973; Saito et al., 1974). GABA is released from brain tissue upon stimulation by a Ca2+-dependent mechanism (Srinivasan et al., 1969; Obata and Takeda, 1969; Iversen et al., 1971; Roberts, 1974) and is eliminated from extracellular spaces by a Na+-dependent uptake into nerve cells or glial cells (Iversen and Neal, 1968; Henn and Hamberger, 1971; Bloom and Iversen, 1971; Schrier and Thompson,1974).

Interaction of Ca-channel blockers and high pressure at the crustacean neuromuscular junction

Exposure to high pressure causes a significant depression of synaptic transmission. We examined the effects of various Ca-channel blockers and their interaction with high pressure on excitatory neuromuscular junction currents (EJCs) of lobster abdominal muscles. Reduced [Ca 2+] o to half of normal concentration or exposure to 40–60 μM CdCl 2, 10–20 μM NiCl 2 and 1 μM ω-conotoxin decreased EJCs by 50%. Nifedipine, Nitrendipine and Bay K-8644 were ineffective. Either Ca-blockers or reduced [Ca 2+] o, enhanced EJC suppression exerted by high pressure. The data suggest that high pressure primarily affects Ca 2+ inflow at the presynaptic terminals through N-type voltage-gated Ca-channel.

Purification and characterisation of a quisqualate sensitive glutamate binding protein.

Glutamate is the major excitatory transmitter at both the crustacean and insect neuromuscular junction [ I , 21. In the vertebrate CNS, four different types of glutamate receptor have been characterised, based on their relative affinites for specific ligands [3]. The pharmacology of the crustacean neuromuscular junction glutamate receptor is similar to one of the vertebrate receptor types, since quisqualate is a potent depolarising agent in crustacean muscle (41. By contrast NMDA has no effect [5], while kainate acts at an extra junctional receptor which is present on the muscle plasma membrane [ 6 1 .

Variation in terminal morphology and presynaptic inhibition at crustacean neuromuscular junctions

Synaptic terminals of excitatory and inhibitory neurons supplying muscle fibers in leg muscles of crabs (Pachygrapsus crassipes and Hyas areneus) were investigated with light and electron microscopy. Terminals responsible for large excitatory postsynaptic potentials (EPSPs) at low frequencies of activation had a compact configuration with clusters of terminal boutons radiating from the main axon branch. Terminals responsible for small EPSPs had a more diffuse organization, with boutons often arranged in series along thin axon branches. Inhibitory neurons, when activated, produced both presynaptic and postsynaptic inhibitory effects, with the former being more potent at low frequencies of activation. Presynaptic inhibition was variable in magnitude but was generally strong in fibers with large EPSPs. Representative terminals from regions of strong and weak presynaptic inhibition were identified by activity-dependent uptake of horseradish peroxidase, serially sectioned, and reconstructed from electron micrographs. Both regions were found to contain axo-axonal synapses from inhibitory to excitatory terminals, with a larger number in the region of strong presynaptic inhibition. In addition, axo-axonal synapses were more uniformly distributed in the latter region. The number of inhibitory presynaptic dense bars (active zones) was somewhat higher in the region of weak inhibition, but larger individual dense bars occurred in the region of strong inhibition. Possible factors contributing to the differences in strength of inhibition include: (1) morphology and electrical properties of terminals; and (2) high probability of transmission at a relatively small number of inhibitory synapses during low frequency activation in the region of strong inhibition.