Invertebrate Synapses Research Articles

GluClR-mediated inhibitory postsynaptic currents reveal targets for ivermectin and potential mechanisms of ivermectin resistance

Glutamate-gated chloride channel receptors (GluClRs) mediate inhibitory neurotransmission at invertebrate synapses and are primary targets of parasites that impact drastically on agriculture and human health. Ivermectin (IVM) is a broad-spectrum pesticide that binds and potentiates GluClR activity. Resistance to IVM is a major economic and health concern, but the molecular and synaptic mechanisms of resistance are ill-defined. Here we focus on GluClRs of the agricultural endoparasite, Haemonchus contortus. We demonstrate that IVM potentiates inhibitory input by inducing a tonic current that plateaus over 15 minutes and by enhancing post-synaptic current peak amplitude and decay times. We further demonstrate that IVM greatly enhances the active durations of single receptors. These effects are greatly attenuated when endogenous IVM-insensitive subunits are incorporated into GluClRs, suggesting a mechanism of IVM resistance that does not affect glutamate sensitivity. We discovered functional groups of IVM that contribute to tuning its potency at different isoforms and show that the dominant mode of access of IVM is via the cell membrane to the receptor.

Pseudopterosin A: Protection of Synaptic Function and Potential as a Neuromodulatory Agent.

Natural products have provided an invaluable source of inspiration in the drug discovery pipeline. The oceans are a vast source of biological and chemical diversity. Recently, this untapped resource has been gaining attention in the search for novel structures and development of new classes of therapeutic agents. Pseudopterosins are group of marine diterpene glycosides that possess an array of potent biological activities in several therapeutic areas. Few studies have examined pseudopterosin effects during cellular stress and, to our knowledge, no studies have explored their ability to protect synaptic function. The present study probes pseudopterosin A (PsA) for its neuromodulatory properties during oxidative stress using the fruit fly, Drosophila melanogaster. We demonstrate that oxidative stress rapidly reduces neuronal activity, resulting in the loss of neurotransmission at a well-characterized invertebrate synapse. PsA mitigates this effect and promotes functional tolerance during oxidative stress by prolonging synaptic transmission in a mechanism that differs from scavenging activity. Furthermore, the distribution of PsA within mammalian biological tissues following single intravenous injection was investigated using a validated bioanalytical method. Comparable exposure of PsA in the mouse brain and plasma indicated good distribution of PsA in the brain, suggesting its potential as a novel neuromodulatory agent.

Presynaptic active zones in invertebrates and vertebrates.

The regulated release of neurotransmitter occurs via the fusion of synaptic vesicles (SVs) at specialized regions of the presynaptic membrane called active zones (AZs). These regions are defined by a cytoskeletal matrix assembled at AZs (CAZ), which functions to direct SVs toward docking and fusion sites and supports their maturation into the readily releasable pool. In addition, CAZ proteins localize voltage-gated Ca(2+) channels at SV release sites, bringing the fusion machinery in close proximity to the calcium source. Proteins of the CAZ therefore ensure that vesicle fusion is temporally and spatially organized, allowing for the precise and reliable release of neurotransmitter. Importantly, AZs are highly dynamic structures, supporting presynaptic remodeling, changes in neurotransmitter release efficacy, and thus presynaptic forms of plasticity. In this review, we discuss recent advances in the study of active zones, highlighting how the CAZ molecularly defines sites of neurotransmitter release, endocytic zones, and the integrity of synapses.

Endocannabinoid-Dependent LTD in a Nociceptive Synapse Requires Activation of a Presynaptic TRPV-Like Receptor

Recent studies have found that some forms of endocannabinoid-dependent synaptic plasticity in the hippocampus are mediated through activation of transient potential receptor vanilloid (TRPV) receptors instead of cannabinoid receptors CB1 or CB2. The potential role for synaptic localization of TRPV receptors during endocannabinoid modulation of nociceptive synapses was examined in the leech CNS where it is possible to record from the same pair of neurons from one preparation to the next. Long-term depression (LTD) in the monosynaptic connection between the nociceptive (N) sensory neuron and the longitudinal (L) motor neuron was found to be endocannabinoid-dependent given that this depression was blocked by RHC-80267, an inhibitor of DAG lipase that is required for 2-arachidonoyl glycerol (2AG) synthesis. Intracellular injection of a second DAG lipase inhibitor, tetrahyrdolipstatin (THL) was also able to block this endocannabinoid-dependent LTD (ecLTD) when injected postsynaptically but not presynaptically. N-to-L ecLTD was also inhibited by the TRPV1 antagonists capsazepine and SB 366791. Bath application of 2AG or the TRPV1 agonists capsaicin and resiniferatoxin mimicked LTD and both capsaicin- and 2AG-induced depression were blocked by capsazepine. In addition, pretreatment with 2AG or capsaicin occluded subsequent expression of LTD induced by repetitive activity. Presynaptic, but not postsynaptic, intracellular injection of capsazepine blocked both activity- and 2AG-induced ecLTD, suggesting that a presynaptic TRPV-like receptor in the leech mediated this form of synaptic plasticity. These findings potentially extend the role ecLTD to nociceptive synapses and suggest that invertebrate synapses, which are thought to lack CB1/CB2 receptor orthologues, utilize a TRPV-like protein as an endocannabinoid receptor.

A Perisynaptic Ménage à Trois between Dlg,DLin-7, and Metro Controls Proper Organization of Drosophila Synaptic Junctions

Structural plasticity of synaptic junctions is a prerequisite to achieve and modulate connectivity within nervous systems, e.g., during learning and memory formation. It demands adequate backup systems that allow remodeling while retaining sufficient stability to prevent unwanted synaptic disintegration. The strength of submembranous scaffold complexes, which are fundamental to the architecture of synaptic junctions, likely constitutes a crucial determinant of synaptic stability. Postsynaptic density protein-95 (PSD-95)/ Discs-large (Dlg)-like membrane-associated guanylate kinases (DLG-MAGUKs) are principal scaffold proteins at both vertebrate and invertebrate synapses. At Drosophila larval glutamatergic neuromuscular junctions (NMJs) DlgA and DlgS97 exert pleiotropic functions, probably reflecting a few known and a number of yet-unknown binding partners. In this study we have identified Metro, a novel p55/MPP-like Drosophila MAGUK as a major binding partner of perisynaptic DlgS97 at larval NMJs. Based on homotypic LIN-2,-7 (L27) domain interactions, Metro stabilizes junctional DlgS97 in a complex with the highly conserved adaptor protein DLin-7. In a remarkably interdependent manner, Metro and DLin-7 act downstream of DlgS97 to control NMJ expansion and proper establishment of synaptic boutons. Using quantitative 3D-imaging we further demonstrate that the complex controls the size of postsynaptic glutamate receptor fields. Our findings accentuate the importance of perisynaptic scaffold complexes for synaptic stabilization and organization.

Synaptotagmin Has an Essential Function in Synaptic Vesicle Positioning for Synchronous Release in Addition to Its Role as a Calcium Sensor

A multitude of synaptic proteins interact at the active zones of nerve terminals to achieve the high temporal precision of neurotransmitter release in synchrony with action potentials. Though synaptotagmin has been recognized as the Ca2+ sensor for synchronous release, it may have additional roles of action. We address this question at the calyx of Held, a giant presynaptic terminal, that allows biophysical dissection of multiple roles of molecules in synaptic transmission. Using high-level expression recombinant adenoviruses, in conjunction with a stereotactic surgery in postnatal day 1 rats, we overcame the previous inability to molecular perturb the calyx by overexpression of a mutated synaptotagmin. We report that this mutation leaves intrinsic Ca2+ sensitivity of vesicles intact while it destabilizes the readily releasable pool of vesicles and loosens the tight coupling between Ca2+ influx and release, most likely by interfering with the correct positioning of vesicles with respect to Ca2+ channels.

Calcineurin and cytoskeleton in low‐frequency depression

Transmitter release at high probability phasic synapses of crayfish neuromuscular junctions depresses by over 50% in 60 min when stimulated at 0.2 Hz. Inhibition of the protein phosphatase calcineurin by intracellular pre-synaptic injection of autoinhibitory peptide inhibited low-frequency depression (LFD) and resulted in facilitation of transmitter release. Since this inhibitor had no major effects when injected into the post-synaptic cell, only pre-synaptic calcineurin activity is necessary for LFD. To examine changes in phosphoproteins during LFD we performed a phosphoproteomic screen on proteins extracted from motor axons and nerve terminals after LFD induction or treatment with various drugs that affect kinase and phosphatase activity. Proteins separated by PAGE were stained with phospho-specific/total protein ratio stains (Pro-Q Diamond/SYPRO Ruby) to identify protein bands for analysis by mass spectrometry. Phosphorylation of actin and tubulin decreased during LFD, but increased when calcineurin was blocked. Tubulin and phosphoactin immunoreactivity in pre-synaptic terminals were also reduced after LFD. The actin depolymerizing drugs cytochalasin and latrunculin and the microtubule stabilizer taxol inhibited LFD. Therefore, dephosphorylation of pre-synaptic actin and tubulin and consequent changes in the cytoskeleton may regulate LFD. LFD is unlike long-term depression found in mammalian synapses because the latter requires in most instances post-synaptic calcineurin activity.Thus, this simpler invertebrate synapse discloses a novel pre-synaptic depression mechanism.

An action potential‐induced and ryanodine sensitive calcium transient dynamically regulates transmitter release at synapses between Lymnaea neurons

The notion that calcium released through ryanodine receptors effects presynaptic neurotransmitter release is gaining acceptance with the observation that this calcium does indeed contribute to both action potential-evoked and spontaneous transmitter release in a variety of preparations. However, the dynamics of this calcium release and its impact on transmitter release has not yet been fully elucidated. Moreover, in contrast to vertebrate synapses, much less is known about the involvement of ryanodine receptors in the regulation of transmitter release at invertebrate synapses. In this study, we reconstructed specific synapses between individually identifiable preand postsynaptic neurons from Lymnaea to demonstrate that although ryanodine reduces the amplitude of the action potential-induced calcium transient, it does not however, alter the resting calcium level. These data suggest that action potential-induced calcium release through ryanodine receptors is fast and highly dynamic and in turn regulates transmitter release at reconstructed synapses between Lymnaea neurons. This study thus provides direct evidence that a dynamic ryanodine receptor-mediated calcium transient occurs with the presynaptic action potential.

Forskolin Induces NMDA Receptor-Dependent Potentiation at a Central Synapse in the Leech

In vertebrate hippocampal neurons, application of forskolin (an adenylyl cyclase activator) and rolipram (a phosphodiesterase inhibitor) is an effective technique for inducing chemical long-term potentiation (cLTP) that is N-methyl-d-aspartate (NMDA) receptor (NMDAR)-dependent. However, it is not known whether forskolin induces a similar potentiation in invertebrate synapses. Therefore, we examined whether forskolin plus rolipram treatment could induce potentiation at a known glutamatergic synapse in the leech (Hirudo sp.), specifically between the pressure (P) mechanosensory and anterior pagoda (AP) neurons. Perfusion of isolated ganglia with forskolin (50 muM) in conjunction with rolipram (0.1 muM) in Mg(2+)-free saline significantly potentiated the P-to-AP excitatory postsynaptic potential. Application of 2-amino-5-phosphonovaleric acid (APV, 100 muM), a competitive NMDAR antagonist, blocked the potentiation, indicating P-to-AP potentiation is NMDAR-dependent. Potentiation was blocked by injection of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA, 1 mM) into the postsynaptic cell, but not by BAPTA injection into the presynaptic neuron, indicating a requirement for postsynaptic elevation of intracellular Ca(2+). Application of db-cAMP mimicked the potentiating effects of forskolin, and Rp-cAMP, an inhibitor of protein kinase A, blocked forskolin-induced potentiation. Potentiation was also blocked by autocamtide-2-related inhibitory peptide (AIP), indicating a requirement for activation of Ca(2+)/calmodulin-dependent kinase II (CaMKII). Finally, potentiation was blocked by botulinum toxin, suggesting that trafficking of glutamate receptors also plays a role in this form of synaptic plasticity. These experiments demonstrate that techniques used to induce cLTP in vertebrate synapses also induce NMDAR-dependent potentiation in the leech CNS and that many of the cellular processes that mediate LTP are conserved between vertebrate and invertebrate phyla.

The Role of Rapid, Local, Postsynaptic Protein Synthesis in Learning-Related Synaptic Facilitation in Aplysia

The discovery that dendrites of neurons in the mammalian brain possess the capacity for protein synthesis stimulated interest in the potential role of local, postsynaptic protein synthesis in learning-related synaptic plasticity. But it remains unclear how local, postsynaptic protein synthesis actually mediates learning and memory in mammals. Accordingly, we examined whether learning in an invertebrate, the marine snail Aplysia, involves local, postsynaptic protein synthesis. Previously, we showed that the dishabituation and sensitization of the defensive withdrawal reflex in Aplysia require elevated postsynaptic Ca(2+), postsynaptic exocytosis, and functional upregulation of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors. Here, we tested whether the synaptic facilitation that underlies dishabituation and sensitization in Aplysia requires local, postsynaptic protein synthesis. We found that the facilitatory transmitter, serotonin (5-HT), enhanced the response of the motor neuron to glutamate, the sensory neuron transmitter, and this enhancement depended on rapid protein synthesis. By using individual motor neurites surgically isolated from their cell bodies, we showed that the 5-HT-dependent protein synthesis occurred locally. Finally, by blocking postsynaptic protein synthesis, we disrupted the facilitation of the sensorimotor synapse. By demonstrating its critical role in a synaptic change that underlies learning and memory in a major model invertebrate system, our study suggests that local, postsynaptic protein synthesis is of fundamental importance to the cell biology of learning.

Presynaptic current changes at the mossy fiber-granule cell synapse of cerebellum during LTP.

The involvement of presynaptic mechanisms in the expression of long-term potentiation (LTP), an enhancement of synaptic transmission suggested to take part in learning and memory in the mammalian brain, has not been fully clarified. Although evidence for enhanced vesicle cycling has been reported, it is unknown whether presynaptic terminal excitability could change as has been observed in invertebrate synapses. To address this question, we performed extracellular focal recordings in cerebellar slices. The extracellular current consisted of a pre- (P(1)/N(1)) and postsynaptic (N(2)/SN) component. In ~50% of cases, N(1) could be subdivided into N(1a) and N(1b). Whereas N(1a) was part of the fiber volley (P(1)/N(1a)), N(1b) corresponded to a Ca(2+)-dependent component accounting for 40-50% of N(1), which could be isolated from individual mossy fiber terminals visualized with fast tetramethylindocarbocyanine perchlorate (DiI). The postsynaptic response, given its timing and sensitivity to glutamate receptor antagonists [N(2) was blocked by 10 microM [1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX) and SN by 100 microM APV +50 microM 7-Cl-kyn], corresponded to granule cell excitation. N(2) and SN could be reduced by 1) Ca(2+) channel blockers, 2) decreasing the Ca(2+) to Mg(2+) ratio, 3) paired-pulse stimulation, and 4) adenosine receptor activation. However, only the first two manipulations, which modify Ca(2+) influx, were associated with N(1) (or N(1b)) reduction. LTP was induced by theta-burst mossy fiber stimulation (8 trains of 10 impulses at 100 Hz separated by 150-ms pauses). Interestingly, during LTP, both N(1) (or N(1b)) and N(2)/SN persistently increased, whereas P(1) (or P(1)/N(1a)) did not change. Average changes were N(1) = 38.1 +/- 31.9, N(2) = 49.6 +/- 48.8, and SN = 59.1 +/- 35.5%. The presynaptic changes were not observed when LTP was prevented by synaptic inhibition, by N-methyl-D-aspartate and metabotropic glutamate receptor blockage, or by protein kinase C blockage. Moreover, the presynaptic changes were sensitive to Ca(2+) channel blockers (1 mM Ni(2+) and 5 microM omega-CTx-MVIIC) and occluded by K(+) channel blockers (1 mM tetraethylammmonium). Thus regulation of presynaptic terminal excitability may take part in LTP expression at a central mammalian synapse.

Monoaminergic long-term facilitation of GABA-mediated inhibitory transmission at cerebellar synapses

Long-term facilitation of neurotransmission by monoaminergic systems is implicated in the cellular mechanism of memory and learning-related processes at invertebrate synapses. Using whole-cell recording and rat cerebellar slices, we have examined whether mammalian monoamine-containing neurons play analogous roles in synaptic plasticity, and our results suggest that serotonin and noradrenaline are critically involved in short- and long-term modulation of GABAergic transmission in the cerebellar cortex. Exogenously applied serotonin and noradrenaline selectively induced a short-term enhancement of GABAergic transmission between cerebellar interneurons and Purkinje cells, their effect subsiding in 30 min. Successive amine applications converted this effect to long-term facilitation lasting more than 2 h. During the monoamine-induced short- and long-term facilitation, spontaneously occurring miniature inhibitory synaptic responses increased in frequency, without significant changes in their mean amplitude and amplitude distribution, as well as the GABA receptor sensitivity of Purkinje cells. The actions of the two amines on the inhibitory transmission were mimicked by forskolin and blocked by kinase inhibitors, H-7, H-89 and Rp-adenosine 3′,5′-cyclic monophosphothioate. Thus, serotonin and noradrenaline are likely to activate cyclic-AMP- and protein kinase-dependent pathways in GABAergic interneurons, thereby reinforcing the inhibitory transmission on to Purkinje cells. Repetitive electrical stimulation within the molecular layer mimicked the facilitatory effect induced by exogenous monoamines: namely, neural stimulation selectively elicited long-lasting enhancement of GABAergic transmission in a manner sensitive to the monoamine receptor antagonists, methiothepin and propranolol, and an uptake inhibitor, imipramine. Synaptically released monoamines thus appear to induce cyclic-AMP- and protein kinase-dependent long-term facilitation of cerebellar GABAergic transmission, thereby providing a likely mechanism of synaptic plasticity associated with motor coordination within the mammalian cerebellar system.

Enhancement of postsynaptic serotonin-activated Cl− currents by depolarization-induced Ca2+ entry into leech neurons

Postsynaptic Ca2+ signals are a precondition for many types of synapse modification, including long-term potentiation. Here we describe a postsynaptic mechanism that augments synaptic currents at the serotonergic Retzius-P cell synapse of the leech. Depolarizations large enough to elicit Ca2+ currents evoked an increase in the amplitude of serotonin-activated Cl- currents in isolated embryonic neurons as well as at the Retzius-P cell synapse. The effect on embryonic whole cell currents lasted for up to one hour and was abolished in solutions containing Cd2+ as a Ca2+ channel blocker. Hence, Ca2+ influx into the postsynaptic neuron may be a common step regulating the plasticity of various vertebrate as well as invertebrate synapses.

Evolutionary conservation of synaptosome-associated protein 25 kDa (SNAP-25) shown by Drosophila and Torpedo cDNA clones.

The neuron-specific proteins SNAP-25 (synaptosome-associated protein 25 kDa), synaptobrevin and syntaxin, are localized to presynaptic terminals in mammals and have been found to associate with proteins involved in vesicle docking and membrane fusion. We describe here SNAP-25 cDNA clones from the fruit fly Drosophila melanogaster and the ray Torpedo marmorata. In situ hybridization showed that SNAP-25 mRNA is exclusively found in brain and ganglia in Drosophila with a pattern suggesting expression in most neurons. The Drosophila and Torpedo proteins show 61 and 81% amino acid identity to mouse SNAP-25, a degree of conservation similar to that previously reported for synaptobrevin. None of the SNAP-25 sequences has a membrane-spanning region, but all contain a cluster of cysteine residues that can be palmitoylated for membrane attachment. SNAP-25 displays sequence similarity to syntaxin A and B. These data show that SNAP-25 and synaptobrevin, which are both implicated in vesicle docking and/or membrane fusion, have both been highly conserved during evolution. This supports the existence of a basic molecular machinery for synaptic vesicle docking in vertebrate and invertebrate synapses.

72. GM-CSF directly induces neutrophil platelet activating factor (PAF) and leukotriene B4 (LTB4) synthesis: activation of the lysoPAF acetyltransferase through a tyrosine ki-nase-dependent signaling cascade

Long-term facilitation of neurotransmission by monoaminergic systems is implicated in the cellular mechanism of memory and learning-related processes at invertebrate synapses. Using whole-cell recording and rat cerebellar slices, we have examined whether mammalian monoamine-containing neurons play analogous roles in synaptic plasticity, and our results suggest that serotonin and noradrenaline are critically involved in short- and long-term modulation of GABAergic transmission in the cerebellar cortex. Exogenously applied serotonin and noradrenaline selectively induced a short-term enhancement of GABAergic transmission between cerebellar interneurons and Purkinje cells, their effect subsiding in 30 min. Successive amine applications converted this effect to long-term facilitation lasting more than 2 h. During the monoamine-induced short- and long-term facilitation, spontaneously occurring miniature inhibitory synaptic responses increased in frequency, without significant changes in their mean amplitude and amplitude distribution, as well as the GABA receptor sensitivity of Purkinje cells. The actions of the two amines on the inhibitory transmission were mimicked by forskolin and blocked by kinase inhibitors, H-7, H-89 and Rp-adenosine 3′,5′-cyclic monophosphothioate. Thus, serotonin and noradrenaline are likely to activate cyclic-AMP- and protein kinase-dependent pathways in GABAergic interneurons, thereby reinforcing the inhibitory transmission on to Purkinje cells.Repetitive electrical stimulation within the molecular layer mimicked the facilitatory effect induced by exogenous monoamines: namely, neural stimulation selectively elicited long-lasting enhancement of GABAergic transmission in a manner sensitive to the monoamine receptor antagonists, methiothepin and propranolol, and an uptake inhibitor, imipramine. Synaptically released monoamines thus appear to induce cyclic-AMP- and protein kinase-dependent long-term facilitation of cerebellar GABAergic transmission, thereby providing a likely mechanism of synaptic plasticity associated with motor coordination within the mammalian cerebellar system.

GABA and GABA receptors in invertebrates

GABA is the major inhibitory transmitter at invertebrate synapses in both the central and peripheral nervous systems. The receptors for GABA are well characterised electrophysiologically in a wide variety of invertebrate organisms but their biochemical and pharmacological profiles are less well defined. In general invertebrate GABA receptors are less sensitive to bicuculline than are vertebrate GABA A receptors. There is much evidence that invertebrate GABA A receptors have benzodiazepine modulatory sites but their pharmacological profile is distinct from that of vertebrate GABA A receptors. The insect neuronal GABA A receptor has been identified as the site of action of the cyclodiene insecticides and the avermectins may also act on these receptors. Molecular cloning experiments now in progress will reveal the detailed relationships between the invertebrate receptors and their mammalian counterparts.

Impulse activity of a crayfish motoneuron regulated its neuromuscular synaptic properties.

1. Previous studies have demonstrated that initial transmitter release, fatigability, and the morphology of identified crayfish neuromuscular synapses adapt to long-term changes in motoneuron impulse activity. 2. Experiments were performed to determine whether these long-term, adaptive alterations in neuromuscular synaptic physiology are triggered by changes in neuromuscular synaptic activity, muscle activity, or neuronal impulse activity. The fast closer excitor of the crayfish claw, a phasic motoneuron, was studied. Either the central or the peripheral region of the motoneuron was selectively stimulated in vivo by blocking impulse activity midway along the motor axon with localized application of tetrodotoxin and stimulating either central or distal to the blocked region. 3. Neither muscle activity nor transmitter release from the neuromuscular synapses was required to trigger the changes in synaptic physiology. Stimulation central to the block induced changes in neuromuscular transmission that included a long-lasting decrease in initial transmitter release and increased fatique resistance. 4. Because peripheral stimulation also produced decreased initial transmitter release, it appears that increased impulse activity in either region of the motoneuron can produce the synaptic changes. These results along with earlier findings suggest that neuronal depolarization induces adaptive, long-term changes in synapses. 5. These results are discussed in relation to findings at vertebrate and invertebrate synapses.

Glutamate regulates adenylate cyclase and guanylate cyclase activities in an isolated membrane preparation from insect muscle.

It is generally believed that glutamate serves as a neurotransmitter at many vertebrate and invertebrate synapses. There is, however, little information concerning the possible involvement of cyclic nucleotides as intracellular second messengers for any of the postsynaptic actions of glutamate. Direct activation of adenylate cyclase has been reported for several neurotransmitters and neuromodulators1, but no such effect has been reported for glutamate. Moreover, although several neurotransmitters including glutamate have been shown to increase cyclic GMP levels in intact preparations of nerve and muscle tissue2,3 apparently by promoting Ca2+ entry into the cells, no neurotransmitter has been shown to activate guanylate cyclase in a cell-free preparation. L-Glutamate is the prime candidate for the neurotransmitter at the excitatory neuromuscular synapses in insects4. Moreover, recent studies have shown that glutamate application raises both cyclic AMP5 and cyclic GMP levels (P.M.C., unpublished observations) in insect muscle. To determine the mechanism underlying these increases, we have studied the effect of glutamate in a membrane fraction prepared from this muscle, and report here that the neurotransmitter activates both adenylate cyclase and guanylate cyclase in the membrane preparation, and that the activation by glutamate of guanylate cyclase is calcium independent.

Inhibitory and excitatory effects of CNS depressants on invertebrate synapses

(1) The effects of pentobarbital were studied on the membrane properties and synaptic activity of crustacean neuromuscular junction preparations and molluscan neurons. (2) Pentobarbital selectively depressed in a dose-dependent, reversible manner the excitatory postsynaptic potentials (EPSPs) recorded at crustacean neuromuscular junctions without altering either inhibitory postsynaptic potentials (IPSPs) or postsynaptic membrane properties. (3)Pentobarbital depressed cholinergic EPSPs recorded in an identified molluscan neuron and depressed the depolarizing phase of a biphasic PSP without affecting the hyperpolarizing phase of the BPSP on the same cell. Facilitation of the EPSP was not affected. (4) Pentobarbital did not appreciably alter the reversal potentials of the EPSP and IPSP. (5) Low concentrations of pentobarbital did not alter the appearance of spontaneously occuring IPSPs, while high concentrations changed the pattern of regular IPSP input to an irregular, burst-like pattern. (6) Pentobarbital and 5 other CNS depressants (chloralose, chloroform, ethanol, and urethane) increased the excitability and altered the current-voltage relations of a cell whose membrane properties have been proposed as a model of presynaptic terminal membranes. The effects were dependent on the species of external divalent cation present. (7) The results in these invertebrate systems may provide insight into the cellular basis of the depressant and excitatory effects of these agents.

Invertebrate synapse: Long-term maintenance of postsynaptic morphology following denervation

Large, electrical, axoaxonal synapses between identified interneurons and motoneurons in the crayfish abdomen can be visualized by injecting cobalt dye into the motoneuron. The dye reveals many fine branches of the post-synaptic cell that make intimate contact with the presynaptic axon. The morphology of these processes remained essentially unchanged for at least 16 months following degeneration of the main presynaptic axon. No sign of new or increased contact with nearby axons was observed.