GABA inactivation at the crayfish neuromuscular junction

Changes in the effective membrane resistance of the abductor muscle of the dactylopodite of the crayfish were used to indicate changes in the GABA concentration in the synaptic cleft. Following bath application of GABA (10^?5 to 5 × 10^?5M), the muscle membrane resistance decreased and then increased slowly over the next few minutes. Renewing the solution or stirring the bath restored the GABA effect. Higher GABA concentrations produced a large stable decrease in membrane resistance. An active uptake system for GABA in the junctional region is suggested by the observation that the slow increase in membrane resistance following GABA application was decreased by cooling to 2°C or by the addition of known GABA uptake blockers such as L -DABA, β-guanidinopropionic acid, or nipecotic acid. The transport inhibitors, PCMBS and chlorpromazine, produced irreversible decreases in muscle membrane resistance, which precluded examining their effects on GABA inactivation. The decrease in GABA effect was not dependent on the external sodium concentration or on the degree of receptor activation. Nipecotic acid, which blocked GABA inactivation, did not affect the decay of the neurally evoked inhibitory junctional potential.     

Release of 3H-gamma-aminobutyric acid (GABA) by inhibitory neurons of the crayfish

Inhibitory neurons innervating the muscle receptor organ (MRO) of crayfish were used to study the uptake and release of tritiated GABA. MROs that have been directly exposed to ³H GABA for 60–75 min release radioactivity during low-frequency electrical stimulation. When ganglia containing the inhibitory cell bodies are exposed to ³H GABA, the isotope travels along a pathway unique to the inhibitory axon, at rates that range between 160 and 240 mm per day. Electrical stimulation of inhibitory axons whose cell bodies have been exposed to ³H GABA for 4–5 hr produces release of isotope from isolated MROs. Low calcium, high magnesium exposure prevents the stimulus-dependent release of radioactivity. Thin layer chromatographic analyses indicate that GABA comprises at least a major fraction of the radioactivity collected from stimulated preparations. A number of unidentified radioactive compounds are usually present with GABA, and it is suggested that most of these are catabolites of GABA.     

GABA and neuroligin signaling: linking synaptic activity and adhesion in inhibitory synapse development

GABA-mediated synaptic inhibition is crucial in neural circuit operations. In mammalian brains, the development of inhibitory synapses and innervation patterns is often a prolonged postnatal process, regulated by neural activity. Emerging evidence indicates that gamma-aminobutyric acid (GABA) acts beyond inhibitory transmission and regulates inhibitory synapse development. Indeed, GABA(A) receptors not only function as chloride channels that regulate membrane voltage and conductance but also play structural roles in synapse maturation and stabilization. The link from GABA(A) receptors to postsynaptic and presynaptic adhesion is probably mediated, partly by neuroligin-reurexin interactions, which are potent in promoting GABAergic synapse formation. Therefore, similar to glutamate signaling at excitatory synapse, GABA signaling may coordinate maturation of presynaptic and postsynaptic sites at inhibitory synapses. Defining the many steps from GABA signaling to receptor trafficking/stability and neuroligin function will provide further mechanistic insights into activity-dependent development and possibly plasticity of inhibitory synapses.     

Intracellular pH of crab and crayfish muscle fibre types during GABA application and inhibitory nerve stimulation

The inhibitory motoneurons of crustaceans form synapses both with the sarcolemma of muscle fibres and with the very distal branchings of the excitatory motoneurons. The transmitter of these synapses is GABA (γ-aminobutyric acid) which is known to open Cl⁻ channels. Studies on the dactyl opener muscle of crayfish suggest that application of GABA not only leads to an increase in the Cl⁻ permeability but also to a considerable HCO⁻ ₃ conductance that causes an intracellular acidification. To investigate possible physiological implications, we measured the intracellular pH of various muscle fibre types of crayfish and crab using pH-sensitive microelectrodes. Independent of the presence or absence of inhibitory innervation, bath application of 10⁻⁵ mol l⁻¹ GABA led to acidification in all fibre types (pH change: 0.14 ± 0.08, n=11). In no preparation was a change in intracellular pH observed upon stimulation of specific or common inhibitory motoneurons with 10–40 pulses s⁻¹ for 2–5 min. The results suggest that HCO⁻ ₃ conductance cannot be activated through synaptic GABA receptors. However, all crustacean muscle fibre types seem to possess extrasynaptic GABA-sensitive channels that exhibit a considerable HCO⁻ ₃ conductance. The physiological importance of these channels remains to be elucidated. Accepted: 13 July 2000     

Compartmentation and regulation of acetylcholine synthesis at the synapse.

Acetylcholine and choline release was measured by using an automated and modified version of the chemiluminescence technique of Israel & Lesbats [(1981) Neurochem. Int. 3, 81-90]. A comparison of acetylcholine and choline release from synaptosomes demonstrated that acetylcholine release was K+-stimulated and inhibited by the Ca2+ ionophore A23187 and cyanide. Choline release, however, did not vary markedly under different conditions, suggesting that it is not associated with acetylcholine release at the nerve ending. Total acetylcholine synthesis in synaptosomal preparations was measured concurrently with the incorporation of [14C]acetyl and [3H]choline moieties by using the chemiluminescence method. Under sub-optimal glucose concentrations or in the absence of treatment of the synaptosomes with the acetylcholinesterase inhibitor phospholine, the incorporation of radioactivity exceeded total synthesis, indicating that cycling between acetylcholine and its precursors may occur. After treatment with phospholine, acetyl-group incorporation from D-[U-14C]glucose occurred without dilution of the precursor at optimal (1.0 mM) and low (0.1 mM) glucose concentrations; however, at very low (0.01 mM) glucose concentrations, dilution by a small endogenous pool occurred. [14C]Acetyl incorporation into acetylcholine was compared with various metabolic parameters. A closer correlation was observed between [14C]acetyl-group incorporation into acetylcholine and the calculated acetyl-carrier efflux from the mitochondria than with the calculated pyruvate-dehydrogenase-complex flux. The results are discussed with respect to the regulation of acetylcholine concentrations at the synapse and the mechanism whereby turnover occurs.     

Exclusion of lipid rafts and decreased mobility of CD94/NKG2A receptors at the inhibitory NK cell synapse

CD94/NKG2A is an inhibitory receptor expressed by most human natural killer (NK) cells and a subset of T cells that recognizes human leukocyte antigen E (HLA-E) on potential target cells. To elucidate the cell surface dynamics of CD94/NKG2A receptors, we have expressed CD94/NKG2A-EGFP receptors in the rat basophilic leukemia (RBL) cell line. Photobleaching experiments revealed that CD94/NKG2A-EGFP receptors move freely within the plasma membrane and accumulate at the site of contact with ligand. The enriched CD94/NKG2A-EGFP is markedly less mobile than the nonligated receptor. We observed that not only are lipid rafts not required for receptor polarization, they are excluded from the site of receptor contact with the ligand. Furthermore, the lipid raft patches normally observed at the sites where FcepsilonR1 activation receptors are cross-linked were not observed when CD94/NKG2A was coengaged along with the activation receptor. These results suggest that immobilization of the CD94/NKG2A receptors at ligation sites not only promote sustenance of the inhibitory signal, but by lipid rafts exclusion prevent formation of activation signaling complexes.     

Evidence against the presence of NMDA receptors at a central glutamatergic synapse in leeches

The N-methyl-D-aspartate (NMDA) receptor, able to detect the coincidence of pre- and postsynaptic events, is considered to be the molecular analogue of associative learning. Associative learning is well known in leeches, particularly for reflexive shortening. The neuronal circuits underlying shortening have been documented and include neurons that release glutamate. Is this type of learning in leeches also mediated by NMDA receptors? The synapse between the P sensory neuron and the motoneuron-like AP cell was examined and: (1) NMDA failed to elicit a response in the AP cell, (2) the NMDA receptor antagonist 2-amino-5-phosphopentanoic acid affected synaptic transmission only at high, non-specific levels, and (3) the antagonist for the glycine-binding site 7-chloro-kynurenic acid at 20 μM did not inhibit transmission. Therefore, there are evidently no NMDA receptors at the P to AP synapse, suggesting other mechanisms of associative learning in leeches. Electronic Publication     

A novel presynaptic inhibitory mechanism underlies paired pulse depression at a fast central synapse.

Several distinct mechanisms may cause synaptic depression, a common form of short-term synaptic plasticity. These include postsynaptic receptor desensitization, presynaptic depletion of releasable vesicles, or other presynaptic mechanisms depressing vesicle release. At the endbulb of Held, a fast central calyceal synapse in the auditory pathway, cyclothiazide (CTZ) abolished marked paired pulse depression (PPD) by acting presynaptically to enhance transmitter release, rather than by blocking postsynaptic receptor desensitization. PPD and its response to CTZ were not altered by prior depletion of the releasable vesicle pool but were blocked by lowering external calcium concentration, while raising external calcium enhanced PPD. We conclude that a major component of PPD at the endbulb is due to a novel, transient depression of release, which is dependent on the level of presynaptic calcium entry and is CTZ sensitive.     

Spillover of glutamate onto synaptic AMPA receptors enhances fast transmission at a cerebellar synapse

Diffusion of glutamate from the synaptic cleft can activate high-affinity receptors, but is not thought to contribute to fast AMPA receptor-mediated transmission. Here, we show that single AMPA receptor EPSCs at the cerebellar mossy fiber-granule cell connection are mediated by both direct release of glutamate and rapid diffusion of glutamate from neighboring synapses. Immunogold localization revealed that AMPA receptors are located exclusively in postsynaptic densities, indicating that spillover of glutamate occurs between synaptic contacts. Spillover currents contributed half the synaptic charge and exhibited little trial-to-trial variability. We propose that spillover of glutamate improves transmission efficacy by both increasing the amplitude and duration of the EPSP and reducing fluctuations arising from the probabilistic nature of transmitter release.     

Magnesium ion exerts a central role in the regulation of inhibitory adenosine receptors.

Guanine nucleotides and Mg2+ differentially regulate agonist binding to adenosine (Ri) receptors in fat-cell plasma membranes. GTP alone decreases binding of the agonist ligand [3H]N6-cyclohexyladenosine (CHA) by increasing the dissociation constant (Kd). Mg2+ alone also decreases [3H]CHA binding, which is associated with a decrease in the number of receptors and in the dissociation constant. In the presence of Mg2+, the effect of GTP is to increase [3H]CHA binding by increasing the total number of receptors. It thus appears that Mg2+ acts specifically at a bivalent-cation site which, with GTP, regulates agonist binding. This putative Mg site is highly sensitive to alkylating agents. Mild treatment with N-ethylmaleimide (NEM) abolishes the characteristic GTP effect on agonist binding in the presence of Mg2+. In addition, the effect of Mg2+ alone is also eliminated. The effect of GTP alone is largely unaltered. Studies of the adenylate cyclase activity indicate that this NEM treatment also abolishes the inhibition of basal activity by adenosine analogues, whereas guanylyl imidodiphosphate inhibition of forskolin-stimulated activity is only slightly impaired at this NEM concentration. These observations indicate that a Mg2+ 'site' or 'component' is required for the integration of receptor (Ri) occupancy with regulation of catalytic activity (C). The regulatory role of Mg2+ is more demonstrable in receptor-GTP-regulatory-protein (Ri-Ni) interactions than in GTP-regulatory-protein-catalytic-unit (Ni-C) interactions.     

Cyclic AMP-regulated GABA release at inhibitory synapses in rat cerebellar slices

We have investigated the effects of agents interfering with the cAMP pathway on the rate of miniature IPSCs in cerebellar slices. Noradrenaline and group II glutamate metabotropic receptor agonists respectively enhance and reduce the rate of miniature IPSCs, presumably because they respectively increase and decrease the presynaptic concentration of cAMP.     

Signal transduction at a protein synapse

Contraction of the bacteriophage T4 tail in the act of host cell penetration represents a massive structural change powered by conformational free energy. A paper in this issue of Cell by compares cryo-electron microscopic reconstructions of the initial and final states and reveals that the basic underlying mechanism is concerted rigid-body movements of the constituent protein subunits, akin to the tumbling of gears in a lock.     

Glycinergic transmission shaped by the corelease of GABA in a mammalian auditory synapse

The firing pattern of neurons is shaped by the convergence of excitation and inhibition, each with finely tuned magnitude and duration. In an auditory brainstem nucleus, glycinergic inhibition features fast decay kinetics, the mechanism of which is unknown. By applying glycine to native or recombinant glycine receptors, we show that response decay times are accelerated by addition of GABA, a weak partial agonist of glycine receptors. Systematic variation in agonist exposure time revealed that fast synaptic time course may be achieved with submillisecond exposures to mixtures of glycine and GABA at physiological concentrations. Accordingly, presynaptic terminals generally contained both transmitters, and depleting terminals of GABA slowed glycinergic synaptic currents. Thus, coreleased GABA accelerates glycinergic transmission by acting directly on glycine receptors, narrowing the time window for effective inhibition. Packaging both weak and strong agonists in vesicles may be a general means by which presynaptic neurons regulate the duration of postsynaptic responses.     

Suppression of inhibitory synaptic potentiation by presynaptic activity through postsynaptic GABA(B) receptors in a Purkinje neuron

At inhibitory synapses on a cerebellar Purkinje neuron, the depolarization caused by heterosynaptic climbing fiber activation induces long-lasting potentiation accompanied by an increase in GABA(A) receptor responsiveness. Here we show that activation of a presynaptic inhibitory interneuron during the conditioning postsynaptic depolarization suppresses the potentiation. The suppression is due to postsynaptic GABA(B) receptor activation by GABA released from presynaptic terminals. The results suggest that GABA(B) receptor activation decreases the activity of cAMP-dependent protein kinase through the G(i)/G(o) proteins. The presynaptic activity-dependent suppression of synaptic plasticity is a novel regulatory mechanism of synaptic efficacy at individual synapses and may contribute to the learning and computational ability of the cerebellar cortex.     

High-resolution quantitative visualization of glutamate and GABA receptors at central synapses

Glutamate and GABA are the main transmitters in the central nervous system and their effects are mediated by ionotropic and metabotropic receptors. Immunogold electron microscopy has revealed the quantitative localization of these receptors at 20-30nm resolution. SDS-digested freeze-fracture replica labeling (SDS-FRL), a newly developed immunogold method, provides an accurate estimate of molecule numbers. Here, we summarize the recent advances in quantitative receptor localization, including use of SDS-FRL analyses to determine numbers of AMPA-type glutamate receptors in the cerebellum. The two-dimensional view and high sensitivity of SDS-FRL have revealed small, irregularly shaped AMPA receptor clusters within cerebellar synapses.