Synaptic vesicle glycoprotein 2A modulates vesicular release and calcium channel function at peripheral sympathetic synapses

Synaptic vesicle glycoprotein (SV)2A is a transmembrane protein found in secretory vesicles and is critical for Ca2+-dependent exocytosis in central neurons, although its mechanism of action remains uncertain. Previous studies have proposed, variously, a role of SV2 in the maintenance and formation of the readily releasable pool (RRP) or in the regulation of Ca2+ responsiveness of primed vesicles. Such previous studies have typically used genetic approaches to ablate SV2 levels; here, we used a strategy involving small interference RNA (siRNA) injection to knockdown solely presynaptic SV2A levels in rat superior cervical ganglion (SCG) neuron synapses. Moreover, we investigated the effects of SV2A knockdown on voltage-dependent Ca2+ channel (VDCC) function in SCG neurons. Thus, we extended the studies of SV2A mechanisms by investigating the effects on vesicular transmitter release and VDCC function in peripheral sympathetic neurons. We first demonstrated an siRNA-mediated SV2A knockdown. We showed that this SV2A knockdown markedly affected presynaptic function, causing an attenuated RRP size, increased paired-pulse depression and delayed RRP recovery after stimulus-dependent depletion. We further demonstrated that the SV2A–siRNA-mediated effects on vesicular release were accompanied by a reduction in VDCC current density in isolated SCG neurons. Together, our data showed that SV2A is required for correct transmitter release at sympathetic neurons. Mechanistically, we demonstrated that presynaptic SV2A: (i) acted to direct normal synaptic transmission by maintaining RRP size, (ii) had a facilitatory role in recovery from synaptic depression, and that (iii) SV2A deficits were associated with aberrant Ca2+ current density, which may contribute to the secretory phenotype in sympathetic peripheral neurons.

The role of the synthetic enzyme GAD65 in the control of neuronal γ-aminobutyric acid release

We have studied GABAergic synaptic transmission in retinal ganglion cells and hippocampal pyramidal cells to determine, at a cellular level, what is the effect of the targeted disruption of the gene encoding the synthetic enzyme GAD65 on the synaptic release of γ-aminobutyric acid (GABA). Neither the size nor the frequency of GABA-mediated spontaneous inhibitory postsynaptic currents (IPSCs) were reduced in retina or hippocampus in GAD65−/− mice. However, the release of GABA during sustained synaptic activation was substantially reduced. In the retina both electrical- and K+-induced increases in IPSC frequency were depressed without a change in IPSC amplitude. In the hippocampus the transient increase in the probability of inhibitory transmitter release associated with posttetanic potentiation was absent in the GAD65−/− mice. These results indicate that during and immediately after sustained stimulation the increase in the probability of transmitter release is not maintained in GAD65−/− mice. Such a finding suggests a decrease in the size or refilling kinetics of the releasable pool of vesicles, and various mechanisms are discussed that could account for such a defect.

Evidence for a voltage-dependent enhancement of neurotransmitter release mediated via the synaptic protein interaction site of N-type Ca2+ channels

Secretion of neurotransmitters is initiated by voltage-gated calcium influx through presynaptic, voltage-gated N-type calcium channels. These channels interact with the SNARE proteins, which are core components of the exocytosis process, via the synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their α1B subunit. Interruption of this interaction by competing synprint peptides inhibits fast, synchronous transmitter release. Here we identify a voltage-dependent, but calcium-independent, enhancement of transmitter release that is elicited by trains of action potentials in the presence of a hyperosmotic extracellular concentration of sucrose. This enhancement of transmitter release requires interaction of SNARE proteins with the synprint site. Our results provide evidence for a voltage-dependent signal that is transmitted by protein–protein interactions from the N-type calcium channel to the SNARE proteins and enhances neurotransmitter release by altering SNARE protein function.

Insulin-like growth factors-I and -II differentially regulate endogenous acetylcholine release from the rat hippocampal formation

Insulin-like growth factors-I and -II (IGF-I and -II) are structurally related mitogenic polypeptides with potent growth promoting effects. These peptides and their corresponding IGF-I and -II receptors are selectively localized in the brain. To date, most of the effects of IGFs are believed to be mediated by IGF-I receptors whereas the significance of IGF-II receptor in mediating biological responses remains unclear. In the present study, we characterized the distribution of IGF-I and IGF-II receptor sites and investigated the effects of both factors on endogenous acetylcholine (ACh) release in adult rat hippocampus. [125I]IGF-I receptor binding sites are recognized by IGF-I> IGF-II> insulin, whereas [125I]IGF-II binding was competed potently by IGF-II> IGF-I but not by insulin. At the cellular level, IGF-I receptor sites were primarily noted in the molecular layer of the dentate gyrus and the CA2-CA3 subfields of the Ammon’s horn whereas IGF-II sites were localized predominantly in the pyramidal cell layer of the CA1-CA3 subfields and in the granular cell layer of the dentate gyrus. IGF-I (10−14–10−8 M) and des(1–3) IGF-I (10−10–10−8 M) were found to inhibit whereas IGF-II (10−14–10−8 M) potentiated K+-evoked ACh release from hippocampal slices. Tetrodotoxin altered the effects of IGF-I but not those of IGF-II suggesting that IGF-I acts indirectly via the release of other modulators whereas IGF-II acts directly on or in close proximity to the cholinergic terminals. The inhibitory effects of IGF-I were also observed in the frontal cortex but not in the striatum. In contrast, the stimulatory effects of IGF-II were evident both in the frontal cortex and striatum. Taken together, these results reveal the differential localization of IGF-I and IGF-II receptor sites in the hippocampal formation and the opposite role for these growth factors in the acute regulation of ACh release likely via two distinct mechanisms. Additionally, these data provide the first evidence for a direct role for IGF-II and its receptors in the regulation of transmitter release in the central nervous system.

Role of the C2B domain of synaptotagmin in vesicular release and recycling as determined by specific antibody injection into the squid giant synapse preterminal.

Synaptotagmin (Syt) is an inositol high-polyphosphate series [IHPS inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate] binding synaptic vesicle protein. A polyclonal antibody against the C2B domain (anti-Syt-C2B), an IHPS binding site, was produced. The specificity of this antibody to the C2B domain was determined by comparing its ability to inhibit IP4 binding to the C2B domain with that to inhibit the Ca2+/phospholipid binding to the C2A domain. Injection of the anti-Syt-C2B IgG into the squid giant presynapse did not block synaptic release. Coinjection of IP4 and anti-Syt-C2B IgG failed to block transmitter release, while IP4 itself was a powerful synpatic release blocker. Repetitive stimulation to presynaptic fiber injected with anti-Syt-C2B IgG demonstrated a rapid decline of the postsynaptic response amplitude probably due to its block of synaptic vesicle recycling. Electron microscopy of the anti-Syt-C2B-injected presynapse showed a 90% reduction of the numbers of synaptic vesicles. These results, taken together, indicate that the Syt molecule is central, in synaptic vesicle fusion by Ca2+ and its regulation by IHPS, as well as in the recycling of synaptic vesicles.

Citrate modulates the regulation by Zn2+ of N-methyl-D-aspartate receptor-mediated channel current and neurotransmitter release.

The effect of the two metal-ion chelators EDTA and citrate on the action of N-methyl-D-aspartate (NMDA) receptors was investigated by use of cultured mouse cerebellar granule neurons and Xenopus oocytes, respectively, to monitor either NMDA-evoked transmitter release or membrane currents. Transmitter release from the glutamatergic neurons was determined by superfusion of the cells after preloading with the glutamate analogue D-[3H]aspartate. The oocytes were injected with mRNA isolated from mouse cerebellum and, after incubation to allow translation to occur, currents mediated by NMDA were recorded electrophysiologically by voltage clamp at a holding potential of -80 mV. It was found that citrate as well as EDTA could attenuate the inhibitory action of Zn2+ on NMDA receptor-mediated transmitter release from the neurons and membrane currents in the oocytes. These effects were specifically related to the NMDA receptor, since the NMDA receptor antagonist MK-801 abolished the action and no effects of Zn2+ and its chelators were observed when kainate was used to selectively activate non-NMDA receptors. Since it was additionally demonstrated that citrate (and EDTA) preferentially chelated Zn2+ rather than Ca2+, the present findings strongly suggest that endogenous citrate released specifically from astrocytes into the extracellular space in the brain may function as a modulator of NMDA receptor activity. This is yet another example of astrocytic influence on neuronal activity.

Calcium channels controlling acetylcholine release from preganglionic nerve terminals in rat autonomic ganglia

Little is known about the nature of the calcium channels controlling neurotransmitter release from preganglionic parasympathetic nerve fibres. In the present study, the effects of selective calcium channel antagonists and amiloride were investigated on ganglionic neurotransmission. Conventional intracellular recording and focal extracellular recording techniques were used in rat submandibular and pelvic ganglia, respectively. Excitatory postsynaptic potentials and excitatory postsynaptic currents preceded by nerve terminal impulses were recorded as a measure of acetylcholine release from parasympathetic and sympathetic preganglionic fibres following nerve stimulation. The calcium channel antagonists omega-conotoxin GVIA (N type), nifedipine and nimodipine (L type), omega-conotoxin MVIIC and omega-agatoxin IVA (P/Q type), and Ni2+ (R type) had no functional inhibitory effects on synaptic transmission in both submandibular and pelvic ganglia. The potassium-sparing diuretic, amiloride, and its analogue, dimethyl amiloride, produced a reversible and concentration-dependent inhibition of excitatory postsynaptic potential amplitude in the rat submandibular ganglion. The amplitude and frequency of spontaneous excitatory postsynaptic potentials and the sensitivity of the postsynaptic membrane to acetylcholine were unaffected by amiloride. In the rat pelvic ganglion, amiloride produced a concentration-dependent inhibition of excitatory postsynaptic currents without causing any detectable effects on the amplitude or configuration of the nerve terminal impulse. These results indicate that neurotransmitter release from preganglionic parasympathetic and sympathetic nerve terminals is resistant to inhibition by specific calcium channel antagonists of N-, L-, P/Q- and R-type calcium channels. Amiloride acts presynaptically to inhibit evoked transmitter release, but does not prevent action potential propagation in the nerve terminals, suggesting that amiloride may block the pharmacologically distinct calcium channel type(s) on rat preganglionic nerve terminals. (C) 1999 IBRO. Published by Elsevier Science Ltd.

Electrically-evoked neurotransmitter release in relation to alcoholism

The present study used electrical stimulation to distinguish the vesicular from the cytoplasmic component of transmitter release in human autopsy synaptosomes. We demonstrate that the present electrical stimulation paradigm can elicit successive release pulses from synaptosome preparation.

Long-term potentiation of synaptic transmission in the avian hippocampus

The avian hippocampus plays a pivotal role in memory required for spatial navigation and food storing. Here we have examined synaptic transmission and plasticity within the hippocampal formation of the domestic chicken using an in vitro slice preparation. With the use of sharp microelectrodes we have shown that excitatory synaptic inputs in this structure are glutamatergic and activate both NMDA-and AMPA-type receptors on the postsynaptic membrane. In response to tetanic stimulation, the EPSP displayed a robust long-term potentiation (LTP) lasting >1 hr. This LTP was unaffected by blockade of NMDA receptors or chelation of postsynaptic calcium. Application of forskolin increased the EPSP and reduced paired-pulse facilitation: (PPF), indicating an increase in release probability. In contrast, LTP was not associated with a change in the PPF ratio. Induction of LTP did not occlude the effects of forskolin. Thus, in contrast to NMDA receptor-independent LTP in the mammalian brain, LTP in the chicken hippocampus is not attributable to a change in the probability of transmitter release and does not require activation of adenylyl cyclase, These findings indicate that a novel form of synaptic plasticity might underlie learning in the avian hippocampus.

H-reflex modulation during passive lengthening and shortening of the human triceps surae

1. The present study investigated the effects of lengthening and shortening actions on IT-reflex amplitude. H-reflexes were evoked in the soleus (SOL) and medial gastroenemius (MG) of human subject, during passive isometric, lengthening and shortening actions performed at angular velocities of 0, +/-2, +/-5 and +/- 15 deg s(-1). 2. H-reflex amplitude, in froth SOL and MG were significantly depressed during passive lengthening actions and facilitated during passive shortening actions, when compared with the isometric R-reflex amplitude. 3. Four experiments were performed in which the latencies front the onset of movement to delivery of the stimulus were altered. Passive H-reflex modulation during lengthening actions was found tee begin at latencies of less than 60 ms suggesting that this inhibition was due to peripheral and/or spinal mechanisms. 4. It is postulated that, the H-reflex modulation seen in the present study is related to the tunic discharge of muscle spindle afferents and the consequent effects of transmission within the la pathway. Inhibition of the H-reflex at less than 60 ms after the onset of muscle lengthening may he attributed to several mechanisms, which cannot be distinguished using the current protocol. These may include the inability to evoke volleys in la fibres that are refractory following muscle spindle discharge during; rapid muscle lengthening, a reduced probability of transmitter release front the presynaptic terminal (homosynaptic post.-activation depression) and presynaptic inhibition of la afferents from plantar flexor agonists. Short latency facilitation of the H-reflex may be attributed to temporal summation of excitatory postsynaptic potentials arising from muscle spindle afferents during rapid muscle lengthening. At longer latencies, presynaptic inhibition of Ia afferents cannot be excluded as a potential inhibitory mechanism.

Calcium-activated potassium channels: Multiple contributions to neuronal function

Calcium-activated potassium channels are a large family of potassium channels that are found throughout the central nervous system and in many other cell types. These channels are activated by rises in cytosolic calcium largely in response to calcium influx via voltage-gated calcium channels that open during action potentials. Activation of these potassium channels is involved in the control of a number of physiological processes from the firing properties of neurons to the control of transmitter release. These channels form the target for modulation for a range of neurotransmitters and have been implicated in the pathogenesis of neurological and psychiatric disorders. Here the authors summarize the varieties of calcium-activated potassium channels present in central neurons and their defining molecular and biophysical properties.

Pre- and postsynaptic actions of ATP on neurotransmission in rat submandibular ganglia

The pre- and postsynaptic actions of exogenously applied ATP were investigated in intact and dissociated parasympathetic neurotics of rat submandibular ganglia. Nerve-evoked excitatory postsynaptic potentials (EPSPs) were not inhibited by the purinergic receptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2 ' ,4 ' -disulphonic acid (PPADS), or the desensitising agonist, alpha,beta -methylene ATP. In contrast. EPSPs were abolished by the nicotinic acetylcholine receptor antagonists, hexamethonium and mecamylamine. Focal application of ATP (100 muM) had no effect on membrane potential of the postsynaptic neurone or on the amplitude of spontaneous EPSPs. Taken together, these results suggest the absence of functional purinergic (P2) receptors on the postganglionic neurone in situ. In contrast, focally applied ATP (100 muM) reversibly inhibited nerve-evoked EPSPs. Similarly, bath application of the non-hydrolysable analogue of ATP, ATP gammaS, reversibly depressed EPSPs amplitude, The inhibitory effects of ATP and ATP gammaS on nerve-evoked transmitter release were antagonised by bath application of either PPADS or suramin, suggesting ATP activates a presynaptic P2 purinoceptor to inhibit acetylcholine release from preganglionic nerves in the submandibular ganglia. In acutely dissociated postganglionic neurotics from rat submandibular ganglia. focal application of ATP (100 LM) evoked an inward current and subsequent excitatory response and action potential firing, which was reversibly inhibited by PPADS (10 muM). The expression of P2X purinoceptors in wholemount and dissociated submandibular ganglion neurones was examined using polyclonal antibodies raised against the extracellular domain of six P2X purinoceptor subtypes (P2X(1-6)). In intact wholemount preparations, only the P2X(5) purinoceptor subtype was found to be expressed in the submandibular ganglion neurones and no P2X immunoreactivity was detected in the nerve fibres innervating the ganglion. Surprisingly, in dissociated submandibular ganglion neurones, high levels of P2X(2) and P2X(4) purinoceptors immunoreactivity were found on the cell surface. This increase in expression of P2X(2) and P2X(4) purinoceptors in dissociated submandibular neurones could explain the increased responsiveness of the neurotics to exogenous ATP. We conclude that disruption of ganglionic transmission in vivo by either nerve damage or synaptic blockade may up-regulate P2X expression or availability and alter neuronal excitability. (C) 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Stressful animal housing conditions and their potential effect on sympathetic neurotransmission in mice

Although the sympathetic nervous system (SNS) plays a major role in mediating the peripheral stress response, due consideration is not usually given to the effects of prolonged stress on the SNS. The present study examined changes in neurotransmission in the SNS after exposure of mice (BALB/c) to stressful housing conditions. Focal extracellular recording of excitatory junction currents (EJCs) was used as a relative measure of neurotransmitter release from different regions of large surface areas of the mouse vas deferens. Mice were either group housed (control), isolation housed (social deprivation), group housed in a room containing rats (rat odor stress), or isolation housed in a room containing rats (concurrent stress). Social deprivation and concurrent stressors induced an increase of 30 and 335% in EJC amplitude, respectively. The success rate of recording EJCs from sets of varicosities in the concurrent stressor group was greater compared with all other groups. The present study has shown that some common animal housing conditions act as stressors and induce significant changes in sympathetic neurotransmission.

A neuron-glia signalling network in the active brain.

Glial cells are active partners of neurons in processing information and synaptic integration. They receive coded signals from synapses and elaborate modulatory responses. The active properties of glia, including long-range signalling and regulated transmitter release, are beginning to be elucidated. Recent insights suggest that the active brain should no longer be regarded as a circuitry of neuronal contacts, but as an integrated network of interactive neurons and glia.

Glutamate exocytosis from astrocytes controls synaptic strength.

The release of transmitters from glia influences synaptic functions. The modalities and physiological functions of glial release are poorly understood. Here we show that glutamate exocytosis from astrocytes of the rat hippocampal dentate molecular layer enhances synaptic strength at excitatory synapses between perforant path afferents and granule cells. The effect is mediated by ifenprodil-sensitive NMDA ionotropic glutamate receptors and involves an increase of transmitter release at the synapse. Correspondingly, we identify NMDA receptor 2B subunits on the extrasynaptic portion of excitatory nerve terminals. The receptor distribution is spatially related to glutamate-containing synaptic-like microvesicles in the apposed astrocytic processes. This glial regulatory pathway is endogenously activated by neuronal activity-dependent stimulation of purinergic P2Y1 receptors on the astrocytes. Thus, we provide the first combined functional and ultrastructural evidence for a physiological control of synaptic activity via exocytosis of glutamate from astrocytes.