Dependent Transmitter Release Research Articles

Dynamin 1 Restrains Vesicular Release to a Subquantal Mode In Mammalian Adrenal Chromaffin Cells.

Dynamin 1 (dyn1) is required for clathrin-mediated endocytosis in most secretory (neuronal and neuroendocrine) cells. There are two modes of Ca2+-dependent catecholamine release from single dense-core vesicles: full-quantal (quantal) and subquantal in adrenal chromaffin cells, but their relative occurrences and impacts on total secretion remain unclear. To address this fundamental question in neurotransmission area using both sexes of animals, here we report the following: (1) dyn1-KO increased quantal size (QS, but not vesicle size/content) by ≥250% in dyn1-KO mice; (2) the KO-increased QS was rescued by dyn1 (but not its deficient mutant or dyn2); (3) the ratio of quantal versus subquantal events was increased by KO; (4) following a release event, more protein contents were retained in WT versus KO vesicles; and (5) the fusion pore size (dp) was increased from ≤9 to ≥9 nm by KO. Therefore, Ca2+-induced exocytosis is generally a subquantal release in sympathetic adrenal chromaffin cells, implying that neurotransmitter release is generally regulated by dynamin in neuronal cells.SIGNIFICANCE STATEMENT Ca2+-dependent neurotransmitter release from a single vesicle is the primary event in all neurotransmission, including synaptic/neuroendocrine forms. To determine whether Ca2+-dependent vesicular neurotransmitter release is "all-or-none" (quantal), we provide compelling evidence that most Ca2+-induced secretory events occur via the subquantal mode in native adrenal chromaffin cells. This subquantal release mode is promoted by dynamin 1, which is universally required for most secretory cells, including neurons and neuroendocrine cells. The present work with dyn1-KO mice further confirms that Ca2+-dependent transmitter release is mainly via subquantal mode, suggesting that subquantal release could be also important in other types of cells.

Gliotransmission: Beyond Black-and-White.

Astrocytes are highly complex cells with many emerging putative roles in brain function. Of these, gliotransmission (active information transfer from glia to neurons) has probably the widest implications on our understanding of how the brain works: do astrocytes really contribute to information processing within the neural circuitry? "Positive evidence" for this stems from work of multiple laboratories reporting many examples of modulatory chemical signaling from astrocytes to neurons in the timeframe of hundreds of milliseconds to several minutes. This signaling involves, but is not limited to, Ca2+-dependent vesicular transmitter release, and results in a variety of regulatory effects at synapses in many circuits that are abolished by preventing Ca2+ elevations or blocking exocytosis selectively in astrocytes. In striking contradiction, methodologically advanced studies by a few laboratories produced "negative evidence," triggering a heated debate on the actual existence and properties of gliotransmission. In this context, a skeptics' camp arose, eager to dismiss the whole positive evidence based on a number of assumptions behind the negative data, such as the following: (1) deleting a single Ca2+ release pathway (IP3R2) removes all the sources for Ca2+-dependent gliotransmission; (2) stimulating a transgenically expressed Gq-GPCR (MrgA1) mimics the physiological Ca2+ signaling underlying gliotransmitter release; (3) age-dependent downregulation of an endogenous GPCR (mGluR5) questions gliotransmitter release in adulthood; and (4) failure by transcriptome analysis to detect vGluts or canonical synaptic SNAREs in astrocytes proves inexistence/functional irrelevance of vesicular gliotransmitter release. We here discuss how the above assumptions are likely wrong and oversimplistic. In light of the most recent literature, we argue that gliotransmission is a more complex phenomenon than originally thought, possibly consisting of multiple forms and signaling processes, whose correct study and understanding require more sophisticated tools and finer scientific experiments than done until today. Under this perspective, the opposing camps can be reconciled and the field moved forward. Along the path, a more cautious mindset and an attitude to open discussion and mutual respect between opponent laboratories will be good companions.Dual Perspectives Companion Paper: Multiple Lines of Evidence Indicate That Gliotransmission Does Not Occur under Physiological Conditions, by Todd A. Fiacco and Ken D. McCarthy.

Mechanisms of Fractional Calcium Currents through TRPV1 Channels in Primary Sensory Neurons

TRPV1 and TRPV2 channels are important molecular sensors on the plasma membrane in mammalian physiology (temperature) and diseases (pain) in sensory neurons. These nonselective cation channels permit Ca2+, Na+ and K+ influxes simultaneously through its pores to regulate intracellular Ca2+ homeostasis and Ca2+-dependent neural transmitter release. The Ca2+ influx can be determined by the fractional Ca2+ current (Pf) through a cation channel (Burnashev et al., 1995; Schneggenburger et al., 1993; Yu et al., 2004; Zhou and Neher, 1993). Here, we report (1) Opposite Ca2+ permeability was found as determined by the classic “Goldman-Hodgkin-Katz equation” of PCa/Na (TRPV1 = 7.6 > TRPV2 = 2.8) under non-physiological intra- and extracellular solutions(Caterina et al., 1999; Caterina et al., 1997), or by the “fractional Ca2+-current” of TRPV1 vs. TRPV2 (Pf = 5.5% vs. 22%) under physiological solutions; (2) The selective filter “GMGX” is similar in TRPV1 and TRPV2, except “X” (X = D for TRPV1 and E for V2). In TRPV1, switching native D to E of TRPV2, Pf(V1, D646E) was greatly increased toward Pf(V2) (from 5.5% to 13%), and vice versa (from 22% to 5.0%); (3) Mutations of two sites outside of the “GMGX”, reduced Pf by half; (4) In native neurons replacing TRPV1-WT (Pf = 5.5%) with TRPV1-D646E (Pf = 13%), the release mode of Ca2+-dependent single vesicle events was dramatically altered from partial (kiss-and-run) to full release as determined by TIRF-imaging, implicating a physilogical relevance of Pf(TRP) studies. Taken together, TRPV1-D646 (or TRPV2-E604) is the dominant site determining fractional Ca2+-influx through thermal sensitive TRP channels—a novel mechanism of TRPV channels in presynaptic neural transmitter release for temperature and pain sensation.

Nanodomain coupling explains Ca²⁺ independence of transmitter release time course at a fast central synapse.

A puzzling property of synaptic transmission, originally established at the neuromuscular junction, is that the time course of transmitter release is independent of the extracellular Ca(2+) concentration ([Ca(2+)]o), whereas the rate of release is highly [Ca(2+)]o-dependent. Here, we examine the time course of release at inhibitory basket cell-Purkinje cell synapses and show that it is independent of [Ca(2+)]o. Modeling of Ca(2+)-dependent transmitter release suggests that the invariant time course of release critically depends on tight coupling between Ca(2+) channels and release sensors. Experiments with exogenous Ca(2+) chelators reveal that channel-sensor coupling at basket cell-Purkinje cell synapses is very tight, with a mean distance of 10-20 nm. Thus, tight channel-sensor coupling provides a mechanistic explanation for the apparent [Ca(2+)]o independence of the time course of release.

What do we know about gliotransmitter release from astrocytes?

Astrocytes participate in information processing by actively modulating synaptic properties via gliotransmitter release. Various mechanisms of astrocytic release have been reported, including release from storage organelles via exocytosis and release from the cytosol via plasma membrane ion channels and pumps. It is still not fully clear which mechanisms operate under which conditions, but some of them, being Ca2+-regulated, may be physiologically relevant. The properties of Ca2+-dependent transmitter release via exocytosis or via ion channels are different and expected to produce different extracellular transmitter concentrations over time and to have distinct functional consequences. The molecular aspects of these two release pathways are still under active investigation. Here, we discuss the existing morphological and functional evidence in support of either of them. Transgenic mouse models, specific antagonists and localization studies have provided insight into regulated exocytosis, albeit not in a systematic fashion. Even more remains to be uncovered about the details of channel-mediated release. Better functional tools and improved ultrastructural approaches are needed in order fully to define specific modalities and effects of astrocytic gliotransmitter release pathways.

An alien divalent ion reveals a major role for Ca²⁺ buffering in controlling slow transmitter release.

Ca(2+)-dependent transmitter release occurs in a fast and in a slow phase, but the differential roles of Ca(2+) buffers and Ca(2+) sensors in shaping release kinetics are still controversial. Replacing extracellular Ca(2+) by Sr(2+) causes decreased fast release but enhanced slow release at many synapses. Here, we established presynaptic Sr(2+) uncaging and made quantitative Sr(2+)- and Ca(2+)-imaging experiments at the mouse calyx of Held synapse, to reveal the interplay between Ca(2+) sensors and Ca(2+) buffers in the control of fast and slow release. We show that Sr(2+) activates the fast, Synaptotagmin-2 (Syt2) sensor for vesicle fusion with sixfold lower affinity but unchanged high cooperativity. Surprisingly, Sr(2+) also activates the slow sensor that remains in Syt2 knock-out synapses with a lower efficiency, and Sr(2+) was less efficient than Ca(2+) in the limit of low concentrations in wild-type synapses. Quantitative imaging experiments show that the buffering capacity of the nerve terminal is markedly lower for Sr(2+) than for Ca(2+) (~5-fold). This, together with an enhanced Sr(2+) permeation through presynaptic Ca(2+) channels (~2-fold), admits a drastically higher spatially averaged Sr(2+) transient compared with Ca(2+). Together, despite the lower affinity of Sr(2+) at the fast and slow sensors, the massively higher amplitudes of spatially averaged Sr(2+) transients explain the enhanced late release. This also allows us to conclude that Ca(2+) buffering normally controls late release and prevents the activation of the fast release sensor by residual Ca(2+).

Ca2+ channels and transmitter release at the active zone

Ca2+-dependent transmitter release is the most important signaling mechanism for fast information transfer between neurons. Transmitter release takes places at highly specialized active zones with sub-micrometer dimension, which contain the molecular machinery for vesicle docking and -fusion, as well as a high density of voltage-gated Ca2+ channels. In the absence of direct evidence for the ultrastructural localization of Ca2+ channels at CNS synapses, important insights into Ca2+ channel–vesicle coupling has come from functional experiments relating presynaptic Ca2+ current and transmitter release, at large and accessible synapses like the calyx of Held. First, high slope values in log–log plots of transmitter release versus presynaptic Ca2+ current indicate that multiple Ca2+ channels are involved in release control of a single vesicle. Second, release kinetics in response to step-like depolarizations revealed fast- and slowly releasable sub-pools of vesicles, FRP and SRP, which, according to the “positional” model, are distinguished by a differential proximity to Ca2+ channels. Considering recent evidence for a rapid conversion of SRP- to FRP vesicles, however, we highlight that multivesicular release events and clearance of vesicle membrane from the active zone must be taken into account when interpreting kinetic release data. We conclude that the careful kinetic analysis of transmitter release at presynaptically accessible and molecularly targeted synapses has the potential to yield important insights into the molecular physiology of transmitter release.

GluK1 inhibits calcium dependent and independent transmitter release at associational/commissural synapses in area CA3 of the hippocampus

CA3 pyramidal cells receive three main excitatory inputs: the first one is the mossy fiber input, synapsing mainly on the proximal apical dendrites. Second, entorhinal cortex cells form excitatory connections with CA3 pyramidal cells via the perforant path in the stratum lacunosum moleculare. The third input involves the ipsi-and contralateral connections, termed the associational/commissural (A/C) pathway terminating in the stratum radiatum of CA3, thus forming a feedback loop within this region. Since this excitatory recurrent synapse makes the CA3 region extremely prone to seizure development, understanding the regulation of synaptic strength of this connection is of crucial interest. Several studies suggest that kainate receptors (KAR) play a role in the regulation of synaptic strength. Our aim was to characterize the influence of KAR on A/C synaptic transmission: application of ATPA, a selective agonist of the GluK1 KAR, depressed the amplitude fEPSP without affecting the size of the fiber volley. Blockade of GABA receptors had no influence on this effect, arguing against the influence of interneuronal KARs. Pharmacological and genetic deletion studies could show that this effect was selectively due to GluK1 receptor activation. Several lines of evidence, such as PPF changes, coefficient of variance-analysis and glutamate uncaging experiments strongly argue for a presynaptic locus of suppression. This is accompanied by an ATPA-mediated reduction in Ca(2+) influx at excitatory synaptic terminals, which is most likely mediated by a G-Protein dependent mechanism, as suggested by application of pertussis toxin. Finally, analysis of miniature EPSCs in the presence and absence of extracellular Ca(2+) suggest that presynaptic KAR can also reduce transmitter release downstream and therefore independent of Ca(2+) influx.

Upregulation of synaptotagmin IV inhibits transmitter release in PC12 cells with targeted synaptotagmin I knockdown

BackgroundThe function of synaptotagmins (syt) in Ca2+-dependent transmitter release has been attributed primarily to Ca2+-dependent isoforms such as syt I. Recently, syt IV, an inducible Ca2+-independent isoform has been implicated in transmitter release. We postulated that the effects of syt IV on transmitter release are dependent on the expression of syt I.ResultsTo test this, we increased syt IV expression in PC12 cells by either upregulation with forskolin treatment or overexpression with transfection. Two separately generated stable PC12 cell lines with syt I expression abolished by RNAi targeting were used and compared to control cells. We measured catecholamine release from single vesicles by amperometry and neuropeptide Y release from populations of cells by an immunoassay. In syt I targeted cells with forskolin-induced syt IV upregulation, amperometry measurements showed a reduction in the number of release events and the total amount of transmitter molecules released per cell. In cells with syt IV overexpressed, similar amperometry results were obtained, except that the rate of expansion for full fusion was slowed. Neuropeptide Y (NPY) release from syt I knockdown cells was decreased, and overexpression of syt IV did not rescue this effect.ConclusionsThese data support an inhibitory effect of syt IV on release of vesicles and their transmitter content. The effect became more pronounced when syt I expression was abolished.

V-ATPase Membrane Sector Associates with Synaptobrevin to Modulate Neurotransmitter Release

Acidification of synaptic vesicles by the vacuolar proton ATPase is essential for loading with neurotransmitter. Debated findings have suggested that V-ATPase membrane domain (V0) also contributes to Ca(2+)-dependent transmitter release via a direct role in vesicle membrane fusion, but the underlying mechanisms remain obscure. We now report a direct interaction between V0 c-subunit and the v-SNARE synaptobrevin, constituting a molecular link between the V-ATPase and SNARE-mediated fusion. Interaction domains were mapped to the membrane-proximal domain of VAMP2 and the cytosolic 3.4 loop of c-subunit. Acute perturbation of this interaction with c-subunit 3.4 loop peptides did not affect synaptic vesicle proton pump activity, but induced a substantial decrease in neurotransmitter release probability, inhibiting glutamatergic as well as cholinergic transmission in cortical slices and cultured sympathetic neurons, respectively. Thus, V-ATPase may ensure two independent functions: proton transport by a fully assembled V-ATPase and a role in SNARE-dependent exocytosis by the V0 sector.

Mitochondria Modulate Ca2+-Dependent Glutamate Release from Rat Cortical Astrocytes

Vesicular glutamate release from astrocytes depends on mobilization of free Ca(2+) from the endoplasmic reticulum (ER), and extracellular space to elevate cytosolic Ca(2+) (Ca(2+)(cyt)). Although mitochondria in neurons, and other secretory cells, have been shown to sequester free Ca(2+) and have been implicated in the modulation of Ca(2+)-dependent transmitter release, the role of mitochondria in Ca(2+)-dependent glutamate release from astrocytes is not known. A pharmacological approach was taken to manipulate Ca(2+) accumulation in mitochondria and thereby affect Ca(2+)(cyt) of solitary astrocytes in response to mechanical stimuli. Ca(2+)(cyt) responses and levels of glutamate release were measured optically in parallel experiments using a fluorescent Ca(2+) indicator and an enzyme-linked assay, respectively. It was observed that inhibiting mitochondrial Ca(2+) accumulation is correlated to increased Ca(2+)(cyt) and glutamate release, whereas enhancing mitochondrial Ca(2+) accumulation is correlated to decreased Ca(2+)(cyt) and glutamate release. These observations suggest that, in addition to the activity of ER and plasma membrane ion channels, mitochondria modulate Ca(2+)(cyt) dynamics in astrocytes and play a role in Ca(2+)-dependent glutamate release from astrocytes.

Regulation of activity dependent transmitter release from chromaffin cells by F‐actin and non‐muscle myosin II

Adrenal chromaffin cells release catecholamines and neuropeptides upon stimulation. Both transmitter classes are packaged in the same secretory organelle. At basal firing rates, highly soluble catecholamines are released from chromaffin cells by ‘kiss and run’ fusion events. Under acute stress, elevated firing leads to full granule collapse releasing both catecholamine as well as a neuropeptide‐containing proteinacious granule core. Thus, activity dependent transmitter release is regulated by a transition in secretion mode. Here we employ electrochemical, electrophysiological and fluorescence based approaches to investigate the role of actin and myosin in regulating this transition. We show that under basal firing rates, F‐actin networks are maintained stabilizing ‘kiss and run’ fusion events. Under the same stimulation conditions, disruption of F‐actin resulted in conversion to full‐collapse exocytosis. Also, under increased cell stimulation, activation of myosin II and disruption of F‐actin is necessary to drive the full collapse of the granule. Under the same conditions stabilization of F‐actin and inhibition of myosin II resulted in a conversion to ‘kiss and run’ exocytosis. Our results demonstrate that the transition from ‘kiss and run’ to ‘full collapse’ exocytosis depends upon both F‐actin and myosin II, helping to provide essential regulation of the fusion mode and a proper stress response.This work was supported by a grant from the National Institutes of Health (NIH) 5/T/32/HL07887

Two Ca 2+-Dependent Steps Controlling Synaptic Vesicle Fusion and Replenishment at the Cerebellar Basket Cell Terminal

Cerebellar basket cells inhibit postsynaptic Purkinje cells in a rapid and precise manner. To investigate the mechanisms of transmitter release underlying this rapid inhibition, Ca(2+) uncaging was employed to measure the intracellular Ca(2+) dependence of transmitter release and the kinetics of synaptic vesicle pool transitions in immature basket cell synapses at room temperature. Vesicle release properties distinct from those previously observed at excitatory synapses were seen, including a relatively high intracellular Ca(2+) sensitivity of vesicle fusion, rapid vesicle pool mobilization with few reluctant vesicles, and vesicle replenishment driven by unusually high Ca(2+) levels from both local and residual Ca(2+) sources during action potential trains. These results suggest that inhibitory basket cell synapses are optimized for rapid and precise temporal and spatial Ca(2+) coordination of synaptic vesicle fusion and replenishment, which may contribute to the unique physiology of inhibitory synaptic transmission, including phasic release during action potential trains and tonic release by residual intracellular Ca(2+).

Anandamide and NADA bi‐directionally modulate presynaptic Ca2+ levels and transmitter release in the hippocampus

Inhibitory CB(1) cannabinoid receptors and excitatory TRPV(1) vanilloid receptors are abundant in the hippocampus. We tested if two known hybrid endocannabinoid/endovanilloid substances, N-arachidonoyl-dopamine (NADA) and anandamide (AEA), presynapticaly increased or decreased intracellular calcium level ([Ca(2+)](i)) and GABA and glutamate release in the hippocampus. Resting and K(+)-evoked levels of [Ca(2+)](i) and the release of [(3)H]GABA and [(3)H]glutamate were measured in rat hippocampal nerve terminals. NADA and AEA per se triggered a rise of [Ca(2+)](i) and the release of both transmitters in a concentration- and external Ca(2+)-dependent fashion, but independently of TRPV(1), CB(1), CB(2), or dopamine receptors, arachidonate-regulated Ca(2+)-currents, intracellular Ca(2+) stores, and fatty acid metabolism. AEA was recently reported to block TASK-3 potassium channels thereby depolarizing membranes. Common inhibitors of TASK-3, Zn(2+), Ruthenium Red, and low pH mimicked the excitatory effects of AEA and NADA, suggesting that their effects on [Ca(2+)](i) and transmitter levels may be attributable to membrane depolarization upon TASK-3 blockade. The K(+)-evoked Ca(2+) entry and Ca(2+)-dependent transmitter release were inhibited by nanomolar concentrations of the CB(1) receptor agonist WIN55212-2; this action was sensitive to the selective CB(1) receptor antagonist AM251. However, in the low micromolar range, WIN55212-2, NADA and AEA inhibited the K(+)-evoked Ca(2+) entry and transmitter release independently of CB(1) receptors, possibly through direct Ca(2+) channel blockade. We report here for hybrid endocannabinoid/endovanilloid ligands novel dual functions which were qualitatively similar to activation of CB(1) or TRPV(1) receptors, but were mediated through interactions with different targets.

Vesicular transmitter release from astrocytes

Astrocytes can release a variety of transmitters, including glutamate and ATP, in response to stimuli that induce increases in intracellular Ca(2+) levels. This release occurs via a regulated, exocytotic pathway. As evidence of this, astrocytes express protein components of the vesicular secretory apparatus, including synaptobrevin 2, syntaxin, and SNAP-23. Additionally, astrocytes possess vesicular organelles, the essential morphological elements required for regulated Ca(2+)-dependent transmitter release. The location of specific exocytotic sites on these cells, however, remains to be unequivocally determined.

Dual roles of the C2B domain of synaptotagmin I in synchronizing Ca2+-dependent neurotransmitter release.

Although the vesicular protein synaptotagmin I contains two Ca2+-binding domains (C2A and C2B), Ca2+ binding to the C2B domain is more important for triggering synchronous neurotransmitter release. We have used point mutagenesis to determine the functional contributions of the five negatively charged aspartate (Asp) residues that constitute the Ca2+-binding sites in the C2B domain of synaptotagmin I. Transfecting wild-type synaptotagmin I DNA into cultured hippocampal neurons from synaptotagmin I knock-out mice rescued Ca2+-dependent synchronous transmitter release and reduced a slower, asynchronous component of release, indicating that synaptotagmin I suppresses asynchronous release. Mutating either the second or third Asp residues of the C2B domain potently inhibited the ability of synaptotagmin I to rescue synchronous release but did not change its ability to suppress asynchronous release. Synaptotagmin I with mutations in the first or fourth Asp residues of the C2B domain partially rescued synchronous release and partially suppressed asynchronous release, whereas neutralizing the fifth Asp residue had no effect on the ability of synaptotagmin I to rescue transmitter release. Thus, we conclude that the C2B domain of synaptotagmin I regulates neurotransmitter release in at least two ways. Synchronous release absolutely requires binding of Ca2+ to the second and third Asp residues in this domain. For the suppression of asynchronous release, Ca2+ binding to the C2B domain of synaptotagmin I apparently is not necessary because mutation of the second Asp residue inhibits Ca2+ binding, yet still allows this protein to suppress asynchronous release.

Suppression of epileptiform activity by GABA B receptors in wild type and weaver hippocampus ‘in vitro’

Inhibition by GABA B receptors comprises activation of K + conductance and inhibition of Ca 2+ conductance, thereby reducing action potential dependent transmitter release and silencing neuronal activity. We compared epileptiform activity and its inhibition by the activation of GABA B receptors in homozygous weaver ( wv/wv) and wild type (+/+) CA3 neurons disinhibited by GABA A receptor blockade. In wv/wv mice GABA B receptors have lost their ability to activate K + conductance (J. Neurosci. 18 (1998) 4001). Spontaneous synchronous burst discharges in elevated [K +] o displayed only subtle differences in +/+ and wv/wv slices, except that the GABA B receptor agonist R-baclofen in low concentration (0.1 μM) strongly reduced the frequency of synchronous bursts in +/+ CA3 neurons, but not in wv/wv CA3 neurons. A high affinity GABA B antagonist, CGP55845A (0.5 μM) promoted the incidence of bursts in low [K +] o. Concentration dependence of the reduction of evoked EPSCs was identical in wv/wv and +/+ neurons (IC 50=0.3 μM). Amplitudes of evoked IPSCs were reduced by 0.01 μM R-baclofen in +/+, but not in wv/wv CA3 neurons. The effect of the low concentration was abolished by Ba 2+, which is known to block Kir conductance. The data suggest that activation of Kir conductance is important for the control of GABA release by GABA B autoreceptors in the CA3 network. We conclude that the loss of a contribution of Kir conductance to GABA B receptor-mediated autoinhibition reduces the inclination towards spontaneous bursts of wv/wv CA3 pyramidal neurons.

Beta-Agkistrodotoxin inhibition of Ca2+-dependent release of glutamate, aspartate, glycine and gamma-aminobutyric acid from cerebrocortical synaptosomes following its binding to synaptic membrane.

The binding properties of beta-AgTX, a snake pre-synaptic toxin, membranes and its effect on transmitter release from cerebrocortical synaptosomes were investigated. Assay of [125I]-beta-AgTX binding to rat synaptic membrane revealed a high affinity binding site for the toxin within the synaptic membrane. Preincubation with beta-AgTX inhibited K+-evoked Ca2+-dependent glutamate release from synaptosomes in a concentration-dependent manner, as determined by an on-line enzyme-linked fluorometric assay. The toxin also blocked the Ca2+-dependent release of other transmitters, aspartate, glycine, and GABA induced by K+-depolarization. However, Ca2+-ionophore, ionomycin-stimulated Ca2+-dependent transmitter release was not significantly affected by beta-AgTX, indicating that the toxin inhibits transmitter release by reducing the entry of Ca2+ into cytoplasm. It is suggested that beta-AgTX-binding site in synaptic membrane is related to the release of a variety of transmitters.

Barbiturates inhibit K +-evoked noradrenaline and dopamine release from rat striatal slices – involvement of voltage sensitive Ca 2+ channels

The cellular target site(s) for anaesthetic action remain unclear. In rat striatal slices we have previously demonstrated that K +-evoked noradrenaline (NA) and dopamine (DA) release is mediated predominantly via P/Q-type voltage sensitive Ca 2+ channels (VSCC). Using this model of Ca 2+ dependent transmitter release we have evaluated the effects of anaesthetic and non-anaesthetic barbiturates. Rat brain striatal slices were incubated in the absence and presence of barbiturate for 10 min at 37°C. The slices were then incubated for 6 min with 40 mM KCl. All anaesthetic barbiturates produced a concentration-dependent inhibition of K +-evoked NA and DA release. Non-anaesthetic barbiturate, barbituric acid was ineffective. The pIC 50 for NA and DA release (thiopental: 4.90±0.13 and 5.00±0.10, pentobarbital: 4.39±0.07 and 4.43±0.14, phenobarbital: 3.85±0.08 and 3.59±0.10, respectively) correlated with lipid solubility (NA: r 2=0.999, DA: r 2=0.987). We therefore suggest that barbiturates inhibit catecholamine release via an interaction with P/Q VSCC further implicating this channel in anaesthetic action.

Cu 2+ induces Ca 2+-dependent neurotransmitter release from brain catecholaminergic nerve terminals

CuCl 2, ZnCl 2 and NiCl 2, but not CdCl 2 or CoCl 2, induced transmitter release from superfused rat hippocampal and striatal synaptosomes preloaded with, respectively, [ 3 H ]noradrenaline and [ 3 H ]dopamine. Cu 2+ was the most potent and effective, acting in a concentration- (0.1–300 μM) and time-dependent (peak effect occurring at 2–3 min) manner. The amount of Cu 2+-induced release over a 5 min period is similar to that induced by depolarization with high KCl or the K + channel blocker 4-aminopyridine. However, the time course of the Cu 2+-induced release is slower and the effect of Cu 2+ is not reversed by washout. Cu 2+-induced catecholamine release requires extracellular calcium (Ca 2+) and is inhibited by the Ca 2+ channel blocker Cd 2+, and in the case of noradrenaline, by the voltage-gated Na + channel blocker tetrodotoxin. The ability of Cu 2+ to induce massive Ca 2+-dependent transmitter release from brain catecholaminergic nerve terminals may contribute to the neuropathological processes associated with Cu 2+ toxicity in Wilson's disease.