Increase In Spontaneous Release Research Articles

Presynaptic store-operated Ca2+ entry drives excitatory spontaneous neurotransmission and augments endoplasmic reticulum stress

Store-operated calcium entry (SOCE) is activated by depletion of Ca2+ from the endoplasmic reticulum (ER) and mediated by stromal interaction molecule (STIM) proteins. Here, we show that in rat and mouse hippocampal neurons, acute ER Ca2+ depletion increases presynaptic Ca2+ levels and glutamate release through a pathway dependent on STIM2 and the synaptic Ca2+ sensor synaptotagmin-7 (syt7). In contrast, synaptotagmin-1 (syt1) can suppress SOCE-mediated spontaneous release, and STIM2 is required for the increase in spontaneous release seen during syt1 loss of function. We also demonstrate that chronic ER stress activates the same pathway leading to syt7-dependent potentiation of spontaneous glutamate release. During ER stress, inhibition of SOCE or syt7-driven fusion partially restored basal neurotransmission and decreased expression of pro-apoptotic markers, indicating that these processes participate in the amplification of ER-stress-related damage. Taken together, we propose that presynaptic SOCE links ER stress and augmented spontaneous neurotransmission, which may, in turn, facilitate neurodegeneration.

Synaptic vesicle pool-specific modification of neurotransmitter release by intravesicular free radical generation.

Synaptic transmission is mediated by the release of neurotransmitters from synaptic vesicles in response to stimulation or through the spontaneous fusion of a synaptic vesicle with the presynaptic plasma membrane. There is growing evidence that synaptic vesicles undergoing spontaneous fusion versus those fusing in response to stimuli are functionally distinct. In this study, we acutely probe the effects of intravesicular free radical generation on synaptic vesicles that fuse spontaneously or in response to stimuli. By targeting vesicles that preferentially release spontaneously, we can dissociate the effects of intravesicular free radical generation on spontaneous neurotransmission from evoked neurotransmission and vice versa. Taken together, these results further advance our knowledge of the synapse and the nature of the different synaptic vesicle pools mediating neurotransmission. Earlier studies suggest that spontaneous and evoked neurotransmitter release processes are maintained by synaptic vesicles which are segregated into functionally distinct pools. However, direct interrogation of the link between this putative synaptic vesicle pool heterogeneity and neurotransmission has been difficult. To examine this link, we tagged vesicles with horseradish peroxidase (HRP) - a haem-containing plant enzyme - or antibodies against synaptotagmin-1 (syt1). Filling recycling vesicles in hippocampal neurons with HRP and subsequent treatment with hydrogen peroxide (H2 O2 ) modified the properties of neurotransmitter release depending on the route of HRP uptake. While strong depolarization-induced uptake of HRP suppressed evoked release and augmented spontaneous release, HRP uptake during mild activity selectively impaired evoked release, whereas HRP uptake at rest solely potentiated spontaneous release. Expression of a luminal HRP-tagged syt1 construct and subsequent H2 O2 application resulted in a similar increase in spontaneous release and suppression as well as desynchronization of evoked release, recapitulating the canonical syt1 loss-of-function phenotype. An antibody targeting the luminal domain of syt1, on the other hand, showed that augmentation of spontaneous release and suppression of evoked release phenotypes are dissociable depending on whether the antibody uptake occurred at rest or during depolarization. Taken together, these findings indicate that vesicles that maintain spontaneous and evoked neurotransmitter release preserve their identity during recycling and syt1 function in suppression of spontaneous neurotransmission can be acutely dissociated from syt1 function to synchronize synaptic vesicle exocytosis upon stimulation.

Spontaneous l-glutamate release enhancement in rat substantia gelatinosa neurons by (−)-carvone and (+)-carvone which activate different types of TRP channel

Transient receptor potential (TRP) channels in the spinal dorsal horn lamina II (substantia gelatinosa; SG), which are involved in the modulation of nociceptive transmission, have not yet been fully examined in property. Activation of the TRP channels by various plant-derived chemicals results in an increase in the spontaneous release of l-glutamate onto the SG neurons. We examined the effects of a monoterpene ketone (−)-carvone (contained in spearmint) and its stereoisomer (+)-carvone (in caraway) on glutamatergic spontaneous excitatory transmission in SG neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. (−)-Carvone and (+)-carvone increased the frequency of spontaneous excitatory postsynaptic current (sEPSC) in a reversible and concentration-dependent manner with a small increase in its amplitude. Half-maximal effective concentrations of (−)-carvone and (+)-carvone in increasing sEPSC frequency were 0.70 mM and 0.72 mM, respectively. The (−)-carvone but not (+)-carvone activity was inhibited by a TRPV1 antagonist capsazepine. On the other hand, the (+)-carvone but not (−)-carvone activity was inhibited by a TRPA1 antagonist HC-030031. These results indicate that (−)-carvone and (+)-carvone activate TRPV1 and TRPA1 channels, respectively, resulting in an increase in spontaneous l-glutamate release onto SG neurons, with almost the same efficacy. Such a difference in TRP activation between the stereoisomers may serve to know the properties of TRP channels in the SG.

Differential Effects of Potassium Channel Blockers on Dopamine Release from Rat Striatal Slices

The effects of different potassium channel blockers on tritiated dopamine [( 3H]DA) release were investigated in rat striatal slices in the presence of pargyline and nomifensine (10 microM each). 4-Aminopyridine (4-AP; 10 and 30 microM) and 3,4-diaminopyridine (3,4-DAP; 30 microM) markedly increased the basal tritium outflow, whereas tetraethylammonium (TEA; 100-1000 microM) was without effect. The facilitating effect of 4-AP (10 microM) on spontaneous release was Ca(2+)- and K(+)-dependent. Moreover, the 4-AP-induced increase in spontaneous release was abolished in the presence of tetrodotoxin, indicating that voltage-dependent Na+ channels were involved in the release mechanism. 4-AP (10 and 30 microM) induced a dose-dependent decrease in K(+)-evoked [3H]DA release. This effect was confirmed with 3,4-DAP (30 microM). When striatal slices were depolarized with veratridine (5 microM), these two aminopyridines increased the evoked release of [3H]DA. TEA increased both K(+)- and veratridine-evoked [3H]DA release. These biochemical results are consistent with electrophysiological differences between the mechanism of action of aminopyridines and that of TEA.

CD4+CD25+ T regulatory cells do not transfer oral tolerance to peanut allergens in a mouse model of peanut allergy

Recent studies have implicated CD4(+) CD25(+) regulatory T cells (nTregs) in the maintenance of tolerance to oral antigens and in the regulation of the food allergic IgE response. The objective was to assess if nTregs can transfer allergen-specific oral tolerance to naïve, non-TCR transgenic mice and regulate peanut extract (PE)-specific hypersensitivity responses. Additionally, the role of the regulatory cytokines IL-10 and TGF-β in the modulation of peanut-allergic sensitization was studied. CD25-enriched T cells from PE-tolerant mice were adoptively transferred to recipient mice, which were subsequently sensitized to PE. Depletion of CD25(+) cells and neutralization of IL-10 and TGF-β were compared in a CH3/HeOuJ mouse model of peanut-allergic sensitization. Transfer of CD25(+) Tregs-enriched cell populations did not affect the PE-specific cytokine production or PE-specific antibody levels compared with control mice but interestingly resulted in a decrease of mast cell responsiveness. On the contrary, transfer of CD25(+) Tregs-depleted cells caused an increase in non-specific cytokine production, in the absence of changes in PE-specific responses. TGF-β neutralization resulted even in a larger increase in spontaneous release of all cytokines measured (IL-4, IL-5, IL-10, IL-13, and IFN-γ), but surprisingly also to a higher PE-specific Th2-associated (IL-4, IL-5, IL-13) cytokine production compared with depletion of CD25 cells or neutralization of IL-10. Similarly, depletion of CD25 cells and TGF-β neutralization but not of IL-10 neutralization lead to an increase in PE-specific antibody levels and elevated mast cell degranulation following a PE challenge. We conclude that CD4(+) CD25(+) Tregs from non-transgenic-tolerant mice cannot transfer specific oral tolerance of exogenous antigens to naïve mice and are more involved in general immune suppressive mechanisms. However, we found evidence that TGF-β secreting Tregs (Th3) may play an important role.

A Novel Role for γ-Secretase: Selective Regulation of Spontaneous Neurotransmitter Release from Hippocampal Neurons

With a multitude of substrates, γ-secretase is poised to control neuronal function through a variety of signaling pathways. Presenilin 1 (PS1) is an integral component of γ-secretase and is also a protein closely linked to the etiology of Alzheimer's disease (AD). To better understand the roles of γ-secretase and PS1 in normal and pathological synaptic transmission, we examined evoked and spontaneous neurotransmitter release in cultured hippocampal neurons derived from PS1 knock-out (KO) mice. We found no changes in the size of evoked synaptic currents, short-term plasticity, or apparent calcium dependence of evoked release. The rate of spontaneous release from PS1 KO neurons was, however, approximately double that observed in wild-type (WT) neurons. This increase in spontaneous neurotransmission depended on calcium influx but did not require activation of voltage-gated calcium channels or presynaptic NMDA receptors or release of calcium from internal stores. The rate of spontaneous release from PS1 KO neurons was significantly reduced by lentivirus-mediated expression of WT PS1 or familial AD-linked M146V PS1, but not the D257A PS1 mutant that does not support γ-secretase activity. Treatment of WT neuronal cultures with γ-secretase inhibitor mimicked the loss of PS1, leading to a selective increase in spontaneous release without any change in the size of evoked synaptic currents. Together, these results identify a novel role for γ-secretase in the control of spontaneous neurotransmission through modulation of low-level tonic calcium influx into presynaptic axon terminals.

Complexin: Does It Deserve Its Name?

Knockout and other perturbations of complexins have provided important insights and elicited controversies about their role in neurotransmitter release. New work by Yang etal. in this issue of Neuron adds important detail and complexity to existing concepts-particularly on the nature of a Ca(2+)-dependent complexin-synaptotagmin switch for the triggering of exocytosis. But it also provokes thoughts about alternative interpretations, which might result in a simpler model of complexin function.

Increases in N‐type calcium current augment transmitter release following three days of chronic intermittent hypoxia

We previously reported changes in synaptic transmission between chemosensory afferent fibers and secondary neurons in NTS in response to chronic intermittent hypoxia (CIH). Ten days (10D) of CIH in rats produces an increase in the frequency of spontaneous miniature release of transmitter (mEPSC) while the evoked EPSC (eEPSC) is significantly reduced (Kline et al., J. Neurosci. 27:4663, 2007). We attributed the latter to a homeostatic response to the increased mEPSC and postulated that the two responses could be separated in time. Here we compared the eEPSC amplitude in rats subjected to a shorter hypoxic exposure, 3 days CIH with NORM and also compared mEPSC frequency in both groups. After 3D CIH, eEPSC amplitude was slightly elevated, 202±26 vs 182±4 pA. To the contrary, mEPSC release had already increased, 11± 3 (CIH) versus 5±1Hz (NORM) similar to values at 10D CIH (11.7±2.6 Hz). To address mechanisms, in voltage clamp studies we recorded calcium currents from soma of the presynaptic neurons. We compared the amplitude of N‐type calcium current (ω‐conotoxin GVIA‐sensitive) responsible for evoked release in cells isolated from 3D CIH or NORM rats. The current was increased (p<.02) after CIH (65±6 pA/pF) compared to control (42±7 pA/pF) suggesting that an increased functional expression of the N‐type channel may be responsible for the increase in spontaneous release as well as the small increase in evoked release at 3D CIH. HL090886

Trans-Synaptic Plasticity: Presynaptic Initiation, Postsynaptic Memory

A novel mechanism of persistent facilitation induced by serotonin at Aplysia synapses depends upon rapid postsynaptic protein synthesis and increased responsiveness to glutamate; whereas the memory for this synaptic change is postsynaptic, the initiating signal may be an increase in spontaneous release of glutamate from the presynaptic terminals.

Genetic analysis of synaptotagmin 2 in spontaneous and Ca2+-triggered neurotransmitter release

Synaptotagmin 2 resembles synaptotagmin 1, the Ca2+ sensor for fast neurotransmitter release in forebrain synapses, but little is known about synaptotagmin 2 function. Here, we describe a severely ataxic mouse strain that harbors a single, destabilizing amino-acid substitution (I377N) in synaptotagmin 2. In Calyx of Held synapses, this mutation causes a delay and a decrease in Ca2+-induced but not in hypertonic sucrose-induced release, suggesting that synaptotagmin 2 mediates Ca2+ triggering of evoked release in brainstem synapses. Unexpectedly, we additionally observed in synaptotagmin 2 mutant synapses a dramatic increase in spontaneous release. Synaptotagmin 1-deficient excitatory and inhibitory cortical synapses also displayed a large increase in spontaneous release, demonstrating that this effect was shared among synaptotagmins 1 and 2. Our data suggest that synaptotagmin 1 and 2 perform equivalent functions in the Ca2+ triggering of action potential-induced release and in the restriction of spontaneous release, consistent with a general role of synaptotagmins in controlling 'release slots' for synaptic vesicles at the active zone.

Cholesterol and synaptic transmitter release at crayfish neuromuscular junctions

During exocytosis of synaptic transmitters, the fusion of highly curved synaptic vesicle membranes with the relatively planar cell membrane requires the coordinated action of several proteins. The role of membrane lipids in the regulation of transmitter release is less well understood. Since it helps to control membrane fluidity, alteration of cholesterol content may alter the fusibility of membranes as well as the function of membrane proteins. We assayed the importance of cholesterol in transmitter release at crayfish neuromuscular junctions where action potentials can be measured in the preterminal axon. Methyl-beta-cyclodextrin (MbetaCD) depleted axons of cholesterol, as shown by reduced filipin labelling, and cholesterol was replenished by cholesterol-MbetaCD complex (Ch-MbetaCD). MbetaCD blocked evoked synaptic transmission. The lack of postsynaptic effects of MbetaCD on the time course and amplitude of spontaneous postsynaptic potentials or on muscle resting potential allowed us to focus on presynaptic mechanisms. Intracellular presynaptic axon recordings and focal extracellular recordings at individual boutons showed that failure of transmitter release was correlated with presynaptic hyperpolarization and failure of action potential propagation. All of these effects were reversed when cholesterol was replenished with Ch-MbetaCD. However, focal depolarization of presynaptic boutons and administration of a Ca2+ ionophore both triggered transmitter release after cholesterol depletion. Therefore, both presynaptic Ca2+ channels and Ca2+-dependent exocytosis functioned after cholesterol depletion. The frequency of spontaneous quantal transmitter release was increased by MbetaCD but recovered when cholesterol was reintroduced. The increase in spontaneous release was not through a calcium-dependent mechanism because it persisted with intense intracellular calcium chelation. In conclusion, cholesterol levels in the presynaptic membrane modulate several key properties of synaptic transmitter release.

Variability of interactions between neuroendocrine and immunological functions in physiological aging and dementia of the Alzheimer's type.

A link between neuroendocrine and immunological changes has been suggested in the pathophysiology of dementia of the Alzheimer's type (DAT). Healthy young and old subjects and patients with DAT were recruited to evaluate the chrononeuroendocrine organization of cortisol, GH, and melatonin (MLT) secretions. The study was carried out together with the evaluation of natural killer (NK) cell function: cytotoxic activity (NKCC) and TNF-alpha and IFN-gamma release after exposure to IL-2 (100 U/mL). Moreover, a cerebral morphometric analysis of hippocampus and temporal lobe (MRI) was performed. The activation of hypothalamo-pituitary-adrenal (HPA) axis and the decrease of GH, and MLT nocturnal peaks were associated with normal NKCC and TNF-alpha/IFN-gamma in healthy elderly subjects, whereas in DAT patients the same neuroendocrine changes occurred together with abnormal NKCC (spontaneous and IL-2/IFN-beta-modulated) and with alterations of TNF-alpha/INF-gamma generation from NK. Moreover significant correlations among the increase of NKCC and TNF-alpha and the decrease of cognitive function were found in the DAT group. These correlations were associated with the impairment of nocturnal GH and MLT levels and with the relatively higher serum cortisol concentrations. Moreover, the impairment of cortisol suppression after dexamethasone (1 mg orally at 23:00) was significantly correlated with the increase of spontaneous release of TNF-alpha and with IL-2-modulated NKCC. Finally the imunoneuroendocrine alterations found in DAT were associated with the reduction of cerebral volume in hippocampus and temporal lobes. Taken together these data indicate that the immunoneuroendocrine balance is maintained in physiological aging, whereas NK immune dysregulation in DAT could contribute to altering the neuroendocrine functions and to extend the progression of neurodegeneration and dementia.

Morphologically Docked Synaptic Vesicles Are Reduced insynaptotagminMutants ofDrosophila

Nerve terminal specializations include mechanisms for maintaining a subpopulation of vesicles in a docked, fusion-ready state. We have investigated the relationship between synaptotagmin and the number of morphologically docked vesicles by an electron microscopic analysis of Drosophila synaptotagmin (syt) mutants. The overall number of synaptic vesicles in a terminal was reduced, although each active zone continued to have a cluster of vesicles in its vicinity. In addition, there was an increase in the number of large vesicles near synapses. Examining the clusters, we found that the pool of synaptic vesicles immediately adjacent to the presynaptic membrane, the pool that includes the docked population, was reduced to 24 +/- 5% (means +/- SEM) of control in the sytnull mutation. To separate contributions of overall vesicle depletion and increased spontaneous release from direct effects of synaptotagmin on morphological docking, we examined syt mutants in an altered genetic background. Recombining syt alleles onto a second chromosome bearing an as yet uncharacterized mutation resulted in the expected decrease in evoked release but suppressed the increase in spontaneous release frequency. Motor nerve terminals in this genotype contained more synaptic vesicles than control, yet the number of vesicles immediately adjacent to the presynaptic membrane near active zones was still reduced (33 +/- 4% of control). Our findings demonstrate that there is a decrease in the number of morphologically docked vesicles seen in syt mutants. The decreases in docking and evoked release are independent of the increase in spontaneous release. These results support the hypothesis that synaptotagmin stabilizes the docked state.

Role of Ca2+Ions in Nicotinic Facilitation of GABA Release in Mouse Thalamus

Presynaptic nicotinic acetylcholine receptors (nAChRs) are present in many regions of the brain and potentially serve as targets for the pharmacological action of nicotine in vivo. To investigate their mechanism of action, we performed patch-clamp recordings in relay neurons from slices of thalamus sensory nuclei. In these nuclei, nAChR activation facilitated the release of the inhibitory neurotransmitter GABA. Micromolar concentrations of nicotinic agonists increased the frequency of miniature GABAergic synaptic currents and decreased the failure rate of evoked synaptic currents. These actions of nicotinic agonists were not observed in knock-out mice lacking the beta 2 nAChR subunit gene. Nicotinic effects were dependent on extracellular calcium ions, and they persisted when calcium was replaced by strontium or barium but not by magnesium. Furthermore, in high extracellular calcium concentrations, nicotinic agonists evoked an increase in spontaneous release lasting for minutes after removal of the agonist. This supports the view that presynaptic nAChRs facilitate the release of neurotransmitter by increasing the calcium concentrations in presynaptic nerve endings. With use of cadmium and nickel ions as selective blockers, it was found that in different sensory nuclei the presynaptic influx of calcium could result either from the activation of voltage-dependent calcium channels or from a direct influx through nAChR channels. Finally, we propose that the nicotinic facilitation of GABAergic transmission may contribute to the increase of signal-to-noise ratio observed in the thalamus in vivo during arousal.

Effect of swimming on vascular reactivity to phenylephrine and KC1 in male rats

1. The present study aimed to examine whether there is any change in vascular responsiveness to phenylephrine and KC1 during exercise, and whether the vascular endothelium plays a role in these changes. 2. Adult male rats were subjected to a swimming schedule every day for 5-6 weeks. Studies were performed in vitro on thoracic aortae. 3. Maximum contractile response to phenylephrine of endothelium-intact thoracic aortic rings (passive tension 1.0 g) obtained from swimming rats (1.2 +/- 0.2 g, n = 8) was lower than of sedentary control rats (2.1 +/- 0.2 g, n = 8). When the endothelium was removed, however, the dose-response curves of both groups of rats were shifted to the left with an increase in maximum responses and they were no longer significantly different (max. tension, swimming rats: 3.2 +/- 0.3 g, n = 6, control rats: 3.4 +/- 0.4 g, n = 5). 4. Indomethacin did not significantly alter the dose-response curves. A similar effect to that obtained by removal of the endothelium was observed when methylene blue and indomethacin were both added. 5. Passive tension in the range of 2.5-3.0 g, caused a significant increase in active tension developed to phenylephrine (1 microM for endothelium-intact and 0.1 microM for endothelium-denuded) of thoracic aortic rings of both swimming and sedentary control rats compared to their corresponding groups when using passive tension of 1.0-1.5 g. 6. The reduction in responses to phenylephrine of endothelium-intact thoracic aortic rings of swimming rats persisted with the use of a passive tension of 3.0 g. The presence of 300 microM N0-nitro-L-arginine (LNOARG)caused a significant leftward shift of the curve with an increase in maximum responses when a passive tension of either 1.0 or 3.0 g was applied to the rings. However, for the rings with a passive tension of 1.0 g, L-NOARG caused a smaller increase in maximal contractile responses to phenylephrine of the rings of sedentary controls than those of swimming rats.7. There was no difference in the dose-response curves to depolarizing concentrations of KCl (20, 40, 80 and 120 mM) of endothelium-intact thoracic aortic rings from swimming and sedentary control rats.When the endothelium was removed, however, the dose-response curves of both groups of animals were shifted to the left with an increase in maximum responses. Moreover, the responses to KCl of endothelium-denuded thoracic aortic rings of swimming rats were greater than those of sedentary control rats.8. These results suggest that there were changes in vascular responsiveness to phenylephrine and KCl during exercise. The fall in sensitivity to phenylephrine with no change in KCl responses, and the increase in maximum responses to phenylephrine in the presence of L-NOARG in endothelium-intact aortae (passive tension 1.0 g) from swimming rats, were due to an increase in spontaneous release and upregulation of phenylephrine-stimulated release of EDRF/NO, and may not be a consequence of an increase in prostaglandins or a decrease in the production of endothelial constrictors by vascular endothelium. EDRF/NO may play an important role in modulating local vasodilatation.

Cytokine production and serum levels in systemic sclerosis

Serum levels of various cytokines, tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL1-β), and interleukin 2 (IL2), and of soluble IL2 receptors (sIL2R) were determined in 30 patients with definite systemic sclerosis (SSc). Spontaneous and lipopolysaccharide- or mitogen-induced production of the cytokines, TNF-α, IL1-β, and IFN-γ, by peripheral blood mononuclear cells (PBMNC) of these SSc patients was measured by immunoassays. The patients were divided into three groups: 12 with limited cutaneous disease (lcSSc), 7 with diffuse cutaneous disease (dcSSc) <3 years duration, and 11 with dcSSc >3 years duration. None were treated with cytotoxic drugs or biologic response modifiers. Sera of patients with SSc had elevated sIL2R levels, and only low levels of IL2 (1–2 U/ml) were detected in 10 29 sera tested. Spontaneous production of TNF-α and IL1-β by PBMNC of patients with SSc (829 pg/ml ± 215 SEM and 728 pg/ml ± 186, respectively) was significantly higher than that by normal PBMNC obtained from 30 volunteers (25 ± 10 and 34 ± 6 pg/ml, respectively) and tested at the same time as patients' PBMNC. The largest increases in spontaneous release of TNF-α or IL1-β were seen in patients with early dcSSc. No significant difference in spontaneous IFN-γ production by patient or control PBMNC was detected. On the other hand, the mean level of mitogen-induced IFN-γ production by PBMNC was significantly depressed in patients with SSc (103 U/ml ± 18 vs 255 ± 33 U/ml in controls). In vitro-induced production of TNF-α or IL1-β by patients' PBMNC was comparable to that of normal PBMNC. These data indicate that in vivo-activated PBMNC of patients with SSc spontaneously secrete excessive amounts of fibrogenic cytokines, which are involved in modulation of connective tissue synthesis.

Presynaptic modulation of ganglionic ACh release by muscarinic and nicotinic receptors

The present experiments were undertaken to investigate the effects of atropine and d-tubocurarine on acetylcholine (ACh) release and ganglionic synaptic transmission in the isolated cat stellate ganglion. Ganglionic release of picomole amounts of ACh was measured by radioenzymatic assay, and ganglionic transmission was estimated on the basis of the compound action potential recorded from the postganglionic stellate cardiac nerve. Atropine (5 microM) produced a significant increase in both spontaneous and evoked ACh release from the ganglion while depressing synaptic transmission. d-Tubocurarine (20 microM) also caused a significant, though smaller, increase in spontaneous release of ACh but had little effect on evoked release of ACh. These results suggest that ACh release and synaptic transmission in the cat stellate ganglion are subject to cholinergic feedback regulation, which appears to be mediated predominantly via muscarinic presynaptic receptors.

Properties of the early phases of crotoxin poisoning at frog neuromuscular junctions

L. Rodrigues-Simioni, B. J. Hawgood and I. C. H. Smith. Properties of the early phases of crotoxin poisoning at frog neuromuscular junctions. Toxicon 28, 1479–1489, 1990.—Addition of crotoxin to the frog neuromuscular junction in either 0.9 mM Ca 2+ plus tubocurarine or 0.5 mM Ca 2+ Ringer solution produced a triphasic change in the amplitude of nerve-evoked endplate potentials (e.p.p.s) and, with 0.5 mM Ca 2+, a biphasic change in miniature endplate potential (m.e.p.p.) frequency. The secondary phase of rising e.p.p. amplitude was associated with an increase in facilitation of e.p.p. amplitude with closely spaced twin impulses; the increase in spontaneous release lagged behind that of evoked release. When a calcium chelator, BAPTA, was loaded into presynaptic nerve terminals to buffer cytosolic free Ca 2+, both e.p.p. amplitude and twin-impulse facilitation were increased by crotoxin to a similar extent relative to that in the control without BAPTA. The duration of the increase in twin-impulse facilitation was reduced but the duration of the increase in e.p.p. amplitude was unaffected by BAPTA loading. The presence of BAPTA did not alter the characteristic changes in spontaneous release in response to crotoxin. These results suggest that the augmentation of evoked and spontaneous transmitter release by crotoxin is not primarily due to changes in cytosolic Ca 2+. The response time between stimulus and e.p.p. peak was lengthened in all phases due to prolongation of the interval between stimulus and e.p.p. onset and, in the secondary and tertiary phases, slowing of e.p.p. rise-time. The protein kinase C inhibitor, H-7, produced complex changes in e.p.p.s. under control conditions but did not alter the triphasic response characteristic of intoxication. These results suggest that crotoxin initiates a primary disturbance in the phasic release process leading to a series of time-gated changes which include transient facilitation then uncoupling of phasic release and generalized acceleration of spontaneous release.

Effects of methylmercury on neurotransmitter release from rat brain synaptosomes

Although the effects of methylmercury (MeHg) at the neuromuscular junction have been well characterized, similar studies employing CNS preparations and transmitters have been limited. We found that MeHg (0.5–5.0 μM) produced a concentration-dependent increase in the spontaneous release of [ 3H]dopamine, γ-[ 3H]aminobutyric acid, and [ 3H]acetylcholine from synaptosomes isolated from rat brain striatum, cortex, and hippocampus, respectively. At these same concentrations MeHg did not attenuate calcium-dependent depolarization-evoked 3H-transmitter release. MeHg did not appear to induce calcium influx into the nerve terminal since the increase in release persists in the absence of extrasynaptosomal calcium. The increase in spontaneous transmitter release induced by MeHg persisted in the presence of low extrasynaptosomal sodium, suggesting that MeHg's effects on release are not mediated by either Na +, K +-ATPase inhibition or selective increases in membrane sodium permeability. MeHg produced only a very small increase in 45Ca efflux from synaptosomes preloaded with 45Ca, whereas these same MeHg concentrations produced large increases in 45Ca efflux from preloaded isolated mitochondria. MeHg did increase the efflux of [ 3H]deoxyglucose phosphate from synaptosomes. An increase in the efflux of [ 3H]deoxyglucose phosphate is believed to reflect an increase in neuronal membrane permeability. The quantitative and temporal aspects of the MeHg-induced [ 3H]-deoxyglucose phosphate efflux were similar to those observed for MeHg-induced neurotransmitter release. These data suggest that the increase in spontaneous transmitter release induced by MeHg is mainly the result of transmitter leakage that occurs subsequent to MeHg-induced increases in synaptosomal membrane permeability. However, these results cannot exclude possible effects of MeHg on intrasynaptosomal calcium homeostasis.

Effect of cerulein on in vivo release of acetylcholine from the rat striatum

An effect of cerulein on in vivo release of acetylcholine (ACh) from the rat striatum was examined by means of intracerebral dialysis. Intraperitoneal administration of cerulein (25–200 μg/kg) enhanced a spontaneous release of ACh in a dose-dependent manner. Intraperitoneal administration of cerulein (100 μg/kg) also enhanced the K +-evoked (30 mM) release of ACh. Bilateral subdiaphragmatic vagotomy reduced the increase of both K +-evoked and that of spontaneous release of ACh induced by cerulein administration. Pretreatment with haloperidol (0.5 mg/kg, i.p.) had no effect on increase in spontaneous release of ACh brought about by cerulein administration (100 μg/kg, i.p.). These results suggested that peripherally administered cerulein stimulated striatal cholinergic neurons, that its stimulatory effect on striatal ACh release was not mediated by striatal dopamine D 2 receptors and that the action of cerulein was, in part, mediated via vagal afferent impulses.