Evoked Transmitter Release Research Articles

The effects of presynaptic calcium channel modulation by roscovitine on transmitter release at the adult frog neuromuscular junction

Calcium (Ca2+) influx through presynaptic calcium channels triggers transmitter release, and any alterations in the gating of these calcium channels results in changes in the magnitude of transmitter released. We used (R)-roscovitine, a cyclin-dependent kinase inhibitor that also appears to act directly on calcium channels, as a tool to modulate presynaptic calcium influx and study effects on transmitter release. We show that this compound increased the quantal content of acetylcholine released from the Rana frog motor nerve terminal (by 149%) without changing paired-pulse facilitation (under low calcium conditions). In contrast, exposure to 3,4-diaminopyridine (DAP; which similarly affects transmitter release by partially blocking potassium channels, altering the shape of the presynaptic action potential, and indirectly increasing calcium entry) increased paired-pulse facilitation (by 23%). In addition, we show that (R)-roscovitine predominately slowed deactivation kinetics of calcium current (by 427%) recorded from Xenopus frog motoneurons, and as a result, increased the integral of calcium channel current evoked by a physiological action potential waveform (by 44%). Because we did not observe any significant effects of structurally related cyclin-dependent kinase inhibitors [(S)-roscovitine or olomoucine] on evoked transmitter release or calcium current kinetics, it appears that these effects of (R)-roscovitine are independent of cyclin-dependent kinases (cdks). In summary, we hypothesize that (R)-roscovitine effects on transmitter release at the adult frog neuromuscular junction (NMJ) are mediated by its effects on calcium channel gating, and these effects increase our understanding of calcium triggered secretion at this synapse.

Combined Analog and Action Potential Coding in Hippocampal Mossy Fibers

In the mammalian cortex, it is generally assumed that the output information of neurons is encoded in the number and the timing of action potentials. Here, we show, by using direct patchclamp recordings from presynaptic hippocampal mossy fiber boutons, that axons transmit analog signals in addition to action potentials. Excitatory presynaptic potentials result from subthreshold dendritic synaptic inputs, which propagate several hundreds of micrometers along the axon and modulate action potential-evoked transmitter release at the mossy fiber-CA3 synapse. This combined analog and action potential coding represents an additional mechanism for information transmission in a major hippocampal pathway.

Ca 2+ ‐Evoked Transmitter Release Is Inhibited in PC12 Cells That Have Synaptotagmin I Silenced By RNAi

Many proteins are postulated to function in vesicle release during synaptic transmission. Synaptotagmin I (syt I) is believed to be a primary Ca2+ sensor protein for Ca2+-dependent release of vesicles during synaptic transmission. To elucidate how a protein functions as a Ca2+ sensor, we have specifically and stably eliminated syt I from a model secretory PC12 cell line using a plasmid-based RNAi system to silence expression of syt I. Immunocytochemistry confirms the specificity and extent of syt I protein knockdown compared to control untransfected cells and cells stably transfected with the plasmid that lacks an insert. In response to 50 mM K+ stimulation, stimulated fractional release of both dopamine and norepinephrine was significantly reduced by ~50% in syt I-shRNA cells when measured by HPLC-electrochemical detection. In addition, stimulated release of ATP was reduced ~50% as measured by HPLC-fluorometric detection. Immunocytochemistry showed that neither tyrosine hydroxylase, the rate-limiting enzyme in the dopamine synthesis pathway, nor vesicular monoamine transporter 2 (VMAT2) which transports dopamine into vesicles, had altered protein expression levels. In contrast to both catecholamine and ATP release, neuropeptide Y stimulated release was abolished in syt I knockdown cells. The differing extent of inhibition of neurotransmitter secretion indicates that syt I may be differentially localized within vesicle populations in PC12 cells.

Modulation of synaptic activity in Purkinje neurons by ATP

Adenosine triphosphate (ATP) is a versatile signalling molecule in the central and peripheral nervous system, where it can be released from both neurons and glial cells. In the cerebellum, ATP is released endogenously from the second postnatal week onwards, and is involved in the up-regulation of spontaneous synaptic input to Purkinje neurons by activation of purinergic P2 receptors. In the cerebellar cortex, ATP presumably acts on presynaptic inhibitory interneurons, which are excited by the activation of both P2X and P2Y receptors. P2 receptors have been reported for Purkinje neurons, where they mediate intracellular Ca(2+) responses. The extracellular concentration of ATP is modulated by its enzymatic degradation by ecto-nucleotidases. Adenosine, which modulates evoked transmitter release, does not influence the spontaneous synaptic activity in Purkinje neurons. Some implications of ATP as a tonically active neuromodulator in the cerebellum are discussed.

Dystrophin Is Required for Appropriate Retrograde Control of Neurotransmitter Release at theDrosophilaNeuromuscular Junction

Mutations in the human dystrophin gene cause the Duchenne and Becker muscular dystrophies. The Dystrophin protein provides a structural link between the muscle cytoskeleton and extracellular matrix to maintain muscle integrity. Recently, Dystrophin has also been found to act as a scaffold for several signaling molecules, but the roles of dystrophin-mediated signaling pathways remain unknown. To further our understanding of this aspect of the function of dystrophin, we have generated Drosophila mutants that lack the large dystrophin isoforms and analyzed their role in synapse function at the neuromuscular junction. In expression and rescue studies, we show that lack of the large dystrophin isoforms in the postsynaptic muscle cell leads to elevated evoked neurotransmitter release from the presynaptic apparatus. Overall synapse size, the size of the readily releasable vesicle pool as assessed with hypertonic shock, and the number of presynaptic neurotransmitter release sites (active zones) are not changed in the mutants. Short-term synaptic facilitation of evoked transmitter release is decreased in the mutants, suggesting that the absence of dystrophin results in increased probability of release. Absence of the large dystrophin isoforms does not lead to changes in muscle cell morphology or alterations in the postsynaptic electrical response to spontaneously released neurotransmitter. Therefore, postsynaptic glutamate receptor function does not appear to be affected. Our results indicate that the postsynaptically localized scaffolding protein Dystrophin is required for appropriate control of neuromuscular synaptic homeostasis.

Calcium From Internal Stores Triggers GABA Release From Retinal Amacrine Cells

The Ca(2+) that promotes transmitter release is generally thought to enter presynaptic terminals through voltage-gated Ca(2+)channels. Using electrophysiology and Ca(2+) imaging, we show that, in amacrine cell dendrites, at least some of the Ca(2+) that triggers transmitter release comes from endoplasmic reticulum Ca(2+) stores. We show that both inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) are present in these dendrites and both participate in the elevation of cytoplasmic [Ca(2+)] during the brief depolarization of a dendrite. Only the Ca(2+) released through IP(3)Rs, however, seems to promote the release of transmitter. Antagonists for the IP(3)R reduced transmitter release, whereas RyR blockers had no effect. Application of an agonist for metabotropic glutamate receptor, known to liberate Ca(2+) from internal stores, enhanced both spontaneous and evoked transmitter release.

Synaptic and Extrasynaptic NMDA Receptor NR2 Subunits in Cultured Hippocampal Neurons

Early in development, neurons only express NR1/NR2B-containing N-methyl-d-aspartate (NMDA) receptors. Later, NR2A subunits are upregulated during a period of rapid synapse formation. This pattern is often interpreted to indicate that NR2A-containing receptors are synaptic and that NR2B-containing receptors are extrasynaptic. We re-examined this issue using whole cell recordings in cultured hippocampal neurons. As expected, the inhibition of whole cell currents by the NR2B-specific antagonist, ifenprodil, progressively decreased from 69.5 +/- 2.4% [6 days in vitro (DIV)] to 54.9 +/- 2.6% (8 DIV), before reaching a plateau in the second week (42.5 +/- 2%, 12-19 DIV). In NR2A-/- neurons, which express only NR1/NR2B-containing NMDA receptors, autaptic excitatory postsynaptic currents (EPSCs; > or =12 DIV) were more sensitive to ifenprodil and decayed more slowly than EPSCs in wild-type neurons. Thus NR2B-containing receptors were not excluded from synapses. We blocked synaptic NMDA receptors with MK-801 during evoked transmitter release, thus allowing us to isolate extrasynaptic receptors. Ifenprodil inhibition of this extrasynaptic population was highly variable in different neurons. Furthermore, extrasynaptic receptors in autaptic cultures were only partially blocked by ifenprodil, indicating that NR2A-containing receptors are not exclusively confined to the synapse. Extrasynaptic NR2A-containing receptors were also detected in NR2A(-/-) neurons transfected with full-length NR2A. Truncation of the NR2A C terminus did not eliminate synaptic expression of NR2A-containing receptors. Our results indicate that NR2A- and NR2B-containing receptors can be located in either synaptic or extrasynaptic compartments.

Muscarinic ACh Receptor Activation Causes Transmitter Release From Isolated Frog Vestibular Hair Cells

In the frog, vestibular efferent fibers innervate only type-II vestibular hair cells. Through this direct contact with hair cells, efferent neurons are capable of modifying transmitter release from hair cells onto primary vestibular afferents. The major efferent transmitter, acetylcholine (ACh), is known to produce distinct pharmacological actions involving several ACh receptors. Previous studies have implicated the presence of muscarinic ACh receptors on vestibular hair cells, although, surprisingly, a muscarinic-mediated electrical response has not been demonstrated in solitary vestibular hair cells. This study demonstrates that muscarinic receptors can evoke transmitter release from vestibular hair cells. Detection of this release was obtained through patch-clamp recordings from catfish cone horizontal cells, serving as glutamate detectors after pairing them with isolated frog semicircular canal hair cells in a two-cell preparation. Although horizontal cells alone failed to respond to carbachol, application of 20 microM carbachol to the two-cell preparation resulted in a horizontal cell response that could be mimicked by exogenous application of glutamate. All of the horizontal cells in the two-cell preparation responded to 20 microM CCh. Furthermore, this presumed transmitter release persisted in the presence of d-tubocurarine at concentrations that block all known hair cell nicotinic ACh receptors. The effect on the detector cell, imparted by the carbachol application to the hair cell-horizontal cell preparation, was blocked both by 2-amino-5-phosphonopentanoic acid, a selective N-methyl-D-aspartate antagonist, and the muscarinic antagonist, atropine. Thus vestibular hair cells from the frog semicircular canal can be stimulated to release transmitter by activating their muscarinic receptors.

Volatile anaesthetic effects on phospholipid binding to synaptotagmin 1, a presynaptic Ca2+ sensor

Volatile anaesthetics have important effects on synaptic transmission in the CNS. Depression of excitatory transmission involves reduced transmitter release via unidentified presynaptic mechanisms. Synaptotagmin 1 is a synaptic vesicle-associated protein that regulates Ca(2+)-evoked transmitter release involving critical Ca(2+)/phospholipid interactions within its C2 domains. We analysed the effects of halothane and isoflurane on the binding of purified recombinant rat synaptotagmin 1 C2A, C2B and C2AB domains to radiolabelled phospholipid liposomes. Halothane and isoflurane had no significant effects on the maximal binding or Ca(2+) dependence of binding of synaptotagmin 1 C2 domains to mixed phospholipid vesicles composed of either phosphatidylserine/phosphatidylcholine or phosphatidylinositol/phosphatidylcholine. Inhibition of synaptic vesicle exocytosis by volatile anaesthetics does not appear to involve an effect on the critical Ca(2+)/phospholipid binding properties of synaptotagmin 1, a Ca(2+) sensor involved in regulating evoked Ca(2+)-dependent neurotransmitter release.

Multiple mechanisms mediate cholesterol-induced synaptogenesis in a CNS neuron

Neurons undergo a complex differentiation process that endows them with the ability to generate electrical signals and to transmit them via synaptic connections. There is increasing evidence that glial cells regulate specific aspects of neuronal differentiation including synapse formation, but the underlying mechanisms are not well understood. Here, we show how glia-derived cholesterol promotes the development of synapses in microcultures of highly purified retinal ganglion cells (RGCs) from postnatal rats. We identify dendrite differentiation as rate limiting step for glia-induced synaptogenesis and we show that this process requires cholesterol. Furthermore, we show that cholesterol enhances directly presynaptic differentiation and that it is essential for continuous synaptogenesis and for the stability of evoked transmitter release. These results reveal new roles of cholesterol in neuronal differentiation and underline the importance of neuron–glia interactions during brain development.

The Multiple Functions of Cysteine-String Protein Analyzed atDrosophilaNerve Terminals

The synaptic vesicle-associated cysteine-string protein (CSP) is important for synaptic transmission. Previous studies revealed multiple defects at neuromuscular junctions (NMJs) of csp null-mutant Drosophila, but whether these defects are independent of each other or mechanistically linked through J domain mediated-interactions with heat-shock cognate protein 70 (Hsc70) has not been established. To resolve this issue, we genetically dissected the individual functions of CSP by an in vivo structure/function analysis. Expression of mutant CSP lacking the J domain at csp null-mutant NMJs fully restored normal thermo-tolerance of evoked transmitter release but did not completely restore evoked release at room temperature and failed to reverse the abnormal intraterminal Ca2+ levels. This suggests that J domain-mediated functions are essential for the regulation of intraterminal Ca2+ levels but only partially required for regulating evoked release and not required for protecting evoked release against thermal stress. Hence, CSP can also act as an Hsc70-independent chaperone protecting evoked release from thermal stress. Expression of mutant CSP lacking the L domain restored neurotransmission and partially reversed the abnormal intraterminal Ca2+ levels, suggesting that the L domain is important, although not essential, for the role of CSP in regulating intraterminal Ca2+ levels. We detected no effects of csp mutations on individual presynaptic Ca2+ signals triggered by action potentials, suggesting that presynaptic Ca2+ entry is not primarily impaired. Both the J and L domains were also required for the role of CSP in synaptic growth. Together, these results suggest that CSP has several independent synaptic functions, affecting synaptic growth, evoked release, thermal protection of evoked release, and intraterminal Ca2+ levels at rest and during stimulation.

Changes in Electrogenesis in the Motor Nerve Terminal with Long-Lasting High-Frequency Activation as a Factor of the Control of Transmitter Release

Using the technique of extracellular recording from the region of the neuromuscular junction in the cutaneous-sternal muscle in the frog under conditions of a reduced concentration of Ca2+ in the surrounding milieu, we demonstrated that long-lasting (10 min) rhythmic stimulation of the motor nerve with a frequency of 10 sec− 1 leads to a gradual increase in the evoked transmitter release. These changes are accompanied by a decrease in the amplitude of electrical responses of the nerve terminal (NT) and by a retardation of its second phase, as well as by a diminution of the third phase. Under conditions of long-lasting (5 min) stimulation with a frequency of 50 sec−1, we observed a two-phase change in the intensity of transmitter release: on the 2nd min, the initial rise was replaced by inhibition. Modifications of the response of the NT with different stimulation frequencies were qualitatively similar, but with a frequency of 10 sec−1 they were clearly expressed. Mathematical simulation of ion currents in the NT demonstrated that voltage-dependent potassium and sodium channels are inactivated in the course of long-lasting high-frequency excitation; the shape of the action potential is modified with changes in the rate of such inactivation. This leads to either an increase or a decrease of the inward calcium current. We conclude that the change in electrogenesis in the NT with long-lasting high-frequency activation of neuromuscular junctions exerts a significant influence on the dynamics of transmitter release.

Effect of prolonged inactivity on skeletal motor nerve terminals during aestivation in the burrowing frog, Cyclorana alboguttata

This study examined the effect of prolonged inactivity, associated with aestivation, on neuromuscular transmission in the green-striped burrowing frog, Cyclorana alboguttata. We compared the structure and function of the neuromuscular junctions on the iliofibularis muscle from active C. alboguttata and from C. alboguttata that had been aestivating for 6 months. Despite the prolonged period of immobility, there was no significant difference in the shape of the terminals (primary, secondary or tertiary branches) or the length of primary terminal branches between aestivators and non-aestivators. Furthermore, there was no significant difference in the membrane potentials of muscle fibres or in miniature end plate potential (EPP) frequency and amplitude. However, there was a significant decrease in evoked transmitter release characterised by a 56% decrease in mean EPP amplitude, and a 29% increase in the failure rate of nerve terminal action potentials to evoke transmitter release. The impact of this suite of neuromuscular characteristics on the locomotor performance of emergent frogs is discussed.

Testosterone modulates Ca v2.2 calcium channels’ functional expression at rat levator ani neuromuscular junction

Spinal nucleus of bulbocavernosus and its target musculature, the bulbocavernosus and levator ani muscles, are sexually dimorphic, and their sexual differentiation depends on plasmatic levels of testosterone. Electrophysiological and immunocytochemical studies have demonstrated that at mammalian adult neuromuscular junctions only P/Q-type Ca 2+ channels (Ca v2.1), mediate evoked transmitter release. Here we report that N-type Ca 2+ channel (Ca v2.2) blocker ω-Conotoxin GVIA, as well as Ca v2.1 blocker ω-Agatoxin IVA, significantly reduced quantal content of transmitter release by ∼ 80% and ∼70% respectively at levator ani muscle of the adult rats, indicating that neuromuscular transmission is jointly mediated by both types of channels. In these synapses, we also observed that castration and restitution of plasmatic testosterone in rats resulted in changes in the sensitivity to ω-Conotoxin GVIA. Castration induced, whereas testosterone treatment avoided, functional loss of Ca v2.2, as mediators of transmitter release in these synapses. Strikingly, the expression and localization of α 1B subunits, which form the pore of the Ca v2.2 channel, were similar at control, gonadectomized and gonadectomized testosterone-treated rats, suggesting that testosterone may regulate the coupling mechanisms between Ca v2.2 and transmitter release at the neuromuscular junctions of these sexually dimorphic motoneurons.

Hippocampal mossy fiber calcium transients are maintained during long-term potentiation and are inhibited by endogenous zinc

The hippocampal mossy fiber long-term potentiation (LTP) is an N-methyl- d-aspartate (NMDA) receptor-independent form of long-lasting synaptic plasticity characteristic of the zinc-enriched mossy fiber synapses. Its expression is generally considered to have a presynaptic locus and to be mediated by a persistent increase of evoked transmitter release. Because the release process is calcium-dependent, the observed increase in synaptic efficacy could be due to a persistent modification of presynaptic calcium mechanisms, triggered by the large calcium influx associated with long-term potentiation induction. Alternatively, it might be caused by an enhancement in the sensitivity to calcium of some components of the synaptic vesicle release system, following the large intraterminal calcium accumulation. We investigated the first hypothesis by measuring presynaptic Fura-2 calcium signals associated with electrically induced mossy fiber long-term potentiation. We have observed that like residual calcium, single presynaptic calcium changes are not enhanced during the maintenance phase of mossy fiber long-term potentiation. This result supports the idea that this form of long-term potentiation may be mediated by persistent changes of some process occurring after calcium entry. It has been established that voltage-dependent calcium channels are inhibited by zinc and that endogenous zinc is released in a calcium-dependent way following intense mossy fiber activation. Because there is evidence that at these synapses zinc is also released following single electrical stimulation, we investigated the effect of endogenous zinc on single presynaptic calcium signals and on field potentials associated with mossy fiber LTP. We have observed that this form of LTP could be induced in the presence of the permeant heavy metal chelator N, N, N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and that application of this chelator, during LTP, caused an enhancement of the presynaptic calcium signals without affecting synaptic transmission. This enhancement is consistent with the idea that mossy fiber zinc, released following individual stimuli, inhibits presynaptic calcium mechanisms.

P2X 7-like receptor subunits enhance excitatory synaptic transmission at central synapses by presynaptic mechanisms

Recent studies demonstrate that P2X 7 receptor subunits (P2X 7RS) are present at central and peripheral synapses and suggest that P2X 7RS can regulate transmitter release. In brainstem slices from 15 to 26 day old pentobarbitone-anesthetized mice, we examined the effect of P2X 7RS activation on excitatory postsynaptic currents (EPSCs) recorded from hypoglossal motoneurons using whole-cell patch clamp techniques. After blockade of most P2X receptors with suramin (which is inactive at P2X 7RS) and of adenosine receptors with 8-phenyltheophylline (8PT), bath application of the P2X receptor agonist 3′-0-(4-benzoyl)ATP (BzATP) elicited a 40.5±16.0% (mean±S.E.M., n=8, P=0.039) increase in evoked EPSC amplitude and significantly reduced paired pulse facilitation of evoked EPSCs. This response to BzATP (with suramin and 8PT present) was completely blocked by prior application of Brilliant Blue G (200 nM or 2 μM), a P2X 7RS antagonist. In contrast, BzATP application with suramin and 8PT present did not alter miniature EPSC frequency or amplitude when action potentials were blocked with tetrodotoxin. These electrophysiological results suggest that P2X 7RS activation increases central excitatory transmitter release via presynaptic mechanisms, confirming previous indirect measures of enhanced transmitter release. We suggest that possible presynaptic mechanisms underlying enhancement of evoked transmitter release by P2X 7RS activation are modulation of action potential width or an increase in presynaptic terminal excitability, due to subthreshold membrane depolarization which increases the number of terminals releasing transmitter in response to stimulation.

R-type voltage-gated ca 2+ channel interacts with synaptic proteins and recruits synaptotagmin to the plasma membrane of xenopus oocytes

It is well established that syntaxin 1A, synaptosomal-associated protein of 25 kDa (SNAP-25) and synaptotagmin either alone or in combination, modulate the kinetic properties of voltage-gated Ca 2+ channels Ca v1.2 (Lc-channel) Ca v2.2 (N-type) and Ca v2.1 (P/Q-type). The interaction interface was found to reside at the cytosolic II–III domain of the α1 subunit of the channels. In this study, we demonstrated a functional coupling of human neuronal Ca v2.3 (R-type channel) with syntaxin 1A, SNAP-25 and synaptotagmin in BAPTA injected Xenopus oocytes. The kinetic properties of Ca v2.3 assembled with syntaxin 1A, SNAP-25 or synaptotagmin individually differed from Ca v2.3 associated with binary complexes syntaxin 1A/SNAP-25, syntaxin 1A/synaptotagmin or SNAP-25/synaptotagmin. Co-expression of Ca v2.3 with syntaxin 1A, SNAP-25 and synaptotagmin together, produced a channel with distinctive kinetic properties analogous to excitosome multiprotein complex generated by Ca v1.2 and Ca v2.2. Exchanging the current-carrying ions altered the kinetics of channel/synaptic proteins interaction, indicating a tight crosstalk formed between the permeation pathway of Ca v2.3 and the fusion apparatus during membrane depolarization. This putative coupling could predict how the release site might be organized to allow a rapid communication between the channel and the release machinery. In vivo confocal imaging of oocytes revealed GFP-synaptotagmin at the plasma membrane when the channel was present, as opposed to random distribution in its absence, consistent with Ca 2+-independent molecular link of synaptotagmin and the channel. Synaptotagmin was detected at the membrane also in oocytes co-expressing the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Both imaging studies and protein-protein interactions in Xenopus oocytes show that channel linkage to synaptotagmin precedes Ca 2+ influx. Altogether, the R-type channel appears to associate with synaptic proteins to generate a multiprotein excitosome complex prior to Ca 2+-entry. We propose that the distinct kinetics of the Ca 2+-channel acquired by the close association with the vesicle and the t-SNAREs within the excitosome complex may be essential for depolarization evoked transmitter release.

Differential expression of α 1 and β subunits of voltage dependent Ca 2+ channel at the neuromuscular junction of normal and p/q Ca 2+ channel knockout mouse

Voltage-dependent calcium channels (VDCC) have a key role in neuronal function transforming the voltage signals into intracellular calcium signals. They are composed of the pore-forming α 1 and the regulatory α 2δ, γ and β subunits. Molecular and functional studies have revealed which α 1 subunit gene product is the molecular constituent of each class of native calcium channel (L, N, P/Q, R and T type). Electrophysiological and immunocytochemical studies have suggested that at adult mouse motor nerve terminal (MNT) only P/Q type channels, formed by α 1A subunit, mediate evoked transmitter release. The generation of α 1A-null mutant mice offers an opportunity to study the expression and localization of calcium channels at a synapse with complete loss of P/Q calcium channel. We have investigated the expression and localization of VDCCs α 1 and β subunits at the wild type (WT) and knockout (KO) mouse neuromuscular junction (NMJ) using fluorescence immunocytochemistry. The α 1A subunit was observed only at WT NMJ and was absent at denervated muscles and at KO NMJ. The subunits α 1B, α 1D and α 1E were also present at WT NMJ and they were over- expressed at KO NMJ suggesting a compensatory expression due to the lack of the α 1A. On the other hand, the β 1b, β 2a and β 4 were present at the same levels in both genotypes. The presence of other types of VDCC at WT NMJ indicate that they may play other roles in the signaling process which have not been elucidated and also shows that other types of VDCC are able to substitute the α 1A subunit, P/Q channel under certain pathological conditions.

Distinct Roles of Kv1 and Kv3 Potassium Channels at the Calyx of Held Presynaptic Terminal

Despite identification of >100 potassium channel subunits, relatively little is known about their roles in synaptic transmission. To address this issue we recorded presynaptic potassium currents (IPK) directly from the calyx of Held terminal in brainstem slices of rats. IPK was composed of a 4-aminopyridine (4-AP)-sensitive component and a smaller 4-AP-insensitive component composed of an iberiotoxin-sensitive current and an unidentified slowly activating potassium current. IPK could also be separated into a tetraethylammonium (TEA; 1 mm)-sensitive high-voltage-activated component and a margatoxin (10 nm)-sensitive low-voltage-activated component, which was also blocked by dendrotoxin-I (200 nm) and tityustoxin-Kalpha (100 nm). In outside-out patches excised from calyceal terminals, TEA (1 mm) consistently and to a large extent attenuated IPK, whereas margatoxin attenuated IPK only in a subset of patches (three of seven). Immunocytochemical examination using Kv subtype-specific antibodies indicated that multiple Kv1 and Kv3 subtypes were present at the calyceal terminal. In paired presynaptic and postsynaptic whole-cell recordings, TEA (1 mm) increased both the duration and peak amplitude of presynaptic action potentials and simultaneously potentiated EPSCs. Margatoxin alone had no such effect but reduced the amount of depolarization required for action potential generation, thereby inducing a burst of spikes when the nerve terminal was depolarized for a prolonged period. Thus, at the calyx of Held terminal, Kv3 channels directly regulate evoked transmitter release, whereas Kv1 channels reduce nerve terminal excitability, thereby preventing aberrant transmitter release. We conclude that both Kv3 and Kv1 channels contribute differentially to maintaining the fidelity of synaptic transmission at the calyx of Held.

Presynaptic Disruption of Transmitter Release by Lead

Low concentrations of inorganic lead ions (Pb 2+) disrupt transmitter release by causing aberrant augmentation of spontaneous and suppression of evoked release. These effects result from high affinity interactions of Pb 2+ with the voltage-gated calcium channels (VGCC) as well as Ca 2+ binding proteins which regulate the synaptic vesicle mobilization, docking, and exocytosis processes. Augmentation of spontaneous release may involve stimulation of vesicle mobilization consequent to Pb 2+ activation of CaMKII-dependent phosphorylation of synapsin I and/or stimulation of asynchronous exocytosis via direct Pb 2+ activation of the putative exocytotic Ca 2+-sensor protein synaptotagmin I. In addition, synergistic stimulation of PLC and DAG/Pb 2+-dependent activation of PKC may enhance the secretagogue effects of Pb 2+ by increasing metal sensitivity of exocytosis and/or modulating calcium channel activity. In contrast to intracellularly-mediated actions of Pb 2+ resulting in augmentation of spontaneous release, the inhibition of evoked transmitter release by Pb 2+ is largely attributable to extracellular block of the voltage-gated calcium channels.