Biphasic actions of topiramate on monoamine exocytosis associated with both soluble N-ethylmaleimide-sensitive factor attachment protein receptors and Ca(2+)-induced Ca(2+)-releasing systems

To explore the pharmacological mechanisms of topiramate (TPM), we determined the effects of TPM on monoamine (dopamine and serotonin) exocytosis associated with N-ethylmaleimide-sensitive factor attachment protein receptors and Ca(2+)-induced Ca(2+)-releasing systems, including inositol-triphosphate receptor and ryanodine receptor in freely moving rat pre-frontal cortex using in vivo microdialysis. During resting stage, Ca(2+) output from endoplasmic reticulum Ca(2+) store via inositol-triphosphate receptor regulates syntaxin-associated monoamine exocytosis mechanism, whereas during neuronal hyperexcitable stage, Ca(2+) output via ryanodine receptor regulates synaptobrevin-associated monoamine exocytosis mechanism. Basal monoamine releases were increased and decreased by therapeutically relevant and supratherapeutic concentration of TPM, respectively. The therapeutic-relevant concentration of TPM increased Ca(2+)-evoked release concentration-dependently; however, its stimulatory effect was attenuated in the supratherapeutic range. The K(+)-evoked releases were reduced by TPM concentration-dependently (from therapeutic to supratherapeutic ranges). The therapeutic-relevant concentration of TPM-induced elevation of basal release was reduced by cleavage with syntaxin and inhibition of inositol-triphosphate receptor predominantly, by cleavage with SNAP-25 and synaptobrevin weakly, but not by ryanodine receptor inhibitor. The therapeutic-relevant concentration of TPM-induced elevation of Ca(2+)-evoked release was reduced by cleavage with syntaxin and inositol-triphosphate receptor inhibitor selectively. The therapeutic-relevant concentration of TPM-induced reduction of K(+)-evoked monoamine release was abolished by cleavage with synaptobrevin, but was not affected by cleavage with SNAP-25 or synaptobrevin. The stimulatory effect of ryanodine receptor agonist on K(+)-evoked monoamine release was reduced by TPM, whereas that of inositol-triphosphate receptor agonist was not affected by TPM. Therefore, these results indicate that the combination of the effects of TPM on exocytosis mechanisms associated with SNARE and Ca(2+)-induced Ca(2+)-releasing systems, enhancement of inositol-triphosphate receptor/syntaxin and inhibition of ryanodine receptor/synaptobrevin in pre-frontal cortex, may be involved in clinical actions of TPM.     

Snapin: a SNARE-associated protein implicated in synaptic transmission

Synaptic vesicle docking and fusion are mediated by the assembly of a stable SNARE core complex of proteins, which include the synaptic vesicle membrane protein VAMP/synaptobrevin and the plasmalemmal proteins syntaxin and SNAP-25. We have now identified another SNAP-25-binding protein, called Snapin. Snapin was enriched in neurons and exclusively located on synaptic vesicle membranes. It associated with the SNARE complex through direct interaction with SNAP-25. Binding of recombinant Snapin-CT to SNAP-25 blocked the association of the SNARE complex with synaptotagmin. Introduction of Snapin-CT and peptides containing the SNAP-25 binding sequence into presynaptic superior cervical ganglion neurons in culture reversibly inhibited synaptic transmission. These results suggest that Snapin is an important component of the neurotransmitter release process through its modulation of the sequential interactions between the SNAREs and synaptotagmin.     

Immunocytochemical identification of proteins involved in dopamine release from the somatodendritic compartment of nigral dopaminergic neurons

We examined the somatodendritic compartment of nigral dopaminergic neurons by immunocytochemistry and confocal microscopy, with the aim of identifying proteins that participate in dopamine packaging and release. Nigral dopaminergic neurons were identified by location, cellular features and tyrosine hydroxylase immunoreactivity. Immunoreactive puncta of vesicular monoamine transporter type 2 and proton ATPase, both involved in the packaging of dopamine for release, were located primarily in dopaminergic cell bodies, but were absent in distal dopaminergic dendrites. Many presynaptic proteins associated with transmitter release at fast synapses were absent in nigral dopaminergic neurons, including synaptotagmin 1, syntaxin1, synaptic vesicle proteins 2a and 2b, synaptophysin and synaptobrevin 1 (VAMP 1). On the other hand, syntaxin 3, synaptobrevin 2 (VAMP 2) and SNAP-25-immunoreactivities were found in dopaminergic somata and dendrites Our data imply that the storage and exocytosis of dopamine from the somatodendritic compartment of nigral dopaminergic neurons is mechanistically distinct from transmitter release at axon terminals utilizing amino acid neurotransmitters.     

Genetic ablation of the t-SNARE SNAP-25 distinguishes mechanisms of neuroexocytosis.

Axon outgrowth during development and neurotransmitter release depends on exocytotic mechanisms, although what protein machinery is common to or differentiates these processes remains unclear. Here we show that the neural t-SNARE (target-membrane-associated-soluble N-ethylmaleimide fusion protein attachment protein (SNAP) receptor) SNAP-25 is not required for nerve growth or stimulus-independent neurotransmitter release, but is essential for evoked synaptic transmission at neuromuscular junctions and central synapses. These results demonstrate that the development of neurotransmission requires the recruitment of a specialized SNARE core complex to meet the demands of regulated exocytosis.     

Development- and activity-dependent regulation of SNAP-25 phosphorylation in rat brain

Synaptosomal-associated protein of 25kDa (SNAP-25), a member of the SNARE proteins essential for neurotransmitter release, is phosphorylated at Ser(187) in PC12 cells and in the rat brain in a protein kinase C-dependent manner. It remains unclear how the phosphorylation of SNAP-25 is regulated during development and by neuronal activity. We studied the mode of SNAP-25 phosphorylation at Ser(187) in the rat brain using an anti-phosphorylated SNAP-25 antibody. Both the expression and phosphorylation of SNAP-25 increased remarkably during the early postnatal period, but their onsets were quite different. SNAP-25 expression was detected as early as embryonic Day 18, whereas the phosphorylation of SNAP-25 could not be detected until postnatal Day 4. A delay in the onset of phosphorylation was also observed in cultured rat hippocampal neurons. The phosphorylation of SNAP-25 was regulated in a neuronal activity-dependent manner and, in the rat hippocampus, decreased by introducing seizures with kainic acid. These results clearly indicated that the phosphorylation of SNAP-25 at Ser(187) is regulated in development- and neuronal activity-dependent manners, and is likely to play important roles in higher brain functions.     

Differential modulation of short-term synaptic dynamics by long-term potentiation at mouse hippocampal mossy fibre synapses

After exocytosis, synaptic vesicle components are selectively retrieved by clathrin-mediated endocytosis and then re-used in future rounds of transmitter release. Under some conditions, synaptic terminals in addition perform bulk endocytosis of large membranous sacs. Bulk endocytosis is less selective than clathrin-mediated endocytosis and probably internalizes components normally targeted to the plasma membrane. Nonetheless, this process plays a major role in some tonic ribbon-type synapses, which release neurotransmitter for prolonged periods of time. We show here, that large endosomes formed after strong and prolonged stimulation undergo stimulated exocytosis in retinal bipolar neurons. The result suggests how cells might return erroneously internalized components to the plasma membrane, and also demonstrates that synaptic vesicles are not the only neuronal organelle that stains with styryl dyes and undergoes stimulated exocytosis.     

Regulation of transmitter release by synapsin II in mouse motor terminals

We investigated quantal release and ultrastructure in the neuromuscular junctions of synapsin II knockout (Syn II KO) mice. Synaptic responses were recorded focally from the diaphragm synapses during electrical stimulation of the phrenic nerve. We found that synapsin II affects transmitter release in a Ca²⁺-dependent manner. At reduced extracellular Ca²⁺ (0.5 m m ), Syn II KO mice demonstrated a significant increase in evoked and spontaneous quantal release, while at the physiological Ca²⁺ concentration (2 m m ), quantal release in Syn II KO synapses was unaffected. Protein kinase inhibitor H7 (100 μ m ) suppressed quantal release significantly stronger in Syn II KO synapses than in wild type (WT), indicating that Syn II KO synapses may compensate for the lack of synapsin II via a phosphorylation-dependent pathway. Electron microscopy analysis demonstrated that the lack of synapsin II results in an approximately 40% decrease in the density of synaptic vesicles in the reserve pool, while the number of vesicles docked to the presynaptic membrane remained unchanged. Synaptic depression in Syn II KO synapses was slightly increased, which is consistent with the depleted vesicle store in these synapses. At reduced Ca²⁺ frequency facilitation of synchronous release was significantly increased in Syn II KO, while facilitation of asynchronous release was unaffected. Thus, at the reduced Ca²⁺ concentration, synapsin II suppressed transmitter release and facilitation. These results demonstrate that synapsin II can regulate vesicle clustering, transmitter release, and facilitation.     

Role of presynaptic L-type Ca2+ channels in GABAergic synaptic transmission in cultured hippocampal neurons

Using dual whole cell patch-clamp recordings of monosynaptic GABAergic inhibitory postsynaptic currents (IPSCs) in cultured rat hippocampal neurons, we have previously demonstrated posttetanic potentiation (PTP) of IPSCs. Tetanic stimulation of the GABAergic neuron leads to accumulation of Ca2+ in the presynaptic terminals. This enhances the probability of GABA-vesicle release for up to 1 min, which underlies PTP. In the present study, we have examined the effect of altering the probability of release on PTP of IPSCs. Baclofen (10 microM), which depresses presynaptic Ca2+ entry through N- and P/Q-type voltage-dependent Ca2+ channels (VDCCs), caused a threefold greater enhancement of PTP than did reducing [Ca2+]o to 1.2 mM, which causes a nonspecific reduction in Ca2+ entry. This finding prompted us to investigate whether presynaptic L-type VDCCs contribute to the Ca2+ accumulation in the boutons during spike activity. The L-type VDCC antagonist, nifedipine (10 microM), had no effect on single IPSCs evoked at 0.2 Hz but reduced the PTP evoked by a train of 40 Hz for 2 s by 60%. Another L-type VDCC antagonist, isradipine (5 microM), similarly inhibited PTP by 65%. Both L-type VDCC blockers also depressed IPSCs during the stimulation (i.e., they increased tetanic depression). The L-type VDCC "agonist" (-)BayK 8644 (4 microM) had no effect on PTP evoked by a train of 40 Hz for 2 s, which probably saturated the PTP process, but enhanced PTP evoked by a train of 1 s by 91%. In conclusion, the results indicate that L-type VDCCs do not participate in low-frequency synchronous transmitter release, but contribute to presynaptic Ca2+ accumulation during high-frequency activity. This helps maintain vesicle release during tetanic stimulation and also enhances the probability of transmitter release during the posttetanic period, which is manifest as PTP. Involvement of L-type channels in these processes represents a novel presynaptic regulatory mechanism at fast CNS synapses.     

Essential role of Ca2+-binding protein 4, a Cav1.4 channel regulator, in photoreceptor synaptic function

CaBP1-8 are neuronal Ca(2+)-binding proteins with similarity to calmodulin (CaM). Here we show that CaBP4 is specifically expressed in photoreceptors, where it is localized to synaptic terminals. The outer plexiform layer, which contains the photoreceptor synapses with secondary neurons, was thinner in the Cabp4(-/-) mice than in control mice. Cabp4(-/-) retinas also had ectopic synapses originating from rod bipolar and horizontal cells tha HJt extended into the outer nuclear layer. Responses of Cabp4(-/-) rod bipolars were reduced in sensitivity about 100-fold. Electroretinograms (ERGs) indicated a reduction in cone and rod synaptic function. The phenotype of Cabp4(-/-) mice shares similarities with that of incomplete congenital stationary night blindness (CSNB2) patients. CaBP4 directly associated with the C-terminal domain of the Ca(v)1.4 alpha(1)-subunit and shifted the activation of Ca(v)1.4 to hyperpolarized voltages in transfected cells. These observations indicate that CaBP4 is important for normal synaptic function, probably through regulation of Ca(2+) influx and neurotransmitter release in photoreceptor synaptic terminals.     

PKC modulation of transmitter release by SNAP-25 at sensory-to-motor synapses in aplysia

Activation of phosphokinase C (PKC) can increase transmitter release at sensory-motor neuron synapses in Aplysia, but the target of PKC phosphorylation has not been determined. One putative target of PKC at synapses is the synaptosomal-associated protein of 25 kDa (SNAP-25), a member of the SNARE protein complex implicated in synaptic vesicle docking and fusion. To determine whether PKC regulated transmitter release through phosphorylation of SNAP-25, we cloned Aplysia SNAP-25 and expressed enhanced green fluorescent protein (EGFP)-coupled SNAP-25 constructs mutated at the PKC phosphorylation site Ser198 in Aplysia sensory neurons. We found several distinct effects of expression of EGFP-SNAP-25 constructs. First, the rates of synaptic depression were slowed when cells contained SNAP-25 with phosphomimetic residues Glu or Asp. Second, PDBu-mediated increases in transmitter release at na?ve synapses were blocked in cells expressing nonphosphorylated-state SNAP-25. Finally, expression of EGFP-coupled SNAP-25 but not uncoupled SNAP-25 inhibited 5-HT-mediated reversal of depression and the ability of EGFP-coupled SNAP-25 to inhibit the reversal of depression was affected by changes at Ser198. These results suggest SNAP-25 and phosphorylation of SNAP-25 by PKC can regulate transmitter release at Aplysia sensory-motor neuron synapses by a number of distinct processes.     

Exocytosis at the hair cell ribbon synapse apparently operates without neuronal SNARE proteins

SNARE proteins mediate membrane fusion. Neurosecretion depends on neuronal soluble NSF attachment protein receptors (SNAREs; SNAP-25, syntaxin-1, and synaptobrevin-1 or synaptobrevin-2) and is blocked by neurotoxin-mediated cleavage or genetic ablation. We found that exocytosis in mouse inner hair cells (IHCs) was insensitive to neurotoxins and genetic ablation of neuronal SNAREs. mRNA, but no synaptically localized protein, of neuronal SNAREs was present in IHCs. Thus, IHC exocytosis is unconventional and may operate independently of neuronal SNAREs.     

K(+)-evoked dopamine release depends on a cytosolic Ca2+ pool regulated by N-type Ca2+ channels.

Membrane depolarization evoked by 25-40 mM K+ elicited an immediate increase of somatic and neuritic [Ca2+]i in cultured dopaminergic neurons as measured by digital fluorescence microscope imaging. The rise of neuritic [Ca2+]i was inhibited by N-type but not L-type Ca2+ channel blockers, while the rise of somatic [Ca2+]i was prevented by both L- and N-type Ca2+ channel blockers. Similarly, depolarization-induced [3H]dopamine release was selectively attenuated by N-type Ca2+ channel blockers. The present results suggest that [3H]dopamine release from mesencephalic neuronal cell cultures relates to a Ca(2+)-dependent mechanism regulated by N-type channels located in the vicinity of the exocytotic sites within neuritic processes.     

Control of neurotransmitter release: From Ca2+ to voltage dependent G-protein coupled receptors

This review discusses two theories that try to explain mechanisms of control of neurotransmitter release in fast synapses: the Ca²⁺ hypothesis and the Ca²⁺ voltage hypothesis. The review summarizes experimental results that are incompatible with predictions from the Ca²⁺ hypothesis and concludes that Ca²⁺ is involved in the control of the amount of release but not in the control of the time course of evoked release, i.e., initiation and termination of evoked release. Results summarizing direct effects of changes in membrane potential on the release machinery are then presented. These changes in membrane potential affect the affinity (for the transmitter) of presynaptic autoinhibitory G-protein coupled receptors (GPCRs). The voltage dependence of these GPCRs and their pivotal role in determining the time course of evoked release is discussed.     

Analysis of SNAP-25 immunoreactivity in hippocampal inhibitory neurons during development in culture and in situ

Synaptosomal associated protein of 25 kDa (SNAP-25) is a component of the soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complex which plays a central role in synaptic vesicle exocytosis. We have previously demonstrated that adult rat hippocampal GABAergic synapses, both in culture and in brain, are virtually devoid of SNAP-25 immunoreactivity and are less sensitive to the action of botulinum toxin type A, which cleaves this SNARE protein [Neuron 41 (2004) 599]. In the present study, we extend our findings to the adult mouse hippocampus and we also provide demonstration that hippocampal inhibitory synapses lacking SNAP-25 labeling belong to parvalbumin-, calretinin- and cholecystokinin-positive interneurons. A partial colocalization between SNAP-25 and glutamic acid decarboxylase is instead detectable in developing mouse hippocampus at P0 and, at a lesser extent, at P5. In rat embryonic hippocampal cultures at early developmental stages, SNAP-25 immunoreactivity is detectable in a percentage of GABAergic neurons, which progressively reduces with time in culture. Consistent with the presence of the substrate, botulinum toxin type A is partially effective in inhibiting synaptic vesicle recycling in immature GABAergic neurons. Since SNAP-25, beside its role as a SNARE protein, is involved in additional processes, such as neurite outgrowth and regulation of calcium dynamics, the presence of higher levels of the protein at specific stages of neuronal differentiation may have implications for the construction and for the functional properties of brain circuits.     

Determining Ca2+-sensor binding time and its variability in evoked neurotransmitter release

The speed and reliability of neuronal reactions are important factors for proper functioning of the nervous system. To understand how organisms use protein molecules to carry out very fast biological actions, we quantified single-molecule reaction time and its variability in synaptic transmission. From the synaptic delay of crayfish neuromuscular synapses the time for a few Ca²⁺ ions to bind with their sensors in evoked neurotransmitter release was estimated. In standard crayfish saline at room temperature, the average Ca²⁺ binding time was 0.12 ms for the first evoked quanta. At elevated extracellular Ca²⁺ concentrations this binding time reached a limit due to saturation of Ca²⁺ influx. Analysis of the synaptic delay variance at various Ca²⁺ concentrations revealed that the variability of the Ca²⁺-sensor binding time is the major source of the temporal variability of synaptic transmission, and that the Ca²⁺-independent molecular reactions after Ca²⁺ influx were less stochastic. The results provide insights into how organisms maximize reaction speed and reliability.     

Facilitated activation of metabotropic glutamate receptors in cerebellar Purkinje cells in glutamate transporter EAAT4-deficient mice

Around excitatory synapses in cerebellar Purkinje cells (PCs), GLAST and EAAT4 are expressed as predominant glial and neuronal glutamate transporters, respectively. EAAC1, another subtype of neuronal glutamate transporter, is also expressed in PCs. EAAT4 is co-localized with metabotropic glutamate receptors (mGluRs) at perisynaptic sites in excitatory synapses in PCs, and this neuronal transporter was reported to be involved in the regulation of mGluR activation induced by the stimulation of parallel fibers (PFs). However, it remains to be elucidated whether only EAAT4 is specifically involved in mGluR activation among the glutamate transporters expressed near excitatory synapses in PCs. Here we examined mGluR-mediated excitatory postsynaptic currents (mGluR-EPSCs) evoked by PF stimulation in cerebellar slices of mice deficient in EAAT4, EAAC1, or GLAST. PF-evoked mGluR-EPSCs showed larger amplitude and faster rising kinetics in EAAT4-deficient mice than in the wild-type mice. In contrast, there was no significant difference in either the amplitude or the rising kinetics of mGluR-EPSCs in GLAST- or EAAC1-deficient mice compared to wild-type mice. We conclude that EAAT4 is most closely involved in mGluR activation in PCs among the glutamate transporters.     

VAMP4 directs synaptic vesicles to a pool that selectively maintains asynchronous neurotransmission

Synaptic vesicles in the brain harbor several soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins. With the exception of synaptobrevin2, or VAMP2 (syb2), which is directly involved in vesicle fusion, the role of these SNAREs in neurotransmission is unclear. Here we show that in mice syb2 drives rapid Ca(2+)-dependent synchronous neurotransmission, whereas the structurally homologous SNARE protein VAMP4 selectively maintains bulk Ca(2+)-dependent asynchronous release. At inhibitory nerve terminals, up- or downregulation of VAMP4 causes a correlated change in asynchronous release. Biochemically, VAMP4 forms a stable complex with SNAREs syntaxin-1 and SNAP-25 that does not interact with complexins or synaptotagmin-1, proteins essential for synchronous neurotransmission. Optical imaging of individual synapses indicates that trafficking of VAMP4 and syb2 show minimal overlap. Taken together, these findings suggest that VAMP4 and syb2 diverge functionally, traffic independently and support distinct forms of neurotransmission. These results provide molecular insight into how synapses diversify their release properties by taking advantage of distinct synaptic vesicle-associated SNAREs.     

Role of vesicle fusion mechanism and cell adhesion molecules in growth cone elongation and synaptogenesis]

Growth cones have been regarded to play central roles in forming complex neural network through target tract selection and target cell recognition. We have found that the vesicle fusion molecular system in growth cones is similar to that in synapses. The cleavage of SNAP25, a protein involved in neurotransmitter release in synapse, resulted in inhibition of neurite elongation. Inversely the overexpression of VAMP2/synaptobrevin, another protein involved in neurotransmitter release in synapse, in PC12 cells resulted in promotion of neurite elongation. We have recently isolated a novel components at cadherin-based cell-cell adherens junctions, named neurabin I, neurabin II, and afadin. Neurabin I is specifically expressed in nervous tissues and localized at synapses. Afadin is uviquitously expressed in various tissues and highly concentrated at cell-cell adherens junctions of small intestine epithelial cells, cardiac muscles, synapses. We have identified the transmembrane protein binding to afadin, and named it nectin, which belongs to immunoglobulin superfamily. Nectin, afadin, neurabin I, and neurabin II may be key molecules to find out various unknown guidance cues which can induce regenerating growth cones to repair the functional neuronal connections in central nervous system after injury.     

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(v)1.2 (Lc-channel) Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type). The interaction interface was found to reside at the cytosolic II-III domain of the alpha1 subunit of the channels. In this study, we demonstrated a functional coupling of human neuronal Ca(v)2.3 (R-type channel) with syntaxin 1A, SNAP-25 and synaptotagmin in BAPTA injected Xenopus oocytes. The kinetic properties of Ca(v)2.3 assembled with syntaxin 1A, SNAP-25 or synaptotagmin individually differed from Ca(v)2.3 associated with binary complexes syntaxin 1A/SNAP-25, syntaxin 1A/synaptotagmin or SNAP-25/synaptotagmin. Co-expression of Ca(v)2.3 with syntaxin 1A, SNAP-25 and synaptotagmin together, produced a channel with distinctive kinetic properties analogous to excitosome multiprotein complex generated by Ca(v)1.2 and Ca(v)2.2. Exchanging the current-carrying ions altered the kinetics of channel/synaptic proteins interaction, indicating a tight crosstalk formed between the permeation pathway of Ca(v)2.3 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.