Effects of A2 type botulinum toxin on spontaneous miniature and evoked transmitter release from the rat spinal excitatory and inhibitory synapses

We observed effects of newly developed A2 type botulinum toxin (A2NTX) on spontaneous miniature and evoked transmitter release from inhibitory (glycinergic or GABAergic), or excitatory (glutamatergic) nerve terminals in rat spinal cord, by use of ‘synaptic bouton’ preparations, under voltage-clamp condition. A2NTX (0.1–1 pM) initially augmented and then decreased amplitude and frequency of spontaneous miniature release of glycine or GABA (mIPSCs) concentration-dependently. At an increased concentration (1–10 pM), A2NTX suppressed the amplitude of glutamatergic mEPSCs. The rank order of the inhibitory effects was glycinergic > GABAergic >> glutamatergic synapses. Focal electrical stimulation of ‘synaptic boutons’ elicited eIPSC or eEPSC with larger amplitude and low failure rate (Rf). A2NTX (0.01–1 pM) initially enhanced the amplitude or decreased the failure rate of eIPSC or eEPSC, and then almost completely abolished the generation of eIPSC or eEPSC. The action of A2NTX on the evoked transmitter release was partially reversible. The rank order of the inhibitory effects on the amplitude or Rf were glycinergic eIPSC ≥ GABAergic eIPSC > glutamatergic eEPSCs. Excess extracellular K⁺ or Ca²⁺ (excess [K⁺]_o or [Ca²⁺]_o), and 4-AP restored spontaneous miniature glycinergic, GABAergic or glutamatergic postsynaptic currents suppressed by A2NTX. We conclude that A2NTX inhibits spontaneous miniature release at 0.1–10 pM and evoked release at 0.01–1 pM in rat spinal cord, and the inhibition was much efficient in the evoked rather than the spontaneous miniature release. Excess [K⁺]_o, 4-AP and excess [Ca²⁺]_o, which can raise the intracellular Ca²⁺ concentration via the activation of voltage-dependent Ca²⁺ channels, rescue the transmission suppressed by A2NTX poisoning, suggesting the transmitter release machinery became less sensitive to intracellular Ca²⁺ in A2NTX poisoned ‘synaptic boutons’.     

Nitric Oxide Modulation of GABAergic Synaptic Transmission in Mechanically Isolated Rat Auditory Cortical Neurons

The auditory cortex (A1) encodes the acquired significance of sound for the perception and interpretation of sound. Nitric oxide (NO) is a gas molecule with free radical properties that functions as a transmitter molecule and can alter neural activity without direct synaptic connections. We used whole-cell recordings under voltage clamp to investigate the effect of NO on spontaneous GABAergic synaptic transmission in mechanically isolated rat auditory cortical neurons preserving functional presynaptic nerve terminals. GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) in the A1 were completely blocked by bicuculline. The NO donor, S-nitroso-N-acetylpenicillamine (SNAP), reduced the GABAergic sIPSC frequency without affecting the mean current amplitude. The SNAP-induced inhibition of sIPSC frequency was mimicked by 8-bromoguanosine cyclic 3'',5''-monophosphate, a membrane permeable cyclic-GMP analogue, and blocked by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a specific NO scavenger. Blockade of presynaptic K⁺ channels by 4-aminopyridine, a K⁺ channel blocker, increased the frequencies of GABAergic sIPSCs, but did not affect the inhibitory effects of SNAP. However, blocking of presynaptic Ca²⁺ channels by Cd²⁺, a general voltage-dependent Ca²⁺ channel blocker, decreased the frequencies of GABAergic sIPSCs, and blocked SNAP-induced reduction of sIPSC frequency. These findings suggest that NO inhibits spontaneous GABA release by activation of cGMP-dependent signaling and inhibition of presynaptic Ca²⁺ channels in the presynaptic nerve terminals of A1 neurons.     

Effects of tetanus toxin on spontaneous and evoked transmitter release at inhibitory and excitatory synapses in the rat SDCN neurons

We observed the effects of tetanus toxin (TeNT) on spontaneous miniature and evoked postsynaptic currents at inhibitory (glycinergic) and excitatory (glutamatergic) synapses in SDCN of rat spinal cord, by use of ‘synaptic bouton’ preparations, under voltage clamp condition. TeNT (>10 pM) dose-dependently decreased the frequency without affecting amplitude of glycinergic spontaneous miniature IPSCs. However, TeNT (100 pM) had no effect on frequency or amplitude of glutamatergic spontaneous EPSCs. Focal paired electrical stimulation of ‘synaptic boutons’ elicited two consecutive glycinergic eIPSCs or glutamatergic eEPSCs with large amplitude and low failure rate (Rf). TeNT (100 pM) reduced the amplitude and increased the failure rate of the first glycinergic eIPSCs and greatly enhanced the ratio of the second to first (P2/P1) eIPSCs. Application of 4-AP restored glycinergic eIPSCs suppressed by TeNT (100 pM). However, TeNT (100 pM) had no effect on the amplitude, Rf or P2/P1 ratio of glutamatergic eEPSCs. These results show that TeNT pre-synaptically affects spontaneous and evoked, and inhibitory and excitatory neurotransmitter release differentially, thereby suggesting that molecular events underlying spontaneous and evoked, inhibitory and excitatory neurotransmitter release may be different in CNS, and that the release machinery becomes less sensitive to Ca²⁺ in TeNT poisoned ‘synaptic boutons’.     

Neurotoxin A2NTX blocks fast inhibitory and excitatory transmitter release from presynaptic terminals

Our recent study showed a possibility that newly developed A2 type botulinum toxin (A2NTX) inhibits both spontaneous and evoked transmitter release from inhibitory (glycinergic or GABAergic) and excitatory (glutamatergic) nerve terminals using rat spinal sacral dorsal commissural nucleus neurons. In the present study, to determine the modulatory effect of A2NTX on glycinergic and glutamatergic release probabilities, we tested the effects of A2NTX on a single inhibitory or excitatory nerve ending adherent to a dissociated neuron that was activated by paired-pulse stimuli by using the focal electrical stimulation technique. The results of the present paired-pulse experiments showed clearly that A2NTX enhanced paired-pulse facilitation of evoked glycinergic inhibitory postsynaptic currents and glutamatergic excitatory postsynaptic currents and increased the failure rate (Rf) of the first postsynaptic currents (P(1)) and both the responses. These effects of A2NTX on the amplitude and Rf of the P(1) and the second postsynaptic currents (P(2)) and paired-pulse ratio were rescued by application of 4-aminophthalimide. In summary, the present results showed that A2NTX acts purely presynaptically and inhibits the release machinery of transmitters such as glycine and glutamate, and the transmitter release machinery became less sensitive to intracellular free-Ca(2+) in A2NTX poisoned nerve terminals.     

三环哌酯对海马脑片神经元烟碱受体及谷氨酸能兴奋性突触传递的阻断作用

目的研究中枢抗胆碱药三环哌酯(TCPN)对海马脑片神经元烟碱受体(nAChR)及谷氨酸能兴奋性突触传递的阻断作用。方法采用海马脑片盲法全细胞记录技术,以自发兴奋性与抑制性突触后电流(sEPSC和sIPSC)为观测指标。结果TCPN(10～500μmol.L-1)浓度依赖地对抗nAChR激动剂碘化二甲基苯基哌嗪(DMPP)增强海马脑片CA1锥体神经元sEPSC的作用,500μmol.L-1完全阻断DMPP的增强作用。同时,TCPN浓度依赖地直接抑制神经元的sEPSC。但TCPN不抑制神经元的sIPSC,也不阻断DMPP对sIPSC的增强作用。结论TCPN对海马脑片神经元突触传递的影响具有双重作用,既能通过阻断谷氨酸能突触前末梢nAChR而抑制sEPSC,同时还能直接抑制sEPSC。     

P- and R-type Ca channels regulating spinal glycinergic nerve terminals

The contributions of P- and R-type Ca²⁺ channels on glycinergic nerve endings (boutons) projecting to the rat spinal sacral commissural nucleus (SDCN) neurons are not understood. Thus, we investigated the functional role of P- and R-type Ca²⁺ channels by measuring the inhibitory postsynaptic currents (eIPSCs) evoked from individual nerve endings (boutons) by focal electrical stimulation. The current amplitude and failure rate (Rf) of glycinergic eIPSCs varied directly with changes in [Ca²⁺]_o. Low concentration of ω-Aga IVA (P-type selective antagonist) suppressed eIPSCs as much as high concentration (both P- and Q-type selective) indicating little contribution of Q-type Ca²⁺ channels. Antagonism of R-type Ca²⁺ channels with SNX-482 and Ni²⁺ greatly decreased the current amplitude and increased failure rate (Rf) of glycinergic eIPSCs. Overall, our results suggest that the dominant control of glycine release depends on Ca²⁺ entry through P- and R-type Ca²⁺ channels that ubiquitously populate spinal glycine release sites.     

Activation of 5-HT6 receptors inhibits corticostriatal glutamatergic transmission

We investigated the effect of 5-HT6 receptor subtype activation on glutamatergic transmission by means of whole-cell patch-clamp electrophysiological recordings from medium spiny neurons of the striatum and layer V pyramidal neurons of the prefrontal cortex. To this aim, we took advantage of a novel ligand, ST1936, showing nM affinity and agonist activity at the 5-HT6 receptor subtype. Our data show that 5-HT6 receptor activation by ST1936 reduces the frequency of spontaneous excitatory postsynaptic currents, with an IC50 of 1.3 μM. Moreover, 5-HT6 receptor activation also reduced the amplitude of spontaneous excitatory postsynaptic currents recorded from medium spiny neurons, suggesting a mechanism of action involving postsynaptic 5-HT6 receptors,as further confirmed by the paired-pulse analysis on evoked excitatory postsynaptic currents and by recordings of miniature glutamatergic events. The inhibitory effect of ST1936 on glutamatergic transmission was prevented by the selective 5-HT6 receptor antagonist SB258585 and mimicked by a different agonist, WAY-181187.Conversely, in the cortex ST1936 reduced the frequency, but not the amplitude, of spontaneous excitatory postsynaptic currents suggesting a presynaptic or indirect effect of the 5-HT6 receptor.     

Inhibition by riluzole of glycinergic postsynaptic currents in rat hypoglossal motoneurones.

1. Riluzole has been shown to have beneficial effects in motoneurone disease, yet its effect on motoneurones is not known. To address this question, we investigated synaptic modulation by riluzole in hypoglossal motoneurones by recording glycinergic inhibitory postsynaptic currents evoked by stimulation of nearby single interneurones. 2. Glycinergic inhibitory postsynaptic currents were evoked by electrical stimulation of single interneurones and were recorded from visually identified hypoglossal motoneurones. Riluzole (10 microM) inhibited mean amplitude of evoked glycinergic inhibitory postsynaptic currents by 87%. 3. We found that riluzole suppressed sodium currents in brainstem interneurones by 23.8%. Riluzole did not modulate barium currents through voltage-activated calcium channels (98% of control). Therefore, the effect of riluzole on synaptic transmission may be mediated, in part, by stabilizing presynaptic neurones through inhibition of voltage-activated sodium currents. 4. In the presence of tetrodotoxin (0.5 microM), riluzole reduced the frequency (1.2 Hz in control to 0.6 Hz in riluzole) of spontaneous transmitter release recorded in motoneurones. 5. Riluzole was found to have no effect on mean miniature inhibitory postsynaptic current amplitude, therefore the reduction in spontaneous transmitter release cannot be due to an action on postsynaptic glycine receptors. 6. We conclude that riluzole inhibits synaptic transmission presynaptically, independent of a reduction in the excitation of presynaptic neurones.     

Multi-walled carbon nanotube inhibits CA1 glutamatergic synaptic transmission in rat’s hippocampal slices

The purpose of the study was to investigate the neurotoxic effect of multi-walled carbon nanotubes (MWCNTs) on the properties of glutamatergic synaptic transmission in rat’s hippocampal slices using whole-cell patch clamp technique. The amplitude and frequency of excitatory postsynaptic current (EPSC) were accessed on the hippocampal pyramidal neurons. The alterations of glutamatergic synaptic transmission in CA3–CA1 were examined by measuring both the amplitude of evoked excitatory postsynaptic current (eEPSC) and paired-pulse ratio (PPR). The data showed that the amplitude of either spontaneous excitatory postsynaptic current (sEPSC) or miniature excitatory postsynaptic current (mEPSC) was significantly inhibited by 1 μg/mL MWCNTs. However, it was found that there was a trend of different change on the frequency index. When 1 μg/mL MWCNTs was applied, there were a decreased frequency of mEPSC and an increased frequency of sEPSC, which might be due to the effect of action potential. Furthermore, the amplitudes of eEPSC at CA3–CA1 synapses were remarkably decreased. And the mean amplitude of AMPAR-mediated eEPSC was significantly reduced as well. Meanwhile, a majority of PPRs data were greater than one. There were no significant differences of PPRs between control and MWCNTs states, but an increased trend of paired-pulse facilitation was found. These results suggested that MWCNT markedly inhibited hippocampal CA1 glutamatergic synaptic transmission in vitro, which provided new insights into the MWCNT toxicology on CNS at cellular level.     

Regional differences in the effects of isoflurane on neurotransmitter release

Stimulus evoked neurotransmitter release requires that Na⁺ channel-dependent nerve terminal depolarization be transduced into synaptic vesicle exocytosis. Inhaled anesthetics block presynaptic Na⁺ channels and selectively inhibit glutamate over GABA release from isolated nerve terminals, indicating mechanistic differences between excitatory and inhibitory transmitter release. We compared the effects of isoflurane on depolarization-evoked [³H]glutamate and [¹⁴C]GABA release from isolated nerve terminals prepared from four regions of rat CNS evoked by 4-aminopyridine (4AP), veratridine (VTD), or elevated K⁺. These mechanistically distinct secretegogues distinguished between Na⁺ channel- and/or Ca²⁺ channel-mediated presynaptic effects. Isoflurane completely inhibited total 4AP-evoked glutamate release (IC₅₀ = 0.42 ± 0.03 mM) more potently than GABA release (IC₅₀ = 0.56 ± 0.02 mM) from cerebral cortex (1.3-fold greater potency), hippocampus and striatum, but inhibited glutamate and GABA release from spinal cord terminals equipotently. Na⁺ channel-specific VTD-evoked glutamate release from cortex was also significantly more sensitive to inhibition by isoflurane than was GABA release. Na⁺ channel-independent K⁺-evoked release was insensitive to isoflurane at clinical concentrations in all four regions, consistent with a target upstream of Ca²⁺ entry. Isoflurane inhibited Na⁺ channel-mediated (tetrodotoxin-sensitive) 4AP-evoked glutamate release (IC₅₀ = 0.30 ± 0.03 mM) more potently than GABA release (IC₅₀ = 0.67 ± 0.04 mM) from cortex (2.2-fold greater potency). The magnitude of inhibition of Na⁺ channel-mediated 4AP-evoked release by a single clinical concentration of isoflurane (0.35 mM) varied by region and transmitter: Inhibition of glutamate release from spinal cord was greater than from the three brain regions and greater than GABA release for each CNS region. These findings indicate that isoflurane selectively inhibits glutamate release compared to GABA release via Na⁺ channel-mediated transduction in the four CNS regions tested, and that differences in presynaptic Na⁺ channel involvement determine differences in anesthetic pharmacology.     

Regulation of spontaneous inhibitory synaptic transmission by endogenous glutamate via non-NMDA receptors in cultured rat hippocampal neurons

The regulation of gamma-aminobutyric acid (GABA)-mediated spontaneous inhibitory synaptic transmission by endogenously released glutamate was studied in cultured rat hippocampal neurons. After 7 days in vitro (DIV), both spontaneous excitatory postsynaptic currents (sEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs) could be detected. After 15 DIV, most postsynaptic spontaneous currents occurred as sEPSC/sIPSC sequences when recorded at a holding voltage of -30 mV. In the presence of the glutamate alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subtype antagonist LY303070, both the frequency and amplitude of sIPSC were strongly and reversibly reduced. The N-methyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonopentanoic acid (AP5), had no effect on sIPSC while cyclothiazide strongly increased sIPSC frequency. Under blockade of AMPA receptors, the kainate- and GluR5-selective kainate receptor agonists, (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid) (ATPA) and (S)-5-iodowillardiine (5IWill), induced a large enhancement of the frequency of small-amplitude sIPSC which was blocked by the non-NMDA receptor antagonist, 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX). All of these effects were sensitive to tetrodotoxin (TTX). In the presence of LY303070 and TTX, kainate could induce a small inward current while GluR5 agonists had no effect. In the presence of NMDA and AMPA receptor antagonists, the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC) could restore sIPSC. When NBQX was used as an AMPA antagonist, the stimulatory effect of t-PDC was blocked while the group I metabotropic glutamate agonist, 3,5-dihydroxyphenylglycine (DHPG), induced a strong enhancement of sIPSC. Therefore, both AMPA and kainate receptors can regulate inhibitory synaptic transmission in cultured hippocampal neurons, the former by tonic activation, the latter when the glutamate concentration is increased by impairing glutonate uptake.     

Modulation of presynaptic GABA(A) receptors by endogenous neurosteroids

BACKGROUND AND PURPOSE

Although 3α-hydroxy, 5α-reduced pregnane steroids, such as allopregnanolone (AlloP) and tetrahydrodeoxycorticosterone, are endogenous positive modulators of postsynaptic GABA_A receptors, the functional roles of endogenous neurosteroids in synaptic transmission are still largely unknown.

EXPERIMENTAL APPROACH

In this study, the effect of AlloP on spontaneous glutamate release was examined in mechanically isolated dentate gyrus hilar neurons by use of the conventional whole-cell patch-clamp technique.

KEY RESULTS

AlloP increased the frequency of glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) in a dose-dependent manner. The AlloP-induced increase in sEPSC frequency was completely blocked by a non-competitive GABA_A receptor blocker, tetrodotoxin or Cd²⁺, suggesting that AlloP acts on presynaptic GABA_A receptors to depolarize presynaptic nerve terminals to increase the probability of spontaneous glutamate release. On the other hand, γ-cyclodextrin (γ-CD) significantly decreased the basal frequency of sEPSCs. However, γ-CD failed to decrease the basal frequency of sEPSCs in the presence of a non-competitive GABA_A receptor antagonist or tetrodotoxin. In addition, γ-CD failed to decrease the basal frequency of sEPSCs after blocking the synthesis of endogenous 5α-reduced pregnane steroids. Furthermore, γ-CD decreased the extent of muscimol-induced increase in sEPSC frequency, suggesting that endogenous neurosteroids can directly activate and/or potentiate presynaptic GABA_A receptors to affect spontaneous glutamate release onto hilar neurons.

CONCLUSIONS AND IMPLICATIONS

The modulation of presynaptic GABA_A receptors by endogenous neurosteroids might affect the excitability of the dentate gyrus-hilus-CA3 network, and thus contribute, at least in part, to some pathological conditions, such as catamenial epilepsy and premenstrual dysphoric disorder.     

Effects of flunarizine on spontaneous synaptic currents in rat neocortex

Flunarizine, a non-selective blocker of voltage-dependent Ca²⁺ and Na⁺ channels, is clinically effective against several neurological disorders, including epilepsy, migraine, and alternating hemiplegia of childhood. We examined the effects of flunarizine on spontaneous post-synaptic currents in acute brain slices maintained in vitro using patch-clamp electrophysiology. Flunarizine significantly attenuated the amplitude of spontaneous currents in pyramidal neurons from juvenile rat neocortex. Flunarizine had no effect on miniature spontaneous events recorded in the presence of tetrodotoxin, a blocker of voltage-dependent sodium channels. In high (9 mM) extracellular potassium, flunarizine reduced the amplitude and frequency of the spontaneous currents. Additionally, dimethyl sulfoxide, the solvent used in our experiments, reduced the amplitude of spontaneous currents, but only in high extracellular potassium. Our data suggest that the clinical activity of flunarizine may in part be a consequence of reducing spontaneous synaptic currents in the neocortex, especially under conditions of heightened neuronal activity.     

Pre- and postsynaptic effects of kainate on layer II/III pyramidal cells in rat neocortex

Kainate receptors mediate both direct excitatory and indirect modulatory actions in the CNS. We report here that kainate has both pre- and postsynaptic actions in layer II/III pyramidal neurons of rat prefrontal cortex. Application of low concentration of kainate (50-500 nM) increased the amplitude of evoked excitatory postsynaptic currents (EPSCs) whereas higher concentrations (3 microM) caused a decrease. The frequency of spontaneous and miniature (action potential-independent) EPSCs was increased by low concentrations of kainate without affecting their amplitudes, suggesting a presynaptic mechanism of action. The facilitatory and inhibitory effects of kainate were mimicked by the GluR5 subunit selective agonist ATPA. In addition to decreasing EPSC amplitudes, high concentrations of kainate and ATPA induced an inward current which was not blocked by AMPA- or NMDA-receptor antagonists GYKI52466 and D-APV, respectively. The inward currents were blocked by the AMPA/KA receptor antagonist CNQX, indicating the presence of postsynaptic kainate receptors. Single shock stimulation in the presence of GYKI52466 and D-APV evoked an EPSC which was blocked by CNQX. The GluR5 antagonist LY382884 changed paired-pulse facilitation to paired pulse depression, indicating that synaptically released glutamate can activate presynaptic kainate receptors. These results suggest that kainate receptors containing GluR5 subunits play a major role in glutamatergic transmission in rat neocortex, having both presynaptic modulatory and direct postsynaptic excitatory actions.     

Effects of the facilitatory compounds catechol,guanidine, noradrenaline and phencyclidine on presynaptic currents of mouse motor nerve terminals

Summary Catechol, guanidine, noradrenaline, and phencyclidine can increase acetylcholine release at neuromuscular junctions. To determine if they act by affecting nerve terminal action potentials, the electrical activity of the terminal regions of motor nerves was recorded with an extracellular electrode inserted in the perineural sheaths of nerves in the mouse triangularis sterni preparation. Catechol (from 10 M) and guanidine (from 1 mM) produced a selective reduction in the component of the perineural waveform associated with voltage-dependent K⁺ currents, without significant effects on Na⁺, Ca⁺, or Ca²⁺-activated K⁺ currents. A selective block of K⁺ channels in nerve terminals would cause a prolonged depolarization and hence a large influx of Ca²⁺ to trigger acetylcholine release; this could explain the facilitatory effects of guanidine and catechol. Noradrenaline produced a slight increase in the amplitude of the. perineural waveform. This is consistent with hyperpolarization of the resting membrane potential of the nerve, which could lead to facilitation of acetylcholine release. Phencyclidine blocked Na⁺- and K⁺-related portions of the signal. Send offprint requests to A. L. Harvey at the above address     

Carisbamate (RWJ-333369) inhibits glutamate transmission in the granule cell of the dentate gyrus

Carisbamate (CRS, RWJ-333369) is a novel antiepileptic drug awaiting approval for use in the treatment of partial and generalized seizures. Our aim was to determine whether CRS modulates synaptic transmission in the dentate gyrus (DG) and the underlying mechanism. The whole-cell patch-clamp method was used to record AMPA receptor- and NMDA receptor-mediated excitatory postsynaptic currents (EPSC(AMPA) and EPSC(NMDA)) and GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSCs) in granule cells of the DG in brain slices prepared from 3- to 5-week-old male Wistar rats. CRS (30-300 μM) inhibited the evoked EPSC(AMPA) and EPSC(NMDA) by the same extent (20%) with significantly altered CV(-2), suggesting presynaptic modulation. It did not significantly change the inward currents induced by AMPA application. The inhibitory effect of CRS on the evoked EPSC(AMPA) was not occluded by selective voltage-gated Ca(2+) channel blockers, ruling out the involvement of presynaptic Ca(2+) channels. The frequency, but not the amplitude, of spontaneous EPSC(AMPA) was significantly reduced by CRS. However, CRS did not alter either the frequency or the amplitude of TTX-insensitive miniature EPSC(AMPA), indicating an action potential-dependent mechanism was involved. In addition, CRS (100 or 300 μM) did not significantly change the amplitude of the evoked IPSCs. To summarize, our results suggest that CRS reduces glutamatergic transmission by an action potential-dependent presynaptic mechanism and consequently inhibits excitatory synaptic strength in the DG without affecting GABAergic transmission. This effect may contribute to the antiepileptic action observed clinically at therapeutic concentrations of CRS.     

Effect of intravenous anesthetic propofol on synaptic vesicle exocytosis at the frog neuromuscular junction

Aim:

To investigate the presynaptic effects of propofol, a short-acting intravenous anesthetic, in the frog neuromuscular junction.

Methods:

Frog cutaneous pectoris nerve muscle preparations were prepared. A fluorescent tool (FM1-43) was used to visualize the effect of propofol on synaptic vesicle exocytosos in the frog neuromuscular junction.

Results:

Low concentrations of propofol, ranging from 10 to 25 μmol/L, enhanced spontaneous vesicle exocytosis monitored by FM1-43 in a Ca²⁺-dependent and Na⁺-independent fashion. Higher concentrations of propofol (50, 100, and 200 μmol/L) had no effect on spontaneous exocytosis. By contrast, higher concentrations of propofol inhibited the Na⁺-dependent exocytosis evoked by 4-aminopyridine but did not affect the Na⁺-independent exocytosis evoked by KCl. This action was similar and non-additive with that observed by tetrodotoxin, a Na⁺ channel blocker.

Conclusion:

Our data suggest that propofol has a dose-dependent presynaptic effect at the neuromuscular transmission which may help to understand some of the clinical effects of this agent on neuromuscular function.     

Hispidulin inhibits the release of glutamate in rat cerebrocortical nerve terminals

Hispidulin, a naturally occurring flavone, has been reported to have an antiepileptic profile. An excessive release of glutamate is considered to be related to neuropathology of epilepsy. We investigated whether hispidulin affected endogenous glutamate release in rat cerebral cortex nerve terminals (synaptosomes) and explored the possible mechanism. Hispidulin inhibited the release of glutamate evoked by the K⁺ channel blocker 4-aminopyridine (4-AP). The effects of hispidulin on the evoked glutamate release were prevented by the chelation of extracellular Ca^2 + ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl-threo-beta-benzyl-oxyaspartate did not have any effect on hispidulin action. Hispidulin reduced the depolarization-induced increase in cytosolic free Ca^2 + concentration ([Ca^2 +]_C), but did not alter 4-AP-mediated depolarization. Furthermore, the effect of hispidulin on evoked glutamate release was abolished by blocking the Ca_v2.2 (N-type) and Ca_v2.1 (P/Q-type) channels, but not by blocking ryanodine receptors or mitochondrial Na⁺/Ca^2 + exchange. Mitogen-activated protein kinase kinase (MEK) inhibition also prevented the inhibitory effect of hispidulin on evoked glutamate release. Western blot analyses showed that hispidulin decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synaptic vesicle-associated protein synapsin I, a major presynaptic substrate for ERK; this decrease was also blocked by the MEK inhibitor. Moreover, the inhibition of glutamate release by hispidulin was strongly attenuated in mice without synapsin I. These results show that hispidulin inhibits glutamate release from cortical synaptosomes in rats through the suppression of presynaptic voltage-dependent Ca^2 + entry and ERK/synapsin I signaling pathway.     

Underlying mechanism of actions of tefluthrin,a pyrethroid insecticide,on voltage-gated ion currents and on action currents in pituitary tumor (GH3) cells and GnRH-secreting (GT1-7) neurons

Tefluthrin is a synthetic pyrethroid and involved in acute neurotoxic effects. How this compound affects ion currents in endocrine or neuroendocrine cells remains unclear. Its effects on membrane ion currents in pituitary tumor (GH₃) cells and in hypothalamic (GT1-7) neurons were investigated. Application of Tef (10 μM) increased the amplitude of voltage-gated Na⁺ current (I_Na), along with a slowing in current inactivation and deactivation in GH₃ cells. The current–voltage relationship of I_Na was shifted to more negative potentials in the presence of this compound. Tef increased I_Na with an EC₅₀ value of 3.2 ± 0.8 μM. It also increased the amplitude of persistent I_Na. Tef reduced the amplitude of L-type Ca²⁺ current. This agent slightly inhibited K⁺ outward current; however, it had no effect on the activity of large-conductance Ca²⁺-activated K⁺ channels. Under cell-attached voltage-clamp recordings, Tef (10 μM) increased amplitude and frequency of spontaneous action currents, along with appearance of oscillatory inward currents. Tef-induced inward currents were suppressed after further application of tetrodotoxin, riluzole or ranolazine. In GT1-7 cells, Tef also increased the amplitude and frequency of action currents. Taken together, the effects of Tef and its structural related pyrethroids on ion currents can contribute to the underlying mechanisms through which they affect endocrine or neuroendocrine function in vivo.     

Analysis of Caribbean ciguatoxin-1 effects on frog myelinated axons and the neuromuscular junction

Caribbean ciguatoxin-1 (C-CTX-1) induced, after about 1 h exposure, muscle membrane depolarisation and repetitive post-synaptic action potentials (APs) in frog neuromuscular preparations. This depolarising effect was also observed in a Ca²⁺-free medium with a strong enhancement of spontaneous quantal transmitter release, compared with control conditions. The ciguatoxin-induced increase in release could be accelerated when Ca²⁺ was present in the extracellular medium. C-CTX-1 also enhanced nerve-evoked quantal acetylcholine (ACh) release. At normal neuromuscular junctions loaded with the fluorescent dye FM1-43, C-CTX-1 induced swelling of nerve terminals, an effect that was reversed by hyperosmotic d-mannitol. In myelinated axons, C-CTX-1 increased nodal membrane excitability, inducing spontaneous and repetitive APs. Also, the toxin enlarged the repolarising phase of APs in control and tetraethylammonium-treated axons. Overall, our data suggest that C-CTX-1 affects nerve excitability and neurotransmitter release at nerve terminals. We conclude that C-CTX-1-induced up-regulation of Na⁺ channels and the inhibition of K⁺ channels, at low nanomolar concentrations, produce a variety of functional dysfunctions that are in part responsible for the human muscle skeletal symptoms observed in ciguatera. All these dysfunctions seem to result from the subtle balance between ionic currents, intracellular Na⁺ and Ca²⁺ concentrations, and engaged second messengers.