Depression of Synaptic N-methyl-D-Aspartate Responses by Xenon and Nitrous Oxide.

In "synapse bouton preparation" of rat hippocampal CA3 neurons, we examined how Xe and N2O modulate N-methyl-D-aspartate (NMDA) receptor-mediated spontaneous and evoked excitatory post-synaptic currents (sEPSCNMDA and eEPSCNMDA). This preparation is a mechanically isolated single neuron attached with nerve endings (boutons) preserving normal physiologic function and promoting the exact evaluation of sEPSCNMDA and eEPSCNMDA responses without influence of extrasynaptic, glial, and other neuronal tonic currents. These sEPSCs and eEPSCs are elicited by spontaneous glutamate release from many homologous glutamatergic boutons and by focal paired-pulse electric stimulation of a single bouton, respectively. The s/eEPSCAMPA/KA and s/eEPSCNMDA were isolated pharmacologically by their specific antagonists. Thus, independent contributions of pre- and postsynaptic responses could also be quantified. All kinetic properties of s/eEPSCAMPA/KA and s/eEPSCNMDA were detected clearly. The s/eEPSCNMDA showed smaller amplitude and slower rise and 1/e decay time constant (τ Decay) than s/eEPSCAMPA/KA Xe (70%) and N2O (70%) significantly decreased the frequency and amplitude without altering the τ Decay of sEPSCNMDA They also decreased the amplitude but increased the Rf and PPR without altering the τ Decay of the eEPSCNMDA These data show clearly that "synapse bouton preparation" can be an accurate model for evaluating s/eEPSCNMDA Such inhibitory effects of gas anesthetics are primarily due to presynaptic mechanisms. Present results may explain partially the powerful analgesic effects of Xe and N2O. SIGNIFICANCE STATEMENT: We could record pharmacologically isolated NMDA receptor-mediated spontaneous and (action potential-evoked) excitatory postsynaptic currents (sEPSCNMDA and eEPSCNMDA) and clearly detect all kinetic parameters of sEPSCNMDA and eEPSCNMDA at synaptic levels by using "synapse bouton preparation" of rat hippocampal CA3 neurons. We found that Xe and N2O clearly suppressed both sEPSCNMDA and eEPSCNMDA. Different from previous studies, present results suggest that Xe and N2O predominantly inhibit the NMDA responses by presynaptic mechanisms.

Protein Kinase A Is Responsible for the Presynaptic Inhibition of Glycinergic and Glutamatergic Transmissions by Xenon in Rat Spinal Cord and Hippocampal CA3 Neurons.

The effects of a general anesthetic xenon (Xe) on spontaneous, miniature, electrically evoked synaptic transmissions were examined using the "synapse bouton preparation," with which we can clearly evaluate pure synaptic responses and accurately quantify pre- and postsynaptic transmissions. Glycinergic and glutamatergic transmissions were investigated in rat spinal sacral dorsal commissural nucleus and hippocampal CA3 neurons, respectively. Xe presynaptically inhibited spontaneous glycinergic transmission, the effect of which was resistant to tetrodotoxin, Cd2+, extracellular Ca2+, thapsigargin (a selective sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor), SQ22536 (an adenylate cyclase inhibitor), 8-Br-cAMP (membrane-permeable cAMP analog), ZD7288 (an hyperpolarization-activated cyclic nucleotide-gated channel blocker), chelerythrine (a PKC inhibitor), and KN-93 (a CaMKII inhibitor) while being sensitive to PKA inhibitors (H-89, KT5720, and Rp-cAMPS). Moreover, Xe inhibited evoked glycinergic transmission, which was canceled by KT5720. Like glycinergic transmission, spontaneous and evoked glutamatergic transmissions were also inhibited by Xe in a KT5720-sensitive manner. Our results suggest that Xe decreases glycinergic and glutamatergic spontaneous and evoked transmissions at the presynaptic level in a PKA-dependent manner. These presynaptic responses are independent of Ca2+ dynamics. We conclude that PKA can be the main molecular target of Xe in the inhibitory effects on both inhibitory and excitatory neurotransmitter release. SIGNIFICANCE STATEMENT: Spontaneous and evoked glycinergic and glutamatergic transmissions were investigated using the whole-cell patch clamp technique in rat spinal sacral dorsal commissural nucleus and hippocampal CA3 neurons, respectively. Xenon (Xe) significantly inhibited glycinergic and glutamatergic transmission presynaptically. As a signaling mechanism, protein kinase A was responsible for the inhibitory effects of Xe on both glycine and glutamate release. These results may help understand how Xe modulates neurotransmitter release and exerts its excellent anesthetic properties.

Effects of TND1128 (a 5-deazaflavin derivative), with self-redox ability, as a mitochondria activator on the mouse brain slice and its comparison with ß-NMN.

We have no definitive treatment for dementia characterized by prolonged neuronal death due to the enormous accumulation of foreign matter, such as ß-amyloid. Since Alzheimer's type dementia develops slowly, we may be able to delay the onset and improve neuronal dysfunction by enhancing the energy metabolism of individual neurons. TND1128, a derivative of 5-deazaflavin, is a chemical known to have an efficient self-redox ability. We expected TND1128 as an activator for mitochondrial energy synthesis. We used brain slices prepared from mice 22 ± 2 h pretreated with TND1128 or ß-NMN. We measured Ca2+ concentrations in the cytoplasm ([Ca2+]cyt) and mitochondria ([Ca2+]mit) by using fluorescence Ca2+ indicators, Fura-4F, and X-Rhod-1, respectively, and examined the protective effects of drugs on [Ca2+]cyt and [Ca2+]mit overloading by repeating 80K exposure. TND1128 (0.01, 0.1, and 1 mg/kg s.c.) mitigates the dynamics of both [Ca2+]cyt and [Ca2+]mit in a dose-dependent manner. ß-NMN (10, 30, and 100 mg/kg s.c.) also showed significant dose-dependent mitigating effects on [Ca2+]cyt, but the effect on the [Ca2+]mit dynamics was insignificant. We confirmed the mitochondria-activating potential of TND1128 in the present study. We expect TND1128 as a drug that rescues deteriorating neurons with aging or disease.

The novel mitochondria activator, 10-ethyl-3-methylpyrimido[4,5-b]quinoline-2,4(3H,10H)-dione (TND1128), promotes the development of hippocampal neuronal morphology.

Adenosine triphosphate (ATP) is the most vital energy source produced mainly in the mitochondria. Age-related mitochondrial dysfunction is associated with brain diseases. Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor for energy production in mitochondria. Here, we examined how the novel NAD+-assisting substance, 10-ethyl-3-methylpyrimido[4,5-b]quinoline-2,4(3H,10H)-dione (TND1128), modulates the morphological growth of cultured mouse hippocampal neurons. The morphological growth effect of TND1128 was also compared with that of ß-nicotinamide mononucleotide (ß-NMN). TND1128 induced the branching of axons and dendrites, and increased the number of excitatory synapses. This study provides new insight into TND1128 as a mitochondria-stimulating drug for improving brain function.

Extracellular pH modulation of excitatory synaptic transmission in hippocampal CA3 neurons.

In this study, the effect of extracellular pH on glutamatergic synaptic transmission was examined in mechanically dissociated rat hippocampal CA3 pyramidal neurons using a whole-cell patch-clamp technique under voltage-clamp conditions. Native synaptic boutons were isolated without using any enzymes, using a so-called "synapse bouton preparation," and preserved for the electrical stimulation of single boutons. Both the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) were found to decrease and increase in response to modest acidic (~pH 6.5) and basic (~pH 8.5) solutions, respectively. These changes in sEPSC frequency were not affected by the addition of TTX but completely disappeared by successive addition of Cd2+. However, changes in sEPSC amplitude induced by acidic and basic extracellular solutions were not affected by the addition of neither TTX nor Cd2+. The glutamate-induced whole-cell currents were decreased and increased by acidic and basic solutions, respectively. Acidic pH also decreased the amplitude and increased the failure rate (Rf) and paired-pulse rate (PPR) of glutamatergic electrically evoked excitatory postsynaptic currents (eEPSCs), while a basic pH increased the amplitude and decreased both the Rf and PPR of eEPSCs. The kinetics of the currents were not affected by changes in pH. Acidic and basic solutions decreased and increased voltage-gated Ca2+ but not Na+ channel currents in the dentate gyrus granule cell bodies. Our results indicate that extracellular pH modulates excitatory transmission via both pre- and postsynaptic sites, with the presynaptic modulation correlated to changes in voltage-gated Ca2+ channel currents.NEW & NOTEWORTHY The effects of external pH changes on spontaneous, miniature, and evoked excitatory synaptic transmission in CA3 hippocampal synapses were examined using the isolated nerve bouton preparation, which allowed for the accurate regulation of extracellular pH at the synapses. Acidification generally reduced transmission, partly via effects on presynaptic Ca2+ channel currents, while alkalization generally enhanced transmission. Both pre- and postsynaptic sites contributed to these effects.

Xenon modulates synaptic transmission to rat hippocampal CA3 neurons at both pre- and postsynaptic sites.

KEY POINTS: Xenon (Xe) non-competitively inhibited whole-cell excitatory glutamatergic current (IGlu ) and whole-cell currents gated by ionotropic glutamate receptors (IAMPA , IKA , INMDA ), but had no effect on inhibitory GABAergic whole-cell current (IGABA ). Xe decreased only the frequency of glutamatergic spontaneous and miniature excitatory postsynaptic currents and GABAergic spontaneous inhibitory postsynaptic currents without changing the amplitude or decay times of these synaptic responses. Xe decreased the amplitude of both the action potential-evoked excitatory and the action potential-evoked inhibitory postsynaptic currents (eEPSCs and eIPSCs, respectively) via a presynaptic inhibition in transmitter release. We conclude that the main site of action of Xe is presynaptic in both excitatory and inhibitory synapses, and that the Xe inhibition is much greater for eEPSCs than for eIPSCs. ABSTRACT: To clarify how xenon (Xe) modulates excitatory and inhibitory whole-cell and synaptic responses, we conducted an electrophysiological experiment using the 'synapse bouton preparation' dissociated mechanically from the rat hippocampal CA3 region. This technique can evaluate pure single- or multi-synapse responses and enabled us to accurately quantify how Xe influences pre- and postsynaptic aspects of synaptic transmission. Xe inhibited whole-cell glutamatergic current (IGlu ) and whole-cell currents gated by the three subtypes of glutamate receptor (IAMPA , IKA and INMDA ). Inhibition of these ionotropic currents occurred in a concentration-dependent, non-competitive and voltage-independent manner. Xe markedly depressed the slow steady current component of IAMPA almost without altering the fast phasic IAMPA component non-desensitized by cyclothiazide. It decreased current frequency without affecting the amplitude and current kinetics of glutamatergic spontaneous excitatory postsynaptic currents and miniature excitatory postsynaptic currents. It decreased the amplitude, increasing the failure rate (Rf) and paired-pulse rate (PPR) without altering the current kinetics of glutamatergic action potential-evoked excitatory postsynaptic currents. Thus, Xe has a clear presynaptic effect on excitatory synaptic transmission. Xe did not alter the GABA-induced whole-cell current (IGABA ). It decreased the frequency of GABAergic spontaneous inhibitory postsynaptic currents without changing the amplitude and current kinetics. It decreased the amplitude and increased the PPR and Rf of the GABAergic action potential-evoked inhibitory postsynaptic currents without altering the current kinetics. Thus, Xe acts exclusively at presynaptic sites at the GABAergic synapse. In conclusion, our data indicate that a presynaptic decrease of excitatory transmission is likely to be the major mechanism by which Xe induces anaesthesia, with little contribution of effects on GABAergic synapses.

Calcium channel subtypes on glutamatergic mossy fiber terminals synapsing onto rat hippocampal CA3 neurons.

The current electrophysiological study investigated the functional roles of high- and low-voltage-activated Ca2+ channel subtypes on glutamatergic small mossy fiber nerve terminals (SMFTs) that synapse onto rat hippocampal CA3 neurons. Experiments combining both the "synapse bouton" preparation and single-pulse focal stimulation technique were performed using the conventional whole cell patch configuration under voltage-clamp conditions. Nifedipine, at a high concentration, and BAY K 8644 inhibited and facilitated the glutamatergic excitatory postsynaptic currents (eEPSCs) that were evoked by 0.2-Hz stimulation, respectively. However, these drugs had no effects on spontaneous EPSCs (sEPSCs). Following the use of a high stimulation frequency of 3 Hz, however, nifedipine markedly inhibited eEPSCs at the low concentration of 0.3 µM. Moreover, ω-conotoxin GVIA and ω-agatoxin IVA significantly inhibited both sEPSCs and eEPSCs. Furthermore, SNX-482 slightly inhibited eEPSCs. R(-)-efonidipine had no effects on either sEPSCs or eEPSCs. It was concluded that glutamate release from SMFTs depends largely on Ca2+ entry through N- and P/Q-type Ca2+ channels and, to a lesser extent, on R-type Ca2+ channels. The contribution of L-type Ca2+ channels to eEPSCs was small at low-firing SMFTs but more significant at high-firing SMFTs. T-type Ca2+ channels did not appear to be involved in neurotransmission at SMFTs. NEW & NOTEWORTHY Action potential-evoked glutamate release from small mossy fiber nerve terminals (SMFTs) that synapse onto rat hippocampal CA3 neurons is regulated by high-threshold but not low-threshold Ca2+ channel subtypes. The functional contribution mainly depends on N- and P/Q-type Ca2+ channels and, to a lesser extent, on R-type Ca2+ channels. However, in SMFTs stimulated at a high 3-Hz frequency, L-type Ca2+ channels contributed significantly to the currents. The present results are consistent with previous findings from fluorometric studies of large mossy fiber boutons.

Effects of triphenyltin on glycinergic transmission on rat spinal neurons.

Glycine is a fast inhibitory transmitter like γ-aminobutyric acid in the mammalian spinal cord and brainstem, and it is involved in motor reflex, nociception, and neuronal development. Triphenyltin (TPT) is an organometallic compound causing environmental hazard to many wild creatures. Our previous findings show that TPT ultimately induces a drain and/or exhaustion of glutamate in excitatory presynaptic nerve terminals, resulted in blockage of neurotransmission as well as methylmercury. Therefore, we have investigated the neurotoxic mechanism how TPT modulates inhibitory glycinergic transmission in the synaptic bouton preparation of rat isolated spinal neurons using a patch clamp technique. TPT at environmentally relevant concentrations (3-300 nM) significantly increased the number of frequency of glycinergic spontaneous and miniature inhibitory postsynaptic currents (sIPSC and mIPSC) without affecting the current amplitude and decay time. The TPT effects were also observed in external Ca2+-free solution containing tetrodotoxin (TTX) but removed in Ca2+-free solution with both TTX and BAPTA-AM (Ca2+ chelator). On the other hand, the amplitude of glycinergic evoked inhibitory postsynaptic currents (eIPSCs) increased with decreasing failure rate (Rf) and paired pulse ratio (PPR) in the presence of 300 nM TPT. At a high concentration (1 µM), TPT completely blocked eIPSCs after a transient facilitation. Overall, these results suggest that TPT directly acts transmitter-releasing machinery in glycinergic nerve terminals. Effects of TPT on the nerve terminals releasing fast transmitters were greater in the order of glycinergic > glutamatergic > GABAergic ones. Thus, TPT is supposed to cause a strong synaptic modulations on glycinergic neurotransmission in wild creatures.

Modulation of inhibitory and excitatory fast neurotransmission in the rat CNS by heavy water (D2O).

The effects of heavy water (deuterium oxide, D2O) on GABAergic and glutamatergic spontaneous and evoked synaptic transmission were investigated in acute brain slice and isolated "synaptic bouton" preparations of rat hippocampal CA3 neurons. The substitution of D2O for H2O reduced the frequency and amplitude of GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) in a concentration-dependent manner but had no effect on glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs). In contrast, for evoked synaptic responses in isolated neurons, the amplitude of both inhibitory and excitatory postsynaptic currents (eIPSCs and eEPSCs) was decreased in a concentration-dependent manner. This was associated with increases of synaptic failure rate (Rf) and paired-pulse ratio (PPR). The effect was larger for eIPSCs compared with eEPSCs. These results clearly indicate that D2O acts differently on inhibitory and excitatory neurotransmitter release machinery. Furthermore, D2O significantly suppressed GABAA receptor-mediated whole cell current (IGABA) but did not affect glutamate receptor-mediated whole cell current (IGlu). The combined effects of D2O at both the pre- and postsynaptic sites may explain the greater inhibition of eIPSCs compared with eEPSCs. Finally, D2O did not enhance or otherwise affect the actions of the general anesthetics nitrous oxide and propofol on spontaneous or evoked GABAergic and glutamatergic neurotransmissions, or on IGABA and IGlu. Our results suggest that previously reported effects of D2O to mimic and/or modulate anesthesia potency result from mechanisms other than modulation of GABAergic and glutamatergic neurotransmission.

Functional changes in piriform cortex pyramidal neurons in the chronic methamphetamine-treated rat.

Chronic treatment of rats with methamphetamine (MAP) causes a range of functional changes to the central nervous system (CNS), including a toxicity that is widespread throughout the brain (Frost and Cadet 2000; Fasihpour et al. 2013). In this report, we examined the effect of chronic MAP treatment on pyramidal neurons of the rat piriform cortex, an area involved in sensory processing, associative learning and a model system for studies on synaptic plasticity. MAP treatment significantly depolarized the membrane potential and decreased neuronal input resistance. Furthermore, the voltage-dependence of both AMPA and NMDA responses was disturbed by chronic MAP treatment, and the extent of long-term potentiation (LTP) was decreased. Morphological changes of MAP-treated rat pyramidal neurons were observed as blebbing of the dendrite trees. The changes we observed represent detrimental effects on the function of piriform cortical neurons further illustrating deficits in synaptic plasticity extend beyond the hippocampus. These changes may contribute to behavioural deficits in chronic MAP-treated animals.

Inhibition of excitatory synaptic transmission in hippocampal neurons by levetiracetam involves Zn²âº-dependent GABA type A receptor-mediated presynaptic modulation.

Levetiracetam (LEV) is an antiepileptic drug with a unique but as yet not fully resolved mechanism of action. Therefore, by use of a simplified rat-isolated nerve-bouton preparation, we have investigated how LEV modulates glutamatergic transmission from mossy fiber terminals to hippocampal CA3 neurons. Action potential-evoked excitatory postsynaptic currents (eEPSCs) were recorded using a conventional whole-cell patch-clamp recording configuration in voltage-clamp mode. The antiepileptic drug phenytoin decreased glutamatergic eEPSCs in a concentration-dependent fashion by inhibiting voltage-dependent Na⁺ and Ca²âº channel currents. In contrast, LEV had no effect on eEPSCs or voltage-dependent Na⁺ or Ca²âº channel currents. Activation of presynaptic GABA type A (GABA(A)) receptors by muscimol induced presynaptic inhibition of eEPSCs, resulting from depolarization block. Low concentrations of Zn²âº, which had no effect on eEPSCs, voltage-dependent Na⁺ or Ca²âº channel currents, or glutamate receptor-mediated whole cell currents, reduced the muscimol-induced presynaptic inhibition. LEV applied in the continuous presence of 1 µM muscimol and 1 µM Zn²âº reversed this Zn²âº modulation on eEPSCs. The antagonizing effect of LEV on Zn²âº-induced presynaptic GABA(A) receptor inhibition was also observed with the Zn²âº chelators Ca-EDTA and RhodZin-3. Our results clearly show that LEV removes the Zn²âº-induced suppression of GABA(A)-mediated presynaptic inhibition, resulting in a presynaptic decrease in glutamate-mediated excitatory transmission. Our results provide a novel mechanism by which LEV may inhibit neuronal activity.

Potent cough suppression by physiologically active substance in human plasma.

Human plasma contains wide variety of bioactive proteins that have proved essential in therapeutic discovery. However many human plasma proteins remain orphans with unknown biological functions. Evidences suggest that some plasma components target the respiratory system. In the present study we adapted heparin affinity chromatography to fractionate human plasma for functional bioassay. Fractions from pooled human plasma yielded particular plasma fractions with strong cough suppressing effects. Purification yielded a fraction that was finally identified as an activated blood coagulation factor fXIa using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF-MS). The fraction almost completely suppressed coughs induced by either chemical or mechanical stimulation applied to larynx or bifurcation of guinea-pig trachea. Cough suppressing effect of the fraction and commercially available fXIa were one million times stronger than codeine and codeine only partially suppressed the mechanically triggered coughing in animal model. Recent reviews highlighted prominent shortcomings of current available antitussives, including narcotic opioids such as codeine and their unpleasant or intolerable side effects. Therefore, safer and more effective cough suppressants would be welcome, and present findings indicate that fXIa in human plasma as a very promising, new therapeutic candidate for effective antitussive action.

Synaptic effects of xenon on NMDA receptor-mediated response in rat spinal neuron.

To investigate the precise mechanism of xenon (Xe), pharmacologically isolated AMPA/KA and NMDA receptor-mediated spontaneous (s) and evoked (e) excitatory postsynaptic currents (s/eEPSCAMPA/KA and s/eEPSCNMDA) were recorded from mechanically isolated single spinal sacral dorsal commissural nucleus (SDCN) neurons attached with glutamatergic nerve endings (boutons) using conventional whole-cell patch-clamp technique. We analysed kinetic properties of both s/eEPSCAMPA/KA and s/eEPSCNMDA by focal single- and/or paired-pulse electrical stimulation to compare them. The s/eEPSCNMDA showed smaller amplitude, slower rise time, and slower 1/e decay time constant (τDecay) than those of s/eEPSCAMPA/KA. We previously examined how Xe modulates s/eEPSCAMPA/KA, therefore, examined the effects on s/eEPSCNMDA in the present study. Xe decreased the frequency and amplitude of sEPSCNMDA, and decreased the amplitude but increased the failure rate and paired-pulse ratio of eEPSCNMDA without affecting their τDecay. It was concluded that Xe might suppress NMDA receptor-mediated synaptic transmission via both presynaptic and postsynaptic mechanisms.

Transsynaptic inhibition of spinal transmission by A2 botulinum toxin.

Type A botulinum toxin blocks not only ACh release from motor nerve terminals but also central synaptic transmission, including glutamate, noradrenaline, dopamine, ATP, GABA and glycine. Neurotoxins (NTXs) are transported by both antero- and retrogradely along either motor or sensory axons for bidirectional delivery between peripheral tissues or the CNS. A newly developed type A2 NTX (A2NTX) injected into one rat foreleg muscle was transported to the contralateral muscle. This finding was consistent with the NTX traveling retrogradely via spinal neurons and then transsynaptically through motor neurons to the contralateral motor neurons within the spinal cord and on to the soleus muscle. In the present study we found that toxin injection into the rat left soleus muscle clearly induced bilateral muscle relaxation in a dose-dependent fashion, although the contralateral muscle relaxation followed the complete inhibition of toxin-injected ipsilateral muscles. The toxin-injected ipsilateral muscle relaxation was faster and stronger in A2NTX-treated rats than A1LL (BOTOX). A1LL was transported almost equally to the contralateral muscle via neural pathways and the bloodstream. In contrast, A2NTX was mainly transported to contralateral muscles via the blood. A1LL was more successfully transported to contralateral spinal neurons than A2NTX. We also demonstrated that A1LL and A2NTX were carried from peripheral to CNS and vice versa by dual antero- and retrograde axonal transport through either motor or sensory neurons.

Effects of selenoprotein P on the contraction and relaxation of the airway smooth muscle.

Selenoprotein P (SeP) not only represents the major selenoprotein in plasma, but also provides more than 50% of the total plasma selenium. However, there is no report concerning the direct action of selenium or selenium-containing compounds on the contraction and relaxation of the airway smooth muscle. Therefore, we investigated the effects of SeP and sodium selenite (SS) on the indirectly induced contraction and relaxation of the cat bronchi, and gel contraction of cultured bovine tracheal smooth muscle cells (BTSMC) induced by ATP. In the present results, SeP or SS suppressed the amplitude of twitch-like contractions of cat bronchiole without affecting the non-adrenergic and non-cholinergic (NANC) relaxations evoked by electrical field stimulation. SeP also suppressed the ATP-induced gel contraction of BTSMC. These results suggest that SeP suppresses the amplitude of twitch-like contraction of cat bronchiole by acting directly on the bronchiolar smooth muscle.

Effects of ethanol on GABA(A) receptors in GABAergic and glutamatergic presynaptic nerve terminals.

Ethanol (EtOH) has a number of behavioral effects, including intoxication, amnesia, and/or sedation, that are thought to relate to the activation of GABA(A) receptors. However, GABA(A) receptors at different cellular locations have different sensitivities to EtOH. The present study used the "synaptic bouton" preparation where we could stimulate nerve endings on mechanically dissociated single rat hippocampal CA1 and CA3 pyramidal neurons and investigate the effects of EtOH on presynaptic and postsynaptic GABA(A) receptors. Low concentrations of EtOH (10 mM) had no effect on postsynaptic GABA(A) and glutamate receptors or voltage-dependent Na(+) and Ca(2+) channels. Higher concentrations (≥100 mM) could significantly inhibit these current responses. EtOH at 10 mM had no direct effect on inhibitory postsynaptic currents (IPSCs) and excitatory postsynaptic currents (EPSCs) evoked by focal stimulation of single boutons [evoked IPSCs (eIPSCs) and evoked EPSCs (eEPSCs)]. However, coapplication of 10 mM EtOH with muscimol decreased the amplitude of eIPSCs and eEPSCs and increased their paired-pulse ratio. The effects on eEPSCs were reversed by bicuculline. Coapplication of muscimol and EtOH significantly increased the frequency of spontaneous IPSCs and EPSCs. The EtOH effects on the postsynaptic responses and eEPSCs were similar in neurons from neonatal and mature rats. These results revealed that low concentrations of EtOH can potentiate the activation of presynaptic GABA(A) receptors to inhibit evoked GABA and glutamate release. These results indicate a high sensitivity of presynaptic GABA(A) receptor to EtOH, which needs to be accounted for when considering the cellular mechanisms of EtOH's physiological responses.

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 (P1) and both the responses. These effects of A2NTX on the amplitude and Rf of the P1 and the second postsynaptic currents (P2) 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-Ca2+ in A2NTX poisoned nerve terminals.

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.

Inhibition of Membrane Na+ Channels by A Type Botulinum Toxin at Femtomolar Concentrations in Central and Peripheral Neurons.

Recent studies have demonstrated that the botulinum neurotoxins inhibit the release of acetylcholine, glutamate, GABA, and glycine in central nerve system (CNS) neurons. The Na+ current (INa) is of major interest because it acts as the trigger for many cellular functions such as transmission, secretion, contraction, and sensation. Thus, these observations raise the possibility that A type neurotoxin might also alter the INa of neuronal excitable membrane. To test our idea, we examined the effects of A type neurotoxins on INa of central and peripheral neurons. The neurotoxins in femtomolar to picomolar concentrations produced substantial decreases of the neuronal INa, but interestingly the current inhibition was saturated at about maximum 50% level of control INa. The inhibitory pattern in the concentration-response curve for the neurotoxins differed from tetrodotoxin (TTX), local anesthetic, and antiepileptic drugs that completely inhibited INa in a concentration-dependent manner. We concluded that A type neurotoxins inhibited membrane Na+-channel activity in CNS neurons and that INa of both TTX-sensitive and-insensitive peripheral dorsal ganglion cells were also inhibited similarly to a maximum 40% of the control by the neurotoxins. The results suggest evidently that A2NTX could be also used as a powerful drug in treating epilepsy and several types of pain.

Inhibition of membrane Na+ channels by A type botulinum toxin at femtomolar concentrations in central and peripheral neurons.

Recent studies have demonstrated that the botulinum neurotoxins inhibit the release of acetylcholine, glutamate, GABA, and glycine in central nerve system (CNS) neurons. The Na(+) current (I(Na)) is of major interest because it acts as the trigger for many cellular functions such as transmission, secretion, contraction, and sensation. Thus, these observations raise the possibility that A type neurotoxin might also alter the I(Na) of neuronal excitable membrane. To test our idea, we examined the effects of A type neurotoxins on I(Na) of central and peripheral neurons. The neurotoxins in femtomolar to picomolar concentrations produced substantial decreases of the neuronal I(Na), but interestingly the current inhibition was saturated at about maximum 50% level of control I(Na). The inhibitory pattern in the concentration-response curve for the neurotoxins differed from tetrodotoxin (TTX), local anesthetic, and antiepileptic drugs that completely inhibited I(Na) in a concentration-dependent manner. We concluded that A type neurotoxins inhibited membrane Na(+)-channel activity in CNS neurons and that I(Na) of both TTX-sensitive and -insensitive peripheral dorsal ganglion cells were also inhibited similarly to a maximum 40% of the control by the neurotoxins. The results suggest evidently that A2NTX could be also used as a powerful drug in treating epilepsy and several types of pain.