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.

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.

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.

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.

Effects of nitrous oxide on glycinergic transmission in rat spinal neurons.

We investigated the effects of nitrous oxide (N2O) on glycinergic inhibitory whole-cell and synaptic responses using a "synapse bouton preparation," dissociated mechanically from rat spinal sacral dorsal commissural nucleus (SDCN) neurons. This technique can evaluate pure single- or multi-synaptic responses from native functional nerve endings and enable us to accurately quantify how N2O influences pre- and postsynaptic transmission. We found that 70 % N2O enhanced exogenous glycine-induced whole-cell currents (IGly) at glycine concentrations lower than 3 × 10-5 M, but did not affect IGly at glycine concentrations higher than 10-4 M. N2O did not affect the amplitude and 1/e decay-time of both spontaneous and miniature glycinergic inhibitory postsynaptic currents recorded in the absence and presence of tetrodotoxin (sIPSCs and mIPSCs, respectively). The decrease in frequency induced by N2O was observed in sIPSCs but not in mIPSCs, which was recorded in the presence of both tetrodotoxin and Cd2+, which block voltage-gated Na+ and Ca2+ channels, respectively. N2O also decreased the amplitude and increased the failure rate and paired-pulse ratio of action potential-evoked glycinergic inhibitory postsynaptic currents. N2O slightly decreased the Ba2+ currents mediated by voltage-gated Ca2+ channels in SDCN neurons. We found that N2O suppresses glycinergic responses at synaptic levels with presynaptic effect having much more predominant role. The difference between glycinergic whole-cell and synaptic responses suggests that extrasynaptic responses seriously modulate whole-cell currents. Our results strongly suggest that these responses may thus in part explain analgesic effects of N2O via marked glutamatergic inhibition by glycinergic responses in the spinal cord.

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 the GABA and glutamate responses at genuine synaptic levels in rat spinal neurons.

Effects of xenon (Xe) on whole-cell currents induced by glutamate (Glu), its three ionotropic subtypes, and GABA, as well as on the fast synaptic glutamatergic and GABAergic transmissions, were studied in the mechanically dissociated "synapse bouton preparation" of rat spinal sacral dorsal commissural nucleus (SDCN) neurons. This technique evaluates pure single or multi-synapse responses from native functional nerve endings and enables us to quantify how Xe influences pre- and postsynaptic transmissions accurately. Effects of Xe on glutamate (Glu)-, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, kainate (KA)- and N-methyl-d-aspartate (NMDA)- and GABAA receptor-mediated whole-cell currents were investigated by the conventional whole-cell patch configuration. Excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) were measured as spontaneous (s) and evoked (e) EPSCs and IPSCs. Evoked synaptic currents were elicited by paired-pulse focal electric stimulation. Xe decreased Glu, AMPA, KA, and NMDA receptor-mediated whole-cell currents but did not change GABAA receptor-mediated whole-cell currents. Xe decreased the frequency and amplitude but did not affect the 1/e decay time of the glutamatergic sEPSCs. Xe decreased the frequency without affecting the amplitude and 1/e decay time of GABAergic sIPSCs. Xe decreased the amplitude and increased the failure rate (Rf) and paired-pulse ratio (PPR) without altering the 1/e decay time of both eEPSC and eIPSC, suggesting that Xe acts on the presynaptic side of the synapse. The presynaptic inhibition was greater in eEPSCs than in eIPSCs. We conclude that Xe decreases glutamatergic and GABAergic spontaneous and evoked transmissions at the presynaptic level. The glutamatergic presynaptic responses are the main target of anesthesia-induced neuronal responses. In contrast, GABAergic responses minimally contribute to Xe anesthesia.

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.

Intra-axonal Ca2+ mobilization contributes to triphenyltin-induced facilitation in glycinergic transmission of rat spinal neurons.

Triphenyltin (TPT) is an organotin compound causing environmental hazard to many wild creatures. Our previous findings show that TPT increases of the frequency of spontaneous glycinergic inhibitory postsynaptic currents (sIPSCs) in rat spinal neurons without changing the amplitude and 1/e decay time. In our study, the effects of 2-aminoethoxydiphenyl borate (2-APB), dantrolene sodium, and thapsigargin on sIPSC frequency were examined to reveal the contribution of intra-axonal Ca2+ mobilization by adding TPT. 2-APB considerably attenuated the TPT-induced facilitation of sIPSC frequency while dantrolene almost completely masked the TPT effects, suggesting that the TPT-induced synaptic facilitation results from the activation of both IP3 and ryanodine receptors on endoplasmic reticulum (ER) membrane, though inositol triphosphate (IP3) receptor is less sensitive to TPT. Thapsigargin itself significantly increased the sIPSC frequency without affecting the current amplitude and decay time. Successive addition of TPT could not further increase the sIPSC frequency in the presence of thapsigargin, indicating that thapsigargin completely masked the facilitatory action of TPT. Results suggest that TPT activates the IP3 and ryanodine receptors while TPT inhibits the Ca2+-pump of ER membranes, resulting in the elevation of intra-axonal Ca2+ levels, leading to the increase of spontaneous glycine release from synaptic vesicles.

Comparison of efficacy and toxicity between botulinum toxin subtypes A1 and A2 in cynomolgus macaques.

Botulinum toxin type A (subtype A1) is used as therapeutic agent for some neurological disorders causing spasticity. The toxin products have an upper dosage limit, and their adverse events, such as side effects of diffusion following high-dose administration, have become serious issues. Therefore, a preparation with greater therapeutic efficacy at lower dosages and less diffusion in the body is desired. We have attempted to produce neurotoxin derived from subtype A2 (A2NTX), which has a different amino acid sequence from that of neurotoxin derived from subtype A1. In this study, to investigate whether A2NTX is applicable for treatment, we compared the muscle relaxation effects and the toxicity between A1LL and A2NTX in adult cynomolgus macaques. In the isometric muscle contraction test elicited by 30 Hz tetanus stimulation, the contractions observed in the 0.4 U/site A1LL-treated group were similar in value to those in the 0.13 U/site A2NTX-treated group. In the toxicity test, the 12 and 24 U/kg A1LL- and A2NTX-treated groups all exhibited similar signs of toxicity regarding symptoms, rate of weight loss, and decrease in the length of the right lower leg perimeter. Thus, A2NTX demonstrated approximately 3.0-times higher muscle relaxation activity than A1LL, and their toxicity was equivalent. This study suggested that A2NTX products are more suitable for the treatment of neurological disorders.

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.

4,5-Dichloro-2-octyl-4-isothiazolin-3-one (DCOIT) modifies synaptic transmission in hippocampal CA3 neurons of rats.

4,5-Dichloro-2-octyl-4-isothiazolin-3-one (DCOIT) is an alternative to organotin antifoulants, such as tributyltin and triphenyltin. Since DCOIT is found in harbors, bays, and coastal areas worldwide, this chemical compound may have some impacts on ecosystems. To determine whether DCOIT possesses neurotoxic activity by modifying synaptic transmission, we examined the effects of DCOIT on synaptic transmission in a 'synaptic bouton' preparation of rat brain. DCOIT at concentrations of 0.03-1 µM increased the amplitudes of evoked synaptic currents mediated by GABA and glutamate, while it reduced the amplitudes of these currents at 3-10 µM. However, the currents elicited by exogenous applications of GABA and glutamate were not affected by DCOIT. DCOIT at 1-10 µM increased the frequency of spontaneous synaptic currents mediated by GABA. It also increased the frequency of glutamate-mediated spontaneous currents at0.3-10 µM. The frequencies of miniature synaptic currents mediated by GABA and glutamate, observed in the presence of tetrodotoxin under external Ca2+-free conditions, were increased by 10 µM DCOIT. With the repetitive applications of DCOIT, the frequency of miniature synaptic currents mediated by glutamate was not increased by the second and third applications of DCOIT. Voltage-dependent Ca2+ channels were not affected by DCOIT, but DCOIT slowed the inactivation of voltage-dependent Na+ channels. These results suggest that DCOIT increases Ca2+ release from intracellular Ca2+ stores, resulting in the facilitation of both action potential-dependent and spontaneous neurotransmission, possibly leading to neurotoxicity.

Effects of propofol on glycinergic neurotransmission in a single spinal nerve synapse preparation.

The effects of the intravenous anesthetic, propofol, on glycinergic transmission and on glycine receptor-mediated whole-cell currents (IGly) were examined in the substantia gelatinosa (SG) neuronal cell body, mechanically dissociated from the rat spinal cord. This "synaptic bouton" preparation, which retains functional native nerve endings, allowed us to evaluate glycinergic inhibitory postsynaptic currents (IPSCs) and whole-cell currents in a preparation in which experimental solution could rapidly access synaptic terminals. Synaptic IPSCs were measured as spontaneous (s) and evoked (e) IPSCs. The eIPSCs were elicited by applying paired-pulse focal electrical stimulation, while IGly was evoked by a bath application of glycine. A concentration-dependent enhancement of IGly was observed for ≥10µM propofol. Propofol (≥3µM) significantly increased the frequency of sIPSCs and prolonged the decay time without altering the current amplitude. However, propofol (≥3µM) also significantly increased the mean amplitude of eIPSCs and decreased the failure rate (Rf). A decrease in the paired-pulse ratio (PPR) was noted at higher concentrations (≥10µM). The decay time of eIPSCs was prolonged only at the maximum concentration tested (30µM). Propofol thus acts at both presynaptic glycine release machinery and postsynaptic glycine receptors. At clinically relevant concentrations (<1µM) there was no effect on IGly, sIPSCs or eIPSCs suggesting that at anesthetic doses propofol does not affect inhibitory glycinergic synapses in the spinal cord.

Nitrous oxide directly inhibits action potential-dependent neurotransmission from single presynaptic boutons adhering to rat hippocampal CA3 neurons.

We evaluated the effects of N2O on synaptic transmission using a preparation of mechanically dissociated rat hippocampal CA3 neurons that allowed assays of single bouton responses evoked from native functional nerve endings. We studied the effects of N2O on GABAA, glutamate, AMPA and NMDA receptor-mediated currents (IGABA, IGlu, IAMPA and INMDA) elicited by exogenous application of GABA, glutamate, (S)-AMPA, and NMDA and spontaneous, miniature, and evoked GABAergic inhibitory and glutamatergic excitatory postsynaptic current (sIPSC, mIPSC, eIPSC, sEPSC, mEPSC and eEPSC) in mechanically dissociated CA3 neurons. eIPSC and eEPSC were evoked by focal electrical stimulation of a single bouton. Administration of 70% N2O altered neither IGABA nor the frequency and amplitude of both sIPSCs and mIPSCs. In contrast, N2O decreased the amplitude of eIPSCs, while increasing failure rates (Rf) and paired-pulse ratios (PPR) in a concentration-dependent manner. On the other hand, N2O decreased IGlu, IAMPA and INMDA. Again N2O did not change the frequency and amplitude of either sEPSCs of mEPSCs. N2O also decreased amplitudes of eEPSCs with increased Rf and PPR. The decay phases of all synaptic responses were unchanged. The present results indicated that N2O inhibits the activation of AMPA/KA and NMDA receptors and also that N2O preferentially depress the action potential-dependent GABA and glutamate releases but had little effects on spontaneous and miniature releases.

Modifications of excitatory and inhibitory transmission in rat hippocampal pyramidal neurons by acute lithium treatment.

The acute effects of high-dose Li(+) treatment on glutamatergic and GABAergic transmissions were studied in the "synaptic bouton" preparation of isolated rat hippocampal pyramidal neurons by using focal electrical stimulation. Both action potential-dependent glutamatergic excitatory and GABAergic inhibitory postsynaptic currents (eEPSC and eIPSC, respectively) were dose-dependently inhibited in the external media containing 30-150 mM Li(+), but the sensitivity for Li(+) was greater tendency for eEPSCs than for eIPSCs. When the effects of Li(+) on glutamate or GABAA receptor-mediated whole-cell responses (IGlu and IGABA) elicited by an exogenous application of glutamate or GABA were examined in the postsynaptic soma membrane of CA3 neurons, Li(+) slightly inhibited both IGlu and IGABA at the 150 mM Li(+) concentration. Present results suggest that acute treatment with high concentrations of Li(+) acts preferentially on presynaptic terminals, and that the Li(+)-induced inhibition may be greater for excitatory than for inhibitory transmission.

Increase in intracellular Ca(2+) level by phenylsulfamide fungicides, tolylfluanid and dichlofluanid, in rat thymic lymphocytes.

Tolylfluanid, a phenylsulfamide fungicide, is one of the many pesticides that are frequently detected in crops. Therefore, its health risk is a concern. Micromolar concentrations of tolylfluanid induce chromosomal aberrations and micronuclei in mammalian lymphocytes. The findings prompted us to study the cellular actions of tolylfluanid and another frequently detected pesticide, dichlofluanid, at submicromolar and micromolar concentrations. Of the cellular actions of chemicals, the action on cellular Ca(2+) homeostasis is important since Ca(2+) is involved in cell signaling and death. Consequently, in this study, the effects of phenylsulfamide fungicides were examined on rat thymocytes by using fluorescent probes in order to further characterize the cellular actions of phenylsulfamide fungicides. Both phenylsulfamide fungicides exhibited biphasic, early and late, increase in intracellular Ca(2+) levels. The early phase was dependent on intracellular Ca(2+) release and increased membrane Ca(2+) permeability. The late phase was owing to Ca(2+) influx via activation of store-operated Ca(2+) channels and the further increase of membrane ionic permeability. Voltage-dependent Ca(2+) channels were not involved. The increases in intracellular Ca(2+) levels by phenylsulfamide fungicides were observed at drug concentrations of 0.1 µM or more (up to 10 µM). Thus, it is plausible that micromolar concentrations of phenylsulfamide fungicides deregulate intracellular Ca(2+) homeostasis in rat thymocytes. Both phenylsulfamide fungicides at 10 µM promoted the transition from intact living cells to living cells with phosphatidylserine-exposed membranes. This was not the case for phenylsulfamide fungicides at 3 µM. The potency of tolylfluanid was similar to that of dichlofluanid. Although the information on residual concentrations of tolylfluanid and dichlofluanid is very limited, their residual concentrations do not reach micromolar levels. It is unlikely that humans will develop adverse effects on exposure to phenylsulfamide fungicides under present environmental conditions.

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.

Tetrodotoxin abruptly blocks excitatory neurotransmission in mammalian CNS.

The present study utilised a 'synaptic bouton' preparation of mechanically isolated rat hippocampal CA3 pyramidal neurons, which permits direct physiological and pharmacological quantitative analyses at the excitatory and inhibitory single synapse level. Evoked excitatory and inhibitory postsynaptic currents (eEPSCs and eIPSCs) were generated by focal paired-pulse electrical stimulation of single boutons. The sensitivity of eEPSC to tetrodotoxin (TTX) was higher than that of the voltage-dependent Na(+) channel whole-cell current (INa) in the postsynaptic CA3 soma membrane. The synaptic transmission was strongly inhibited by 3 nM TTX, at which concentration the INa was hardly suppressed. The IC50 values of eEPSC and INa for TTX were 2.8 and 37.9 nM, respectively, and complete inhibition was 3-10 nM for eEPSC and 1000 nM for INa. On the other hand, both eEPSC and eIPSC were equally and gradually inhibited by decreasing the external Na(+) concentration ([Na]o), which decreases the Na(+)gradient across the cell membrane. The results indicate that TTX at 3-10 nM could block most of voltage-dependent Na(+) channels on presynaptic nerve terminal, resulting in abruptly inhibition of action potential dependent excitatory neurotransmission.