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.

Wakita M ，Kotani N ，Shoudai K ，Yamaga T ，Akaike N ... -  《-》

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.

Wakita M ，Kotani N ，Yamaga T ，Akaike N ... -  《-》

Effects of propofol on GABAergic and glutamatergic transmission in isolated hippocampal single nerve-synapse preparations.

We evaluated the effects of propofol on synaptic transmission using a mechanically dissociated preparation of rat hippocampal CA3 neurons to allow assays of single bouton responses evoked from retained functional native nerve endings. We studied synaptic and extrasynaptic GABAA and glutamate receptor responses in a preparation in which experimental solutions rapidly accessed synaptic terminals. Whole-cell responses were evoked by bath application of GABA and glutamate. Synaptic inhibitory and excitatory postsynaptic currents (IPSC and EPSC) were measured as spontaneous and evoked postsynaptic responses. Evoked currents were elicited by focal electrical stimulation. Propofol (1-100 μM) enhanced extrasynaptic GABAA-receptor mediated responses but the increase at clinically relevant concentrations (1 μM) were minor. In contrast, 1 μM propofol significantly increased both the amplitude and frequency of spontaneous IPSCs (sIPSCs) and increased the amplitudes of evoked IPSCs (eIPSCs) while decreasing failure rates (Rf) and paired-pulse ratios (PPR). Decay times of sIPSCs and eIPSCs were significantly prolonged. Although propofol had no effect on extrasynaptic glutamate responses, only supra-clinical propofol concentrations (≥ 10 µM) increased the spontaneous EPSCs (sEPSCs, amplitudes and frequencies) but suppressed evoked EPSCs (eEPSCs decreased amplitudes with increased Rf and PPR). The decay phases of sEPSCs and eEPSCs were not changed. The propofol-induced changes in sEPSCs and eEPSCs resulted from presynaptic GABAA receptor-mediated depolarization, because these actions were blocked by bicuculline. These results suggest that propofol acts at presynaptic and postsynaptic GABAA receptors within GABAergic synapses, but also increases extrasynaptic GABA responses. Our results expand the locus of propofol actions to GABAergic and glutamatergic synapses.

Wakita M ，Kotani N ，Nonaka K ，Shin MC ，Akaike N ... -  《-》

Comparative effects of pentobarbital on spontaneous and evoked transmitter release from inhibitory and excitatory nerve terminals in rat CA3 neurons.

Pentobarbital (PB) modulates GABA(A) receptor-mediated postsynaptic responses through various mechanisms, and can directly activate the channel at higher doses. These channels exist both pre- and postsynaptically, and on the soma outside the synapse. PB also inhibits voltage-dependent Na⁺ and Ca²⁺ channels to decrease excitatory synaptic transmission. Just how these different sites of action combine to contribute to the overall effects of PB on inhibitory and excitatory synaptic transmission is less clear. To compare these pre- and postsynaptic actions of PB, we used a 'synaptic bouton' preparation of isolated rat hippocampal CA3 pyramidal neurons where we could measure in single neurons the effects of PB on spontaneous and single bouton evoked GABAergic inhibitory and glutamatergic excitatory postsynaptic currents (sIPSCs, sEPSCs, eIPSCs and eEPSCs), respectively. Low (sedative) concentrations (3-10 μM) of PB increased the frequency and amplitude of sIPSCs and sEPSCs, and also presynaptically increased the amplitude of both eIPSCs and eEPSCs. There was no change in current kinetics at this low concentration. At higher concentrations (30-300 μM), PB decreased the frequency, and increased the amplitude of sIPSCs, and presynaptically decreased the amplitude of eIPSCs. The current decay phase of sIPSCs and eIPSCs was increased. An increase in both frequency and amplitude was seen for sEPSCs, while the eIPSCs was also decreased by a bicuculline-sensitive presynaptic effect. The results confirm the multiple sites of action of PB on inhibitory and excitatory transmission and demonstrate that the most sensitive site of action is on transmitter release, via effects on presynaptic GABA(A) receptors. At low concentrations, however, both glutamate and GABA release is similarly enhanced, making the final effects on neuronal excitability difficult to predict and dependent on the particular systems involved and/or on subtle differences in susceptibility amongst individuals. At higher concentrations, release of both transmitters is decreased, while the postsynaptic effects to increase IPSPs and decrease EPSCs would be expected to both results in reduced neuronal excitability.

Shin MC ，Wakita M ，Iwata S ，Nonaka K ，Kotani N ，Akaike N ... -  《-》

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.

Wakita M ，Nagami H ，Takase Y ，Nakanishi R ，Kotani N ，Akaike N ... -  《-》