Modulation of allopregnanolone on excitatory transmitters release from single glutamatergic terminal.

Neurosteroids such as allopregnanolone (Allo) are widely distributed in the brain and may modulate neuronal excitability under physiological or pathological states. Allo modulates GABAA receptor responses, and in this study we investigated the functional effects of Allo on presynaptic GABAA receptors on single glutamatergic nerve terminal projecting on CA3 neurons. In the present study, we measured spontaneous and evoked excitatory postsynaptic currents (sEPSCs and eEPSCs), the latter was elicited with single or paired-pulse focal electrical stimulation, using mechanically isolated 'synaptic bouton' preparation. Allo (10 nM) increased significantly eEPSC amplitude while decreasing the failure rate (Rf) and the paired-pulse response ratio (PPR). Conversely high concentration (100 nM) of Allo decreased eEPSC amplitude and increased Rf and PPR. Allo also increased significantly the frequency and amplitude of sEPSCs at low concentrations (10-30 nM) but at high concentration (100 nM) it had no effect on current amplitude but modestly decreased sEPSC frequency. Application of Allo at nanomolar concentrations facilitated exogenous muscimol-induced outward postsynaptic currents but had no effect on glutamate-induced inward postsynaptic currents. Our results demonstrate that Allo modulates glutamate release via presynaptic GABAA receptors, in addition to its better characterized effects to modulate postsynaptic GABAA responses. Both pre- and postsynaptic GABAA receptor modulation is likely to contribute to the physiological actions of neurosteroids.

Iwata S ，Wakita M ，Shin MC ，Fukuda A ，Akaike N ... -  《-》

GABA(A) receptor-mediated presynaptic inhibition on glutamatergic transmission.

We investigated the functional roles of presynaptic GABA(A) receptors on excitatory nerve terminals in contributing to spontaneous and action potential-evoked glutamatergic transmission to rat hippocampal CA3 pyramidal neurons. Single CA3 neurons were mechanically isolated with adherent nerve terminals, namely the 'synaptic bouton preparation', and spontaneous glutamatergic excitatory synaptic potentials (sEPSCs) and EPSCs evoked by focal electrical stimuli of a single presynaptic glutamatergic boutons (eEPSCs) were recorded using conventional whole-cell patch recordings. Selective activation of presynaptic GABA(A) receptors on these excitatory nerve terminals by muscimol, markedly facilitated sEPSCs frequency but inhibited eEPSC amplitude. The facilitation of sEPSC frequency was completely occluded by GABA(A) receptor-Cl⁻ channel blockers bicuculline or penicillin (PN). PN itself concentration-dependently inhibited the GABA(A) receptor response induced by bath application of muscimol, but had no effect on the glutamate receptor response. In addition, pretreatment with a blocker of the Na(+), K(+), 2Cl⁻ co-transporter type 1 (NKCC-1), bumetanide, prevented the muscimol-induced inhibition of eEPSCs. The results indicate that activation of presynaptic GABA(A) receptors directly depolarizes glutamatergic excitatory nerve terminals and thereby differentially modulates sEPSCs and eEPSCs.

Yamamoto S ，Yoshimura M ，Shin MC ，Wakita M ，Nonaka K ，Akaike N ... -  《-》

Synergic effect of diazepam and muscimol via presynaptic GABA(A) receptors on glutamatergic evoked EPSCs.

We investigated the functional roles of diazepam (DZP) at presynaptic GABA(A) receptors on glutamatergic nerve terminals in contributing to glutamatergic transmission evoked by single and/or paired-pulse focal electrical stimulation. In mechanically dissociated rat hippocampal CA3 neurons with adherent glutamatergic nerve terminals (boutons), namely 'synaptic bouton' preparation, action potential-evoked excitatory postsynaptic currents (eEPSCs) were recorded using conventional whole-cell patch configuration under voltage-clamp condition. Selective activation of presynaptic GABA(A) receptors by muscimol (3-30μM) induced presynaptic inhibition: i.e. the decrease of amplitude and increase of failure rate (Rf) and paired-pulse ratio (PPR) of eEPSCs which are sensitive to bicuculline. DZP (10-100μM) also induced such presynaptic inhibition, but the bicuculline-insensitive effects were caused by inhibition of both voltage-dependent Na(+) and Ca(2+) channels. Muscimol (0.01-0.3μM) or DZP (0.1-3μM) itself did not induce any currents at the low concentration used. However, simultaneous application of muscimol and DZP at low concentrations induced a significant bicuculline-sensitive presynaptic inhibition. Marked desensitization of presynaptic inhibition was also caused by muscimol at higher concentrations than 10μM. The results suggest that in vivo conditions, activation of presynaptic GABA(A) receptors could be readily available with a tiny amount of DZP.

Shin MC ，Wakita M ，Xie DJ ，Iwata S ，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 ... -  《-》

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 ... -  《-》