Inhibitory effects of intravenous anaesthetic agents on K(+)-evoked glutamate release from rat cerebrocortical slices. Involvement of voltage-sensitive Ca(2+) channels and GABA(A) receptors.
ABSTRACT It is widely accepted that most general anaesthetic agents depress the central nervous system (CNS) by potentiation or activation of the GABA(A) receptor-mediated Cl(-) conductance. These agents also reportedly inhibit voltage-sensitive Ca(2+) channels (VSCCs), thus depressing excitatory transmission in the CNS. However, in this regard there are few functional data at the level of neurotransmitter release. In this study we examined the effects of VSCC antagonists and a range of intravenous anaesthetic agents on K(+)(40 mM)-evoked glutamate release from rat cerebrocortical slices in the absence and presence of the GABA(A) receptor antagonist bicuculline (100 microM). We employed both selective and non-selective VSCC antagonists, the anaesthetic barbiturates thiopental, pentobarbital and phenobarbital, the non-anaesthetic barbiturate barbituric acid, the non-barbiturate anaesthetics alphaxalone, propofol and ketamine and the GABA(A) receptor agonist, muscimol. Glutamate released into the incubation medium was determined by a glutamate dehydrogenase-coupled assay. Omega-agatoxin IV(A) (P-type VSCC), omega-conotoxin MVII(C) (P/Q-type VSCC) and Cd(2+) (non-selective) essentially abolished glutamate release whilst nifedipine (L-type VSCC) and omega-conotoxin GVI(A) (N-type VSCC) reduced release by less than 30%. The concentrations producing 50% of the maximum inhibition (IC(50)) for thiopental, pentobarbital, phenobarbital, alphaxalone, propofol and ketamine were (in microM) 8.3, 22, 112, 6.3, 83 and 120, respectively. Barbituric acid produced a small (about 20%) inhibition. With the exception of ketamine, the IC(50) values for these anaesthetic agents were increased threefold by bicuculline (100 microM). In addition, muscimol significantly inhibited release by 26% with an IC(50) of 1.1 microM. In summary, a range of anaesthetic agents at clinically achievable concentrations inhibit glutamate release and this inhibition of release appears to be due mainly to direct inhibition of P/Q-type VSCCs, although activation of the GABA(A) receptor plays a role in this response.
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ABSTRACT: 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 Ca(2+) 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 (3-10μM) 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 (30-300μM), 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.Brain research bulletin 09/2012; · 2.97 Impact Factor
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ABSTRACT: Numerous reports suggest that intravenously administered (IV) anesthetics affect postsynaptic events in the central nervous system. However, there is little evidence about how general anesthetics influence the presynaptic processes. The level of presynaptic calcium (Ca(2+)) concentration ([Ca(2+)](pre)) regulates neurotransmitter release. In this study, we investigated the effects of anesthetic propofol IV and the barbiturate pentobarbital on neurotransmitter release by measuring [Ca(2+)](pre) in the presynaptic nerve terminals (boutons) on a dissociated single hippocampal rat neuron. Sprague-Dawley rats 10-14 days old were decapitated under pentobarbital anesthesia, and brain slices were prepared. The hippocampal CA1 area was touched with a fire-polished glass pipette, which vibrated horizontally, and neurons were dissociated, along with the attached presynaptic boutons. The presynaptic boutons were visualized under a confocal laser-scanning microscope after staining with FM1-43 dye, and [Ca(2+)](pre) was measured with acetoxymethyl ester of fluo-3 (fluo-3 AM). High potassium (K(+)) (15-90 mM) increased the [Ca(2+)](pre) in the Ca(2+)-containing solution in a concentration-dependent manner. Whereas propofol (10 μM) and pentobarbital (300 μM) suppressed the high K(+) (60 mM)-induced increase in [Ca(2+)](pre) in the boutons attached to the dendrite, they did not affect [Ca(2+)](pre) in the boutons attached to the soma or dendrite base. As a large majority of excitatory synapses are located on dendritic spines, these agents may affect Ca(2+) mobilization in the excitatory presynaptic boutons. Propofol and pentobarbital may affect neurotransmitter release from the excitatory presynaptic nerve terminals due to inhibition of increase in [Ca(2+)](pre).Journal of Anesthesia 07/2011; 25(5):727-33. · 0.87 Impact Factor
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ABSTRACT: The barbiturate phenobarbital has been in use in the treatment of epilepsy for 100 years. It has long been recognized that barbiturates act by prolonging and potentiating the action of γ-aminobutyric acid (GABA) on GABAA receptors and at higher concentrations directly activating the receptors. A large body of data supports the concept that GABAA receptors are the primary central nervous system target for barbiturates, including the finding that transgenic mice with a point mutation in the β3 GABAA-receptor subunit exhibit diminished sensitivity to the sedative and immobilizing actions of the anesthetic barbiturate pentobarbital. Although phenobarbital is only modestly less potent as a GABAA-receptor modulator than pentobarbital, phenobarbital is minimally sedating at effective anticonvulsant doses. Possible explanations for the reduced sedative effect of phenobarbital include more regionally restricted action; partial agonist activity; reduced propensity to directly activate GABAA receptors (possibly including extrasynaptic receptors containing δ subunits); and reduced activity at other ion channel targets, including voltage-gated calcium channels. In recent years, substantial progress has been made in defining the structural features of GABAA receptors responsible for gating and allosteric modulation by drugs. Although the precise sites of action of barbiturates have not yet been defined, the second and third transmembrane domains of the β subunit appear to be critical; binding may involve a pocket formed by β-subunit methionine 286 as well as α-subunit methionine 236. In addition to effects on GABAA receptors, barbiturates block AMPA/kainate receptors, and they inhibit glutamate release through an effect on P/Q-type high-voltage activated calcium channels. The combination of these various actions likely accounts for their diverse clinical activities. Despite the remarkable progress of the last century, there is still much to learn about the actions of barbiturates that can be applied to the discovery of new, more therapeutically useful agents.Epilepsia 12/2012; 53(s8). · 3.96 Impact Factor