Research reportDifferential effects of midazolam on inhibitory postsynaptic currents in CA1 pyramidal cells and dentate gyrus granule cells of rat hippocampal slices
Introduction
Regional differences in γ-aminobutyric acid (GABA) inhibitory synaptic transmission in the hippocampus have been reported in several studies. Investigations using in vitro preparations have shown that CA1 pyramidal cells (CA1-PCs) and dentate gyrus granule cells (DG-GCs) exhibit regional differences in their membrane properties, membrane time constants and specific membrane resistivity [5], [38], [39]. It is well known that differential responses to ischemic insult are shown by CA1-PCs and DG-GCs in vitro [1], [3], [13], [32]. In addition, an immunohistochemical study [27], [37], which used a specific antibody against the purified GABAA receptor and a monoclonal antibody against benzodiazepines, demonstrated a greater density of GABAA receptors in the stratum pyramidale than in other regions of the hippocampus. Furthermore, it has been reported that different subtypes of GABAA receptors occur in CA1-PCs and DG-GCs [19]. The main GABAA receptor subtypes in CA1-PCs are the α2βγ2 and α5β2γ2 receptors, whereas the α4βδ receptor is predominant in DG-GC [6], [19], [33], [40]. Quirk et al. [25] have reported that receptors immunoprecipitated from the cerebellum with δ subunit-specific antisera do not bind benzodiazepines. Other investigators [30] have noted that recombinant receptors composed of αβδ subunits are not modulated by benzodiazepines. A coexpression of the γ2 subunit with α and β subunits is essential for modulation of the expressed receptors by benzodiazepines [21]. The α4 subunits contain δ receptors and are insensitive to benzodiazepine [9], [30]. These findings suggest the possibility that the effects of the benzodiazepines, which are considered to enhance GABAA-mediated inhibition [10], [17], [31], could be different in CA1-PCs and DG-GCs. The different effects could be due to a different sensitivity to benzodiazepines among the GABAA receptor subtypes [9].
In a previous experiment [28] which used extracellular recordings we demonstrated that midazolam depressed the amplitude of population spikes and the excitatory postsynaptic potential (EPSP) slope of CA1-PCs. To a lesser extent those of DG-GCs were also depressed [28]. In an earlier intracellular study benzodiazepine (diazepam) increased inhibitory postsynaptic potentials (IPSPs) in CA1-PCs [2]. In our intracellular study [29] midazolam (75 μM) significantly enhanced the monosynaptic IPSPs in CA1-PCs, whereas it slightly decreased those in DG-GCs. It could be suggested that the differential effects of midazolam on CA1-PCs and DG-GCs may be due to the different types and/or density of GABAA receptors. However, the previous intracellular recordings were performed under conditions where the resting membrane potentials of the recorded neurons were variable (−63.0±1.7 mV in CA1-PCs; −75.5±1.5 mV in DG-GCs) and significantly changed (approximately 3.0 mV, P<0.05 for 65–79% of the recorded neurons) following midazolam application [29].
In the present study, we aimed for a more direct comparison of the differential effects of midazolam on inhibitory synaptic transmissions in CA1-DGs and DG-GCs. Consequently, inhibitory postsynaptic currents (IPSCs) were recorded at a variety of clamping membrane potentials by using whole cell patch-clamp techniques. Monosynaptic IPSCs in CA1-PCs and DG-GCs were evoked by electrical stimulation of GABAergic interneurons [8] near the CA1-PCs or DG-GCs. The IPSCs in the CA1-PCs and DG-GCs were recorded and compared in various concentrations of midazolam at specific clamping membrane potentials. The present results therefore extend previous data about the differential effects of midazolam on IPSCs in CA1-PCs and DG-GCs. Part of the present data has been previously reported in abstract form [14].
Section snippets
Slice preparation
The experiments were performed on young Wistar rats (14–28 postnatal days). All of the surgical and animal care methods conformed to the Guidelines for the Use of Animals of the International Brain Research Organization. The experimental protocol was approved by the Sapporo Medical University Animal Care and Use Committee (Sapporo, Japan). The rats were anesthetized with halothane and decapitated. Each rat brain was rapidly removed after the decapitation, and then placed in pre-cooled
IPSCs in CA1-PC
Whole cell patch-clamp recordings were successfully performed in a total of 20 neurons (10 CA1-PCs and 10 DG-GCs) in 20 slices. The mean resting membrane potential in the CA1-PCs was −63.0±0.8 mV (n=7). The recordings of the IPSCs were obtained at various voltage commands, in 20 mV steps, from −120 to +40 mV (holding potential −60 mV). A representative record of an evoked IPSC before midazolam application is shown in Fig. 1A. At the clamped membrane potential of 0 mV an IPSC with a fast,
IPSCs in CA1-PCs and DG-GCs
The main finding of the present study was that midazolam had differential effects on GABAergic IPSCs in rat hippocampal neurons in vitro. Midazolam was observed to increase the conductance of the IPSCs in CA1-PCs, but did not either significantly increase or decrease the amplitude of the IPSCs in DG-GCs. The results confirm and extend our previous intracellular data [29].
The normalized conductance for the IPSCs of the CA1-PCs were enhanced by midazolam in a dose dependent manner but the effect
Acknowledgements
We thank Dr. T. Isa (National Institute for Physiological Sciences) for his valuable advice and Dr. H. Takagi (Shinshu University School of Medicine) for technical advice during this research. The work was supported, in part, by a Grant-in-Aid (13780639) for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
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