Developmental study of miniature IPSCs of CA3 hippocampal cells: modulation by midazolam

Abstract

Maturation of GABA_A/benzodiazepine receptors is associated with changes in their subunit composition. We have investigated whether these changes are accompanied by a developmental modification in the kinetic properties of miniature IPSCs (mIPSCs) and sensitivity to midazolam, a benzodiazepine agonist. In the presence of TTX (10 μM) and excitatory amino acid antagonists, AP5 (50 μM) and CNQX (50 μM), we whole-cell recorded mIPSCs in CA3 cells of hippocampal slices of adult and young (4–8 days) rats. mIPSCs were mediated by GABA_A receptors as they were suppressed by bicuculline (10 μM). In both the adult and young rats, mIPSCs were similar in amplitude and kinetic properties. However, the mIPSCs frequency markedly increased with age from 4±3 Hz in the young rats to 20±9 Hz in the adult rats. In both age groups, midazolam (0.01 μM–10 μM) and pentobarbital (30 μM) did not affect the amplitude, frequency and rise time of the mIPSCs but they increased to a similar extent their decay time constant. The current responses to isoguvacine, a GABA_A agonist, were potentiated by 0.1 μM midazolam in both age groups. It is concluded that in immature and adult rats, synaptic GABA_A receptors of CA3 were not different in their kinetic properties and sensitivity to midazolam.

Introduction

In the hippocampus, GABAergic interneurons may strongly influence synaptogenesis since they differentiate before the principal neurons including pyramidal cells and granule cells 44, 48. GABA is expressed early in foetal life [30]and released from growth cones [51]. There are several indications that GABA_A receptor mediated synapses mature prior to glutamatergic AMPA receptor mediated ones 3, 16. In the CA3 pyramidal cells GABA_A receptor mediated inhibition has a late developmental onset since synaptically released or exogenously applied GABA excites and depolarizes neurons by an outward flux of Cl⁻ and generates action potentials 3, 8. This depolarizing action of GABA which in turn increases intracellular calcium 21, 33, 56and releases trophic factor [29]may account for GABA effects on neurite outgrowth 1, 5, 19, 36. The excitatory effects of GABA at this early stage play an important role in activity dependent modulation of synaptogenesis and circuit formation 1, 4, 5, 19, 36.

At the nicotinic and glycinergic receptors 43, 50, synaptogenesis involves not only morphological changes with respect to the formation of postsynaptic receptor density but also changes in the transmitter receptor subunit composition which may lead to differences between synaptic and extrasynaptic receptors. Developmental regulation of the expression of GABA_A receptor subunits has been demonstrated in the hippocampus and other brain structures 13, 17, 20, 24, 34, 35. However, it is not clear whether GABA_A receptor maturation occurs before or after synapse formation. In the immature hippocampal granule cells, it has been recently shown that mIPSCs had slower kinetic properties and a lower sensitivity to benzodiazepine [15]while in cultured neurons from the colliculus there is no evidence that synaptic contact induces developmental changes in the amplitude and duration of the mIPSC [18]. On the other hand there seems to be a developmental regulation of the pharmacological properties of GABA_A receptors. For example, progressive loss of the Zn²⁺ ion blockade of GABA currents in the hippocampal neurons during development may be indicative of changes in the GABA_A receptor subunits 9, 27, 28, 46, 49. Receptors to benzodiazepines, the allosteric modulators of GABA_A receptors also undergo developmental changes in various brain structures 14, 31. As we have previously reported [40], zolpidem, a specific agonist of benzodiazepine receptors types I, enlarged the IPSPs of hippocampal pyramidal cells in the adult rats but not in the young rats. These data corroborate the late developmental onset of expression of the α1 subunit/benzodiazepine type I receptors 13, 15, 20, 22, 24, 34, 35. Midazolam, a type I–II benzodiazepine receptor agonist [45], did not increase the membrane current responses to exogenously applied GABA 27, 39, 40, but enhanced the evoked IPSPs in young and adult rats [40]. Therefore, the possibility exists that synaptic and extrasynaptic GABA_A receptors have a different developmental profile. In the present study, we used whole-cell voltage-clamp recording techniques in hippocampal slices to examine whether in immature neurons the occurrence and kinetic properties of GABA_A receptor mediated events were different in young and adult rats. We also tested the effects of midazolam on mIPSCs and on the current responses to exogenously applied isoguvacine, a GABA_A receptor agonist.