Research reportDevelopmental study of miniature IPSCs of CA3 hippocampal cells: modulation by 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 GABAA receptor mediated synapses mature prior to glutamatergic AMPA receptor mediated ones 3, 16. In the CA3 pyramidal cells GABAA 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 GABAA receptor subunits has been demonstrated in the hippocampus and other brain structures 13, 17, 20, 24, 34, 35. However, it is not clear whether GABAA 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 GABAA 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 GABAA receptor subunits 9, 27, 28, 46, 49. Receptors to benzodiazepines, the allosteric modulators of GABAA 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 GABAA 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 GABAA 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 GABAA receptor agonist.
Section snippets
Slices preparation
Experiments were performed on hippocampal slices of young (4–8 days old, n=29) and adult (26–35 days old, n=10) male Wistar rats. Slices were prepared as described before [40]. Briefly, rats were decapitated under ether anesthesia, the brain removed and the hippocampi were rapidly dissected out in oxygenated, ice-cooled artificial cerebrospinal fluid (ACSF), composed of (in mM): 126 NaCl, 3.5 KCl, 2.0 CaCl2, 1.3 MgCl2, 25 NaHCO3, 1.2 NaH2PO4 and 11 glucose; gassed with 95% O2–5% CO2. Transverse
Developmental changes in the mIPSCs of CA3 cells
In the presence of tetrodotoxin (TTX, 1 μM), CNQX (10 μM) and AP5 (50 μM) to block spontaneous excitatory currents, spontaneous transient inward currents were recorded both in adult (n=6) and immature (n=7) slices (Fig. 1A,B). At holding potential of −80 mV, these miniature events were inward currents that reversed polarity at −4.6±3.0 mV. These currents were blocked by bicuculline (10 μM), and were therefore, mIPSCs mediated by GABAA receptors (Fig. 1A,B).
As shown in the control traces in Fig.
Discussion
Our results therefore suggest that the amplitude and kinetic properties of mIPSCs as well as the sensitivity to midazolam are similar in young and adult CA3 cells. Contradictory observations have been reported earlier, evidence in favor of developmental changes in hippocampal and cerebellar granule cells 15, 53in which immature cells mIPSCs had a slower kinetic properties whereas the rates of activation and deactivation of mIPSC of superior colliculus neurons in culture were not altered with
Acknowledgements
We thank J. Mariani for his critical reading of the manuscript.
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