Developmental Changes of Inhibitory Synaptic Currents in Cerebellar Granule Neurons: Role of GABA_AReceptor α6 Subunit

Abstract

Eye opening and increased motor activity after the second postnatal week in rats imply an extensive development of motor control and coordination. We show a parallel development change in spontaneous IPSC (sIPSC) kinetics in cerebellar granule neurons. sIPSCs were studied by whole-cell recordings in cerebellar slices, prepared from 7-30 postnatal day old rats. Early in development, sIPSCs had slow decay kinetics whereas in older rats faster decaying sIPSCs were found in larger proportion. Currents elicited by 1 mM GABA pulses (GABACs) in nucleated patches excised from cerebellar granule neurons revealed that GABACs kinetics better approximate sIPSC decay in young but not in more developed rats. The expression of alpha 6 subunit of GABAA receptors, unique in cerebellar granule neurons, has been shown to increase during development. Therefore, we took advantage of the recently reported selective inhibition of GABAA receptors by furosemide to characterize the relative contribution of alpha 6 subunits to native receptors in inhibitory synapses of cerebellar granule neurons. Although furosemide inhibition of sIPSCs amplitude was highly variable among distinct granule cells, it increased during development. At the same time, furosemide failed to inhibit sIPSCs recorded from Purkinje neurons. From the comparison of furosemide inhibition and kinetics of sIPSCs with GABACs recorded from mammalian HEK293 cells transfected with combinations of alpha 1 and alpha 6 GABAA receptor subunits together with beta 2 gamma 2 subunits, we propose that an increased alpha 6 subunit contribution in the molecular assembly of postsynaptic receptors in cerebellar glomeruli is responsible for the developmental changes observed.

Properties of GABAAReceptors Underlying Inhibitory Synaptic Currents in Neocortical Pyramidal Neurons

“…Recent experimental results led to the proposal that the biexponential decay of I PSC s recorded from cultured hippocampal cells (Jones and Westbrook, 1995) and granule cerebellar neurons (Tia et al, 1996) could be generated by a rapid entry and exit of the GABA A receptors through desensitized states (i.e., nonresponsive states). To test if this model also could apply to the mIPSCs recorded from neocortical pyramidal neurons, we studied the properties of their GABA A receptors, using rapid applications of GABA to excised patches.…”

“…To test this issue, we used a paired-pulse protocol (Jones and Westbrook, 1995;Tia et al, 1996). If GABA A receptors are desensitized by the first pulse of agonist, then the availability of receptors to a second activation would be diminished.…”

“…Previous studies on the mechanisms underlying the time course of I PSC s led to the proposal that these mostly reflect the functional properties of the postsynaptic GABA A receptors rather than the clearance of GABA from the synaptic cleft (Maconochie et al, 1994;Puia et al, 1994;Jones and Westbrook, 1995;Tia et al, 1996). The GABA A receptor channel is thought to be a heteropentameric complex assembled from at least 16 potential subunits (Macdonald and Olsen, 1994;Sieghart, 1995), and different neuronal types have been shown to express distinct combinations of GABA A receptor subunits (Fritschy et al, 1992;Wisden et al, 1992;Ruano et al, 1997) (for review, see McKernan and Whiting, 1996).…”

“…Assuming that brief pulses of agonist would mimic transmitter transients during synaptic transmission better than prolonged ones, we have examined the kinetic properties of pyramidal GABA A receptors in response to brief pulses of GABA. Under these conditions we have studied the possible role of receptor desensitization in shaping the decay of the IPSCs (Jones and Westbrook, 1995;Tia et al, 1996), the kinetics of the outside-out patch responses at different concentrations of GABA, and the levels of agonist required for receptor saturation.…”

Effect of the α subunit subtype on the macroscopic kinetic properties of recombinant GABAA receptors

“…Some of these differences might be due to differences in the subunit composition of the receptors. Previous results from recombinant receptors suggest that the α subtype influences desensitization and deactivation kinetics, although these studies used a variety of experimental protocols and findings are often inconsistent among different laboratories (Gingrich et al, 1995;Tia et al, 1996aTia et al, , 1996bLavoie et al, 1997;Burgard et al, 1999;McClellan and Twyman, 1999;Bianchi et al, 2002a;Lagrange et al, 2007). In order to directly compare the effect of the α subtype on channel properties, we examined the macroscopic kinetic properties of recombinant receptors containing each of the different α subtypes combined with the same β and γ subunit subtypes and under the same recording conditions.…”