Developmental-Dependent Action of Microtubule Depolymerization on the Function and Structure of Synaptic Glycine Receptor Clusters in Spinal Neurons

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Abstract
Microtubules have been proposed to interact with gephyrin/glycine receptors (GlyRs) in synaptic aggregates. However, the consequence of microtubule disruption on the structure of postsynaptic GlyR/gephyrin clusters is controversial and possible alterations in function are largely unknown. In this study, we have examined the physiological and morphological properties of GlyR/gephyrin clusters after colchicine treatment in cultured spinal neurons during development. In immature neurons (5-7 DIV), disruption of microtubules resulted in a 33 ± 4% decrease in the peak amplitude and a 72 ± 15% reduction in the frequency of spontaneous glycinergic miniature postsynaptic currents (mIPSCs) recorded in whole cell mode. However, similar colchicine treatments resulted in smaller effects on 10-12 DIV neurons and no effect on mature neurons (15-17 DIV). The decrease in glycinergic mIPSC amplitude and frequency reflects postsynaptic actions of colchicine, since postsynaptic stabilization of microtubules with GTP prevented both actions and similar reductions in mIPSC frequency were obtained by modifying the Cl-driving force to obtain parallel reductions in mIPSC amplitude. Confocal microscopy revealed that colchicine reduced the average length and immunofluorescence intensity of synaptic gephyrin/GlyR clusters in immature (approximately 30%) and intermediate (approximately 15%) neurons, but not in mature clusters. Thus the structural and functional changes of postsynaptic gephyrin/GlyR clusters after colchicine treatment were tightly correlated. Finally, RT-PCR, kinetic analysis and picrotoxin blockade of glycinergic mIPSCs indicated a reorganization of the postsynaptic region from containing both α2β and α1β GlyRs in immature neurons to only α1β GlyRs in mature neurons. Microtubule disruption preferentially affected postsynaptic sites containing α2β-containing synaptic receptors.