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J Neurophysiol (September 10, 2003). doi:10.1152/jn.00364.2003
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Submitted on April 14, 2003
Accepted on September 2, 2003

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

Brigitte van Zundert1, Francisco J. Alvarez2, Juan C. Tapia1, Hermes H. Yeh3, Emilio Diaz1, and Luis G. Aguayo1*

1 Physiology, University of Concepcion, Concepcion, Chile
2 Anatomy, Wright State Univresity, Dayton, Ohio, USA
3 Pharmacology and Physiology, University of Rochester School of Medicine, Rochester, New York, USA

* To whom correspondence should be addressed. E-mail: laguayo{at}udec.cl.

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 (~30%) and intermediate (~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, RTPCR, kinetic analysis and picrotoxin blockade of glycinergic mIPSCs indicated a reorganization of the postsynaptic region from containing both {alpha}2{beta} and {alpha}1{beta} GlyRs in immature neurons to only {alpha}1{beta} GlyRs in mature neurons. Microtubule disruption preferentially affected {alpha}2{beta}-containing synaptic receptors.




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