Abstract

Using fluorometric and immunocytochemical techniques, we found that high glycine concentrations or blockade of glycine receptors increases neurite outgrowth in developing mouse spinal cord neurons. Glycine- and GABAA-activated currents were demonstrated during applications of glycine and GABA (50-100 ?M) in 5 days in vitro (DIV) neurons. Long application (?10 min) of 100 ?M glycine desensitized the membrane response by more than 95%. Application of glutamate in the absence of external Mg2+, at several membrane potentials, did not produce any detectable membrane response in these cells. Immunocytochemical studies with NR1 and GluR1 antibodies showed a delayed appearance of N-methyl-D-aspartate (NMDA) and ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors respectively. Spontaneous synaptic activity was readily observed in 5 DIV neurons. The use of various receptor antagonists (strychnine, bicuculline, DL-2-amino-5-phosphonovalerate [APV], 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX]) revealed that this activity was predominantly glycinergic, and to a smaller extent, GABAergic. In the presence of bicuculline, APV and CNQX, we detected abundant spontaneous depolarizing potentials which often reached the action potential threshold. Further evidence for functional synaptic activity was provided by the detection of co-localization of gephyrin and synaptophysin at 5 DIV using confocal microscopy. Fluorometric studies with Fluo-3, a Ca2+ indicator, in 5 DIV cultures showed the presence of spontaneous fluctuations associated with tetrodotoxin-sensitive synaptic events. The number of neurons displaying these fluctuations was significantly increased (> 100%) when the cells were bathed in a strychnine-containing solution. On the other hand, these synaptically mediated Ca2+ events were blocked by the co-application of strychnine and bicuculline. This suggests that glycine and GABAA receptors provide a fundamental regulation of both neuronal excitability and intracellular Ca2+ at this early time of development. The neurotrophic effects of agonists and antagonists for glycine, GABAA and glutamate receptors were examined in neurons cultured for 2 or 5 DIV. From all the agonists used, only high concentrations of glycine increased neurite outgrowth in 5 DIV neurons. We found that strychnine also increased neurite outgrowth, whereas tetrodotoxin (1 ?M), nimodipine (4 ?M) and bicuculline (20 ?M) completely blocked it. On the other hand, APV (50 ?M) and CNQX (20 ?M) were unable to affect neurite outgrowth. These data suggest that spinal glycine receptors depress neurite outgrowth by shunting neuronal excitability. Outgrowth induction possibly results from the enhanced activity found after the inhibition of glycinergic activity. We postulate that this resets the intracellular calcium at a concentration that favors neurite outgrowth. © 2001 IBRO. Publihed by Elsevier Science Ltd. All rights reserved.