Role for calbindin-D28K in in vitro classical conditioning of abducens nerve responses in turtles

Intracellular calcium has a pivotal role in synaptic modifications that may underlie learning and memory. The present study examined whether there were changes in immunoreactivity levels of the AMPA receptor subunits GluR2/3 and calcium binding proteins during classical conditioning recorded in the abducens nerve of in vitro brain stem preparations from turtles. The results showed that abducens motor neurons in unconditioned turtle brain stems were immunopositive for GluR2/3, calbindin-D28K, and calmodulin, but were immunonegative for parvalbumin. After classical conditioning, immunoreactivity for calbindin-D28K in the abducens motor nuclei was significantly reduced, whereas there were no significant changes in GluR2/3, calmodulin, or parvalbumin. This reduction in calbindin-D28K immunoreactivity was not observed following conditioning in the NMDA receptor antagonist AP-5, which blocked conditioned responses, suggesting that these changes are NMDA receptor-dependent. Moreover, the degree of the decrease in calbindin-D28K immunoreactivity was negatively correlated with the level of conditioning. Consistent with the immunocytochemical findings, Western blot analysis showed that calbindin-D28K protein levels were reduced after classical conditioning. The results support the hypothesis that in vitro classical conditioning of abducens nerve responses utilizes intracellular calcium-dependent signaling pathways that require NMDA receptor function and suggest a specific role for the calcium binding protein calbindin-D28K.