Synaptic Mechanisms of Delay Eyeblink Classical Conditioning: AMPAR Trafficking and Gene Regulation in an In Vitro...

An in vitro model of delay eyeblink classical conditioning was developed to investigate synaptic plasticity mechanisms underlying acquisition of associative learning. This was achieved by replacing real stimuli, such as an airpuff and tone, with patterned stimulation of the cranial nerves using an isolated brainstem preparation from turtle. Here, our primary findings regarding cellular and molecular mechanisms for learning acquisition using this unique approach are reviewed. The neural correlate of the in vitro eyeblink response is a replica of the actual behavior, and features of conditioned responses (CRs) resemble those observed in behavioral studies. Importantly, it was shown that acquisition of CRs did not require the intact cerebellum, but the appropriate timing did. Studies of synaptic mechanisms indicate that conditioning involves two stages of AMPA receptor (AMPAR) trafficking. Initially, GluA1-containing AMPARs are targeted to synapses followed later by replacement by GluA4 subunits that support CR expression. This two-stage process is regulated by specific signal transduction cascades involving PKA and PKC and is guided by distinct protein chaperones. The expression of the brain-derived neurotrophic factor (BDNF) protein is central to AMPAR trafficking and conditioning. BDNF gene expression is regulated by coordinated epigenetic mechanisms involving DNA methylation/demethylation and chromatin modifications that control access of promoters to transcription factors. Finally, a hypothesis is proposed that learning genes like BDNF are poised by dual chromatin features that allow rapid activation or repression in response to environmental stimuli. These in vitro studies have advanced our understanding of the cellular and molecular mechanisms that underlie associative learning.