The classically conditioned eyeblink response is a well-studied model for understanding the neural mechanisms underlying associative learning. Classical conditioning is an example of associative learning that occurs when a reinforcing unconditioned stimulus (US) is contingent on the occurrence of a preceding conditioned stimulus (CS). In humans and other vertebrates, an eyeblink reflex in response to a tone can be learned when the tone is repeatedly paired with an airpuff to the cornea. We have developed an in vitro model of vertebrate associative learning using a turtle brain stem preparation that generates a neural analog of eyeblink classical conditioning to examine cellular mechanisms of conditioned response (CR) acquisition. In place of using tone and airpuff stimuli as in behaving animals we use paired stimulation of the auditory nerve (the """"""""tone"""""""" CS) with the trigeminal nerve (the """"""""airpuff"""""""" US) that results in burst discharge in the abducens nerve, which controls blinking in this species, characteristic of conditioned eyeblink responses. Our studies in the last funding period have focused on detailing molecular events that take place during in vitro conditioning related to protein kinase activation and AMPAR trafficking. These have allowed us to construct a two-stage model for conditioning in which synaptic delivery of GluR1-containing AMPARs initially activate silent synapses, followed by synaptic incorporation GluR4 subunits that support the acquisition of CRs. The third renewal of our project will further investigate the molecular mechanisms that underlie associative learning in vertebrates using this in vitro model of eyeblink classical conditioning. The following Specific Aims will be examined: 1) Specific elements of our model for conditioning will be tested directly using selective knockdown of gene targets by the RNAi approach. 2) To investigate the signal transduction mechanisms for coincidence detection and initiation of acquisition. 3) Our previous studies indicated that synaptic delivery of GluR1 was PKA-dependent while GluR4 was PKC- dependent. However, both require ERK. In this Aim, we will examine whether intracellular compartmentalization maintains the functional specificity of these kinases. 4) We have shown that brain-derived neurotrophic factor (BDNF) is required for synaptic AMPAR incorporation and conditioning, and morphological alterations of presynaptic terminals. Here, we will assess whether the coordinate pre- and postsynaptic modifications that occur during conditioning are mediated by the trans-synaptic eph/ephrin signaling system working in combination with BDNF/TrkB. Detailed investigation of the mechanisms that underlie learning and memory are fundamental to understanding disease states affecting these processes and will contribute to an overall effort to understand and treat the cognitive decline associated with normal aging and Alzheimer's disease.

Public Health Relevance

Using our in vitro model system of eyeblink classical conditioning, we have been able to directly link the function of metalloproteinases, brain-derived neurotrophic factor (BDNF) expression, synaptic AMPAR delivery, and associative learning. The studies performed in this proposal will contribute to a greater understanding of the molecular events that lead to associative learning and provide a potentially fruitful basis for future research into molecular mechanisms that underlie the cognitive decline associated with normal aging and Alzheimer's disease.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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Neurobiology of Learning and Memory Study Section (LAM)
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Babcock, Debra J
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University of South Dakota
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Zheng, Zhaoqing; Ambigapathy, Ganesh; Keifer, Joyce (2017) MeCP2 regulates Tet1-catalyzed demethylation, CTCF binding, and learning-dependent alternative splicing of the BDNF gene in Turtle. Elife 6:
Keifer, Joyce; Tiwari, Neeraj K; Buse, Leah et al. (2017) Subunit-specific synaptic delivery of AMPA receptors by auxiliary chaperone proteins TARP?8 and GSG1L in classical conditioning. Neurosci Lett 645:53-59
Keifer, Joyce; Zheng, Zhaoqing (2015) Coincidence detection in a neural correlate of classical conditioning is initiated by bidirectional 3-phosphoinositide-dependent kinase-1 signalling and modulated by adenosine receptors. J Physiol 593:1581-95
Ambigapathy, Ganesh; Zheng, Zhaoqing; Keifer, Joyce (2015) Regulation of BDNF chromatin status and promoter accessibility in a neural correlate of associative learning. Epigenetics 10:981-93
Keifer, Joyce; Zheng, Zhaoqing; Ambigapathy, Ganesh (2015) A MicroRNA-BDNF Negative Feedback Signaling Loop in Brain: Implications for Alzheimer's Disease. Microrna 4:101-8
Zheng, Zhaoqing; Keifer, Joyce (2014) Sequential delivery of synaptic GluA1- and GluA4-containing AMPA receptors (AMPARs) by SAP97 anchored protein complexes in classical conditioning. J Biol Chem 289:10540-50
Ambigapathy, Ganesh; Zheng, Zhaoqing; Keifer, Joyce (2014) Genomic organization and identification of promoter regions for the BDNF gene in the pond turtle Trachemys scripta elegans. J Mol Neurosci 53:626-36
Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei et al. (2013) Identification of a functionally distinct truncated BDNF mRNA splice variant and protein in Trachemys scripta elegans. PLoS One 8:e67141
Zheng, Zhaoqing; Sabirzhanov, Boris; Keifer, Joyce (2012) Two-stage AMPA receptor trafficking in classical conditioning and selective role for glutamate receptor subunit 4 (tGluA4) flop splice variant. J Neurophysiol 108:101-11
Li, W; Keifer, J (2012) Rapid enrichment of presynaptic protein in boutons undergoing classical conditioning is mediated by brain-derived neurotrophic factor. Neuroscience 203:50-8

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