Insights into the processes of learning and memory have fundamental implications for understanding memory disorders such as occur during amnesia and Alzheimer's disease. Conditioned response learning is a simple form of associative learning. A promising model to study the neural mechanisms underlying associative learning is the classically conditioned eye-blink reflex. The classically conditioned eye-blink reflex has been widely adopted for physiologic studies of the mechanisms of associative learning. Simple in vitro model systems of associative learning have been established in several species of marine mollusks. These preparations have greatly facilitated our understanding of the neural mechanisms of learning and memory. Similar model systems of learning in vertebrates have previously been hampered by the technical limitations of maintaining the viability of large portions of intact neural tissue in isolation. This problem was recently overcome by the development of an in vitro brainstem-cerebellum preparation from the turtle which takes advantage of this species' extraordinary resistance to anoxia. Using this preparation, conditioning of reflex pathways was undertaken simply by using electrical stimulation of sensory nerves rather than more natural stimuli such as a tone or airpuff. Preliminary data show that a neural correlate of the conditioned eye-blink reflex, the abducens nerve response, can be generated entirely in vitro. Using this model, studies of the neural mechanisms underlying this form of motor learning will complement those studies using whole animal preparations. The goals of the present project are to elucidate the synaptic organization of the abducens eye-blink reflex circuitry and the mechanisms that may underlie associative learning in this in vitro preparation.
The Specific Aims are: to evaluate whether NMDA receptors are required for classical conditioning by training the preparation in the presence or absence of the NMDA receptor antagonist APV, to examine the morphological distribution of synaptic inputs from nerves V (US) and pVIII (CS) onto abducens motoneurons using anterograde transport of Fluoro-Ruby and intracellular Lucifer yellow fills in fixed slices, to examine the morphological distribution of NMDA and non-NMDA glutamate receptors on LY-filled abducens motoneurons using immunocytochemistry, to identify the premotor pathways afferent to the abducens motoneurons using retrograde and anterograde tract tracing methods, and to determine the behavioral correlate of the abducens nerve CR by recording EMGs in a reduced preparation and by classical conditioning of the eye-blink reflex in alert turtles.
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