Application) This project contributes to the Center's overall objectives by examining the roles of the NMDA receptor and calcineurin (PP2B) functions in hippocampal CA1 pyramidal cells in hippocampus-dependent learning. How synaptic plasticity in the hippocampus contributes to hippocampus-dependent learning is one of the major goals of the Center. Activation of NMDA receptors in CA1 neurons is known to play a pivotal role in the induction of several forms of synaptic plasticity such as LTP and LTD at CA1 synapses. On the other hand, calcineurin is known to play a critical role in the induction of LTD. Our previous studies on CA1-specific NR1 KO (CA1 KO NR1) mice which were conducted collaboratively between our and Wilson's laboratories showed that NMDA- dependent synaptic plasticity at CA1 synapses plays a crucial role in spatial learning. These studies also suggested that the spatial learning impairment exhibited by the NR1 KO mice is probably due to the animal's inability to generate normal place fields in the CAI area which may be required for place-specific, coordinated firing of neurons in downstream brain areas. We will further test this hypothesis by recording the activities of neuron ensembles in the subiculum and the deep layers of the entorhinal cortex of CA1 KO NR1 mice. While NMDA receptor-mediated synaptic plasticity seems to play a crucial role in spatial learning, the relative contributions of LTP and LTD in this cognitive process remain unknown. We predict that a loss of calcineurin will result in a selective loss of LTD with preservation of LTP. We therefore plan to construct CA1-specific PP2B knockout mice (called CA 1 KO Crib 1) by the method already available in Tonegawa's laboratory. These mice will be analyzed by subjecting them to electrophysiological and behavioral studies. In addition to the procedures already utilized in our laboratories during the analysis of the CA1-specific N-R1 KO mice, expertise in Bear's laboratory on LTD will also be applied (Specific Aim #3). Another set of experiments revisits the unsettled issue of whether the hippocampus plays a crucial role not only in spatial learning but also in non-spatial learning. We will address this issue by subjecting the CAIKNR1 mice to a non-spatial learning paradigm, social transmission of food preference (Specific Aim #2). Individual Project #2 also contributes to the Center's overall objectives by examining the role of cAMP-dependent transcriptional factor CREB in the hippocampal CA1 area as well as in the neocortex in memory consolidation and maintenance (Specific Aim #4). Earlier studies by others with Drosophila and Aplysia implicated CREB-dependent protein synthesis in long term memory or long term facilitation. Although an effort was made by others to extend this notion to mammals using unrestricted CREB KO mice, the lack of regional and temporal restriction in the gene knockout and the incomplete inactivation of CREB activity prevented a firm conclusion. In addition, no information could be obtained as to where, for instance in hippocampus or parietal cortex, CREB is required in the consolidation and maintenance of hippocampus-dependent long term memory. We plan to address this central issue in memory research by creating and analyzing a new set of CREB KO mice in which gene knockout is complete and is restricted to the CA1 area or to the parietal and temporal cortices (see Core #1 for the construction of the latter mice). We also propose to produce and analyze CREB KO mice in which the gene knockout can be induced in a specific area of the brain after maturation in order to study adult function of CREB independent of its developmental function. Another CREB mouse to be made using a dominant negative form of CREB (dn CREB) will allow spatially-restricted inducible inhibition of endogenous CREB function. The inducible knockout or inhibition system will enable us to study the roles of a specific gene product (in this case CREB) in different phases of themnemonic process such as acquisition vs. consolidation (or maintenance) of memory. With all of these CREB gene manipulated mice, our or Bear's laboratory will analyze LTP and LTD (or depotentiation), particularly late LTP (L-LTP) at CA1 synapses of the hippocampus or at Te-2 synapses of the neocortex. Both our laboratory and Wilson's will subject the same mice to spatial learning and memory paradigms to assess possible impairments in the learning and/or memory phases of the behavior. In a closely related study as part of Individual Project #6, Bear's laboratory will extend the behavioral analysis to include visual recognition memory. All these studies may permit correlations of physiological and behavioral defects and also may identify brain site(s) and phases(s) of the mnemonic process in which CREB plays a crucial role. Finally, Wilson's laboratory will analyze some of these CREB mice by large-scale recordings in order to correlate defects in synaptic plasticity and behaviors to defects in the activity of neuronal ensembles.
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