Synaptic And Circuitric Mechanisms Underlying Hippocampus-depedent Learning And Memory

[Background]Emotion, learning and memory are the core components of cognitive functions. The function of hippocampus, an encephalic region highly relative with learning and memory, is mediated by both the outside neurons which are projected to hippocampus directly or indirectly and the inside hippocampus neural circuit. The previous study showed that amygdala, a region closely correlated to emotion, has direct fiber projections to hippocampus and generates monosynapic connections. However, the contribution of the emotion to the hippocampus-dependent spatial learning and memory and the possible molecular mechanisms remain unknown. The inner hippocampal neural circuits are mediated by genetic and epigenetic regulations. In the brain, a major cellular signaling molecule that is linked with gene expression is cyclic AMP (cAMP), known to activate EPAC (Exchange Protein Activated by Cyclic AMP) besides PKA. EPAC gene is shown to be abnormally expressed in the brain of AD patients, whose main clinical manifestation is the deficits of learning and memory. Whether the EPAC signal pathway mediates the synaptic plasticity of hippocampal circuit and the formation of learning and memory through the regulation of the transcription and translation of a certain gene is unclear.[Objective]1. To study the neural circuitry linking amygdala-associated learning to hippocampal synaptic morphology and functions, and as well as spatial learning and memory. 2. To explore EPAC-coupled genetic and epigenetic pathways underlying hippocampal synaptic plasticity and memory.[Methods]We used step-down test and simple shock pattern to replicate the animal model of emotional stress and explore the effects of the emotion on hippocampal synaptic morphology, synaptic transmission plasticity and learning and memory. Spatial learning and memory abilities of rats were tested by Morris Water Maze. The expression level of synapse-related proteins was measured by immunoblotting. The neuronal activities of amygdala and hippocampus were tested by immunohistochemistry. Synaptic transmission plasticity of hippocampus was tested by electrophysiological technique of patch clamp. Synaptic morphologic plasticity of hippocampus was tested by Golgi-cox staining and electron microscopy. D, L-2-amino-5-phosphonovaleric acid (AP5), a receptor blocking pharmacon of N-methyl-D-aspartate (NMDA), was used to inhibit basolateral nucleus of the amygdala to clarify the role of basolateral nucleus of the amygdala in the regulation of emotion on hippocampus-dependent spatial learning and memory. Hippocampal GluR2 was knocked down by gene silencing to clarify the role of GluR2 in the regulation of emotion on hippocampus-dependent spatial learning and memory.We used EPAC1-/-,EPAC2-/-,EPAC1/2-/- and IN-EPAC mice to study the regulation of EPAC-coupled genetic and epigenetic pathway on the synaptic plasticity of the hippocampal neural circuits and learning and memory. The spatial learning and memory abilities of the mutant mice were tested by Morris Water Maze. Juvenile play tests were used to investigate the social abilities. The expression levels of synapse-related proteins and Zif268 protein were measured by immunoblotting. Synaptic transmission plasticity of hippocampus was tested by Whole cell patch clamp. Synaptic morphologic plasticity of hippocampus was tested by Golgi-cox staining and electron microscopy. miRNA microarrays were used to test the levels of miRNA. q-PCR was used to test the levels of miR-124 and Zif268. Luciferase Reporters were used to test the interactions of miR-124 and Zif268. A construct of LNA-miR-124 was used to inhibit the expression of miR-124 in the brain. Generation of rAAV1/2 was used to express miR-124. Immunoprecipitation by anti-Rap1 was used to test the interactions of Rapl and miR-124.[Results]1. Amygdala-associated learning facilitates hippocampus-dependent spatial memory via upregulating GluR2 and enhancing synaptic plasticity.To study the neural circuitry linking amygdala and hippocampus, we used step-down test or simple electric shock to replicate rat models of emotion-associated learning or emotional stress, and then, measured the effects of amygdala-associated emotion learning on hippocampus-dependent spatial memory by water maze test. We observed that both of models showed significant activation of basolateral nucleus of the amygdala. The emotion-associated learning selectively activated the neurons in hippocampal CA1 and CA3 regions with a remarkable facilitation of the hippocampus-dependent spatial learning and memory. By using patch clamp, we found that the emotion-associated learning facilitates the formation of late LTP in hippocampal CA1 neurons. To further study the role of basolateral nucleus of the amygdala in the effects of emotion on the hippocampal functions, we used NMDA receptor blocking pharmacon AP5 to inhibit basolateral nucleus of the amygdala and tested the spatial learning and memory abilities of the rats. We found that inhibition of basolateral nucleus of the amygdala could reverse the facilitation of emotion-associated learning on hippocampus-dependent spatial learning and memory. The silence of GluR2 that was up-regulated by emotion-associated learning decreases the accuracy of rat's spatial memory, but does not affect the learning progress. 2. EPAC null mutation impairs learning and social interactions via aberrant regulation of miR-124 and Zif268 translation.To investigate the mechanisms of the regulation of EPAC-coupled genetic and epigenetic pathway on the synaptic plasticity of hippocampal neural circuits and learning and memory, we used EPAC null mutant mice and the control mice in the following studies. EPAC null mutant mice exhibit severe deficits in spatial learning and cortex-dependent social interactions. The electrophysiologic analysis shows that EPAC null mutation reduces glutamate release from presynaptic terminals without altering the receptor channel conductance on he postsynaptic sites. Synaptic Structures are normal in EPAC null alleles. Western-blot shows that EPAC null mutation does not affect the expression of synapse-related proteins. To further study the mechanisms of the regulation of EPAC-coupled genetic and epigenetic pathway on the synaptic plasticity and learning and memory, we conducted the miRNA arrays and found EPAC null mutation significantly increases miR-124. We identify that EPAC proteins via activation of Rapl directly interact with the regulatory element upstream of miR-124 gene and restricts miR-124. We further show that miR-124 directly binds to and inhibits Zif268 translation.[Conclusion]1. Amygdala-associated learning facilitates hippocampus-dependent spatial memory via upregulating GluR2 and enhancing synaptic plasticity.2. EPAC null mutation impairs learning and social interactions via aberrant regulation of miR-124 and Zif268 translation.