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Aversive classical conditioning and the expression of neuronal nitric oxide (nNOS) in the hippocampus


The time-of-day oscillations of specific molecules after learning and circadian regulation of cellular signaling in the hippocampus are required for proper memory consolidation and persistence. Work with fear conditioning in mice suggests that memory processes, or the recall of the behavioral task, may be particularly sensitive to disruptions in circadian timing. In fact, repeated rounds of NMDA receptor synthesis are required for memory consolidation. This may support consideration of a similar regulation of nitric oxide (NO) and of nitric oxide synthase (NOS), which is mediated by NMDA receptor. Evidence from several studies indicates learning and memory deficits resulting from inhibition of the activity of nitric oxide synthases (NOS), and gives support to the fundamental role of nitric oxide in these processes. The present study intends to investigate the circadian variation of the expression of the neural isoform of NOS (nNOS) in the hippocampus (Experiment 1), its relation with contextual aversive conditioning (Experiment 2) and the effects of nNOS inhibition on the aversive contextual memory (Experiment 3). In Experiment 1 different groups of pigeons will be used for the analysis of the expression of nNOS in the hippocampus at six times points: ZT02, ZT06, ZT10, ZT14, ZT18, ZT22. In Experiment 2, groups of pigeons will be trained and tested in a contextual aversive situation during the morning or during the night. The time of the sessions will be defined according to the oscillation in nNOS expression observed in Experiment 1. Experimental groups will have a 20 min session of training in contextual aversive conditioning with three context-shock associations and, 24 h later, a 5 min session of exposure to the context (test). Control pigeons will have two sessions of exposure to the experimental chamber without shock presentation, or will be submitted to daily handling (Naïve). Experiment 3 intend to investigate how the administration of 7-nitroindazole, i.c.v., imediately after training, affects the retrieval of memory during the test and the expression of nNOS in the hippocampus. Five groups of pigeons will be used: 7-NI group, Vehicle group, no-treatment group, context control group and naive group. The pigeons' behavior during the sessions will be recorded with a digital video-system. Western blotting and immunohistochemistry analysis will be used in order to analyze nNOS expresion in the hippocampus. Statistical analysis of both behavioral and molecular data (nNOS-positive cell counting) will be conducted with ANOVA followed by multiple comparisons tests when appropriate. (AU)

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