--This fiscal year, we continued our studies of our gene knockouts (KO) of the mouse oxytocin (Oxt) and vasopressin (Avp) 1b receptors (Oxtr and Avpr1b, respectively) to investigate their specific roles in mediating behavior. Avp and Oxt are neurohormones that are best known for their peripheral actions in regulating salt and water balance, blood pressure, lactation and parturition. However, numerous pharmacological studies have implicated these hormones in various behaviors as well, including aggressive, affiliative, social recognition, fear conditioning and maternal. --We continued our studies of the CA2 region of the hippocampus where the Oxtr and Avpr1b are located. The CA2 area is an important, although relatively unexplored, component of the hippocampus. We used various tracers to provide a comprehensive analysis of CA2 connections in C57BL/6J mice. Using various adeno-associated viruses that express fluorescent proteins, we found a vasopressinergic projection from the paraventricular nuclei of the hypothalamus (PVN) to the CA2, as well as a projection from pyramidal neurons of the CA2 to the supramammillary nuclei. These projections were confirmed by retrograde tracing. As expected, we observed CA2 afferent projections from neurons in ipsilateral entorhinal cortical layer II as well as from bilateral dorsal CA2 and CA3 using retrograde tracers. Additionally, we saw CA2 neuronal input from bilateral medial septal nuclei, vertical and horizontal limbs of the nucleus of diagonal band of Broca, supramammillary nuclei (SUM) and median raphe nucleus. Dorsal CA2 injections of adeno-associated virus expressing green fluorescent protein revealed axonal projections primarily to dorsal CA1, CA2 and CA3 bilaterally. No projection was detected to the entorhinal cortex from the dorsal CA2. These results are consistent with recent observations that the dorsal CA2 forms disynaptic connections with the entorhinal cortex to influence dynamic memory processing. Mouse dorsal CA2 neurons send bilateral projections to the medial and lateral septal nuclei, vertical and horizontal limbs of the diagonal band of Broca and the SUM. Novel connections from the PVN and to the SUM suggest important regulatory roles for CA2 in mediating social and emotional input for memory processing. --We looked for microRNAs (miRNAs) that might control Oxt expression within the CNS. MiRNAs, endogenous regulators of many genes, are a class of small non-coding RNAs that mediate post-transcriptional gene silencing. We performed miRNA expression profiling of the mouse hypothalamus by deep sequencing. Among the sequenced and cross-mapped small RNAs, expression of known miRNAs and unknown miRNAs candidates were analyzed. We investigated in detail one miRNA, miR-24, and found that it is a novel regulator of Oxt and controls both transcript and peptide levels of Oxt. These results provide insights into potential neurohypophysial hormone regulation mediated by miRNAs. --Our laboratory is interested in carefully delineating the in vivo expression patterns between three closely related miR-7 miRNA family members in the mouse. The miR-7 family of miRNAs is conserved between humans and invertebrates. The miR-7 family also shows conserved expression in neural tissues, and members of this family are involved in the development of sensory structures in flies, associated with many types of cancer, and likely play a role in osmoregulation in the mouse brain. In this study, we used multiple methods to differentiate the expression of the miR-7 family members in the mouse brain. Our results suggest that although there are useful tools for exploring miRNA expression in vivo, better tools and/or methods are still needed for thorough in vivo studies of closely related family members.
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