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The use of viral vector technology to deliver short hairpin RNAs (shRNAs) to cells of the nervous system of many model organisms has been widely utilized by neuroscientists to study the influence of genes on behavior. However, there have been numerous reports that delivering shRNAs to the nervous system can lead to neurotoxicity. Here we report the results of a series of experiments where adeno-associated viruses (AAV), that were engineered to express shRNAs designed to target known plasticity associated genes (i.e. Arc, Egr1 and GluN2A) or control shRNAs that were designed not to target any rat gene product for depletion, were delivered to the rat basal and lateral nuclei of the amygdala (BLA), and auditory Pavlovian fear conditioning was examined. In our first set of experiments we found that animals that received AAV (3.16E13 – 1E13 GC/mL; 1ul/side), designed to knockdown Arc (shArc), or control shRNAs targeting either luciferase (shLuc), or nothing (shCntrl), exhibited impaired fear conditioning compared to animals that received viruses that did not express shRNAs. Notably, animals that received shArc did not exhibit differences in fear conditioning compared to animals that received control shRNAs despite gene knockdown of Arc. Viruses designed to harbor shRNAs did not induce obvious morphological changes to the cells/tissue of the BLA at any dose of virus tested, but at the highest dose of shRNA virus examined (3.16E13 GC/mL; 1ul/side), a significant increase in microglia activation occurred as measured by an increase in IBA1 immunoreactivity. In our final set of experiments we infused viruses into the BLA at a titer of (1.60E+12 GC/mL; 1ul/side), designed to express shRNAs designed to target Egr1 (shEgr1), GluN2A (shGluN2A), shArc, shLuc, shCntrl, or a virus which did not express an shRNA, and found that all groups exhibited impaired fear conditioning compared to the group which received a virus that did not express an shRNA. The shEgr1 and shGluN2A groups exhibited gene knockdown of Egr1 and GluN2A compared to the other groups examined respectively, but Arc was not knocked down in the shArc group under these conditions. Differences in fear conditioning among the shLuc, shCntrl, shArc and shEgr1 groups were not detected under these circumstances, however the shGluN2A group exhibited significantly impaired fear conditioning compared to most of the groups, indicating that gene specific deficits in fear conditioning could be observed utilizing viral mediated delivery of shRNA. Collectively, these data indicate that viral mediated shRNA expression was toxic to neurons in vivo, under all viral titers examined and this toxicity in some cases may be masking gene specific changes in learning. Therefore, the use of this technology in behavioral neuroscience warrants a heightened level of careful consideration and study design and potential methods to alleviate shRNA induced toxicity are discussed.

The amygdala is a heterogeneous, medial temporal lobe structure that has been implicated in the formation, expression and extinction of emotional memories. This structure is composed of numerous nuclei that vary in cytoarchitectonics and neural connections. In particular the Lateral nucleus of the Amygdala (LA), Central nucleus of the Amygdala (CeA), and the Basal (B) nucleus contribute an essential role to emotional learning. However, to date it is still unclear to what extent these nuclei differ at the molecular level. Therefore we have performed whole genome gene expression analysis on these nuclei to gain a better understanding of the molecular differences and similarities among these nuclei. Specifically the LA, CeA and B nuclei were laser microdissected from the rat brain, and total RNA was isolated from these nuclei and subjected to RNA amplification. Amplified RNA was analyzed by whole genome microarray analysis which revealed that 129 genes are differentially expressed among these nuclei. Notably gene expression patterns differed between the CeA nucleus and the LA and B nuclei. However gene expression differences were not considerably different between the LA and B nuclei. Secondary confirmation of numerous genes was performed by in situ hybridization to validate the microarray findings, which also revealed that for many genes, expression differences among these nuclei were consistent with the embryological origins of these nuclei. Knowing the stable gene expression differences among these nuclei will provide novel avenues of investigation into how these nuclei contribute to emotional arousal and emotional learning, and potentially offer new genetic targets to manipulate emotional learning and memory.