Viral Tool Development for Investigations of Learning and Memory

Developing new viral tools to investigate biological and behavioral phenomena is important to push neuroscience research to new avenues that were once limited by expensive and time consuming transgenic and knockout rodent models. This dissertation is a collection of experiments that outline the development and optimization of several viral vector systems for application of studying a gene’s function in the brain and the role it plays in behavioral phenomena. First, I show that knockdown of a gene’s transcript using short hairpin RNAs (shRNAs) delivered to neurons using adeno associated virus (AAV), is toxic to neurons and results in an impairment of Pavlovian auditory fear conditioning. Next, I outline the development of a doxycycline dependent CRISPR/Cas genome editing system for knockdown of genes in the brain using AAV as an alternative to gene knockdown with shRNAs. I then examine the efficiency of several AAV serotypes for their ability to function in a specific cell type, inhibitory neurons. Next, I sought to look for important genes that might be potentially important for memory consolidation in the hippocampus, with respect to transport of mRNA transcripts to the distal ends of dendrites of dentate gyrus granule cells following the induction of long-term potentiation (LTP). I found that Arc (Activity related cytoskeletal protein) mRNA was the most present in the dendrites following neuronal activation compared to all other known transcripts in the rat genome. Interestingly, another gene that was highly upregulated was an unprocessed microRNA (pri-miRNA), pri-miRNA-132. Finally, I use lentiviral vectors to investigate the role of GluN2B, an NMDA receptor subunit, in the updating of strong memories and found that increasing the level of GluN2B within the mouse basal and lateral complex of the amygdala (BLA) allows for the modification of an existing strong fear memory via reconsolidation, a process typically not initiated in stronger memories.