A relatively new approach to genetically modify organisms involves the use of recombinant viruses to infect discrete populations of cells in vivo to alter gene expression by overexpressing a transgene of interest. If the virus is designed to express a short hairpin RNA (shRNA), RNA interference technology (RNAi) can be utilized to silence specific genes of interest in the targeted population. This technology is especially useful for learning and memory research, since the genetic manipulations can be introduced acutely in specific brain regions of interest and these manipulations can be created before or after learning. However, for viral mediated gene silencing to truly be effective it must be able to knockdown gene expression more than 50% across a population of pertinent target cells in vivo, which is not a trivial feat since targeted cells need to each be infected by multiple viruses consistently across regions of the rat brain that can span millimeters to effectively knockdown the target gene. Therefore this proposal is designed to improve upon this approach to facilitate the study of genes in amygdala-dependent learning and memory. In our first aim we will identify the Adeno-associated virus (AAV), viral serotypes that maximally infect the rat Basal Lateral and Central nuclei of the Amygdala (BLA and CeA). Additionally we will determine which AAV serotypes infect excitatory glutamatergic, aCaMKII expressing neurons and inhibitory gabaergic GAD65 expressing neurons within these nuclei using custom-made viruses that will tag these differing neuronal types, allowing them to be distinguished from other cell types within the amygdala. In our second aim we will examine if viral-mediated in vivo gene knockdown can be enhanced by increasing the number of shRNA transgenes included within the virus and if this modification leads to a greater impairment in learning and memory. Specifically AAV will be designed to harbor varying numbers of shRNA expression cassettes designed to target the Activity Regulated Cytoskeletal protein (Arc). Additionally, a set of control viruses will be designed to harbor a similar number of shRNA expression cassettes that do not target any transcript for knockdown. Separate groups of rats will receive bilateral infusions of these viruses into the BLA. The rats will be fear conditioned and tested for retention of auditory fear memory at both 3 hours and 24 hours after training to evaluate the effect of Arc knockdown on the retention of short-term and long-term fear memory, respectively. Additional experiments will be conducted to measure locomotor behavior, innate fear, expression of fear and Arc protein levels within the BLA. We hypothesize that LTM will be impaired by Arc knockdown and the impairment in LTM will correlate with the degree of Arc knockdown and therefore the number of shRNA transgenes that were present in the virus targeting Arc. This RO3 research project will allow us to improve upon technology that will facilitate learning and memory research.
This project aims to optimize tools to study amygdala dependent learning and memory. The amygdala is critical for proper functioning of the fear system within the brain. Dysregulation of the fear system is associated with many psychiatric disorders, underscoring the need for developing better treatments for pathological fear.
|Holehonnur, Roopashri; Luong, Jonathan A; Chaturvedi, Dushyant et al. (2014) Adeno-associated viral serotypes produce differing titers and differentially transduce neurons within the rat basal and lateral amygdala. BMC Neurosci 15:28|