Chromatin Regulatory Mechanisms of Bdnf expression During Cocaine Abstinence

Abstract

Sensory experience leads to long-term changes in neuronal structure and function, which are important for learning and memory. These long-term changes occur through activity-dependent gene regulatory mechanisms, driving dynamic stimulus-dependent changes in gene expression, and long-term epigenetic mechanisms, which can fine-tune gene expression levels. This temporal regulation of gene expression is important for the formation and maintenance of long-term memories, such as the persistent drug memories that exist long after cocaine abstinence. Animals trained to self-administer cocaine display incubation of cocaine craving behaviors during abstinence, which is paralleled by the increase in BDNF protein in the VTA during this period. Previous studies have demonstrated the importance of this BDNF increase on synaptic potentiation of dopaminergic neurons, but the regulatory mechanisms that drive this abstinence-dependent increase have not been tested. In this dissertation, I utilized CRISPR/dCas9 technologies to test the functionality of different Bdnf regulatory elements in mediating its stimulus-dependent expression. I first repressed putative distal enhancer elements that have been previously observed to interact with Bdnf upon neuronal activation and determined that these regions were shadow enhancers. Next, I characterized Bdnf expression in different cell types in the VTA upon forced cocaine abstinence and observed Bdnf upregulation occur in a non-cell-type specific manner. Then, I recruited dCas9-KRAB to different Bdnf regulatory elements and determined their contribution to abstinence-dependent Bdnf upregulation in different VTA cell types. In the process of these experiments, I discovered a previously unknown in vivo role of the Bdnf intragenic enhancer in regulating cocaine-abstinence dependent upregulation of Bdnf I. Finally, I repressed Bdnf expression in mice trained to self-administer cocaine and observed an abolition of incubation of cocaine craving. From these experiments, I demonstrate the contributions of different Bdnf regulatory elements in mediating its dynamic and persistent expression in response to stimulus, which can impact memory.