Date of Award
Doctor of Philosophy (PhD)
Mammalian neocortex is the most complex structure in the brain. Considered the seat of executive function, the neocortex subserves diverse cognitive processes such as decision-making, motor planning, sensory discrimination and memory consolidation. The cellular heterogeneity of the neocortex makes it difficult to understand the molecular and cellular substrate of cognition during normal brain function and disease related dysfunction. A deep understanding of the contribution of the neocortex to behavior can best be achieved with reproducible access to genetically defined and functionally coherent cell populations. Using BAC modification strategy, we targeted distinct cell types in the neocortex for expressing effector molecules such as EGFPtagged ribosomal protein L10 and Cre recombinase. We generated bacTRAP transgenic mice for six pyramidal cell types in the neocortex distinct by marker expression, areal/laminar topography, connectivity and cell morphology. These include corticocortical neurons in upper layer 2 of neocortex defined by genes Ntf3, Pdyn and Wfs1, corticostriatal neurons in layer 5a of lateral cortex expressing Etv1 and corticothalamic neurons in layer 6 of neocortex defined by Ntsr1 and Syt6. We generated translation profiles of actively translated mRNAs in the targeted cell types with affinity purification of tagged polysomes and carried out quantitative comparative analysis of the cell types using the Specificity Index algorithm to identify cell type specific molecular repertoire. Besides the molecular profile and cellular morphology, pyramidal cell types can be characterized by connectivity and function at the level of circuitry as well as global behavior. Using Cre-inducible viral transduction of fluorophore (EGFP or mCherry), we were able to map the circuit of focal populations of three cell types defined by Ntsr1, Syt6 and Wfs1 in the visual cortex and anterior cingulate cortex respectively. We then applied viral mediated silencing of neural transmission using viral tethered toxins that target voltage gated calcium channels (VGCCs) Cav2.1 and Cav2.2. We found that the Wfs1 cell type in the anterior cingulate cortex is necessary for the affective pain circuitry. Compared to control mice, the Wfs1 silenced mice exhibited a significantly reduced aversion to formalin-paired context in the formalin-conditioned place aversion paradigm but have intact pain sensitivity. Anterior Cingulate has previously been shown to be connected to the emotional center of the brain, the amygdale, and neural responses during pain perception have been recorded in the ACC. Hence we present supportive evidence that layer 2 Wfs1 cells in the anterior cingulate cortex are important for mediating affective pain. In addition, we investigated a candidate gene, Wfs1, enriched in the translational profile for Ntf3 cell type in the neocortex and studied behavioral phenotype of mice harboring cortex specific deletion of Wfs1. The Wfs1 Cortex KO mice were anhedonic and were impaired in their preference for sucrose, which is normally considered more pleasurable than water. The KO mice also exhibited compulsive responding in the three-choice operant conditioning task. In conclusion, this dissertation presents evidence that BAC transgenesis can be applied to genetically access pyramidal cell types in the neocortex for molecular profiling as well as functional characterization using silencing viruses and conditional alleles. We also showed direct utility of TRAP data in elucidating cortex specific contribution to the complex neurological disorder, Wolfram Syndrome. Therefore, a combination of specific gene driver selection, BAC transgenesis methods and functional modulatory strategies can give us valuable insight into the underlying neuronal circuitry in the cortex responsible for distinct behavioral outputs, physiology and pathophysiology.
Shrestha, Prerana, "Molecular and Functional Characterization of Pyramidal Cell Types in the Frontal Cortex of Mouse Brain" (2012). Student Theses and Dissertations. 246.