The Role of Neuronal Nitric Oxide Synthase in Regulating Cerebellar Network Formation Across Murine Development
Abstract
The cerebellum is a region of the central nervous system widely known for its role in motor learning and coordination. In the past, mutant mice have been critical in discerning key pathways associated with cerebellar dysfunction and motor deficits. Although a variety of mouse models exist that model the symptoms and pathogenesis of cerebellar ataxia, some have yet to be characterized at the molecular level. Nitric oxide (NO), specifically derived from neuronal nitric oxide synthase (nNOS), is a well-established regulator of synaptic transmission in Purkinje neurons (PNs), governing fundamental processes such as motor learning and coordination. Previous morphological analyses showed similar gross cerebellar structures between neuronal nitric oxide null (nNOS-/-) and wild-type (WT) adult male mice, despite prominent ataxic behaviour within nNOS-/- mice. However, a study has yet to characterize potential differences in cerebellar network formation during development in nNOS-/- mice. This thesis study is the first to determine morphological and functional deficits within the cerebellum of nNOS-/- mice using immunostaining, immunoblotting, ex vivo slice culturing, calcium- and sodium-imaging, colourmetric assays and ELISAs. Results from Chapter 2 showed stark PN dendritic abnormalities in nNOS-/- mice compared to WT across development. Specifically, we noted that PN dendritic abnormalities are associated with elevated levels of intracellular calcium via overactivation of metabotropic glutamate receptor type 1 (mGluR1)-initiated store operated calcium entry. Chapter 3 analyses linked the overactivation of mGluR1 in nNOS-/- mice with decreased glutamate uptake via glutamate aspartate transporters (GLAST) on Bergmann glia (BG). Specifically, a lack of NO production resulted in decreased GLAST expression on BG and decreased glutamate uptake. Importantly, Chapter 4 results further demonstrated that the effects of mGluR1 overactivation on PNs specific to nNOS-/- mice resulted in increases in endocannabinoid levels. Specifically, our group noted increases in enzymes downstream of mGluR1 activation and subsequent increases in the endocannabinoid 2-arachidonoylglycerol, as well as decreases in endocannabinoid hydrolyzing enzymes in nNOS-/- cerebella compared to WT. Together, these results are foundational in establishing the nNOS-/- mouse as a model of cerebellar ataxia. Understanding the role of nNOS/NO signaling in cerebellar development may be beneficial in uncovering novel therapeutics for cerebellar disorders.