Neuronal Nitric Oxide Synthase Critically Regulates the Endocannabinoid Pathway in the Murine Cerebellum During Development

The cerebellum is a major site of endocannabinoid (eCB) production and signaling. The predominant eCB within the cerebellum, 2-arachidonoylglycerol (2-AG), is produced by a metabotropic glutamate receptor type 1 (mGluR1)-initiated signaling cascade within Purkinje neurons (PNs). 2-AG retrogradely stimulates cannabinoid 1 receptors (CB1Rs) located on presynaptic terminals. The activated CB1R decreases neurotransmitter release and leads to the production of nitric oxide (NO), a gaseous molecule. Recently, our group discovered that during development in mice lacking neuronal nitric oxide synthase (nNOS^-/-), PNs display an excitotoxic phenotype associated with overactivated mGluR1. Considering the importance of mGluR1 in 2-AG synthesis, the present study explored the role of nNOS-derived NO in regulating the eCB pathway within the cerebella of wildtype (WT) and nNOS^-/- mice at postnatal day 7 (PD7), 2 weeks (2 W), and 7 weeks (7 W). Our analysis showed that diacylglycerol lipase α, the enzyme that catalyzes 2-AG production, was elevated at early postnatal ages, and followed by elevated levels of 2-AG in nNOS^-/- cerebella compared to WT. CB1R expression in nNOS^-/- cerebella was upregulated at PD7 but decreased at 2 W and 7 W when compared to age-matched WT mice cerebella. Importantly, treating organotypic nNOS^-/- cerebellar slice cultures with an NO-donor-attenuated CB1R levels after 7 days in vitro. In addition, expression of the eCB hydrolases fatty acid amide hydrolase and monoacylglycerol lipase were significantly downregulated in nNOS^-/- cerebella compared to WT cerebella at 7 W. Together, these results reveal a novel role for nNOS/NO in regulating eCB signaling in the cerebellum.