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.