Modulation of CaMKII and endocannabinoid signaling by Calcium Channels

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DESCRIPTION (provided by applicant): Normal habit learning, motor control, and decision making requires striatal GABA-ergic medium spiny neurons to integrate excitatory inputs with modulatory inputs from other basal ganglia structures. One major mechanism playing a key role in these processes is endocannabinoid (eCB) dependent plasticity. The major striatal eCB, 2-arachidonylglycerol (2-AG), is synthesized postsynaptically by diacylglycerol lipase-a (DGL) in response to synaptic activity, but acts retrogradely to inhibit presynaptic glutamate release and induce synaptic depression. Striatal L-Type voltage gated calcium channels (LTCCs) mediate calcium influx and are thought to drive synthesis of 2-AG to promote eCB-dependent synaptic depression. Another regulator of striatal calcium levels are T-type voltage gated calcium channels (TTCCs), which can make substantial contributions to calcium entry and have also been linked to eCB plasticity. However, few studies have directly measured changes in 2-AG levels following modulation of voltage gated calcium channel activity, nor have the calcium signaling pathways coupling LTCCs and TTCCs to striatal eCB plasticity been well characterized. Our lab has recently made the exciting discovery that Ca2+/calmodulin-dependent protein kinase-IIa (CaMKII) restrains DGL activity and 2-AG production to limit eCB-dependent synaptic depression (Nat Neurosci, 2013; 16(4):456- 63). Furthermore, work from our lab and others shows that CaMKII associates with and propagates signaling from LTCCs and TTCCs. My preliminary studies found that high striatal CaMKII activity and localization to the postsynaptic density under basal conditions requires ongoing entry of extracellular calcium entry, partially mediated by TTCCs. The goal of the proposed 1 year project is to test the overarching hypothesis that LTCCs and TTCCs differentially activate CaMKII to regulate eCB-dependent synaptic signaling with two specific aims: 1. Test the hypothesis that striatal CaMKII is differentially coupled to TTCCs and LTCCs. Acutely isolated striatal slices will be incubated with agonists and antagonists of these channels. Extracts and subcellular fractions will be analyzed by western blotting using phospho-site specific antibodies to monitor the CaMKII activation by autophosphorylation at Thr286 and by phosphorylation of established synaptic substrates (Ser831 in GluR1; Ser1303 in NR2B), and by immunohistochemical analyses of Thr286 autophosphorylation. 2. Test the hypothesis that LTCCs and TTCCs differentially modulate striatal DGL and 2-AG levels. I will assay DGL activity and 2-AG levels in extracts of striatal slices incubated under conditions similar to those defined in Aim 1 using a liquid chromatography/mass spectrometry based method. In combination, these studies will be the first to directly investigate how the modulation of striatal CaMKII Thr286 autophosphorylation by LTCCs and TTCCs regulates synaptic signaling and 2-AG synthesis. This will provide a firm foundation for future studies of potential disruptions of these pathways in striatal-based neurological and psychiatric diseases.