The effects of cannabinoids are sometimes biphasic, for example, low doses produce anxiolysis whereas high doses induce anxiety; similar effects have been seen on memory and cognition. However, the reasons for this are not clear. At the synapse level, the cannabinoid receptor 1 (CB1R) is known to impact transmitter release through inhibition of presynaptic calcium channels, including the N-type (CaV2.2) channels that are paramount in coupling neuronal activity to transmitter release in the hippocampus (HPC). This brain area is important for emotional processing and learning, and CaV2.2 channels have been shown to be essential in one of the best-studied circuits in the brain. Our long-term goal is to decipher the regulation and function of CaV2.2 channels at specific HPC synapses to inform basic mechanisms of HPC activity, and enable novel therapies based on CB1R signaling. Alternative splicing is a cell-specific mechanism that impacts the function and regulation of CaV2.2 channels. Splicing of the cassette exon 18a generates the +18a-CaV2.2 and Δ18a-CaV2.2 splice variants. Neurotransmission in synapses that express +18a-CaV2.2 variants exhibit enhanced transmitter release, and reduced modulation by CB1R agonists compared to those that express the Δ18a-CaV2.2 variants, but the underlying mechanisms are unknown. +18-CaV2.2 channels contain a 21 aminoacid insertion in the region that interacts with proteins of the release machinery. Our central hypothesis is CaV2.2 splice variants containing exon 18a increase synaptic activity and prevent CB1R regulation of neurotransmission by generating CaV2.2 channels that uncouple to proteins of the transmitter release machinery. To test this, we will use validated mouse models with restricted splice choice (+18a-only or Δ18a-only) and recombinase-based labeling of specific neurons in HPC for electrophysiology in acute slices, and biochemical assays to evaluate protein interaction. The specific aims of the project are: 1) To determine the functional impact of +18a-CaV2.2 and Δ18a-CaV2.2 splice variants on synaptic activity of HPC, and 2) To determine how +18a-CaV2.2 and Δ18a-CaV2.2 splice variants couple to CB1R modulation of neurotransmission in HPC. The results of the project are expected to provide insights into the underlying mechanisms for the biphasic effects of cannabinoids. Novel cell-specific effectors of CB1R signaling could positively impact development of cannabinoid-based therapeutics
