Phospholipase Cgamma1 in the Nucleus Accumbens Reduces Heroin-seeking: Effects on Synaptic Phospholipids and the Synaptoproteome

Chronic drug use leads to long-lasting increases in drug-seeking behavior, however, the causal molecular and cellular mechanisms responsible are not fully understood. One group of molecules that are dynamically and dramatically altered by opiate exposure and withdrawal are phospholipids of membrane bilayers including phosphatidylinositol (PI) and phosphatidylserine (PS). However, though phospholipid changes have been examined in whole striatal synaptosomes following chronic morphine exposure and withdrawal, specific changes in the nucleus accumbens (NAc) synaptosomes following heroin self-administration behavior have never been examined. One protein that regulates both phospholipids in neurons and drug-seeking behavior in the NAc is phospholipase Cgamma1 (PLCg1). I recently showed that PLCg1 overexpression in the NAc greatly reduces the motivation to take cocaine and I have unpublished preliminary data showing that endogenous NAc PLCg1 limits heroin-seeking relapse-like behaviors. However, the mechanism of how endogenous PLCg1 exerts these strong effects on drug-seeking behavior is still unknown. What is known is that PLCg1 normally cleaves phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) into inositol triphosphate (IP3) and diacylglycerol (DAG), allowing them to act as second messengers in cells. This suggests that heroin-induced changes in PLCg1 could have its effects through regulating phospholipids like PI(4,5)P2. Specifically, I hypothesize that heroin self-administration normally increases PI(4,5)P2 signaling in synapses to promote drug-seeking behavior; however endogenous PLCg1 activity reduces PI(4,5)P2 levels and therefore limits drug-seeking behavior. I also hypothesize that these effects are coupled to changes in the synaptic proteome that lead to reduced drug-seeking behavior since both PI(4,5)P2 and PLCg1 interact with many actin-binding and synaptic proteins. In this pilot grant, I propose to examine these changes using a 2x2 design in both PLCg1 knockdown and control NAc neurons from both saline and heroin self-administering rats. In Aim 1, I will determine the phosphoinositide signaling changes in NAc synaptosomes during heroin-taking, heroin withdrawal, and heroin-seeking timepoints. In Aim 2, I will determine the proteomic changes in NAc synaptosomes during heroin-seeking using the Yale/NIDA Neuroproteomics Center. These two aims will be completed using a within-subject analysis in order to correlate the phospholipid and proteomic changes in heroin-induced synapses in order to better understand the changes responsible for long-lasting heroin-seeking behaviors and the mechanism of PLCg1’s reduction of relapse-like effects.