Investigating PKCε as a Target for Opioid Withdrawal and Pain

Opioid use disorder (OUD) has become much more prevalent in the United States over the past two decades. Withdrawal symptoms from both opioids and OUD medications used as replacement therapies can perpetuate the addiction cycle via negative reinforcement. Thus, there is a need to discover new, non-addictive medications for treatment. Protein kinase C epsilon (PKCε) has emerged as a target for treating both nicotine and alcohol use disorders. More recently, our group found that inhibiting PKCε reduces conditioned place aversion in morphine-dependent mice following naloxone-precipitated withdrawal, suggesting that PKCε is important for negative emotional signs of opioid withdrawal. Our current goal is to investigate whether other behavioral aspects of opioid withdrawal are associated with PKCε and determine which brain regions are important for these behaviors. This was accomplished by identifying the neuronal ensembles that modulate opioid withdrawal via PKCε using Fos immunohistochemistry. I found reductions in Fos expression between mice treated with the PKCε inhibitor in brain regions previously associated with negative emotional signs of withdrawal. However, inherent flaws with immunohistochemistry, which include asymmetrical sectioning, mechanical distortion, and random sub-sampling, may have confounded these findings. Recently, novel tissue-clearing methods have been developed which render millimeter-thick sections transparent and preserve the integrity of the brain’s natural cytoarchitecture, allowing analysis of Fos counts across brain regions in an unbiased fashion. I was able to successfully clear brains using FACT and FDISCO, which are techniques that can clear brains while preserving endogenous fluorescence. These can be used in future experiments investigating brain regions important for PKCε modulation of opioid withdrawal.