Identifying Diazepam Induced Alterations in GABA A R Synaptic Plasticity

Despite 50+ years of clinical use as anxiolytics, anti‐convulsants, and sedative/hypnotic agents, the mechanisms underlying benzodiazepine (BZD) tolerance are poorly understood. BZDs potentiate the actions of gamma‐aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, through positive allosteric modulation of γ2 subunit containing GABA type A receptors (GABA A R). Our work utilizes in‐vitro and in‐vivo BZD treatment combined with novel live imaging approaches, biochemical methods and mass spectrometry to identify BZD‐induced changes in GABA A R signaling. Previous findings from our lab and others have implicated trafficking mechanisms in the loss of BZD potentiation. To identify cellular mechanisms contributing to the initial loss of BZD potentiation, we engineered a novel GABA A R γ2 subunit that is capable of tracking receptors through nearly all phases of trafficking. An N terminal fluorogen‐activating peptide dual L5 (DL5) was genetically inserted into a γ2 subunit tagged with pH‐sensitive green fluorescent protein (γ2 pH ‐DL5). DL5 selectively binds and activates malachite green (MG) dyes that are otherwise non‐fluorescent in solution. We find that γ2 pH ‐DL5 containing GABA A Rs are expressed at the cell surface in transfected cortical neurons, form synaptic clusters, and do not perturb neuronal development. Electrophysiological studies show GABA A Rs incorporating γ2 pH ‐DL5 respond to the endogenous ligand GABA and exhibit positive modulation by the BZD diazepam (DZP). Imaging studies with γ2 pH ‐DL5 transfected neurons show time‐dependent receptor accumulation in endosomal and lysosomal vesicles, revealing constitutive endolysosomal trafficking. Ongoing experiments are investigating BZD‐induced modifications of receptor trafficking using this novel optical reporter. Complementary studies on endogenous GABA A Rs in rat cortical neurons suggest 24 hr exposure to 1μM DZP may reduce synaptic levels of the gephyrin scaffolding protein, while synaptic or total γ2 levels appear unchanged. Pilot biochemical studies of mice injected with 10mg/kg DZP vs vehicle show a significant decrease in total γ2 subunit cortical levels 12 hr post DZP injection. In contrast, mice treated with DZP daily for 1 week show sedative tolerance, but show no change in overall cortical γ2‐subunit levels. Consistent with DZP tolerance, acute slices from 7 day treated mice show loss of DZP potentiation of miniature inhibitory postsynaptic currents (mIPSCs). Overall, our work suggests DZP treatment induces a decrease in γ2 GABA A R levels and gephyrin scaffolding function only during the initial stages of prolonged BZD exposure, while subsequent compensatory mechanisms restore synaptic GABA A R levels once BZD tolerance has been established. These results highlight the complex nature of neuronal adaptations occurring with BZD tolerance. Support or Funding Information This work was generously funded by T32GM008424, the Whitehall Foundation, the William C. deGroat Neuropharmacology departmental fellowship and Pharmacology and Chemical Biology Startup Funds. Quantitative mass spectrometry experiments were performed by the MacDonald lab at the University of Pittsburgh. The DL5 peptide vector, MG dyes, and critical guidance was provided by Ming Zhang and the Waggoner Lab at Carnegie Melon University. HEK293 cell electrophysiology experiments were performed by Maddy Wilcox and the Johnson Lab at the University of Pittsburgh.