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Identifying Diazepam Induced Alterations in GABA Type A Receptor Synaptic Plasticity
Author(s) -
LorenzGuertin Joshua,
Das Sabyasachi,
Pardo Sammy,
Molleur Dana,
Weintraub Susan,
Jacob Tija C.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.680.12
Subject(s) - gephyrin , gabaa receptor , inhibitory postsynaptic potential , fluorescence recovery after photobleaching , gamma aminobutyric acid , chemistry , long term potentiation , protein subunit , glycine receptor , neurotransmitter , diazepam , benzodiazepine , receptor , pharmacology , allosteric regulation , gaba receptor , microbiology and biotechnology , biophysics , neuroscience , biochemistry , biology , glycine , amino acid , membrane , gene
The mechanisms underlying tolerance to the benzodiazepine (BZD) drug class 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 uses the classical BZD, diazepam (DZP), for in‐vitro and in‐vivo experiments combined with novel live‐imaging and biochemical methods 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 associated with tolerance. Microscopy studies of endogenous GABA A Rs in rat cortical neurons indicate 24 hr exposure of 1μM DZP causes a reduction in extrasynaptic γ2 levels and modification of the inhibitory synaptic scaffolding protein gephyrin. Immunoprecipitation experiments uncover enhanced ubiquitination of the γ2 subunit and reduced total levels in‐vitro following DZP. Biochemical studies of mice injected with 10 mg/kg DZP vs vehicle also show a significant decrease in γ2 subunit and total gephyrin cortical levels 12 hr post DZP injection. Live‐imaging studies using a novel GABA A R γ2 subunit encoding a N terminal fluorogen‐activating peptide (FAP) and pH‐sensitive green fluorescent protein (γ2 pH FAP) identified elevated lysosomal targeting of γ2 following DZP. Furthermore, RFP‐gephyrin and γ2‐GABA A Rs exhibit enhanced synaptic diffusion rates after 24 h DZP as measured by fluorescence recovery after photobleaching experiments. Overall, our work suggests DZP treatment decreases γ2 GABA A R inhibitory function by reducing synaptic dwell time and surface levels after one day of exposure, which may contribute to the formation of DZP 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. The FAP, MG dyes, and critical guidance was provided by Ming Zhang, Brigitte Schmidt, and the Waggoner Lab at Carnegie Melon University. S. Das performed animal injections and tissue collection. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .