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REGULATION OF GABAAR SIGNALING AND NEUROADAPTATIONS IN RESPONSE TO DIAZEPAM

Lorenz-Guertin, Joshua (2019) REGULATION OF GABAAR SIGNALING AND NEUROADAPTATIONS IN RESPONSE TO DIAZEPAM. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Despite 50+ years of use as anxiolytics, anti-convulsants, and sedative/hypnotic agents, the mechanisms underlying benzodiazepine (BZD) tolerance are poorly understood. BZDs potentiate the actions of GABA, the primary inhibitory neurotransmitter in the adult brain, through positive allosteric modulation of γ2 subunit containing GABA type A receptors (GABAARs). Sustained treatment with BZD drugs is intimately associated with the development of tolerance, dependence, withdrawal and addiction. BZD efficacy diminishes after prolonged or high dose acute exposure, with tolerance to the sedative/hypnotic effects forming most quickly. We investigated the adaptive mechanisms occurring during initial exposure to the classical BZD, Diazepam (DZP), and the molecular signature of the mouse brain during established sedative tolerance. We found cultured neurons treated 24 h with DZP presented no change in surface or synaptic levels of γ2-GABAARs. In contrast, both γ2 and the key inhibitory synaptic scaffolding protein gephyrin levels were decreased after a single DZP treatment in vitro and in vivo. Live-imaging and label-free quantitative proteomics further revealed alterations in γ2 subunit surface trafficking, internalization and lysosomal targeting. In comparison, mice treated seven days with DZP had altered GABAAR subunit composition, reduced responsiveness to DZP, and tonic inhibition was diminished. Furthermore, DZP increased excitatory NMDA receptor subunit levels and function. State of the art mass spectrometry experiments revealed increased CaMKII subunits, which are positive regulators of NMDA receptors and involved in tolerance to other drugs. Downstream bioinformatics analysis confirmed robust synaptic plasticity after DZP. Together, we describe a time-dependent downregulation of synaptic GABAAR function after initial DZP exposure followed by an adaptive increase in excitatory neurotransmission, neuronal remodeling and altered synaptic GABAAR composition.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Lorenz-Guertin, Joshuajol73@pitt.edujol730000-0001-9686-947X
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorJacob, Tijatcj11@pitt.edu
Committee ChairPalladino, Michaelmjp44@pitt.edu
Committee MemberWatkins, Simonsimon.watkins@pitt.edu
Committee MemberHomanics, Gregghomanicsge@anes.upmc.edu
Committee MemberSorkin, Alexandersorkin@pitt.edu
Date: 3 December 2019
Date Type: Publication
Defense Date: 26 July 2019
Approval Date: 3 December 2019
Submission Date: 5 September 2019
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 194
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Pharmacology and Chemical Biology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Benzodiazepine Diazepam GABA Type A Receptor Gephyrin Inhibition Trafficking
Date Deposited: 03 Dec 2019 16:55
Last Modified: 03 Dec 2019 16:55
URI: http://d-scholarship.pitt.edu/id/eprint/37627

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