HIPPOCAMPAL BENZODIAZEPINE TOLERANCE

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Benzodiazepines (BZs) which act through the GABA/A receptor (GABAR), are potent anti-convulsant for a variety of epilepsies. Their clinical usefulness is limited by the appearance of functional tolerance, commonly-held to be mediated by changes at the post-synaptic on CA1 pyramidal cells in the in vitro hippocampus. In addition to changes in post-synaptic GABAR structure (alpha1 and beta3 subunit mRNA and protein down-regulation), measured in situ, and function (decreased mIPSC amplitude and C1-channel conductance), changes in GABAergic interneuron activity are proposed to contribute to BZ tolerance. Rapid BZ antagonist-induced restoration of GABAR subunit protein levels and mIPSC amplitude suggested that both translational and post-translational mechanisms may interdependently contribute to BZ tolerance. Preliminary studies of PKA-mediated modulation of GABAR currents and the ability of a cAMP analogue to partially restore mIPSC amplitude in BZ-treated rats suggests that a modulation of PKA-mediated events, in part, contribute to GABAR system dysfunction. Additional studies have identified changes in excitatory amino acid part, contribute to GABAR system dysfunction. Additional studies have identified changes in excitatory amino acid receptor (EAAR) subunit mRNAs and protein suggesting that excitatory systems are also regulated by chronic BZ treatment in response to reduced GABA inhibition. Three central hypotheses were generated from these findings in in vitro and in situ hippocampus which will be addressed by 3 SPECIFIC AIMS: SPECIFIC AIM 1: to examine the role of intrinsic and extrinsic interneuron function in reducing GABA tone by sampling subpopulations of visualized CA1 interneurons using intracellular and whole-cell patch techniques. Specific Aim 2: is to explore the role of both 2A) translational, i.e., GABAR alpha1 subunit protein redistribution following chronic agonist and acute antagonist administration, using fluorescent co-localization and quantitative EM immunogold techniques and 2B) post-translational mechanisms, i.e., the effect of exogenous and endogenous stimulators of PKA-mediated protein phosphorylation to modulate mIPSC amplitude in CA1 pyramidal cells. SPECIFIC AIM 3: is to examine the compensatory changes which occur in excitatory amino acid receptor (EAAR) structure and function in CA1 pyramidal cells: 3A) Structural measures include: quantitative immunohistochemical techniques and Western blot analysis of microdissected hippocampus and changes in EAAR receptor binding using autoradiographic techniques. 3B) Whole-cell slice patch techniques will e used to measure EAAR receptor-mediated, evoked and miniature EPSC amplitude and decay kinetics. EAAR function will also be assessed using microfluorometric measurements of Ca2+ uptake into acutely dissociated CA1 pyramidal cells. Understanding the nature of the changes at inhibitory and excitatory synapses may allow the design of drugs and approaches to circumvent tolerance and allow us to gain a better understanding of basic mechanisms involved in the dysfunction of these receptor systems during anticonvulsant drug treatment of epileptic patients.