Traumatic brain injury and the effects of diazepam , diltiazem , and MK-801 on GABAA receptor subunit expression in rat hippocampus

Background: Excitatory amino acid release and subsequent biochemical cascades following traumatic brain injury (TBI) have been well documented, especially glutamate-related excitotoxicity. The effects of TBI on the essential functions of inhibitory GABA-A receptors, however, are poorly understood. Methods: We used Western blot procedures to test whether in vivo TBI in rat altered the protein expression of hippocampal GABA-A receptor subunits α1, α2, α3, α5, β3, and γ2 at 3 h, 6 h, 24 h, and 7 days post-injuy. We then used pre-injury injections of MK-801 to block calcium influx through the NMDA receptor, diltiazem to block L-type voltagegated calcium influx, or diazepam to enhance chloride conductance, and re-examined the protein expressions of α1, α2, α3, and γ2, all of which were altered by TBI in the first study and all of which are important constituents in benzodiazepine-sensitive GABA-A receptors. Results: Western blot analysis revealed no injury-induced alterations in protein expression for GABA-A receptor α2 or α5 subunits at any time point post-injury. Significant time-dependent changes in α1, α3, β3, and γ2 protein expression. The pattern of alterations to GABA-A subunits was nearly identical after diltiazem and diazepam treatment, and MK-801 normalized expression of all subunits 24 hours post-TBI. Conclusions: These studies are the first to demonstrate that GABA-A receptor subunit expression is altered by TBI in vivo, and these alterations may be driven by calcium-mediated cascades in hippocampal neurons. Changes in GABA-A receptors in the hippocampus after TBI may have far-reaching consequences considering their essential importance in maintaining inhibitory balance and their extensive impact on neuronal function. Background Traumatic brain injury (TBI) disrupts neuronal ionic balance and is known to produce glutamate-mediated neurotoxicity [1-3]. Glutamate related activation of Nmethyl-D-aspartate (NMDA) receptors and the resulting elevations in intracellular calcium concentration ([Ca]i) are important components in synaptic and cellular degeneration and dysfunction after both in vivo [1,4,5] and in vitro neuronal injury [6-8]. Disruption of calcium (Ca2+) homeostasis after TBI has been implicated in a wide range of intracellular changes in gene expression, signaling pathways, enzymatic activation and even cellular death [see [9] for review]. Voltage gated calcium channels (VGCCs) also contribute to the increases in [Ca]i identified in glutamate related neurotoxicity due to TBI [10]. Although glutamate-related neurotoxic mechanisms after TBI have been studied extensively, relatively little is understood about inhibitory changes and the role of GABA receptors. Normal neuronal function relies on the constant orchestration and integration of excitatory and inhibitory potentials. GABA-A receptors (GABAAR) mediate the majority of inhibitory neurotransmission in the central nervous system by ligand gating of fast-acting chloride (Cl-) channels [11]. The impact of TBI on GABAAR is poorly understood even though changes in the composition and function of these receptors may have extensive consequences after injury. * Correspondence: cgibson2@washcoll.edu 1 Department of Psychology, Washington College, Chestertown, MD, 21620,