Elsevier

Experimental Neurology

Volume 277, March 2016, Pages 178-189
Experimental Neurology

Enduring changes in tonic GABAA receptor signaling in dentate granule cells after controlled cortical impact brain injury in mice

https://doi.org/10.1016/j.expneurol.2016.01.005Get rights and content

Highlights

  • GABAAR currents in DGCs contralateral to CCI are unchanged after brain injury.

  • Resting tonic GABAAR currents are unchanged in DGCs ipsilateral to CCI injury.

  • THIP and THDOC-evoked tonic GABAAR currents are reduced in injured DGCs.

  • L655,708-evoked tonic GABAAR currents are unchanged in injured DGCs.

  • Gene expression of GABAAR α4, α5 and δ subunits is unchanged in injured DG.

Abstract

Changes in functional GABAAR signaling in hippocampus have previously been evaluated using pre-clinical animal models of either diffuse brain injury or extreme focal brain injury that precludes measurement of cells located ipsilateral to injury. As a result, there is little information about the status of functional GABAAR signaling in dentate granule cells (DGCs) located ipsilateral to focal brain injury, where significant cellular changes have been documented. We used whole-cell patch-clamp recordings from hippocampal slices to measure changes in GABAARs in dentate granule cells (DGCs) at 1–2, 3–5, and 8–13 weeks after controlled cortical impact (CCI) brain injury. Synaptic and tonic GABAAR currents (ITonicGABA) were measured in DGCs at baseline conditions and during application of the GABAAR agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol hydrochloride (THIP) to assess in the function of δ subunit-containing GABAARs. DGCs ipsilateral to CCI exhibited no changes in the amplitude of resting ITonicGABA relative to DGCs after sham-injury or contralateral to CCI. In contrast, there was a significant reduction in the THIP-evoked ITonicGABA in DGCs ipsilateral to CCI at both time-points. Tonic GABAergic inhibition of DGCs ipsilateral to injury also exhibited reduced responsiveness to the neurosteroid THDOC. ITonicGABA in DGCs ipsilateral to CCI did not exhibit a change in sensitivity to L655,708, an inverse agonist with selectivity for α5 subunit-containing GABAARs, suggesting a lack of functional change in GABAARs containing this subunit. At the 8–13 week time-point, gene expression of GABAAR subunits expected to contribute to ITonicGABA (i.e., α4, α5 and δ) was not significantly altered by CCI injury in isolated dentate gyrus. Collectively, these results demonstrate enduring functional changes in ITonicGABA in DGCs ipsilateral to focal brain injury that occur independent of altered gene expression.

Introduction

Type A GABA receptors (GABAARs) are prominent inhibitory signaling components of the central nervous system and are formed as heteropentamers of α1–6, β1–4, γ1–3, δ, ε, θ and π subunits (summarized in Barnard et al., 1998, Kapur and Macdonald, 1999, Laurie et al., 1992, Macdonald and Olsen, 1994, Whiting et al., 1999). The subunit composition of these pentamers varies by cell type and brain location and confers receptor kinetics and pharmacological response profiles (summarized in Davies et al., 1997, Draguhn et al., 1990, Farrant and Nusser, 2005, Glykys and Mody, 2007, Lüddens and Wisden, 1991, Verdoorn et al., 1990). In adult rodents, dentate granule cells (DGCs) express functional subunit compositions that are either predominantly α1βxγ2 for synaptic signaling or predominantly α4βxδ for tonic current signaling, although other subunit combinations can occur (summarized in Brown et al., 2002, Chang et al., 1996, Farrant and Nusser, 2005, Glykys et al., 2008, Mody, 2001, Mtchedlishvili and Kapur, 2006, Semyanov et al., 2004, Stell et al., 2003, Wei et al., 2003).

Pathology-induced changes in the activity or pharmacological responsiveness of GABAARs may contribute to cellular and network hyperexcitability and disrupted information encoding that accompanies cognitive impairment, behavioral and motor dysfunction, and epileptogenesis after brain injury. Further, changes in functional GABAAR signaling have been reported to reflect rearrangements in GABAAR subunit composition (Brooks-Kayal et al., 1998, Cossart et al., 2001, Gibbs et al., 1997, Leroy et al., 2004). Aberrant GABA signaling and pharmacological responsiveness of GABAARs have been reported after a variety of brain insults in animal models, including cerebral ischemia (Kharlamov et al., 2008, Redecker et al., 2002), fluid percussion injury (Drexel et al., 2015, Gupta et al., 2012, Pavlov et al., 2011, Raible et al., 2012), kindling-induced status epilepticus (Sun et al., 2007) and chemoconvulsant-induced status epilepticus (Peng et al., 2004, Zhan and Nadler, 2009, Zhang et al., 2007, Zhang et al., 2012). Acute seizures alone may also drive GABAAR subunit reorganization (Kapur and Macdonald, 1997).

Traumatic brain injury (TBI) leads to reorganization of brain networks, which can include selective cell loss, aberrant axonal arrangements, and synaptic reorganization (Hunt et al., 2009, Hunt et al., 2010, Hunt et al., 2011, Lowenstein et al., 1992, Santhakumar et al., 2000, Santhakumar et al., 2001, Scheff et al., 2005, Swartz et al., 2006). Controlled cortical impact (CCI) is a model of focal TBI used to investigate mechanisms of brain repair (Dixon et al., 1991, Hall et al., 2005, Lighthall, 1988, Scheff et al., 1997) and posttraumatic epilepsy (Hunt et al., 2009, Hunt et al., 2010, Hunt et al., 2011, Hunt et al., 2012, Hunt et al., 2013). At present, functional changes in GABAAR subunits after focal brain injury have been characterized after extreme CCI that results in a loss of ipsilateral hippocampus, rendering it impractical to perform electrophysiological recordings or molecular analysis near the injury epicenter (Kharlamov et al., 2011, Mtchedlishvili et al., 2010), where many cellular changes in DG circuitry occur (Hunt et al., 2009, Hunt et al., 2010, Hunt et al., 2011, Hunt et al., 2012). As a result, understanding GABAAR subunit composition and signaling in the context of posttraumatic epileptogenesis and brain reorganization, requires assessment of changes in the injured hemisphere.

Here, functional changes in GABAAR populations in DGCs after focal brain trauma were assessed using whole-cell patch-clamp recordings of DGCs 1–2, 3–5, or 8–12 weeks after CCI injury. Effects of pharmacological agents having relative selectivity for GABAARs containing α5 and δ subunits were assessed, since these subunits contribute predominantly to tonic GABAAR-mediated currents in DGCs (summarized in Brown et al., 2002, Chang et al., 1996, Farrant and Nusser, 2005, Glykys et al., 2008, Mody, 2001, Mtchedlishvili and Kapur, 2006, Semyanov et al., 2004, Stell et al., 2003, Wei et al., 2003). Brain injury-induced changes in gene expression of GABAAR subunits in the dentate gyrus were also examined using quantitative real-time polymerase chain reaction (qRT-PCR). We tested the hypotheses that functional GABAAR signaling and GABAAR subunit composition are altered in DGCs located ipsilateral to CCI and maintained for months after injury.

Section snippets

Animals

Six-to-eight-week-old adult male CD-1 mice were housed under a normal 14/10 h light/dark cycle and given food and water ad libitum. All animals were acclimated to the University of Kentucky vivarium for at least one week prior to experimentation. All procedures were approved by the University of Kentucky Animal Care and Use Committee and adhered to NIH guidelines for the care and use of laboratory animals.

Brain injury

Mice were administered unilateral, focal contusion injury using controlled cortical impact

Results

Synaptic and tonic GABAAR-mediated currents were assessed at 1–2, 3–5, and 8–13 weeks following CCI or sham injury. Comparisons were made between DGCs from sham animals (i.e., sham), DGCs contralateral to CCI injury (i.e., contralateral DGCs) and DGCs ipsilateral to CCI injury (i.e., ipsilateral DGCs).

Discussion

Although altered expression and distribution of GABAAR subunits has been reported in animal models of epilepsy, little is known about GABAAR function in DGCs ipsilateral to CCI brain injury, which also results in post-traumatic epileptogenesis (Brooks-Kayal et al., 1998, Cossart et al., 2001, Gibbs et al., 1997, Hunt et al., 2009, Leroy et al., 2004). Similar to results after focal brain injury reported here, ITonicGABA in DGCs from mice that survive pilocarpine-induced status epilepticus

Conclusions

Results here demonstrate that long-lasting functional reductions in ITonicGABA responsiveness to THIP and THDOC occur in DGCs located ipsilateral to focal brain injury. These changes in pharmacological responsiveness may represent functional changes in GABAAR subunit composition in DGCs and interneurons after focal brain injury, but do not appear to involve changes in gene transcription for the α4, α5, or δ subunits. Many of the changes in GABAAR signaling ipsilateral to CCI share similarity

Role of the funding source

Funding sources had no involvement in study design, collection, analysis, and interpretation of data, writing of the reporter in the decision to submit the article for publication.

Author contributions

JAB, CRB, KCH and BNS contributed to the conception and design of the work; acquisition and analysis were performed by JAB, CRB and KCH; interpretation of the data was performed by JAB, CRB, KCH and BNS. JAB, CRB and KCH and BNS drafted and revised manuscript and approve of the version to be published.

Acknowledgments

Supported by Department of Defense (USAMRMC) Grant W81XWH-11-1-0502 (BNS) and a Fellowship from the American Epilepsy Society and Lennox and Lombroso Trust for Epilepsy Research and Training (JAB).

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