Alterations in Purkinje cell GABA_A receptor pharmacology following oxygen and glucose deprivation and cerebral ischemia reveal novel contribution of β₁‐subunit‐containing receptors

Abstract

Cerebellar Purkinje cells (PCs) are particularly sensitive to cerebral ischemia, and decreased GABAA receptor function following injury is thought to contribute to PC sensitivity to ischemia-induced excitotoxicity. Here we examined the functional properties of the GABAA receptors that are spared following ischemia in cultured Purkinje cells from rat and in vivo ischemia in mouse. Using subunit-specific positive modulators of GABAA receptors, we observed that oxygen and glucose deprivation (OGD) and cardiac arrest-induced cerebral ischemia cause a decrease in sensitivity to the β2/3-subunit-preferring compound, etomidate. However, sensitivity to propofol, a β-subunit-acting compound that modulates β1–3-subunits, was not affected by OGD. The α/γ-subunit-act-ing compounds, diazepam and zolpidem, were also unaffected by OGD. We performed single-cell reverse transcription–polymerase chain reaction on isolated PCs from acutely dissociated cerebellar tissue and observed that PCs expressed the β1-subunit, contrary to previous reports examining GABAA receptor subunit expression in PCs. GABAA receptor β1-subunit protein was also detected in cultured PCs by western blot and by immunohistochemistry in the adult mouse cerebellum and levels remained unaffected by ischemia. High concentrations of loreclezole (30 µm) inhibited PC GABA-mediated currents, as previously demonstrated with β1-subunit-containing GABAA receptors expressed in heterologous systems. From our data we conclude that PCs express the β1-subunit and that there is a greater contribution of β1-subunit-containing GABAA receptors following OGD.

Neurosteroids as Selective Inhibitors of Glycine Receptor Activity: Structure-Activity Relationship Study on Endogenous Androstanes and Androstenes

“…The experiments with I Gly were conducted on rat hippocampal neurons, and experiments with I GABA were conducted on rat cerebellar Purkinje cells. The I Gly is larger in amplitude and more stable on hippocampal cells, and, conversely, the I GABA is more convenient to study on Purkinje cells, since GABA receptors on Purkinje cells are more homogeneous (Kelley et al, 2013). Glycine (100 µM) and GABA (5 µM) were applied to the neurons through an application pipette during 600-1000 ms and compounds 1-9 were added to the same pipette in different concentrations (0.01-100 µM).…”

Baclofen mediates neuroprotection on hippocampal CA1 pyramidal cells through the regulation of autophagy under chronic cerebral hypoperfusion

“…There are three main types of GABA receptors, the ionotropic GABA A receptor and GABA C receptor, and the metabotropic GABA B receptor. GABA A and GABA B receptors have been shown to play a neuroprotective role in experimental models of middle cerebral artery occlusion (MCAO) 30 , transient brain ischemia 31 32 33 34 35 36 and oxygen-glucose deprivation (OGD) 31 32 37 38 39 40 . Besides, GABA B receptors can regulate the GABA A receptors function in bullfrog DRG neurons 41 and dentate gyrus granule cells 42 .…”

Examining the GABA_A receptor alpha-subunits in the neonatal pig brain: changes across development and the effect of seizures after hypoxic-ischaemic brain injury

“…Several antiepileptics used to treat neonatal seizures target the GABAAR and include the barbiturate phenobarbital and the benzodiazepines midazolam, diazepam and clonazepam (Vento et al 2009). In the developing brain, normal changes in GABAAR subunit expression coupled with pathological disturbances that alter receptor composition may influence receptor pharmacology potentially altering antiepileptic drug efficacy (Kelley et al 2013, Loddenkemper et al 2014). …”

“…In addition, the authors concluded that dephosphorylation of the GABAAR under ischemic conditions may regulate surface localisation and that internalisation may contribute to neuronal death (Mele et al 2014). In several studies by Kelley et al, they suggested that the ischaemia-induced reduction in α1 protein expression was attributable to protein degradation (Kelley et al 2008, Kelley et al 2013). In our HI-Sz animals at 24 h, the α1 protein was noticeably absent from cell processes.…”

“…The α3 protein predominates in immature brain whilst α1 is most abundant in the mature brain, a transition that is postulated to occur around birth but may continue for several months in the human , Herlenius and Lagercrantz 2001. Receptor composition influences pharmacological properties including channel kinetics and ligand binding efficacy with alterations to receptor makeup resulting in changes to receptor function (Kelley et al 2013). GABA is the first In adult patients with focal epilepsies reductions in GABAAR α1 and α3 protein expression have been observed (Loup et al 2000, Loup et al 2006.…”