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Subunit composition of glutamate and gamma-aminobutyric acid receptors in status epilepticus.

ABSTRACT: PURPOSE:To describe the subunit composition of glutamate and gamma-aminobutyric acid (GABA) receptors in brain tissue from patients with different types of status epilepticus. PATIENTS AND METHODS:The subunit composition of glutamate and GABA receptors was analyzed in: (1) surgical brain samples from three patients with refractory convulsive status epilepticus, three patients with electrical status epilepticus in sleep, and six patients with refractory epilepsy, and (2) brain autopsy samples from four controls who died without neurological disorders. Subunit expression was quantified with Western blotting and messenger ribonucleic acid (mRNA) expression was quantified with reverse polymerase chain reaction. RESULTS:Western blot analysis demonstrated the following patterns (as compared to controls): (1) alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors: elevated GluA1/GluA2 ratio in electrical status epilepticus in sleep (465%±119) and refractory epilepsy (329%±125; p<0.01); (2) N-methyl-d-aspartate (NMDA) receptors: increased GluN2B/GluN2A ratio in electrical status epilepticus in sleep (3682%±1000) and refractory convulsive status epilepticus (3520%±751; p<0.05); (3) GABA receptors: elevated ?2/?1 ratio in refractory epilepsy (321%±138; p<0.05) and refractory convulsive status epilepticus (346%±74; p<0.05); and (4) patients with underlying malformation of cortical development had increased ratios in GluA1/GluA2 (382%±149; p<0.01), GluN2B/GluN2A (3321%±1581; p<0.05) and ?2/?1 (303%±86; p<0.01). Quantification of mRNA demonstrated an elevated GABRA2/GABRA1 ratio in refractory epilepsy (712; p<0.05) as compared to controls. CONCLUSIONS:The subunit composition of glutamate and GABA receptors in patients with status epilepticus mirrors that found in animal models of refractory status epilepticus and may promote self-sustaining seizures. Receptor subunit changes may provide additional targets for improved treatment.

SUBMITTER: Loddenkemper T

PROVIDER: S-EPMC6294571 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Subunit composition of glutamate and gamma-aminobutyric acid receptors in status epilepticus.

Loddenkemper Tobias T Talos Delia M DM Cleary Ryan T RT Joseph Annelise A Sánchez Fernández Iván I Alexopoulos Andreas A Kotagal Prakash P Najm Imad I Jensen Frances E FE

Epilepsy research 20140202 4

<h4>Purpose</h4>To describe the subunit composition of glutamate and gamma-aminobutyric acid (GABA) receptors in brain tissue from patients with different types of status epilepticus.<h4>Patients and methods</h4>The subunit composition of glutamate and GABA receptors was analyzed in: (1) surgical brain samples from three patients with refractory convulsive status epilepticus, three patients with electrical status epilepticus in sleep, and six patients with refractory epilepsy, and (2) brain auto ...[more]

PMID: 24613745

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Project description:OBJECTIVE:Sex differences are evident in the antiseizure activity of neurosteroids; however, the potential mechanisms remain unclear. In this study, we sought to determine whether differences in target extrasynaptic δ-subunit γ-aminobutyric acid type A (GABA-A) receptor expression and function underlie the sex differences in seizure susceptibility and the antiseizure activity of neurosteroids. METHODS:Sex differences in seizure susceptibility and protective activity of three distinct neurosteroids-allopregnanolone (AP), androstanediol (AD), and ganaxolone-were evaluated in the pilocarpine model of status epilepticus (SE) and kindling seizure test in mice. Immunocytochemistry was used for δGABA-A receptor expression analysis, and patch-clamp recordings in brain slices evaluated its functional currents. RESULTS:Sex differences were apparent in kindling epileptogenic seizures, with males exhibiting a faster progression to a fully kindled state. Neurosteroids AP, AD, or ganaxolone produced dose-dependent protection against SE and acute partial seizures. However, female mice exhibited strikingly enhanced sensitivity to the antiseizure activity of neurosteroids compared to males. Sex differences in neurosteroid protection were unrelated to pharmacokinetic factors, as plasma levels of neurosteroids associated with seizure protection were similar between sexes. Mice lacking extrasynaptic δGABA-A receptors did not exhibit sex differences in neurosteroid protection. Consistent with a greater abundance of extrasynaptic δGABA-A receptors, AP produced a significantly greater potentiation of tonic currents in dentate gyrus granule cells in females than males; however, such enhanced AP sensitivity was diminished in δGABA-A receptor knockout female mice. SIGNIFICANCE:Neurosteroids exhibit greater antiseizure potency in females than males, likely due to a greater abundance of extrasynaptic δGABA-A receptors that mediate neurosteroid-sensitive tonic currents and seizure protection. These findings indicate the potential to develop personalized gender-specific neurosteroid treatments for SE and epilepsy in men and women, including catamenial epilepsy.

Project description:γ-aminobutyric acid type A receptors (GABAARs), the major inhibitory neurotransmitter receptors in the mammalian central nervous system, are arguably the most challenging member of the pentameric Cys-loop receptors to study due to their heteromeric structure. When two or more subunits are expressed together in heterologous systems, receptors of variable subunit type, ratio, and orientation can form, precluding accurate interpretation of data from functional studies. Subunit concatenation is a technique that involves the linking of individual subunits and in theory allows the precise control of the uniformity of expressed receptors. In reality, the resulting concatemers from widely used constructs are flexible in their orientation and may therefore assemble with themselves or free GABAAR subunits in unexpected ways. In this study, we examine functional responses of receptors from existing concatenated constructs and describe refinements necessary to allow expression of uniform receptor populations. We find that dimers from two commonly used concatenated constructs, β-23-α and α-10-β, assemble readily in both the clockwise and the counterclockwise orientations when coexpressed with free subunits. Furthermore, we show that concatemers formed from new tetrameric α-10-β-α-β and α-10-β-α-γ constructs also assemble in both orientations with free subunits to give canonical αβγ receptors. To restrict linker flexibility, we systematically shorten linker lengths of dimeric and pentameric constructs and find optimized constructs that direct the assembly of GABAARs only in one orientation, thus eliminating the ambiguity associated with previously described concatemers. Based on our data, we revisit some noncanonical GABAAR configurations proposed in recent years and explain how the use of some concatenated constructs may have led to wrong conclusions. Our results help clarify current contradictions in the literature regarding GABAAR subunit stoichiometry and arrangement. The lessons learned from this study may guide future efforts in understanding other related heteromeric receptors.

Project description:ObjectiveHuman hypothalamic hamartomas (HHs) are highly associated with treatment-resistant gelastic seizures. HHs are intrinsically epileptogenic, although the basic cellular mechanisms responsible for seizure activity are unknown. Altered gamma-aminobutyric acid (GABA) function can contribute to epileptogenesis in humans and animal models. Recently, functional GABA(A) receptor (GABA(A) R) rundown has been described in surgically resected human temporal lobe epilepsy tissue. We asked whether functional GABA(A) R rundown also occurs in human HH neurons.MethodsGABA(A) R-mediated currents were measured using perforated patch-clamp recordings in single neurons acutely dissociated from surgically resected HH tissue. In addition, functional GABA(A) Rs were expressed in Xenopus oocytes after microinjection with membrane fractions from either HH or control hypothalamus, and were studied with 2-electrode voltage-clamp recordings.ResultsPerforated patch-clamp recordings in dissociated HH neurons showed that repetitive exposure to GABA (5 consecutive exposures to 0.1 mM GABA with 1-second duration and at 20-second intervals) induced a time-dependent rundown of whole-cell currents in small HH neurons, whereas large HH neurons showed much less rundown using the same protocol. Functional rundown was not observed in HH neurons with repetitive exposure to glycine or glutamate. Two-electrode voltage-clamp recordings (6 consecutive exposures to 1 mM GABA with 10-second duration and at 40-second intervals) induced GABA current rundown in Xenopus oocytes microinjected with HH membrane proteins, but not in the oocytes expressing hypothalamic membrane proteins derived from human autopsy controls. Functional rundown of GABA currents was significantly attenuated by intracellular application of adenosine triphosphate or the nonspecific phosphatase inhibitor, okadaic acid.InterpretationNeurons from surgically resected human HH demonstrate functional rundown of GABA(A) R-mediated transmembrane currents in response to GABA agonist exposure. Rundown may be a marker for impaired GABAergic function and a contributing mechanism for seizure genesis within HH tissue.

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Subunit composition of glutamate and gamma-aminobutyric acid receptors in status epilepticus.

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Subunit composition of glutamate and gamma-aminobutyric acid receptors in status epilepticus.

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