Human GABA Research Articles

(R)-(3-Amino-2-fluoropropyl) Phosphinic Acid (AZD3355), a Novel GABAB Receptor Agonist, Inhibits Transient Lower Esophageal Sphincter Relaxation through a Peripheral Mode of Action

Gastroesophageal reflux disease (GERD) affects >10% of the Western population. Conventionally, GERD is treated by reducing gastric acid secretion, which is effective in most patients but inadequate in a significant minority. We describe a new therapeutic approach for GERD, based on inhibition of transient lower esophageal sphincter relaxation (TLESR) with a proposed peripherally acting GABA(B) receptor agonist, (R)-(3-amino-2-fluoropropyl)phosphinic acid (AZD3355). AZD3355 potently stimulated recombinant human GABA(B) receptors and inhibited TLESR in dogs, with a biphasic dose-response curve. In mice, AZD3355 produced considerably less central side effects than the prototypical GABA(B) receptor agonist baclofen but evoked hypothermia at very high doses (blocked by a GABA(B) receptor antagonist and absent in GABA(B)-/- mice). AZD3355 and baclofen differed markedly in their distribution in rat brain; AZD3355, but not baclofen, was concentrated in circumventricular organs as a result of active uptake (shown by avid intracellular sequestration) and related to binding of AZD3355 to native GABA transporters in rat cerebrocortical membranes. AZD3355 was also shown to be transported by all four recombinant human GABA transporters. AR-H061719 [(R/S)-(3-amino-2-fluoropropyl)phosphinic acid], (the racemate of AZD3355) inhibited the response of ferret mechanoreceptors to gastric distension, further supporting its peripheral site of action on TLESR. In summary, AZD3355 probably inhibits TLESR through stimulation of peripheral GABA(B) receptors and may offer a potential new approach to treatment of GERD.

The Four Human γ-Aminobutyric Acid (GABA) Transporters: Pharmacological Characterization and Validation of a Highly Efficient Screening Assay

The neurotransmission mediated by gamma-aminobutyric acid (GABA) in the mammalian brain is terminated by a family of four GABA transporters (GATs). Inhibition of GATs is currently used in the treatment of epilepsy and these proteins are generally considered as important drug targets. In this study, we perform the first elaborate pharmacological characterization of all four human GAT subtypes. We conduct the experiments in parallel in a [3H]GABA uptake assay using 14 standard GAT substrates and inhibitors. This setup enables direct comparison of the absolute values of inhibitory activities of the compounds between the different GAT subtypes. The results are overall in agreement with data reported by other groups for the orthologous murine GATs. However, there do seem to be some minor variations among species. In contrast to the several subtype selective ligands identified for the GAT-1 subtype, no subtype selective ligands have been reported for the three remaining GATs. Given the potential therapeutic relevance of the individual GAT subtypes, a search for novel structures displaying selectivities for specific GAT subtypes is important. In this study, we validate our [3H]GABA uptake assay for use in high throughput screening. We find that the assay is categorized by high Z'-factors (Z' > 0.5) for all four GAT subtypes, demonstrating that the assay is excellent for a high throughput screen. This [3H]GABA uptake assay therefore enables future high throughput screening of compound libraries at the four human GATs.

Homology modeling of human α1β2γ2 and house fly β3 GABA receptor channels and Surflex-docking of fipronil

To further explore the mechanism of selective binding of the representative gamma-aminobutyric acid receptors (GABARs) noncompetitive antagonist (NCA) fipronil to insect over mammalian GABARs, three-dimensional models of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABAR were generated by homology modeling, using the cryo-electron microscopy structure of the nicotinic acetylcholine receptor (nAChR) of Torpedo marmorata as a template. Fipronil was docked into the putative binding site of the human alpha 1 beta 2 gamma 2 and house fly beta 3 receptors by Surflex-docking, and the calculated docking energies are in agreement with experimental results. The GABA receptor antagonist fipronil exhibited higher potency with house fly beta 3 GABAR than with human alpha 1 beta 2 gamma 2 GABAR. Furthermore, analyses of Surflex-docking suggest that the H-bond interaction of fipronil with Ala2 and Thr6 in the second transmembrane segment (TM2) of these GABARs plays a relatively important role in ligand selective binding. The different subunit assemblies of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABARs may result in differential selectivity for fipronil.

Carisoprodol-Mediated Modulation of GABAA Receptors: In Vitro and in Vivo Studies

Carisoprodol is a frequently prescribed muscle relaxant. In recent years, this drug has been increasingly abused. The effects of carisoprodol have been attributed to its metabolite, meprobamate, a controlled substance that produces sedation via GABA(A) receptors (GABA(A)Rs). Given the structural similarities between carisoprodol and meprobamate, we used electrophysiological and behavioral approaches to investigate whether carisoprodol directly affects GABA(A)R function. In whole-cell patch-clamp studies, carisoprodol allosterically modulated and directly activated human alpha1beta2gamma2 GABA(A)R function in a barbiturate-like manner. At millimolar concentrations, inhibitory effects were apparent. Similar allosteric effects were not observed for homomeric rho1 GABA or glycine alpha1 receptors. In the absence of GABA, carisoprodol produced picrotoxin-sensitive, inward currents that were significantly larger than those produced by meprobamate, suggesting carisoprodol may directly produce GABAergic effects in vivo. When administered to mice via intraperitoneal or oral routes, carisoprodol elicited locomotor depression within 8 to 12 min after injection. Intraperitoneal administration of meprobamate depressed locomotor activity in the same time frame. In drug discrimination studies with carisoprodol-trained rats, the GABAergic ligands pentobarbital, chlordiazepoxide, and meprobamate each substituted for carisoprodol in a dose-dependent manner. In accordance with findings in vitro, the discriminative stimulus effects of carisoprodol were antagonized by a barbiturate antagonist, bemegride, but not by the benzodiazepine site antagonist, flumazenil. The results of our studies in vivo and in vitro collectively suggest the barbiturate-like effects of carisoprodol may not be due solely to its metabolite, meprobamate. Furthermore, the functional traits we have identified probably contribute to the abuse potential of carisoprodol.

Linkage analysis between idiopathic generalized epilepsies and the GABAA receptor α5, β3 and γ3 subunit gene cluster on chromosome 15

We tested the hypothesis that genetic variants within the GABA(A) alpha5, beta3 and gamma3 subunit gene cluster on chromosome 15q11-q13 confer genetic susceptibility to common subtypes of idiopathic generalized epilepsy (IGE).Ninety-four families were selected from IGE patients with either juvenile myoclonic epilepsy (JME), juvenile (JAE) or childhood absence epilepsy (CAE). Cosegregation was tested between dinucleotide polymorphisms associated with the human GABA(A) alpha5, beta3 and gamma3 subunit gene cluster and three different IGE trait models.Evidence against linkage to the GABA(A) alpha5, beta3 and gamma3 subunit gene cluster was found in the entire family set and subsets selected from either CAE or JAE. In 61 families of JME patients, a maximum lod score (Zmax=1.40 at Theta(max)=0.00) was obtained for a broad IGE spectrum ("idiopathic" generalized seizure or generalized spike and wave discharges in the electroencephalogram) assuming genetic heterogeneity (alpha=0.37; P=0.06) and an autosomal recessive mode of inheritance.The possible hint of linkage in families of JME patients emphasizes the need for further studies to determine whether a recessively inherited gene variant within the GABA(A) alpha5, beta3 and gamma3 subunit gene cluster contributes to the pathogenesis of "idiopathic" generalized seizures and associated EEG abnormalities in a proportion of families.

Novel, Potent, and Selective GABACAntagonists Inhibit Myopia Development and Facilitate Learning and Memory

This study reports pharmacological and physiological effects of cis- and trans-(3-aminocyclopentanyl)butylphosphinic acid (cis- and trans-3-ACPBPA). These compounds are conformationally restricted analogs of the orally active GABA(B/C) receptor antagonist (3-aminopropyl)-n-butylphosphinic acid (CGP36742 or SGS742). cis-[IC(50)(rho1) = 5.06 microM and IC(50)(rho2) = 11.08 microM; n = 4] and trans-3-ACPMPA [IC(50)(rho1) = 72.58 microM and IC(50)(rho2) = 189.7 microM; n = 4] seem competitive at GABA(C) receptors expressed in Xenopus laevis oocytes, having no effect as agonists (1 mM) but exerting weak antagonist (1 mM) effects on human GABA(A) and GABA(B) receptors. cis-3-ACPBPA was more potent and selective than the trans-compound, being more than 100 times more potent at GABA(C) than GABA(A) or GABA(B) receptors. cis-3-ACPBPA was further evaluated on dissociated rat retinal bipolar cells and dose-dependently inhibited the native GABA(C) receptor (IC(50) = 47 +/- 4.5 microM; n = 6). When applied to the eye as intravitreal injections, cis- and trans-3-ACPBPA prevented experimental myopia development and inhibited the associated vitreous chamber elongation, in a dose-dependent manner in the chick model. Doses only 10 times greater than required to inhibit recombinant GABA(C) receptors caused the antimyopia effects. Using intraperitoneal administration, cis- (30 mg/kg) and trans-3-ACPBPA (100 mg/kg) enhanced learning and memory in male Wistar rats; compared with vehicle there was a significant reduction in time for rats to find the platform in the Morris water maze task (p < 0.05; n = 10). As the physiological effects of cis- and trans-3-ACPBPA are similar to those reported for CGP36742, the memory and refractive effects of CGP36742 may be due in part to its GABA(C) activity.

Focus on Molecules: The GABAC Receptor

γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the retina and CNS, exerts its effects by activating three pharmacologically distinct types of GABA receptor (GABAR), each of which is expressed on retinal neurons. The GABACR is a member of the Cys-loop superfamily of ligand-gated ion channels that include the nicotinic acetylcholine, glycine and 5-HT3 receptors. The pentomeric structure of native GABAC receptors is composed of GABA ρ subunits, and three of which (ρ1 – ρ3) have been cloned from mammalian retinal cDNA libraries (NCBI accession number for human GABA ρ1, ρ2, and ρ3 subunit: NM_002042, NM_002043, and NM_001105580, respectively). In humans, the ρ1 and ρ2 subunits are located on chromosome 6q15; the ρ3 subunit is on chromosome 3q11. Each of the subunits is capable of forming a GABA-sensitive homooligomeric receptor when expressed in Xenopus oocytes, and co-assembly with the γ subunit of the GABAAR results in the formation of a receptor with functional properties that closely mimic the behavior of GABACR on bipolar cells (Qian and Ripps, 1999).

Developmental impact of a familial GABAA receptor epilepsy mutation

A major goal of epilepsy research is to understand the molecular and functional basis of seizure genesis. A human GABA(A) gamma2 gene mutation (R43Q) is associated with generalized epilepsy. Introduction of this mutation into a mouse by gene targeting recapitulates the human phenotype demonstrating a strong genotype to phenotype link. GABA(A) receptors play a role in the moment-to-moment control of brain function and also on the long-term wiring of the brain by directing neuronal development. Our objective was to determine whether developmental expression of the mutation alters seizure susceptibility later in life. A tetracycline-based conditional model for activation of a hypomorphic Q43 disease allele was created and validated. Seizure susceptibility was assessed using the subcutaneous pentylenetetrazole model. Seizure susceptibility was significantly reduced in mice where the Q43 allele was suppressed during development. These results demonstrate that a human epilepsy-causing mutation impacts network stability during a critical developmental period. These data suggest that identification of presymptomatic children may provide a window for therapeutic intervention before overt symptoms are observed, potentially altering the course of epileptogenesis.

NS11394 [3′-[5-(1-Hydroxy-1-methyl-ethyl)-benzoimidazol-1-yl]-biphenyl-2-carbonitrile], a Unique Subtype-Selective GABAA Receptor Positive Allosteric Modulator: In Vitro Actions, Pharmacokinetic Properties and in Vivo Anxiolytic Efficacy

The novel positive allosteric modulator NS11394 [3'-[5-(1-hydroxy-1-methyl-ethyl)-benzoimidazol-1-yl]-biphenyl-2-carbonitrile] possesses a functional selectivity profile at GABA(A) receptors of alpha(5) > alpha(3) > alpha(2) > alpha(1) based on oocyte electrophysiology with human GABA(A) receptors. Compared with other subtype-selective ligands, NS11394 is unique in having superior efficacy at GABA(A)-alpha(3) receptors while maintaining low efficacy at GABA(A)-alpha(1) receptors. NS11394 has an excellent pharmacokinetic profile, which correlates with pharmacodynamic endpoints (CNS receptor occupancy), yielding a high level of confidence in deriving in vivo conclusions anchored to an in vitro selectivity profile and allowing for translation to higher species. Specifically, we show that NS11394 is potent and highly effective in rodent anxiety models. The anxiolytic efficacy of NS11394 is most probably mediated through its high efficacy at GABA(A)-alpha(3) receptors, although a contributory role of GABA(A)-alpha(2) receptors cannot be excluded. Compared with benzodiazepines, NS11394 has a significantly reduced side effect profile in rat (sedation, ataxia, and ethanol interaction) and mouse (sedation), even at full CNS receptor occupancy. We attribute this benign side effect profile to very low efficacy of NS11394 at GABA(A)-alpha(1) receptors and an overall partial agonist profile across receptor subtypes. However, NS11394 impairs memory in both rats and mice, which is possibly attributable to its efficacy at GABA(A)-alpha(5) receptors, albeit activity at this receptor might be relevant to its antinociceptive effects (J Pharmacol Exp Ther 327:doi;10.1124/jpet.108.144, 2008). In conclusion, NS11394 has a unique subtype-selective GABA(A) receptor profile and represents an excellent pharmacological tool to further our understanding on the relative contributions of GABA(A) receptor subtypes in various therapeutic areas.

GABAA receptor properties in catastrophic infantile epilepsy

Catastrophic epilepsy due to cortical dysplasia is often intractable to anticonvulsant treatment. Many of the medications used unsuccessfully in treating this disorder are thought to exert at least a portion of their action through enhancement of inhibitory GABA(A) neurotransmission. In the present study, GABA(A) receptor properties in resected brain tissue from four infants with infantile spasms and intractable epilepsy due to cortical dysplasia were measured to determine if this clinical resistance to pharmacologic treatment correlates with alterations in receptor function. Results from epileptic cortex were compared with those from autopsy control samples. To perform these studies, we utilized the technique of injection of brain cellular membrane preparations into the Xenopus oocyte, which results in the incorporation of human GABA(A) receptors in their native configuration into the oocyte plasma membrane. Two-electrode voltage-clamp electrophysiology analysis was then performed to assess GABA(A) receptor pharmacologic properties. The intrinsic properties of affinity, reversal potential, current decay, and current rundown were unchanged in the epileptic infants. Current enhancement by benzodiazepines was also unaltered, as was the response to barbiturates. However, a significant decrease was found in the degree of GABA(A) current enhancement by neurosteroids in the epileptic infants, along with an increase in current inhibition by zinc. These findings may contribute to the mechanisms of intractability in catastrophic infantile epilepsy due to cortical dysplasia, and suggest alternative therapeutic approaches.

Ligand Binding Mode of GABA A Receptor-Associated Protein

The γ-aminobutyric acid type A (GABA A) receptor-associated protein is a versatile adaptor protein playing an important role in intracellular vesicle trafficking, particularly in neuronal cells. We present the X-ray structure of the soluble form of human GABA A receptor-associated protein complexed with a high-affinity synthetic peptide at 1.3 Å resolution. The data shed light on the probable binding modes of key interaction partners, including the GABA A receptor and the cysteine protease Atg4. The resulting models provide a structural background for further investigation of the unique biological properties of this protein.

Hyperglycosylation and Reduced GABA Currents of Mutated GABRB3 Polypeptide in Remitting Childhood Absence Epilepsy

Childhood absence epilepsy (CAE) accounts for 10% to 12% of epilepsy in children under 16 years of age. We screened for mutations in the GABA(A) receptor (GABAR) beta 3 subunit gene (GABRB3) in 48 probands and families with remitting CAE. We found that four out of 48 families (8%) had mutations in GABRB3. One heterozygous missense mutation (P11S) in exon 1a segregated with four CAE-affected persons in one multiplex, two-generation Mexican family. P11S was also found in a singleton from Mexico. Another heterozygous missense mutation (S15F) was present in a singleton from Honduras. An exon 2 heterozygous missense mutation (G32R) was present in two CAE-affected persons and two persons affected with EEG-recorded spike and/or sharp wave in a two-generation Honduran family. All mutations were absent in 630 controls. We studied functions and possible pathogenicity by expressing mutations in HeLa cells with the use of Western blots and an in vitro translation and translocation system. Expression levels did not differ from those of controls, but all mutations showed hyperglycosylation in the in vitro translation and translocation system with canine microsomes. Functional analysis of human GABA(A) receptors (alpha 1 beta 3-v2 gamma 2S, alpha 1 beta 3-v2[P11S]gamma 2S, alpha 1 beta 3-v2[S15F]gamma 2S, and alpha 1 beta 3-v2[G32R]gamma 2S) transiently expressed in HEK293T cells with the use of rapid agonist application showed that each amino acid transversion in the beta 3-v2 subunit (P11S, S15F, and G32R) reduced GABA-evoked current density from whole cells. Mutated beta 3 subunit protein could thus cause absence seizures through a gain in glycosylation of mutated exon 1a and exon 2, affecting maturation and trafficking of GABAR from endoplasmic reticulum to cell surface and resulting in reduced GABA-evoked currents.

Levetiracetam has no acute effects on brain γ-aminobutyric acid levels

Objective The mechanism of action of levetiracetam (LEV), an antiepileptic drug, is related to a novel binding site, SV2, but LEV acts on GABA-A receptors. The objective of the study described here was to determine if LEV modulates brain GABA in vivo. Methods Concentrations of cerebral GABA and serum LEV were obtained in seven healthy individuals using 1H magnetic resonance spectroscopy at baseline and 3 and 6 hours following oral administration of 1 g of LEV. Results Brain cerebral GABA acutely concentrations did not change from baseline. Conclusion The results indicate that LEV does not increase human cerebral GABA concentrations acutely in healthy individuals.

Relative impact of residues at the intracellular and extracellular ends of the human GABA C ρ1 receptor M2 domain on picrotoxinin activity

The relative impact on picrotoxinin activity of residues at the intracellular (2′ and 6′ residues) and extracellular (15′ and 17′ residues) ends of the second transmembrane (M2) domain of the human γ-aminobutyric acid-C (GABA C) ρ1 receptor was investigated. A series of GABA C ρ1 subunits were produced containing either single or multiple mutations at the positions of interest. Wild-type and mutant subunits (containing one or more of the following mutations: P2′S, T6′M, I15′N, G17′H) were expressed in Xenopus oocytes and characterized using agonists, partial agonists and antagonists. Changes in agonist activity were observed for mutant receptors. Most notably, mutation at the 2′ position resulted in decreased agonist potency, while mutation at the 15′ and 17′ residues increased agonist potency. The affinity of the competitive antagonist (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA) was unchanged compared to wild-type at all mutant receptors. Of the four residues studied, mutation of residues at the 2′ and 6′ positions had the greatest impact on picrotoxinin activity. Inclusion of the P2′S mutation typically produced receptors with increased picrotoxinin potency, while the T6′M mutation reduced picrotoxinin potency. Picrotoxinin is a mixed antagonist at wild-type and all mutant receptors, with the exception of the double mutant ρ1P2′S/T6′M receptors at which the non-competitive component was isolated. It is proposed that the contribution of M2 domain residues to picrotoxinin activity is potentially two-fold: (1) their role as a potential picrotoxinin binding site within the pore; and (2) they are critical for receptor activation properties of the receptor, thus may alter the allosteric mechanism of picrotoxinin.

Enantioselective actions of 4-amino-3-hydroxybutanoic acid and (3-amino-2-hydroxypropyl)methylphosphinic acid at recombinant GABAC receptors

The R- and S-enantiomers of 4-amino-3-hydroxybutanoic acid (GABOB) were full agonists at human recombinant rho1 GABA(C) receptors. Their enantioselectivity (R>S) matched that reported for their agonist actions at GABA(B) receptors, but was the opposite to that reported at GABA(A) receptors (S>R). The corresponding methylphosphinic acid analogues proved to be rho1 GABA(C) receptor antagonists with R(+)-CGP44533 being more potent than S(-)-CGP44532, thus showing the opposite enantioselectivity to the agonists R(-)- and S(+)-GABOB. These studies highlight the different stereochemical requirements for the hydroxy group in these analogues at GABA(A), GABA(B) and GABA(C) receptors.

Exploring ligand recognition and ion flow in comparative models of the human GABA type A receptor

We present two comparative models of the GABA A receptor. Model 1 is based on the 4-Å resolution structure of the nicotinic acetylcholine receptor from Torpedo marmorata and represents the unliganded receptor. Two agonists, GABA and muscimol, two benzodiazepines, flunitrazepam and alprazolam, together with the general anaesthetic halothane, have been docked to this model. The ion flow is also explored in model 1 by evaluating the interaction energy of a chloride ion as it traverses the extracellular, transmembrane and intracellular domains of the protein. Model 2 differs from model 1 only in the extracellular domain and represents the liganded receptor. Comparison between the two models not only allows us to explore commonalities and differences with comparative models of the nicotinic acetylcholine receptor, but also suggests possible protein sub-domain interactions with the GABA A receptor not previously addressed.

Phosphonic acid analogs of GABA through reductive dealkylation of phosphonic diesters with lithium trialkylborohydrides

Lithium trialkylborohydrides were found to effect rapid monodealkylation of phosphonic diesters, and this reaction was applied to the synthesis of alkylphosphonic acid 2-aminoethyl esters [H 2N(CH 2) 2OP(OH)R, 4], a little-explored class of analogs of the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Compound 4a (R = Me) proved to be a potent antagonist at human ρ1 GABA C receptors (expressed in Xenopus laevis oocytes), with an IC 50 of 11.1 μM, but is inactive at α 1β 2γ 2 GABA A receptors.

Characterization of [35S]t‐butylbicyclophosphorothionate ([35S]TBPS) binding to GABAA receptors in postmortem human brain

The aim of the present study was to determine whether binding of [(35)S]t-butylbicyclophosphorothionate ([(35)S]TBPS) to the convulsant binding site of GABA(A) receptors in human postmortem brain samples can be used as an in vitro index of the functional activation of these receptors. Postmortem stability of [(35)S]TBPS binding was assessed in rat brain samples harvested at various times after death and the binding properties of [(35)S]TBPS binding (K(D) and B(max)) were determined in human postmortem brain using radioligand binding studies. In addition, the ability of human brain [(35)S]TBPS binding to be allosterically modulated by compounds that bind at recognition sites distinct from the convulsant binding site was measured. Whereas binding of [(3)H]Ro 15-1788 to the benzodiazepine binding site and [(3)H]muscimol to the agonist (GABA) binding site were retained over a 20 h postmortem interval, there was a significant decrease in the affinity and number of [(35)S]TBPS binding sites. Nevertheless, [(35)S]TBPS binding in human brain could be inhibited by TBPS, picrotoxin, loreclezole and pentobarbital and modulated by GABA with potencies comparable to those observed in rats. In addition, the GABA-induced reduction in human brain [(35)S]TBPS binding could be modulated by benzodiazepine site ligands in a manner that reflected their intrinsic efficacies. These results suggest that allosteric coupling between the [(35)S]TBPS, GABA and benzodiazepine binding sites is preserved in postmortem human brain and that [(35)S]TBPS binding in this tissue may be used to study functional characteristics of native human GABA(A) receptors.

A human anti-neuronal autoantibody against GABA B receptor induces experimental autoimmune agrypnia

In the serum and cerebrospinal fluid of a patient with recurrent acute episodes of respiratory crises, autonomic symptoms and total insomnia (agrypnia), we identified a novel anti-neural complement fixing antibody directed against GABA B receptor (GABA BR). Patient purified IgG recognized a band of approximately 110 kDa on protein extracts of mouse cerebellum, cortex and brainstem and immunolabelled cultured Chinese hamster ovary (CHO) cells, transfected with human GABA BR1 and rat GABA BR2 receptors. Western blot analysis of transfected CHO homogenates showed the same band using both patient purified IgG and anti-GABA BR1 antibody. In order to verify the pathogenic role of these purified antibodies, we injected patient IgG intrathecally into cisterna magna of C57BL/6 mice pre-implanted with EEG electrodes and we observed severe ataxia followed by breathing depression and total suppression of slow wave sleep, as evidenced by EEG recording, in a dose-dependent manner. Immunohistochemistry on brain sections of mice injected with patient IgG showed the simultaneous presence of bound human IgG and C5b-9 deposits on Purkinje cells and cerebellar granular layer. After incubation with anti-GABA BR antibody, a marked reduction of receptor immunostaining was found with relative sparing of neuronal architecture. In conclusion we recognized an anti-neuronal autoantibody directed against GABA BR that is associated with autoimmune agrypnia and we showed that our patient purified IgG was able to induce in mice experimental autoimmune agrypnia characterized by a complex neurological syndrome affecting several CNS functions.

Androstenol (5α-androst-16-en-3α-ol) Is a Novel Neurosteroid Positive Modulator of GABA A Receptors: In Vitro and In Vivo Studies

Androstenol (5α-androst-16-en-3α-ol) is a volatile steroid compound belonging to the group of 16-androstenes found in human plasma. It is structurally similar to endogenous A-ring reduced steroids that act as positive modulators of GABA A receptors, i.e., neurosteroids. Thus, we have hypothesized that androstenol may have electrophysiological and behavioral traits characteristic for neurosteroids. The influence of androstenol on GABA A receptors currents under voltage clamp conditions in mouse cerebellar granule cell cultures, rat brain slices, and HEK cells expressing human GABA A receptor subunits has been assessed. Additionally, the effect of androstenol on 4-aminopyridine–induced epileptiform activity in rat hippocampal slices was studied. Assessment of anticonvulsant, anxiolytic, and antidepressant effects of androstenol in the 6 Hz, PTZ, open field, and forced swim models has also been performed. We have found that androstenol potentiates GABA-evoked currents in HEK cells as well as in cerebellar cultures and slices. Androstenol inhibited epileptiform activity induced by 4-aminopyridine in rat brain slices and had strong anticonvulsant effects against PTZ- and 6 Hz-induced seizures in mice. In addition, we have found that androstenol has anxiolytic and antidepressant effects in animal models. These results for the first time demonstrate that androstenol acts as a positive modulator of GABA A receptors and has behavioral properties compatible with this physiological action. Androstenol may act as an endogenous modulator of GABA A receptors, and it may be useful in treatment of epilepsy and possibly other neurological disorders, i.e., anxiety and depression.