Docking of 1,4-benzodiazepines in the alpha1/gamma2 GABA(A) receptor modulator site.

Positive allosteric modulation of the GABA(A) receptor (GABA(A)R) via the benzodiazepine recognition site is the mechanism whereby diverse chemical classes of therapeutic agents act to reduce anxiety, induce and maintain sleep, reduce seizures, and induce conscious sedation. The binding of such therapeutic agents to this allosteric modulatory site increases the affinity of GABA for the agonist recognition site. A major unanswered question, however, relates to how positive allosteric modulators dock in the 1,4-benzodiazepine (BZD) recognition site. In the present study, the X-ray structure of an acetylcholine binding protein from the snail Lymnea stagnalis and the results from site-directed affinity-labeling studies were used as the basis for modeling of the BZD binding pocket at the alpha(1)/gamma(2) subunit interface. A tethered BZD was introduced into the binding pocket, and molecular simulations were carried out to yield a set of candidate orientations of the BZD ligand in the binding pocket. Candidate orientations were refined based on known structure-activity and stereospecificity characteristics of BZDs and the impact of the alpha(1)H101R mutation. Results favor a model in which the BZD molecule is oriented such that the C5-phenyl substituent extends approximately parallel to the plane of the membrane rather than parallel to the ion channel. Application of this computational modeling strategy, which integrates site-directed affinity labeling with structure-activity knowledge to create a molecular model of the docking of active ligands in the binding pocket, may provide a basis for the design of more selective GABA(A)R modulators with enhanced therapeutic potential.

Uptake and release of [3H]gamma-aminobutyric acid by embryonic spinal cord neurons in dissociated cell culture.

We have investigated the uptake and release of [3H]gamma-aminobutyric acid (GABA) by embryonic chick spinal cord cells maintained in culture. Cells dissociated from 4- or 7-d-old embryos were studied between 1 and 3 wk after plating. At 3 degrees C, [3H]GABA was accumulated by a high affinity (Km approximately equal to 4 microM) and a low affinity (Km approximately equal to 100 microM) mechanism. The high affinity transport was markedly inhibited in low Na+ media, by ouabain, at 0 degrees C, and by 2,4-diaminobutyric acid. Autoradiography, after incubation in 0.1 microM [3H]GABA, showed that approximately 50% (range = 30-70%) of the multipolar cells were labeled. These cells were neurons rather than glia; action potentials and/or synaptic potentials were recorded in cells subsequently found to be labeled. Non-neuronal, fibroblast-like cells and co-cultured myotubes were not labeled under the same conditions. The fact that not all of the neurons were labeled is consistent with the suggestion, based on studies of intact adult tissue, that high affinity transport of [3H]GABA may be unique to neurons that use GABA as a neurotransmitter. Our finding that none of fifteen physiologically identified cholinergic neurons, i.e., cells that innervated nearby myotubes, were heavily labeled after incubation in 0.1 microM [3H]GABA is significant in this regard. The newly taken up [3H]GABA was not metabolized in the short run. It was stored in a form that could be released when the neurons were depolarized in a high K+ (100 mM) medium. As expected for a neurotransmitter, the K+-evoked release was reversibly inhibited by reducing the extracellular Ca++/Mg++ ratio.

Benzodiazepine receptor synthesis and degradation by neurons in culture.

The benzodiazepine-gamma-aminobutyric acid receptor complex was used to study functional receptor synthesis and degradation in primary cultures of neurons. Fifty percent of the receptors turned over with an unusually rapid half-life (4 hours); this was followed by a second, slower phase (32 hours). These results provide the basis for elucidating the mechanism by which neurons derived from the central nervous system control neurotransmitter receptor number, an important problem in cellular neurobiology. The findings may be of significance in the study of neurological and psychiatric disorders.

Intrathecal capsaicin depletes substance P in the rat spinal cord and produces prolonged thermal analgesia.

A single intrathecal injection of capsaicin depletes substance P from primary sensory neurons and causes a prolonged increase in the thermal and chemical pain thresholds of the rat but no apparent change in responses to noxious mechanical stimuli.

Nanomolar concentrations of pregnenolone sulfate enhance striatal dopamine overflow in vivo.

The balance between GABA-mediated inhibitory and glutamate-mediated excitatory synaptic transmission represents a fundamental mechanism for controlling nervous system function, and modulators that can alter this balance may participate in the pathophysiology of neuropsychiatric disorders. Pregnenolone sulfate (PS) is a neuroactive steroid that can modulate the activity of ionotropic glutamate and GABA(A) receptors either positively or negatively, depending upon the particular receptor subtype, and modulates synaptic transmission in a variety of experimental systems. To evaluate the modulatory effect of PS in vivo, we infused PS into rat striatum for 20 min via a microdialysis probe while monitoring local extracellular dopamine (DA) levels. The results demonstrate that PS at low nanomolar concentrations significantly increases extracellular DA levels. The PS-induced increase in extracellular DA is antagonized by the N-methyl-d-aspartate (NMDA) receptor antagonist, d-AP5 [d-(-)-2-amino-5-phosphonopentanoic acid], but not by the sigma receptor antagonist, BD 1063 [1(-)[2-(3,4-dichlorophenyl)-ethyl]-4-methylpiperazine]. The results demonstrate that exogenous PS, at nanomolar concentrations, is able to increase DA overflow in the striatum through an NMDA receptor-mediated pathway.

Dueling enigmas: neurosteroids and sigma receptors in the limelight.

Neurosteroids can be positive or negative regulators of neurotransmitter receptor action, depending on the receptor and the chemical structure of the neurosteroid. This Perspective by Gibbs and Farb is one of two on the subject of neurosteroids. The authors address the possible role of sigma receptors in mediating neurosteroid action and describe how the regulation of inhibitory and excitatory ion channels by neurosteroids has implications for the role of these molecules in learning and memory, nociception, and excitotoxicity.

Human GABA(B)R genomic structure: evidence for splice variants in GABA(B)R1 but not GABA(B)R2.

The type B gamma-aminobutryic acid receptor (GABA(B)R) is a G protein coupled receptor that mediates slow pre- and post-synaptic inhibition in the nervous system. We find that the human GABA(B)R2 gene spans greater than 350 kb and contains 2.8 kb of coding region in 19 exons. The overall similarity in genomic structure with regard to conservation of intron position and exon size between human or Drosophila GABA(B)R1 and GABA(B)R2 genes suggests a common ancestral origin. Multiple transcripts GABA(B)R1a-c and GABA(B)R2a-c have been described and alternative splicing has been proposed to result in GABA(B)R1c, GABA(B)R2b and GABA(B)R2c. The results described here provide support for the existence of GABA(B)R1c but not for GABA(B)R2b and GABA(B)R2c. Splice junctions present in the GABA(B)R1 gene sequence are consistent with the formation of GABA(B)R1c by exon skipping of one sushi domain module. The GABA(B)R2 gene lacks canonical splice junctions for the reported variants. Consistent with this, RNA analysis demonstrates the presence of GABA(B)R1c and GABA(B)R2 transcripts in fetal and adult human brain RNA but GABA(B)R2b and GABA(B)R2c transcripts are not detected. These results provide insight into the evolution and transcript diversity of the mammalian GABA(B)R genes.

gamma-aminobutyric acidA receptor regulation: heterologous uncoupling of modulatory site interactions induced by chronic steroid, barbiturate, benzodiazepine, or GABA treatment in culture.

Prolonged administration of anxiolytic, sedative, and anticonvulsant drugs that act through the GABAA receptor (GABAAR) can evoke tolerance and dependence, suggesting the existence of an endogenous mechanism(s) for altering the ability of such agents to interact with the GABAAR. Uncoupling appears to be one such mechanism. This is a decrease in the allosteric interactions between the benzodiazepine (BZD) recognition site and other agonist or modulator sites on the GABAAR, as measured by potentiation of [3H]flunitrazepam ([3H]FNZ) binding. To investigate the mechanism(s) of uncoupling, neuronal cultures were treated chronically with 3 alpha-hydroxy-5 beta-pregnan-20-one (pregnanolone), pentobarbital, flurazepam, or GABA, then tested for enhancement of [3H]FNZ binding by these substances. The results indicate that BZDs, barbiturates, and steroids, as well as GABA itself, are capable of inducing both heterologous and homologous uncoupling. Surprisingly, different chronic drug treatments produce different patterns of homologous and heterologous uncoupling. Chronic exposure to pregnanolone, GABA, flurazepam or pentobarbital induces complete uncoupling of barbiturate-BZD site interactions, partial uncoupling of GABA-BZD site interactions, but different amounts of uncoupling of steroid-BZD site interactions. In addition, the EC50 for pregnanolone-induced homologous uncoupling (1.7 microM) is over an order of magnitude greater than that for heterologous uncoupling of GABA and BZD sites (82 nM). Moreover, heterologous uncoupling by pregnanolone is inhibited by the GABA site antagonist SR-95531, whereas homologous uncoupling by pregnanolone is resistant to SR-95531. Therefore, there are at least two distinct ways in which GABAAR modulatory site interactions can be regulated by chronic drug treatment.

Ethanol potentiates GABA- and glycine-induced chloride currents in chick spinal cord neurons.

The effects of acute ethanol exposure of chick spinal cord neurons were studied in tissue culture, using whole-cell voltage-clamp techniques. Results indicate that ethanol produces a persistent increase in the sensitivity of spinal neurons to GABA and glycine, with no change in input resistance or resting membrane potential. Glutamate responses, in contrast, are unaffected by ethanol.

17beta-Estradiol protects against NMDA-induced excitotoxicity by direct inhibition of NMDA receptors.

Several lines of evidence suggest that 17beta-estradiol (betaE2) has neuroprotective properties. The risk and severity of dementia are decreased in women who have received estrogen therapy, and betaE2 protects neurons in vitro against death from a variety of stressors. Neuroprotection by betaE2 has been suggested to be due to free radical scavenging. We demonstrate an additional neuroprotective mechanism whereby betaE2 protects against NMDA-induced neuronal death by directly inhibiting the NMDA receptor.

Pregnenolone sulfate exacerbates NMDA-induced death of hippocampal neurons.

Excessive stimulation of the N-methyl-d-aspartate (NMDA)-type glutamate receptor has been implicated in the neuronal death resulting from focal hypoxia-ischemia. Certain neurosteroids, steroids synthesized de novo in the central nervous system (CNS), have been shown to modulate the action of neurotransmitters at their cellular receptors. Pregnenolone sulfate (PS) is an abundant neurosteroid that enhances the current evoked by NMDA. Using the Ca2+-sensitive fluorescent dye, Fluo-3, AM, and a trypan blue exclusion assay, we evaluated the ability of PS to modulate NMDA-induced changes in intracellular free calcium concentration ([Ca2+]i) and neuronal death in primary cultures of rat hippocampal neurons. The results demonstrate that PS potentiates NMDA-induced increases in [Ca2+]i by 150%. Further, PS exacerbates the MK-801-sensitive neuronal death produced by acute (PS EC50=37 microM) or chronic NMDA exposure, reducing the EC50 of NMDA from 13 to 4 microM under chronic exposure conditions, whereas pregnenolone is ineffective. Our results show that PS, or related sulfated neurosteroids, may play a role in the onset of excitotoxic neuronal death in vivo.

Neurosteroid modulation of recombinant ionotropic glutamate receptors.

Pregnenolone sulfate (PS) is an abundant neurosteroid that can potentiate or inhibit ligand gated ion channel activity and thereby alter neuronal excitability. Whereas PS is known to inhibit kainate and AMPA responses while potentiating NMDA responses, the dependence of modulation on receptor subunit composition remains to be determined. Toward this end, the effect of PS on recombinant kainate (GluR6), AMPA (GluR1 or GluR3), and NMDA (NR1(100)+NR2A) receptors was characterized electrophysiologically with respect to efficacy and potency of modulation. With Xenopus oocytes expressing GluR1, GluR3 or GluR6 receptors, PS reduces the efficacy of kainate without affecting its potency, indicative of a noncompetitive mechanism of action. Conversely, with oocytes expressing NR1(100)+NR2A subunits, PS enhances the efficacy of NMDA without affecting its potency. Whereas the modulatory efficacy, but not the potency, of PS is increased two-fold by co-injection of NR1(100)+NR2A cRNAs as compared with NR1(100) cRNA alone, there is little or no effect of the NR2A subunit on efficacy or potency of pregnanolone (or epipregnanolone) sulfate as an inhibitor of the NMDA response. This suggests that the NR2A subunit controls the efficacy of neurosteroid enhancement, but not inhibition, which is consistent with our previous finding that potentiating and inhibitory steroids act at distinct sites on the NMDA receptor. This represents a first step towards understanding the role of subunit composition in determining neurosteroid modulation of ionotropic glutamate receptor function.

Sulfated and unsulfated steroids modulate gamma-aminobutyric acidA receptor function through distinct sites.

Sulfated and unsulfated neurosteroids such as pregnenolone sulfate, dehydroepiandrosterone sulfate (DHEAS), pregnanolone, and allopregnanolone, modulate ionotropic amino acid neurotransmitter receptors, and may function as endogenous neuromodulators. The gamma-aminobutyric acid type A (GABAA) receptor exhibits both negative and positive modulation by neurosteroids, but the interaction between negative and positive modulators is not well-understood. For a number of neuroactive steroids, sulfation at C-3 reverses the direction of modulation from positive to negative, suggesting that sulfation could be an important control point for the activity of endogenous neurosteroids. Modulation by endogenous and synthetic steroids of the response to exogenous or synaptically released GABA was examined in primary chick spinal cord and rat hippocampal neurons, and in Xenopus laevis oocytes expressing alpha1beta2gamma2S GABAA receptors. Inhibitory activity is retained when hemisuccinate is substituted for sulfate at C-3, suggesting that it is the negative charge, rather than the sulfate group, that confers inhibitory efficacy. The interaction between steroid negative and positive modulators is not competitive, indicating that steroid negative and positive modulators act through distinct sites. Some steroids, such as 11-ketopregnenolone sulfate, appear to act at both negative and positive modulatory sites, as indicated by an 'off-response' upon washout. A similar off-response is also observed after co-application of the negative modulator DHEAS and the positive modulator allopregnanolone. The observation that simultaneous application of sulfated and unsulfated steroids, such as DHEAS and allopregnanolone, act at distinct sites implies that steroid negative and positive modulators can act independently or coordinately to regulate GABA-mediated inhibition in the central nervous system.

Dual activation of GABAA and glycine receptors by beta-alanine: inverse modulation by progesterone and 5 alpha-pregnan-3 alpha-ol-20-one.

The differential sensitivity of the glycine and GABAA receptors to modulation by progesterone and 5 alpha-pregnan-3 alpha-ol-20-one (5 alpha 3 alpha) was used to determine whether beta-alanine acts through its own receptor, or through the glycine and/or GABAA receptor(s). The response to beta-alanine resembles the glycine response as it is inhibited by strychnine (a competitive glycine antagonist) or progesterone (a negative modulator of the glycine response). Significantly, the response to beta-alanine also resembles the GABA response in that it is inhibited by 2-(carboxy-3'-propyl)-3-amino-6-paramethoxy-phenylpyridazinium+ ++ bromide (SR-95531; a competitive GABA antagonist) and potentiated by 5 alpha 3 alpha (a positive modulator of the GABA response). The efficacy of beta-alanine at the GABAA receptor is comparable to that of GABA. Similarly, the efficacy of beta-alanine at the glycine receptor is comparable to that of glycine. The greater potency of beta-alanine at the glycine receptor indicates that, if beta-alanine is a neurotransmitter, its effects are more likely to be mediated by glycine receptors than by GABAA receptors. However, activation of the GABAA receptor by beta-alanine may become important in the presence of steroid modulators such as progesterone or 5 alpha 3 alpha.

Benzodiazepine receptor photoaffinity labeling: correlation of function with binding.

Exhaustive photoaffinity coupling of flunitrazepam to living spinal cord neurons reduced the capacity of benzodiazepines to potentiate the electrophysiologically measured GABA response. In qualitative agreement with reversible binding data the dose-response curve for enhancement of the GABA response by benzodiazepines was shifted to the right, indicating that the remaining reversible benzodiazepine binding sites have lower affinity for benzodiazepines. Photoaffinity labeling did not reduce inhibition of the GABA response by beta-carbolines and there was only a small decrease in beta-carboline binding. In both control and photoaffinity-labeled cultures, the inhibitory effect of beta-carbolines on the GABA response was reversed in the presence of excess benzodiazepine. The results indicate that the effects of photoaffinity labeling are confined to the BZD recognition site, and that coupling between benzodiazepine receptors and GABA receptors remains intact.

Pregnenolone sulfate augments NMDA receptor mediated increases in intracellular Ca2+ in cultured rat hippocampal neurons.

The ability of the neuroactive steroid pregnenolone sulfate to alter N-methyl-D-aspartate (NMDA) receptor-mediated elevations in intracellular Ca2+ ([Ca2+]i) was studied in cultured fetal rat hippocampal neurons using microspectrofluorimetry and the Ca2+ sensitive indicator fura-2. Pregnenolone sulfate (5-250 microM) caused a concentration-dependent and reversible potentiation of the rise (up to approximately 800%) in [Ca2+]i induced by NMDA. In contrast, the steroid failed to alter basal (unstimulated) [Ca2+]i or to modify the rise in [Ca2+]i that occurs when hippocampal neurons are depolarized by high K+ in the presence of the NMDA receptor antagonist CPP. These data suggest that the previously reported excitatory properties of pregnenolone sulfate may be due, in part, to an augmentation of the action of glutamic acid at the NMDA receptor.

Multiple embryonic benzodiazepine binding sites: evidence for functionality.

We have found high-affinity binding (site-A) and low-affinity binding (site-B) of benzodiazepines to membrane homogenates of embryonic chick brain and spinal cord. A new technique was developed to permit the determination of complete electrophysiological dose-response curves on single neurons in cell culture, eliminating cell-to-cell variability as a problem that complicates the interpretation of pooled data. The electrophysiological potencies and binding affinities of a series of benzodiazepines correlate well for site-A but not for site-B or the micromolar site reported in adult rat brain. Site-A and the electrophysiological response are sensitive to photo-affinity blockade with flunitrazepam (FNZM) by about 75% while site-B is resistant to blockade. The FNZM-photolinked benzodiazepine receptor/GABA receptor complex is not chronically potentiated and thus exists in an 'unpotentiated' state. These experiments suggest that site-A in embryonic CNS membranes corresponds to a functional benzodiazepine receptor/GABA receptor complex in spinal cord cell cultures.

Genetic disruption of the autism spectrum disorder risk gene PLAUR induces GABAA receptor subunit changes.

Disruption of the GABAergic system has been implicated in multiple developmental disorders, including epilepsy, autism spectrum disorder and schizophrenia. The human gene encoding uPAR (PLAUR) has been shown recently to be associated with the risk of autism. The uPAR(-/-) mouse exhibits a regionally-selective reduction in GABAergic interneurons in frontal and parietal regions of the cerebral cortex as well as in the CA1 and dentate gyrus subfields of the hippocampus. Behaviorally, these mice exhibit increased sensitivity to pharmacologically-induced seizures, heightened anxiety, and atypical social behavior. Here, we explore potential alterations in GABAergic circuitry that may occur in the context of altered interneuron development. Analysis of gene expression for 13 GABA(A) receptor subunits using quantitative real-time polymerase chain reaction (PCR) indicates seven subunit mRNAs (alpha(1), alpha(2), alpha(3), beta(2), beta(3), gamma(2S) and gamma(2L)) of interest. Semi-quantitative in situ hybridization analysis focusing on these subunit mRNAs reveals a complex pattern of potential gene regulatory adaptations. The levels of alpha(2) subunit mRNAs increase in frontal cortex, CA1 and CA3, while those of alpha3 decrease in frontal cortex and CA1. In contrast, alpha(1) subunit mRNAs are unaltered in any region examined. beta(2) subunit mRNAs are increased in frontal cortex whereas beta(3) subunit mRNAs are decreased in parietal cortex. Finally, gamma(2S) subunit mRNAs are increased in parietal cortex while gamma(2L) subunit mRNAs are increased in the dentate gyrus, potentially altering the gamma(2S):gamma(2L) ratio in these two regions. For all subunits, no changes were observed in forebrain regions where GABAergic interneuron numbers are normal. We propose that disrupted differentiation of GABAergic neurons specifically in frontal and parietal cortices leads to regionally-selective alterations in local circuitry and subsequent adaptive changes in receptor subunit composition. Future electrophysiological studies will be useful in determining how alterations in network activity in the cortex and hippocampus relate to the observed behavioral phenotype.

Selective anxiolysis produced by ocinaplon, a GABA(A) receptor modulator.

Benzodiazepines remain widely used for the treatment of anxiety disorders despite prominent, often limiting side effects including sedation, muscle relaxation, and ataxia. A compound producing a robust anxiolytic action comparable to benzodiazepines, but lacking these limiting side effects at therapeutic doses (an anxioselective agent), would represent an important advance in the treatment of generalized anxiety disorder, and perhaps other anxiety disorders. Here we report that the pyrazolo[1,5-a]-pyrimidine, ocinaplon, exhibits an anxioselective profile in both preclinical procedures and in patients with generalized anxiety disorder, the most common of the anxiety disorders. In rats, ocinaplon produces significant muscle relaxation, ataxia, and sedation only at doses >25-fold higher than the minimum effective dose (3.1 mg/kg) in the Vogel "conflict" test. This anticonflict effect is blocked by flumazenil (Ro 15-1788), indicating that like benzodiazepines, ocinaplon produces an anxiolytic action through allosteric modulation of GABA(A) receptors. Nonetheless, in eight recombinant GABA(A) receptor isoforms expressed in Xenopus oocytes, the potency and efficacy of ocinaplon to potentiate GABA responses varied with subunit composition not only in an absolute sense, but also relative to the prototypical benzodiazepine, diazepam. In a double blind, placebo controlled clinical trial, a 2-week regimen of ocinaplon (total daily dose of 180-240 mg) produced statistically significant reductions in the Hamilton rating scale for anxiety scores. In this study, the incidence of benzodiazepine-like side effects (e.g., sedation, dizziness) in ocinaplon-treated patients did not differ from placebo. These findings indicate that ocinaplon represents a unique approach both for the treatment and understanding of anxiety disorders.