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ObjectiveTemporal lobe epilepsy (TLE) is a progressive disorder mediated by pathological changes in molecular cascades and hippocampal neural circuit remodeling that results in spontaneous seizures and cognitive dysfunction. Targeting these cascades may provide disease-modifying treatments for TLE patients. Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) inhibitors have emerged as potential disease-modifying therapies; a more detailed understanding of JAK/STAT participation in epileptogenic responses is required, however, to increase the therapeutic efficacy and reduce adverse effects associated with global inhibition.MethodsWe developed a mouse line in which tamoxifen treatment conditionally abolishes STAT3 signaling from forebrain excitatory neurons (nSTAT3KO). Seizure frequency (continuous in vivo electroencephalography) and memory (contextual fear conditioning and motor learning) were analyzed in wild-type and nSTAT3KO mice after intrahippocampal kainate (IHKA) injection as a model of TLE. Hippocampal RNA was obtained 24 h after IHKA and subjected to deep sequencing.ResultsSelective STAT3 knock-out in excitatory neurons reduced seizure progression and hippocampal memory deficits without reducing the extent of cell death or mossy fiber sprouting induced by IHKA injection. Gene expression was rescued in major networks associated with response to brain injury, neuronal plasticity, and learning and memory. We also provide the first evidence that neuronal STAT3 may directly influence brain inflammation.InterpretationInhibiting neuronal STAT3 signaling improved outcomes in an animal model of TLE, prevented progression of seizures and cognitive co-morbidities while rescuing pathogenic changes in gene expression of major networks associated with epileptogenesis. Specifically targeting neuronal STAT3 may be an effective disease-modifying strategy for TLE. ANN NEUROL 2023;94:106-122.

Advancements in genetically based technologies have begun to allow us to better understand the relationships between underlying neural activity and the patterns of measurable behavior that can be reproducibly studied in the laboratory. As this field develops, there are key limitations to the currently available technologies hindering their full potential to deliver meaningful datasets. The limitations which are most critical to advancement of these technologies in behavioral neuroscience are: the temporal resolution at which physiological events can be windowed, the divergent molecular pathways in signal transduction that introduce ambiguity into the output of activity sensors, and the impractical size of the genetic material that requires 3-4 separate AAV vectors to deliver a fully functional system into a cell. To address these limitations and help bring the potential of these types of technologies into better realization, we have engineered a nucleus localized light-sensitive Ca2+-dependent gene expression system based on AsLOV2 and the downstream responsive element antagonist modulator (DREAM). The design and engineering of each component was performed in such a way to: 1) preserve behaviorally relevant temporal dynamics, 2) preserve signal fidelity appropriate for studying experience-driven neural activity patterns and their relationship to specific animal responses, and 3) have full delivery of the genetic material via a single AAV vector. The system was tested in vitro and subsequently in vivo with neural activity induced by Channelrhodopsin and could be used in the future with behaviorally-driven neural activity. To our knowledge this is the first optogenetic tool for the practical use of linking activity-dependent gene activation in response to direct nuclear calcium transduction.

BackgroundBrain-derived neurotrophic factor (BDNF) is a major signaling molecule that the brain uses to control a vast network of intracellular cascades fundamental to properties of learning and memory, and cognition. While much is known about BDNF signaling in the healthy nervous system where it controls the mitogen activated protein kinase (MAPK) and cyclic-AMP pathways, less is known about its role in multiple brain disorders where it contributes to the dysregulated neuroplasticity seen in epilepsy and traumatic brain injury (TBI). We previously found that neurons respond to prolonged BDNF exposure (bothin vivo(in models of epilepsy and TBI) andin vitro(in BDNF treated primary neuronal cultures)) by activating the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway. This pathway is best known for its association with inflammatory cytokines in non-neuronal cells.ResultsHere, using deep RNA-sequencing of neurons exposed to BDNF in the presence and absence of well characterized JAK/STAT inhibitors, and without non-neuronal cells, we determine the BDNF transcriptome that is specifically reliant on JAK/STAT signaling. Surprisingly, the transcriptome contains ion channels and neurotransmitter receptors coming from all the major classes expressed in the brain, along with key modulators of synaptic plasticity, neurogenesis, and axonal remodeling. Analysis of this dataset has also provided a window on the unique mechanism of JAK/STATs in neurons as differential gene expression mediated by STAT3 does not appear to be dependent upon phosphorylation at residue 705.ConclusionsOur findings strengthen and expand the role that BDNF plays in the regulation of brain excitability at the transcriptional level. They also suggest that a majority of such signaling in neurons is tied to the activation of the JAK/STAT pathway which may be non-canonical, not based on phosphorylation of STAT3 proteins at Tyrosine 705.

Tonic inhibition mediated by extrasynaptic GABAAreceptors (GABARs) sensing ambient levels of GABA can profoundly alter the membrane input resistance to affect cellular excitability. Therefore, regulation of tonic inhibition is an attractive mechanism to control the levels of cortical firing. In cortical pyramidal cells, tonic inhibition is regulated by age and several neurotransmitters and is affected by stroke and epilepsy. However, the possible role of sensory experience has not been examined. Here, we report that a brief 2-day exposure to dark reduces by 1/3 the inhibitory tonic conductance recorded in layer II/III pyramidal cells of the mouse juvenile (postnatal day 12–27) visual cortex. In these cells, tonic inhibition is carried primarily by GABARs containing the δ subunit. Consistently, the dark exposure reduction in conductance was associated with a reduction in δ subunit levels, which were not affected in control frontal cortex. We propose that a deprivation-induced reduction in tonic inhibition might serve a homeostatic function by increasing the firing levels of cells in deprived cortical circuits.SIGNIFICANCE STATEMENTPreviousin vivostudies reported rapid increases in spontaneous activity after visual deprivation. These adaptive responses to deprivation are believed to reflect a reduction in the recruitment of inhibitory circuits. Notably, the possible role of tonic GABAergic inhibition, which strongly limits cellular and network excitability, has not been examined. We report that a brief 2-day exposure to dark reduces both the conductance of tonic inhibition in layer 2/3 pyramidal cells and the expression of receptors containing the δ-GABAAreceptor subunit, the principal carrier of tonic inhibition in these cells. These results suggest that the early phases of homeostatic adaptations to sensory deprivation might result from modulation of GABAergic function at multiple levels.

RNA binding proteins are a diverse class of proteins that regulate all aspects of RNA metabolism. Accumulating studies indicate that heterogeneous nuclear ribonucleoproteins are associated with cellular adaptations in response to drugs of abuse. We recently mapped and validated heterogeneous nuclear ribonucleoprotein H1 (Hnrnph1) as a quantitative trait gene underlying differential behavioral sensitivity to methamphetamine. The molecular mechanisms by which hnRNP H1 alters methamphetamine behaviors are unknown but could involve preand/or post-synaptic changes in protein localization and function. Methamphetamine initiates post-synaptic D1 dopamine receptor signaling indirectly by binding to pre-synaptic dopamine transporters and vesicular monoamine transporters of midbrain dopaminergic neurons which triggers revers e transport and accumulation of dopamine at the synapse. Here, we examined changes in neuronal localization of hnRNP H in primary rat cortical neurons that express dopamine receptors that can be modulated by the D1 or D2 dopamine receptor agonists SKF38393 and (-)-Quinpirole HCl, respectively. Basal immunostaining of hnRNP H was localized primarily to the nucleus. D1 dopamine receptor activation induced an increase in hnRNP H nuclear immunostaining as detected by immunocytochemistry with a C-domain directed antibody containing epitope near the glycine-rich domain but not with an N-domain specific antibody. Although there was no change in hnRNP H protein in the nucleus or cytoplasm, there was a decrease in Hnrnph1 transcript following D1 receptor stimulation. Taken together, these results suggest that D1 receptor activation increases availability of the hnRNP H C-terminal epitope, which could potentially reflect changes in protein-protein interactions. Thus, D1 receptor signaling could represent a key molecular post-synaptic event linking Hnrnph1 polymorphisms to drug-induced behavior.HighlightsWe investigated immunofluorescence and localization of hnRNP H following dopamine receptor activation in primary rat cortical neurons.Activation of D1 dopamine receptor increased nuclear immunofluorescence of the C-terminal domain but not the N-terminal domain of hnRNP H with no change in either nuclear or cytoplasmic levels of hnRNP H protein.D1 dopamine signaling could alter interaction of hnRNP H with other proteins.

Summary Objective Temporal lobe epilepsy (TLE) is frequently medically intractable and often progressive. Compromised inhibitory neurotransmission due to altered γ-aminobutyric acid (GABA)A receptor α4 subunit (GABAARα4) expression has been emphasized as a potential contributor to the initial development of epilepsy following a brain insult (primary epileptogenesis), but the regulation of GABAARα4 during chronic epilepsy, specifically, how expression is altered following spontaneous seizures, is less well understood. Methods Continuous video–electroencephalography (EEG) recordings from rats with pilocarpine-induced TLE were used to capture epileptic animals within 3 h of a spontaneous seizure (SS), or >24 h after the last SS, to determine whether recent occurrence of a seizure was associated with altered levels of GABAARα4 expression. We further evaluated whether this GABAARα4 plasticity is regulated by signaling mechanisms active in primary epileptogenesis, specifically, increases in brain-derived neurotrophic factor (BDNF) and early growth response factor 3 (Egr3). Results Elevated levels of GABAARα4 messenger RNA (mRNA) and protein were observed following spontaneous seizures, and were associated with higher levels of BDNF and Egr3 mRNA. Significance These data suggest that spontaneous, recurrent seizures that define chronic epilepsy may influence changes in GABAARα4 expression, and that signaling pathways known to regulate GABAARα4 expression after status epilepticus may also be activated after spontaneous seizures in chronically epileptic animals.

Neurotrophins, such as brain-derived neurotrophic factor (BDNF), are initially expressed in a precursor form (e.g., pro-BDNF) and cleaved to form mature BDNF (mBDNF). After pilocarpine-induced status epilepticus (SE), increases in neurotrophins regulate a wide variety of cell-signaling pathways, including prosurvival and cell-death machinery in a receptor-specific manner. Pro-BDNF preferentially binds to the p75 neurotrophin receptor (p75NTR), whereas mBDNF is the major ligand of the tropomyosin-related kinase receptor. To elucidate a potential role for p75NTR in acute stages of epileptogenesis, rats were injected prior to and at onset of SE with LM11A-31, a small-molecule ligand that binds to p75NTR to promote survival signaling and inhibit neuronal cell death. Modulation of early p75NTR signaling and its effects on electrographic SE, SE-induced neurodegeneration, and subsequent spontaneous seizures were examined after LM11A-31 administration. Despite an established neuroprotective effect of LM11A-31 in several animal models of neurodegenerative disorders (e.g., Alzheimer's disease, traumatic brain injury, and spinal cord injury), high-dose LM11A-31 administration prior to and at onset of SE did not reduce the intensity of electrographic SE, prevent SE-induced neuronal cell injury, or inhibit the progression of epileptogenesis. Further studies are required to understand the role of p75NTR activation during epileptogenesis and in seizure-induced cell injury in the hippocampus, among other potential cellular pathologies contributing to the onset of spontaneous seizures. Additional studies utilizing more prolonged treatment with LM11A-31 are required to reach a definite conclusion on its potential neuroprotective role in epilepsy. © 2014 Wiley Periodicals, Inc.

N-methyl D-aspartate (NMDA) receptors (NMDARs) mediate fast excitatory synaptic transmission and play a critical role in synaptic plasticity associated with learning and memory. NMDAR hypoactivity has been implicated in the pathophysiology of schizophrenia, and clinical studies have revealed reduced negative symptoms of schizophrenia with a dose of pregnenolone that elevates serum levels of the neuroactive steroid pregnenolone sulfate (PregS). This report describes a novel process of delayed-onset potentiation whereby PregS approximately doubles the cell's response to NMDA via a mechanism that is pharmacologically and kinetically distinct from rapid positive allosteric modulation by PregS. The number of functional cell-surface NMDARs in cortical neurons increases 60-100% within 10 minutes of exposure to PregS, as shown by surface biotinylation and affinity purification. Delayed-onset potentiation is reversible and selective for expressed receptors containing the NMDAR subunit subtype 2A (NR2A) or NR2B, but not the NR2C or NR2D, subunits. Moreover, substitution of NR2B J/K helices and M4 domain with the corresponding region of NR2D ablates rapid allosteric potentiation of the NMDA response by PregS but not delayed-onset potentiation. This demonstrates that the initial phase of rapid positive allosteric modulation is not a first step in NMDAR upregulation. Delayed-onset potentiation by PregS occurs via a noncanonical, pertussis toxin-sensitive, G protein-coupled, and Ca(2+)-dependent mechanism that is independent of NMDAR ion channel activation. Further investigation into the sequelae for PregS-stimulated trafficking of NMDARs to the neuronal cell surface may uncover a new target for the pharmacological treatment of disorders in which NMDAR hypofunction has been implicated.

Epilepsy is a disease of complex etiology and multiple molecular mechanisms contribute to its development. Temporal lobe epilepsy (TLE) may result from an initial precipitating event such as hypoxia, head injury, or prolonged seizure (i.e., status epilepticus (SE)), that is followed by a latent period of months to years before spontaneous seizures occur. GABAA receptor (GABAAR) subunits changes occur during this latent period and may persist following the onset of spontaneous seizures. Research into the molecular mechanisms regulating these changes and potential targets for intervention to reverse GABAAR subunit alterations have uncovered seizure-induced pathways that contribute to epileptogenesis. Several growth or transcription factors are known to be activated by SE, including (but not limited to): Brain Derived Neurotrophic Factor (BDNF), cAMP response element binding protein (CREB), Inducible cAMP Early Repressor (ICER), and Early Growth Response factors (Egrs). Results of multiple studies suggest that these factors transcriptionally regulate GABAAR subunit gene expression in a way that is pertinent to the development of epilepsy. This article will focus on these signaling elements and describe their possible roles in gene regulatory pathways that may be critical in the development of chronic epilepsy.

Regulation of gene expression via brain-derived neurotrophic factor (BDNF) is critical to the development of the nervous system and may well underlie cognitive performance throughout life. We now describe a mechanism by which BDNF can exert its effects on postsynaptic receptor populations that may have relevance to both the normal and diseased brain where BDNF levels either rise or fall in association with changes in excitatory neurotransmission. Increased levels of NMDA receptors (NMDARs) occur in rat cortical neurons via synthesis of new NMDA receptor 1 (NR1) subunits. The majority of synthesis is controlled by binding of cAMP response element binding protein (CREB) and early growth response factor 3 (Egr3) to the core NR1 promoter (NR1-p) region. BDNF-mediated NR1 transcription depends upon induction of the mitogen-activated protein kinase (MAPK) pathway through activation of the TrK-B receptor. Taken together with the fact that NMDAR activation stimulates BDNF synthesis, our results uncover a feed-forward gene regulatory network that may enhance excitatory neurotransmission to change neuronal behavior over time.

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 GABAA receptor subunits using quantitative real-time polymerase chain reaction (PCR) indicates seven subunit mRNAs (α1, α2, α3, β2, β3, γ2S and γ2L) 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 α2 subunit mRNAs increase in frontal cortex, CA1 and CA3, while those of α3 decrease in frontal cortex and CA1. In contrast, α1 subunit mRNAs are unaltered in any region examined. β2 subunit mRNAs are increased in frontal cortex whereas β3 subunit mRNAs are decreased in parietal cortex. Finally, γ2S subunit mRNAs are increased in parietal cortex while γ2L subunit mRNAs are increased in the dentate gyrus, potentially altering the γ2S:γ2L 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. Fil: Eagleson, K. L.. University of Southern California; Estados Unidos Fil: Gravielle, Maria Clara. Boston University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Schlueter McFadyen Ketchum, L. J.. University of Southern California; Estados Unidos Fil: Russek, S. J.. Boston University; Estados Unidos Fil: Farb, D. H.. Boston University; Estados Unidos Fil: Levitt, P.. University of Southern California; Estados Unidos

Retrospective studies suggest that precipitating events such as prolonged seizures, stroke, or head trauma increase the risk of developing epilepsy later in life. The process of epilepsy development, known as epileptogenesis, is associated with changes in the expression of a myriad of genes. One of the major challenges for the epilepsy research community has been to determine which of these changes contributes to epileptogenesis, which may be compensatory, and which may be noncontributory. Establishing this for any given gene is essential if it is to be considered a therapeutic target for the prevention or treatment of epilepsy. Our laboratories have examined alterations in gene expression related to inhibitory neurotransmission that have been proposed as contributing factors in epileptogenesis. The GABA(A) receptor mediates most fast synaptic inhibition, and changes in GABA(A) receptor subunit expression and function have been reported in adult animals beginning immediately after prolonged seizures (status epilepticus [SE]) and continue as animals become chronically epileptic. Prevention of GABA(A) receptor subunit changes after SE using viral gene transfer inhibits development of epilepsy in an animal model, suggesting that these changes directly contribute to epileptogenesis. The mechanisms that regulate differential expression of GABA(A) receptor subunits in hippocampus after SE have recently been identified, and include the CREB-ICER, JAK-STAT, BDNF, and Egr3 signaling pathways. Targeting signaling pathways that alter the expression of genes involved in epileptogenesis may provide novel therapeutic approaches for preventing or inhibiting the development of epilepsy after a precipitating insult.

The ability of nerve cells to regulate the expression of specific neurotransmitter receptors is of central importance to nervous system function, but little is known about the DNA elements that mediate neuron specific gene expression. The type A gamma-aminobutyric acid (GABA(A)) receptor alpha6-subunit gene, which is expressed exclusively in cerebellar granule cells, presents a unique opportunity to study the cis elements involved in restricting gene expression to a distinct neuronal population. In an effort to identify the regulatory elements that govern cerebellar granule cell-specific gene expression, the proximal 5' flanking regions for the human, rat, and mouse alpha6 genes were cloned and sequenced, and a major transcriptional initiation site was identified in the rodent genes. Functional analysis of rat alpha6 gene-reporter constructs in primary neuronal cultures reveals that a 155-bp TATA-less promoter region (-130 to +25 bp) constitutes a minimal promoter that can drive cerebellar granule cell-specific expression. Internal deletion and decoy competition studies demonstrate that the minimal promoter contains a 60-bp region (-130 to -70 bp) that is critical for enhanced promoter activity in cerebellar granule cells. Activity of the compromised promoter containing the deletion cannot be rescued by placing the 60-bp region downstream of the reporter gene, demonstrating that it is not a classical enhancer but rather a positionally dependent regulator. An additional cerebellar-specific activating sequence is located between -324 and -130 bp, and a downstream negative regulatory region (+158 to +294) has been shown to be active in fibroblasts but inactive in cerebellar granule cells. Taken together, the results suggest a possible mechanism for the control of cerebellar granule cell-specific expression of the GABA(A) receptor alpha6 subunit gene.

The vesicular glutamate (GLU) transporter (VGLUT1) is a critical component of glutamatergic neurons that regulates GLU release. Despite the likely role of GLU release in drug abuse pathology, there is no information that links VGLUT1 with drugs of abuse. This study provides the first evidence that methamphetamine (METH) alters the dynamic regulation of striatal VGLUT1 function and expression through a polysynaptic pathway. METH increases cortical VGLUT1 mRNA, striatal VGLUT1 protein in subcellular fractions, and the V(max) of striatal vesicular GLU uptake. METH also increases glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in the crude vesicle fraction. METH-induced increases in cortical VGLUT1 mRNA, as well as striatal VGLUT1 and GAPDH, are GABA(A) receptor-dependent because they are blocked by GABA(A) receptor antagonism in the substantia nigra. These results show that VGLUT1 can be dynamically regulated via a polysynaptic pathway to facilitate vesicular accumulation of GLU for subsequent release after METH.

Synaptic inhibition in the adult brain is primarily mediated by the γ-aminobutyric acid (GABA) type A receptor (GABA(A)R). The distribution, properties, and dynamics of these receptors are largely determined by their subunit composition. Alteration of subunit composition after a traumatic brain injury (TBI) may result in abnormal increased synaptic firing and possibly contribute to injury-related pathology. Several studies have shown that the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway can alter GABA(A)R subunit expression. The present study investigated changes in JAK/STAT pathway activation after two different severities of experimental TBI in the mouse using the controlled cortical impact (CCI) model. It also investigated whether modulating the activation of the JAK/STAT pathway after severe controlled cortical impact (CCI-S) with a JAK/STAT inhibitor (WP1066) alters post-traumatic epilepsy development and/or neurological recovery after injury. Our results demonstrated differential changes in both the activation of STAT3 and the expression of the GABA(A)R α1 and γ2 subunit levels that were dependent on the severity of the injury. The change in the GABA(A)R α1 subunit levels appeared to be at least partly transcriptionally mediated. We were able to selectively reverse the decrease in GABA(A)R α1 protein levels with WP1066 treatment after CCI injury. WP1066 treatment also improved the degree of recovery of vestibular motor function after injury. These findings suggest that the magnitude of JAK/STAT pathway activation and GABA(A)R α1 subunit level decrease is dependent on injury severity in this mouse model of TBI. In addition, reducing JAK/STAT pathway activation after severe experimental TBI reverses the decrease in the GABA(A)R α1 protein levels and improves vestibular motor recovery.

Brain-derived neurotrophic factor (BDNF) levels are elevated after status epilepticus (SE), leading to activation of multiple signaling pathways, including the janus kinase/signal transducer and activator of transcription pathway that mediates a decrease in GABA A receptor α1 subunits in the hippocampus (Lund et al., 2008). While BDNF can signal via its pro or mature form, the relative contribution of these forms to signaling after SE is not fully known. In the current study, we investigate changes in proBDNF levels acutely after SE in C57BL/6J mice. In contrast to previous reports (Unsain et al., 2008; Volosin et al., 2008; VonDran et al., 2014), our studies found that levels of proBDNF in the hippocampus are markedly elevated as early as 3 h after SE onset and remain elevated for 7 d. Immunohistochemistry studies indicate that seizure-induced BDNF localizes to all hippocampal subfields, predominantly in principal neurons and also in astrocytes. Analysis of the proteolytic machinery that cleaves proBDNF to produce mature BDNF demonstrates that acutely after SE there is a decrease in tissue plasminogen activator and an increase in plasminogen activator inhibitor-1 (PAI-1), an inhibitor of extracellular and intracellular cleavage, which normalizes over the first week after SE. In vitro treatment of hippocampal slices from animals 24 h after SE with a PAI-1 inhibitor reduces proBDNF levels. These findings suggest that rapid proBDNF increases following SE are due in part to reduced cleavage, and that proBDNF may be part of the initial neurotrophin response driving intracellular signaling during the acute phase of epileptogenesis.

Changes in the function of type A γ-aminobutyric acid receptors (GABAARs) are associated with neuronal development and tolerance to the sedative-hypnotic effects of GABAAR positive modulators. Persistent activation of GABAARs by millimolar concentrations of GABA occurs under physiological conditions as GABAergic fast-spiking neurons in neocortex and cerebellum exhibit basal firing rates of 5 to 50 Hz and intermittent rates up to 250 Hz, leaving a substantial fraction of synaptic receptors occupied persistently by GABA. Persistent exposure of neurons to GABA has been shown to cause a down-regulation of receptor number and an uncoupling of GABA/benzodiazepine (BZD) site interactions with a half-life of ∼24 h. Here, we report that a single brief exposure of neocortical neurons in primary culture to GABA for 5-10 min (t1/2 = 3.2 ± 0.2 min) initiates a process that results in uncoupling hours later (t1/2 = 12.1 ± 2.2 h). Initiation of delayed-onset uncoupling is blocked by co-incubation with picrotoxin or α-amanitin but is insensitive to nifedipine, indicating that uncoupling is contingent upon receptor activation and transcription but is not dependent on voltage-gated Ca2+ influx. Delayed-onset uncoupling occurs without a change in receptor number or a change in the proportion of α1 subunit pharmacology, as zolpidem binding affinity is unaltered. Such activity dependent latent modulation of GABAAR function that manifests as delayed-onset uncoupling may be relevant to physiological, pathophysiological, and pharmacological conditions where synaptic receptors are transiently exposed to GABA agonists for several minutes. Fil: Gravielle, Maria Clara. Boston University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Faris, Ramona. Boston University; Estados Unidos Fil: Russek, Shelley J.. Boston University; Estados Unidos Fil: Farb, David Howard. Boston University; Estados Unidos

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 GABAA receptors. Nonetheless, in eight recombinant GABAA 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 Fil: A. Lippa. FACULTAD; Argentina Fil: P. Czobor. FACULTAD; Argentina Fil: J. Stark. FACULTAD; Argentina Fil: B. Beer. FACULTAD; Argentina Fil: E. Kostakis. FACULTAD; Argentina Fil: Gravielle, Maria Clara. ININFA; Argentina Fil: S. Bandyopadhyay. FACULTAD; Argentina Fil: S. J. Russek. FACULTAD; Argentina Fil: T. T. Gibbs. FACULTAD; Argentina Fil: D. H. Farb. FACULTAD; Argentina Fil: P. Skolnick. FACULTAD; Argentina

Transcription mediated by protein kinase A and the cAMP response element binding protein (CREB) has been linked to the establishment of long-term memory and cell survival. However, all of the major targets for activated CREB have yet to be identified. Given the fact that CREB-mediated transcription is intimately involved in cellular processes of learning and memory and that CREB activity can be regulated by synaptic N-methyl-d-aspartate receptors (NMDARs) and metabotropic GABA receptors, we have studied the role of the cAMP-dependent signaling pathway in the regulation of the NMDA receptor subunit 1 (NMDAR1), a subunit required for functional receptor formation. We now report that levels of NMDAR1 subunit protein in primary neocortical cultures are increased 66% in response to forskolin, an activator of adenylyl cyclase. Up-regulation of NMDAR1 is paralleled by a twofold increase in mRNA levels and an 83% increase in NMDAR1 promoter/luciferase reporter activity that is dependent on protein kinase A. Three cAMP regulatory elements (CREs) in the rat NMDAR1 promoter (− 228, − 67, and − 39) bind CREB in vitro and forskolin increases binding to two of the sites (− 228 and – 67). Chromatin immunoprecipitation of neuronal rat genomic DNA reveals that CREB is bound in vivo to the endogenous NMDAR1 gene. Increased presence of the activated Ser133 phosphorylated form is dependent on the length of exposure to forskolin. Taken together with the results of mutational analysis, the findings strongly suggest that transcription of NMDAR1 is regulated by the c-AMP signaling pathway, most likely through the binding of CREB and its activation by signal-dependent phosphorylation.

Fast excitatory synaptic transmission that is contingent upon N-methyl d-aspartate receptor (NMDAR) function contributes to core information flow in the central nervous system and to the plasticity of neural circuits that underlie cognition. Hypoactivity of excitatory NMDAR-mediated neurotransmission is hypothesized to underlie the pathophysiology of schizophrenia, including the associated cognitive deficits. The neurosteroid pregnenolone (PREG) and its metabolites pregnenolone sulfate (PregS) and allopregnanolone in serum are inversely associated with cognitive improvements after oral PREG therapy, raising the possibility that brain neurosteroid levels may be modulated therapeutically. PregS is derived from PREG, the precursor of all neurosteroids, via a single sulfation step and is present at low nanomolar concentrations in the central nervous system. PregS, but not PREG, augments long-term potentiation and cognitive performance in animal models of learning and memory. In this report, we communicate the first observation that PregS, but not PREG, is a potent (EC50 ∼2 pM) enhancer of intracellular Ca(2+) that is contingent upon neuronal activity, NMDAR-mediated synaptic activity, and L-type Ca(2+) channel activity. Low picomolar PregS similarly activates cAMP response element-binding protein (CREB) phosphorylation (within 10 minutes), an essential memory molecule, via an extracellular-signal-regulated kinase/mitogen-activated protein kinase signal transduction pathway. Taken together, the results are consistent with a novel biologic role for the neurosteroid PregS that acts at picomolar concentrations to intensify the intracellular response to glutamatergic signaling at synaptic but not extrasynaptic, NMDARs by differentially augmenting CREB activation. This provides a genomic signal transduction mechanism by which PregS could participate in memory consolidation of relevance to cognitive function.

Pilocarpine-induced status epilepticus (SE), which results in temporal lobe epilepsy (TLE) in rodents, activates the JAK/STAT pathway. In the current study, we evaluate whether brief exposure to a selective inhibitor of the JAK/STAT pathway (WP1066) early after the onset of SE affects the severity of SE or reduces later spontaneous seizure frequency via inhibition of STAT3-regulated gene transcription. Rats that received systemic WP1066 or vehicle at the onset of SE were continuously video-EEG monitored during SE and for one month to assess seizure frequency over time. Protein and/or mRNA levels for pSTAT3, and STAT3-regulated genes including: ICER, Gabra1, c-myc, mcl-1, cyclin D1, and bcl-xl were evaluated in WP1066 and vehicle-treated rats during stages of epileptogenesis to determine the acute effects of WP1066 administration on SE and chronic epilepsy. WP1066 (two 50 mg/kg doses) administered within the first hour after onset of SE results in transient inhibition of pSTAT3 and long-term reduction in spontaneous seizure frequency. WP1066 alters the severity of chronic epilepsy without affecting SE or cell death. Early WP1066 administration reduces known downstream targets of STAT3 transcription 24 h after SE including cyclin D1 and mcl-1 levels, known for their roles in cell-cycle progression and cell survival, respectively. These findings uncover a potential effect of the JAK/STAT pathway after brain injury that is physiologically important and may provide a new therapeutic target that can be harnessed for the prevention of epilepsy development and/or progression.

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.

The regulated expression of type A γ-aminobutyric acid receptor (GABA A R) subunit genes is postulated to play a role in neuronal maturation, synaptogenesis, and predisposition to neurological disease. Increases in GABA levels and changes in GABA A R subunit gene expression, including decreased β1 mRNA levels, have been observed in animal models of epilepsy. Persistent exposure to GABA down-regulates GABA A R number in primary cultures of neocortical neurons, but the regulatory mechanisms remain unknown. Here, we report the identification of a TATA-less minimal promoter of 296 bp for the human GABA A R β1 subunit gene that is neuron specific and autologously down-regulated by GABA. β1 promoter activity, mRNA levels, and subunit protein are decreased by persistent GABA A R activation. The core promoter, 270 bp, contains an initiator element (Inr) at the major transcriptional start site. Three concatenated copies of the 10-bp Inr and its immediate 3′ flanking sequence produce full neural specific activity that is down-regulated by GABA in transiently transfected neocortical neurons. Taking these results together with those of DNase I footprinting, electrophoretic mobility shift analysis, and 2-bp mutagenesis, we conclude that GABA-induced down-regulation of β1 subunit mRNAs involves the differential binding of a sequence-specific basal transcription factor(s) to the Inr. The results support a transcriptional mechanism for the down-regulation of β1 subunit GABA A R gene expression and raises the possibility that altered levels of sequence-specific basal transcription factors may contribute to neurological disorders such as epilepsy.

We have identified a gene encoding a GABAB receptor, the human GABABR2, located on chromosome 9q22.1, that is distinct from the recently reported rat GABABR1. GABABR2 structurally resembles GABABR1 (35% identity), having seven transmembrane domains and a large extracellular region, but differs in having a longer carboxy-terminal tail. GABABR2 is localized to the cell surface in transfected COS cells, and negatively couples to adenylyl cyclase in response to GABA, baclofen, and 3-aminopropyl(methyl)phosphinic acid in CHO cells lacking GABABR1. Baclofen action is inhibited by the GABABR antagonist, 2-hydroxysaclofen. The human GABABR2 and GABABR1 genes are differentially expressed in the nervous system, with the greatest difference being detected in the striatum in which GABABR1 but not GABABR2 mRNA transcripts are detected. GABABR2 and GABABR1 mRNAs are also coexpressed in various brain regions such as the Purkinje cell layer of the cerebellum. Identification of a functional homomeric GABABR2 coupled to adenylyl cyclase suggests that the complexity of GABAB pharmacological data is at least in part due to the presence of more than one receptor and opens avenues for future research leading to an understanding of metabotropic GABA receptor signal transduction mechanisms.

Background The γ-aminobutyric acid (GABA) type A receptor (GABAAR) contains the recognition sites for a variety of agents used in the treatment of brain disorders, including anxiety and epilepsy. A better understanding of how receptor expression is regulated in individual neurons may provide novel opportunities for therapeutic intervention. Towards this goal we have studied transcription of a GABAAR subunit gene (GABRB1) whose activity is autologously regulated by GABA via a 10 base pair initiator-like element (β1-INR). Methods By screening a human cDNA brain library with a yeast one-hybrid assay, the Polycomblike (PCL) gene product PHD finger protein transcript b (PHF1b) was identified as a β1-INR associated protein. Promoter/reporter assays in primary rat cortical cells demonstrate that PHF1b is an activator at GABRB1, and chromatin immunoprecipitation assays reveal that presence of PHF1 at endogenous Gabrb1 is regulated by GABAAR activation. Results PCL is a member of the Polycomb group required for correct spatial expression of homeotic genes in Drosophila. We now show that PHF1b recognition of β1-INR is dependent on a plant homeodomain, an adjacent helix-loop-helix, and short glycine rich motif. In neurons, it co-immunoprecipitates with SUZ12, a key component of the Polycomb Repressive Complex 2 (PRC2) that regulates a number of important cellular processes, including gene silencing via histone H3 lysine 27 trimethylation (H3K27me3). Conclusions The observation that chronic exposure to GABA reduces PHF1 binding and H3K27 monomethylation, which is associated with transcriptional activation, strongly suggests that PHF1b may be a molecular transducer of GABAAR function and thus GABA-mediated neurotransmission in the central nervous system.

The gamma-aminobutyric acid (GABA) type A receptor (GABA(A)R) is responsible for most fast synaptic inhibition in the adult brain. The GABA(A)R protein is composed of multiple subunits that determine the distribution, properties, and dynamics of the receptor. Several studies have shown that the Janus kinase/signal transducer and activator of transcription (JaK/STAT) and early growth response 3 (Egr3) signaling pathways can alter GABA(A)R subunit expression after status epilepticus (SE). In this study we investigated changes in these pathways after experimental TBI in the rat using a lateral fluid percussion injury (FPI) model. Our results demonstrated changes in the expression of several GABA(A)R subunit levels after injury, including GABA(A)R α1 and α4 subunits. This change appears to be transcriptional, and there is an associated increase in the phosphorylation of STAT3, and an increase in the expression of Egr3 and inducible cAMP element repressor (ICER) after FPI. These findings suggest that the activation of the JaK/STAT and Egr3 pathways after TBI may regulate injury-related changes in GABA(A)R subunit expression.

We demonstrated previously that an alpha 1-beta 2-gamma 2 gene cluster of the gamma-aminobutyric acid (GABAA) receptor is located on human chromosome 5q34-q35 and that an ancestral alpha-beta-gamma gene cluster probably spawned clusters on chromosomes 4, 5, and 15. Here, we report that the alpha 4 gene (GABRA4) maps to human chromosome 4p14-q12, defining a cluster comprising the alpha 2, alpha 4, beta 1, and gamma 1 genes. The existence of an alpha 2-alpha 4-beta 1-gamma 1 cluster on chromosome 4 and an alpha 1-alpha 6-beta 2-gamma 2 cluster on chromosome 5 provides further evidence that the number of ancestral GABAA receptor subunit genes has been expanded by duplication within an ancestral gene cluster. Moreover, if duplication of the alpha gene occurred before duplication of the ancestral gene cluster, then a heretofore undiscovered subtype of alpha subunit should be located on human chromosome 15q11-q13 within an alpha 5-alpha x-beta 3-gamma 3 gene cluster at the locus for Angelman and Prader-Willi syndromes.

The gamma-aminobutyric acid receptor (GABAAR) is a multisubunit Cl- channel that mediates most fast inhibitory synaptic transmission in the central nervous system. Molecular evolution has given rise to many genetic variants of GABAAR subunits, including alpha 1-6, beta 1-4, gamma 1-4, delta, and rho 1-2, suggesting that an enormous number of combinations of subunits are possible. Here we report that the beta 2 gene is located on chromosome 5q34-q35, defining a cluster comprising alpha 1, beta 2, and gamma 2 genes that together code for the most abundant GABAAR isoform. The fact that intron position is conserved in the beta 1-3 genes, taken together with the observation that chromosomes 4 and 15 also contain distinct alpha-beta-gamma gene clusters, strongly suggests that an ancestral alpha-beta-gamma cluster was duplicated and translocated to at least two different chromosomes. This organization of GABAAR gene clusters may have been preserved as linkage provides a mechanism for facilitating coordinate gene expression.

The gamma-aminobutyric acid (GABA) type A receptor (GABA(A)R) is the major inhibitory neurotransmitter receptor in the brain. Its multiple subunits show regional, developmental, and disease-related plasticity of expression; however, the regulatory networks controlling GABA(A)R subunit expression remain poorly understood. We report that the seizure-induced decrease in GABA(A)R alpha1 subunit expression associated with epilepsy is mediated by the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway regulated by brain-derived neurotrophic factor (BDNF). BDNF- and seizure-dependent phosphorylation of STAT3 cause the adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB) family member ICER (inducible cAMP early repressor) to bind with phosphorylated CREB at the Gabra1:CRE site. JAK/STAT pathway inhibition prevents the seizure-induced decrease in GABA(A)R alpha1 abundance in vivo and, given that BDNF is known to increase the abundance of GABA(A)R alpha4 in a JAK/STAT-independent manner, indicates that BDNF acts through at least two distinct pathways to influence GABA(A)R-dependent synaptic inhibition.

The regulated expression of type A gamma-aminobutyric acid (GABA) receptor (GABA(A)R) subunit genes plays a critical role in neuronal maturation and synaptogenesis. It is also associated with a variety of neurological diseases. Changes in GABA(A) receptor alpha1 subunit gene (GABRA1) expression have been reported in animal models of epilepsy, alcohol abuse, withdrawal, and stress. Understanding the genetic mechanism behind such changes in alpha subunit expression will lead to a better understanding of the role that signal transduction plays in control over GABA(A)R function and brings with it the promise of providing new therapeutic tools for the prevention or cure of a variety of neurological disorders. Here we show that activation of protein kinase C increases alpha1 subunit levels via phosphorylation of CREB (pCREB) that is bound to the GABRA1 promoter (GABRA1p). In contrast, activation of protein kinase A decreases levels of alpha1 even in the presence of pCREB. Decrease of alpha1 is dependent upon the inducible cAMP early repressor (ICER) as directly demonstrated by ICER-induced down-regulation of endogenous alpha1-containing GABA(A)Rs at the cell surface of cortical neurons. Taken together with the fact that there are less alpha1gamma2-containing GABA(A)Rs in neurons after protein kinase A stimulation and that activation of endogenous dopamine receptors down-regulates alpha1 subunit mRNA levels subsequent to induction of ICER, our studies identify a transcriptional mechanism for regulating the cell surface expression of alpha1-containing GABA(A)Rs that is dependent upon the formation of CREB heterodimers.

Differential expression of GABAAreceptor (GABR) subunits has been demonstrated in hippocampus from patients and animals with temporal lobe epilepsy (TLE), but whether these changes are important for epileptogenesis remains unknown. Previous studies in the adult rat pilocarpine model of TLE found reduced expression of GABR α1 subunits and increased expression of α4 subunits in dentate gyrus (DG) of epileptic rats compared with controls. To investigate whether this altered subunit expression is a critical determinant of spontaneous seizure development, we used adeno-associated virus type 2 containing the α4 subunit gene (GABRA4) promoter to drive transgene expression in DG after status epilepticus (SE). This novel use of a condition-dependent promoter upregulated after SE successfully increased expression of GABR α1 subunit mRNA and protein in DG at 1–2 weeks after SE. Enhanced α1 expression in DG resulted in a threefold increase in mean seizure-free time after SE and a 60% decrease in the number of rats developing epilepsy (recurrent spontaneous seizures) in the first 4 weeks after SE. These findings provide the first direct evidence that altering GABR subunit expression can affect the development of epilepsy and suggest that α1 subunit levels are important determinants of inhibitory function in hippocampus.

Altered function of gamma-aminobutyric acid type A receptors (GABA(A)Rs) in dentate granule cells of the hippocampus has been associated with temporal lobe epilepsy (TLE) in humans and in animal models of TLE. Such altered receptor function (including increased inhibition by zinc and lack of modulation by benzodiazepines) is related, in part, to changes in the mRNA levels of certain GABA(A)R subunits, including alpha4, and may play a role in epileptogenesis. The majority of GABA(A)Rs that contain alpha4 subunits are extra-synaptic due to lack of the gamma2 subunit and presence of delta. However, it has been hypothesized that seizure activity may result in expression of synaptic receptors with altered properties driven by an increased pool of alpha4 subunits. Results of our previous work suggests that signaling via protein kinase C (PKC) and early growth response factor 3 (Egr3) is the plasticity trigger for aberrant alpha4 subunit gene (GABRA4) expression after status epilepticus. We now report that brain derived neurotrophic factor (BDNF) is the endogenous signal that induces Egr3 expression via a PKC/MAPK-dependent pathway. Taken together with the fact that blockade of tyrosine kinase (Trk) neurotrophin receptors reduces basal GABRA4 promoter activity by 50%, our findings support a role for BDNF as the mediator of Egr3-induced GABRA4 regulation in developing neurons and epilepsy and, moreover, suggest that BDNF may alter inhibitory processing in the brain by regulating the balance between phasic and tonic inhibition.

Expression of metabotropic GABABreceptors is essential for slow inhibitory synaptic transmission in the CNS, and disruption of GABABreceptor-mediated responses has been associated with several disorders, including neuropathic pain and epilepsy. The location of GABABreceptors in neurons determines their specific role in synaptic transmission, and it is believed that sorting of subunit isoforms, GABABR1a and GABABR1b, to presynaptic or postsynaptic membranes helps to determine this role. GABABR1a and GABABR1b are thought to arise by alternative splicing of heteronuclear RNA. We now demonstrate that alternative promoters, rather than alternative splicing, produce GABABR1a and GABABR1b isoforms. Our data further show that subunit gene expression in hippocampal neurons is mediated by the cAMP response element-binding protein (CREB) by binding to unique cAMP response elements in the alternative promoter regions. Double-stranded oligonucleotide decoys selectively alter levels of endogenous GABABR1a and GABABR1b in primary hippocampal neurons, and CREB knock-out mice show changes in levels of GABABR1a and GABABR1b transcripts, consistent with decoy competition experiments. These results demonstrate a critical role of CREB in transcriptional mechanisms that control GABABR1 subunit levelsin vivo. In addition, the CREB-related factor activating transcription factor-4 (ATF4) has been shown to interact directly with GABABR1 in neurons, and we show that ATF4 differentially regulates GABABR1a and GABABR1b promoter activity. These results, together with our finding that the depolarization-sensitive upstream stimulatory factor (USF) binds to a composite CREB/ATF4/USF regulatory element only in the absence of CREB binding, indicate that selective control of alternative GABABR1 promoters by CREB, ATF4, and USF may dynamically regulate expression of their gene products in the nervous system.

N -methyl- d -aspartate (NMDA) receptor function is modulated by several endogenous molecules, including zinc, polyamines, protons, and sulfated neurosteroids. Zinc, polyamines, and phenylethanolamines exert their respective modulatory effects by exacerbating or relieving tonic proton inhibition. Here, we report that pregnenolone sulfate (PS) uses a unique mechanism for enhancement of NMDA receptor function that is independent of the proton sensor. We identify a steroid modulatory domain, SMD1, on the NMDA receptor NR2B subunit that is critical for both PS enhancement and proton sensitivity. This domain includes the J/K helices in the S2 region of the glutamate recognition site and the fourth membrane transmembrane region (M4). A molecular model based on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor structure suggests that steroid modulatory domain 1 contributes residues to a hydrophobic pocket that is capable of accommodating PS. The results demonstrate that the J/K helices and the fourth membrane transmembrane region participate in transducing allosteric interactions induced by steroid and proton binding to their respective sites.

Nowhere is the record of receptor evolution more accessible than in the organization of the 19 vertebrate genes coding for subunits of the major inhibitory neurotransmitter receptor in the central nervous system, the gamma-aminobutyric acid receptor (GABAAR). Co-expression of alpha, beta, and gamma subunit genes is necessary for the formation of a GABAAR that is potentiated by widely used anxiolytics, anticonvulsants, and hypnotics. The identification of alpha, beta, and gamma genes on chromosomes 4, 5, and 15 suggests that co-localization of a gamma gene with an alpha and beta may be important for brain function. We have now directly examined the organization of GABAAR subunit genes on human chromosomes. Estimates of physical distance using in situ hybridization to cells in interphase, and gene localization using hybridization to cells in metaphase demonstrate the existence of beta-alpha-alpha-gamma gene clusters in cytogenetic bands on chromosomes 4(p12) and 5(q34). Sequencing of PAC clones establishes intercluster conservation of a unique head-to-head configuration for alpha and beta genes on chromosomes 4, 5, and 15. Remarkably, phylogenetic tree analysis predicts the existence of a beta-alpha-gamma ancestral gene cluster in which internal duplication of an ancestral alpha was followed by cluster duplication, resulting in the relative chromosomal positions of modern GABAAR subunit genes in the human genome.

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.

Although multiple genes drive epileptogenesis, uncovering the mechanisms controlling expression of GABA A receptors (GABA A Rs), the brain's major inhibitory receptors, may have far-reaching therapeutic implications. In this review, we describe how seizure-induced changes in GABA A Rs result from brain-derived neurotrophic factor (BDNF)-induced changes in Janus kinase/ Signal Transducer and Activators of Transcription (JAK/ STAT), cAMP response element binding protein/inducible cAMP early repressor (CREB/ICER), and early growth factor (Egr3) signaling, and discuss implications for prevention and treatment. For an expanded treatment of this topic see Jasper's Basic Mechanisms of the Epilepsies, Fourth Edition (Noebels JL, Avoli M, Rogawski MA, Olsen RW, Delgado-Escueta AV, eds) published by Oxford University Press (available on the National Library of Medicine Bookshelf [NCBI] at www.ncbi.nlm.nih.gov/books).

This review discusses our present knowledge of the structure and activities of the mast cell degranulating peptide (MCDP). This peptide is a basic, 22 amino acid residue component of honey bee venom with striking immunological and pharmacological activities. MCDP is a potent anti-inflammatory agent, but at low concentrations it is a strong mediator of mast cell degranulation and histamine release. MCDP is also an epileptogenic neurotoxin, an avid blocker of the potassium channels and can cause a significant lowering of the blood pressure in rats. Some of the biological activities of MCDP appear to have distinct mechanisms and may represent a good illustration of the structure-function relationship.

Understanding transcription factor (TF) mediated control of gene expression remains a major challenge at the interface of computational and experimental biology. Computational techniques predicting TF-binding site specificity are frequently unreliable. On the other hand, comprehensive experimental validation is difficult and time consuming. We introduce a simple strategy that dramatically improves robustness and accuracy of computational binding site prediction. First, we evaluate the rate of recurrence of computational TFBS predictions by commonly used sampling procedures. We find that the vast majority of results are biologically meaningless. However clustering results based on nucleotide position improves predictive power. Additionally, we find that positional clustering increases robustness to long or imperfectly selected input sequences. Positional clustering can also be used as a mechanism to integrate results from multiple sampling approaches for improvements in accuracy over each one alone. Finally, we predict and validate regulatory sequences partially responsible for transcriptional control of the mammalian type A gamma-aminobutyric acid receptor (GABA(A)R) subunit genes. Positional clustering is useful for improving computational binding site predictions, with potential application to improving our understanding of mammalian gene expression. In particular, predicted regulatory mechanisms in the mammalian GABA(A)R subunit gene family may open new avenues of research towards understanding this pharmacologically important neurotransmitter receptor system.

When a presented stimulus includes three pure‐tone primaries of frequencies f1, f2, and f3, a combination tone generated by two of the primaries may combine with the third primary in generating further combination tones: for instance, 2f1 − (2f2 minus; f3) = 2f1 minus; 2f2 + f3. If the parent combination tone (in this case, 2f2−f3) is eliminated by canceling it with a pure tone of appropriate frequency, the “second‐generation” tone (2f1 − f2 + f3) disappears along with it. The process of combination‐tone generation thus appears to be a recursive one in which a combination tone, once generated, may participate in the generation of other combination tones. This suggests that distortion products in the ear are coupled back to the input of the distorting stage and themselves undergo distortion. Similar experiments using only two primaries suggest that all combination tones of odd order arise mainly from the recursive generation of cubic distortion products. The combination tone 2f2−f1, as well as the tone 2f1...