Spreading depolarization (SD) is a slowly propagating wave of near complete depolarizations of neurons and glia. Previous studies have reported large GABA releases during SD, but there is limited understanding of how GABA release and receptor activation are regulated and influence the propagating SD wavefront, as well as an excitatory phase immediately following the passage of SD. The present study characterized GABA-A type receptor (GABAAR) currents during SD generated by KCl microinjection in acute hippocampal slices from adult mice. Spontaneous GABAAR-mediated currents (sIPSCs) were initially enhanced, and were followed by a large outward current at the wavefront. sIPSC were then transiently supressed during the late SD phase, resulting in a significant reduction of the sIPSC/sEPSC ratio. The large outward current generated during SD was eliminated by the GABAAR antagonist gabazine, but the channel potentiator/agonist propofol failed to potentiate the current, likely because of a ceiling effect. Extracellular Cl- decreases recorded during SD were reduced by the antagonist but were not increased by the potentiator. Together with effects of GABAAR modulators on SD propagation rate, these results demonstrate a significant inhibitory role of the initial GABAAR activation and suggest that intracellular Cl- loading is insufficient to generate excitatory GABAAR responses during SD propagation. These results provide a mechanistic explanation for facilitating effects of GABAAR antagonists, and the lack of inhibitory effect of GABAAR potentiators on SD propagation. In addition, selective suppression of GABA transmission in the late SD period and the lack of effect of GABAA modulators on the duration of SD suggests that GABA modulation may not be effective approach to protect neurons during the vulnerable phase of SD.

Insulin signaling to the brain is important not only for metabolic homeostasis but also for higher brain functions such as cognition. GABA (γ-aminobutyric acid) decreases neuronal excitability by activating GABA(A) channels that generate phasic and tonic currents. The level of tonic inhibition in neurons varies. In the hippocampus, interneurons and dentate gyrus granule cells normally have significant tonic currents under basal conditions in contrast to the CA1 pyramidal neurons where it is minimal. Here we show in acute rat hippocampal slices that insulin (1 nM) "turns on" new extrasynaptic GABA(A) channels in CA1 pyramidal neurons resulting in decreased frequency of action potential firing. The channels are activated by more than million times lower GABA concentrations than synaptic channels, generate tonic currents and show outward rectification. The single-channel current amplitude is related to the GABA concentration resulting in a single-channel GABA affinity (EC(50)) in intact CA1 neurons of 17 pM with the maximal current amplitude reached with 1 nM GABA. They are inhibited by GABA(A) antagonists but have novel pharmacology as the benzodiazepine flumazenil and zolpidem are inverse agonists. The results show that tonic rather than synaptic conductances regulate basal neuronal excitability when significant tonic conductance is expressed and demonstrate an unexpected hormonal control of the inhibitory channel subtypes and excitability of hippocampal neurons. The insulin-induced new channels provide a specific target for rescuing cognition in health and disease.

γ-Aminobutyric acid (GABA) is the most prominent neuroinhibitory transmitter in the brain, where it activates neuronal GABA-A receptors (GABA-A channels) located at synapses and outside of synapses. The GABA-A receptors are primary targets of many clinically useful drugs. In recent years, GABA has been shown to act as an immunomodulatory molecule. We have examined in human, mouse and rat CD4(+) and CD8(+) T cells which subunit isoforms of the GABA-A channels are expressed. The channel physiology and drug specificity is dictated by the GABA-A receptor subtype, which in turn is determined by the subunit isoforms that make the channel. There were 5, 8 and 13 different GABA-A subunit isoforms identified in human, mouse and rat CD4(+) and CD8(+) T cells, respectively. Importantly, the γ2 subunit that imposes benzodiazepine sensitivity on the GABA-A receptors, was only detected in the mouse T cells. Immunoblots and immunocytochemistry showed abundant GABA-A channel proteins in the T cells from all three species. GABA-activated whole-cell transient and tonic currents were recorded. The currents were inhibited by picrotoxin, SR95531 and bicuculline, antagonists of GABA-A channels. Clearly, in both humans and rodents T cells, functional GABA-A channels are expressed but the subtypes vary. It is important to bear in mind the interspecies difference when selecting the appropriate animal models to study the physiological role and pharmacological properties of GABA-A channels in CD4(+) and CD8(+) T cells and when selecting drugs aimed at modulating the human T cells function.

GABA is more than the main inhibitory neurotransmitter found in the adult CNS. Several studies have shown that GABA regulates the proliferation of progenitor and stem cells. This work examined the effects of the GABA(A) receptor system on the proliferation of retinal progenitors and non-pigmented ciliary epithelial (NPE) cells. qRT-PCR and whole-cell patch-clamp electrophysiology were used to characterize the GABA(A) receptor system. To quantify the effects on proliferation by GABA(A) receptor agonists and antagonists, incorporation of thymidine analogues was used. The results showed that the NPE cells express functional extrasynaptic GABA(A) receptors with tonic properties and that low concentration of GABA is required for a baseline level of proliferation. Antagonists of the GABA(A) receptors decreased the proliferation of dissociated E12 NPE cells. Bicuculline also had effects on progenitor cell proliferation in intact E8 and E12 developing retina. The NPE cells had low levels of the Cl-transporter KCC2 compared to the mature retina, suggesting a depolarising role for the GABA(A) receptors. Treatment with KCl, which is known to depolarise membranes, prevented some of the decreased proliferation caused by inhibition of the GABA(A) receptors. This supported the depolarising role for the GABA(A) receptors. Inhibition of L-type voltage-gated Ca(2+) channels (VGCCs) reduced the proliferation in the same way as inhibition of the GABA(A) receptors. Inhibition of the channels increased the expression of the cyclin-dependent kinase inhibitor p27(KIP1), along with the reduced proliferation. These results are consistent with that when the membrane potential indirectly regulates cell proliferation with hyperpolarisation of the membrane potential resulting in decreased cell division. The increased expression of p27(KIP1) after inhibition of either the GABA(A) receptors or the L-type VGCCs suggests a link between the GABA(A) receptors, membrane potential, and intracellular Ca(2+) in regulating the cell cycle.

GABAergic inhibition in the central nervous system (CNS) can occur via rapid, transient postsynaptic currents and via a tonic increase in membrane conductance, mediated by synaptic and extrasynaptic GABA(A) receptors (GABA(A)Rs) respectively. Retinal bipolar cells (BCs) exhibit a tonic current mediated by GABA(C)Rs in their axon terminal, in addition to synaptic GABA(A)R and GABA(C)R currents, which strongly regulate BC output. The tonic GABA(C)R current in BC terminals (BCTs) is not dependent on vesicular GABA release, but properties such as the alternative source of GABA and the identity of the GABA(C)Rs remain unknown. Following a recent report that tonic GABA release from cerebellar glial cells is mediated by Bestrophin 1 anion channels, we have investigated their role in non-vesicular GABA release in the retina. Using patch-clamp recordings from BCTs in goldfish retinal slices, we find that the tonic GABA(C)R current is not reduced by the anion channel inhibitors NPPB or flufenamic acid but is reduced by DIDS, which decreases the tonic current without directly affecting GABA(C)Rs. All three drugs also exhibit non-specific effects including inhibition of GABA transporters. GABA(C)R ρ subunits can form homomeric and heteromeric receptors that differ in their properties, but BC GABA(C)Rs are thought to be ρ1-ρ2 heteromers. To investigate whether GABA(C)Rs mediating tonic and synaptic currents may differ in their subunit composition, as is the case for GABA(A)Rs, we have examined the effects of two antagonists that show partial ρ subunit selectivity: picrotoxin and cyclothiazide. Tonic and synaptic GABA(C)R currents were differentially affected by both drugs, suggesting that a population of homomeric ρ1 receptors contributes to the tonic current. These results extend our understanding of the multiple forms of GABAergic inhibition that exist in the CNS and contribute to visual signal processing in the retina.

In the brain, seizures lead to release of large amounts of polyunsaturated fatty acids including arachidonic acid (ARA). ARA is a substrate for three major enzymatic routes of metabolism by cyclooxygenase, lipoxygenase and cytochrome P450 enzymes. These enzymes convert ARA to potent lipid mediators including prostanoids, leukotrienes and epoxyeicosatrienoic acids (EETs). The prostanoids and leukotrienes are largely pro-inflammatory molecules that sensitize neurons whereas EETs are anti-inflammatory and reduce the excitability of neurons. Recent evidence suggests a GABA-related mode of action potentially mediated by neurosteroids. Here we tested this hypothesis using models of chemically induced seizures. The level of EETs in the brain was modulated by inhibiting the soluble epoxide hydrolase (sEH), the major enzyme that metabolizes EETs to inactive molecules, by genetic deletion of sEH and by direct administration of EETs into the brain. All three approaches delayed onset of seizures instigated by GABA antagonists but not seizures through other mechanisms. Inhibition of neurosteroid synthesis by finasteride partially blocked the anticonvulsant effects of sEH inhibitors while the efficacy of an inactive dose of neurosteroid allopregnanolone was enhanced by sEH inhibition. Consistent with earlier findings, levels of prostanoids in the brain were elevated. In contrast, levels of bioactive EpFAs were decreased following seizures. Overall these results demonstrate that EETs are natural molecules which suppress the tonic component of seizure related excitability through modulating the GABA activity and that exploration of the EET mediated signaling in the brain could yield alternative approaches to treat convulsive disorders.

Glucocorticoids influence vagal parasympathetic output to the viscera via mechanisms that include modulation of neural circuitry in the dorsal vagal complex, a principal autonomic regulatory center. Glucocorticoids can modulate synaptic neurotransmitter release elsewhere in the brain by inducing release of retrograde signalling molecules. We tested the hypothesis that the glucocorticoid agonist dexamethasone (DEX) modulates GABA release in the rat dorsal motor nucleus of the vagus (DMV). Whole-cell patch-clamp recordings revealed that DEX (1-10 µM) rapidly (i.e. within three minutes) increased the frequency of tetrodotoxin-resistant, miniature IPSCs (mIPSCs) in 67% of DMV neurons recorded in acutely prepared slices. Glutamate-mediated mEPSCs were also enhanced by DEX (10 µM), and blockade of ionotropic glutamate receptors reduced the DEX effect on mIPSC frequency. Antagonists of type I or II corticosteroid receptors blocked the effect of DEX on mIPSCs. The effect was mimicked by application of the membrane-impermeant BSA-conjugated DEX, and intracellular blockade of G protein function with GDP βS in the recorded cell prevented the effect of DEX. The enhancement of GABA release was blocked by the TRPV1 antagonists, 5'-iodoresiniferatoxin or capsazepine, but was not altered by the cannabinoid type 1 receptor antagonist AM251. The DEX effect was prevented by blocking fatty acid amide hydrolysis or by inhibiting anandamide transport, implicating involvement of the endocannabinoid system in the response. These findings indicate that DEX induces an enhancement of GABA release in the DMV, which is mediated by activation of TRPV1 receptors on afferent terminals. The effect is likely induced by anandamide or other 'endovanilloid', suggesting activation of a local retrograde signal originating from DMV neurons to enhance synaptic inhibition locally in response to glucocorticoids.

Ionotropic GABA receptors are the targets for several classes of insecticides. One of the most widely-studied insect GABA receptors is RDL (resistance to dieldrin), originally isolated from Drosophila melanogaster. RDL undergoes alternative splicing and RNA editing, which influence the potency of GABA. Most work has focussed on minority isoforms. Here, we report the first characterisation of the predominant native splice variant and RNA edit, combining functional characterisation with molecular modelling of the agonist-binding region. The relative order of agonist potency is GABA> muscimol> TACA> β-alanine. The I/V edit does not alter the potency of GABA compared to RDLbd. Docking calculations suggest that these agonists bind and activate RDLbdI/V through a similar binding mode. TACA and β-alanine are predicted to bind with lower affinity than GABA, potentially explaining their lower potency, whereas the lower potency of muscimol and isoguvacine cannot be explained structurally from the docking calculations. The A301S (resistance to dieldrin) mutation reduced the potency of antagonists picrotoxin, fipronil and pyrafluprole but the I/V edit had no measurable effect. Ivermectin suppressed responses to GABA of RDLbdI/V, RDLbd and RDLbdI/VA301S. The dieldrin resistant variant also showed reduced sensitivity to Ivermectin. This study of a highly abundant insect GABA receptor isoform will help the design of new insecticides.

Cholinergic modulation of hippocampal network function is implicated in multiple behavioral and cognitive states. Activation of nicotinic and muscarinic acetylcholine receptors affects neuronal excitability, synaptic transmission and rhythmic oscillations in the hippocampus. In this work, we studied the ability of the cholinergic system to sustain hippocampal epileptiform activity independently from glutamate and GABA transmission. Simultaneous CA3 and CA1 field potential recordings were obtained during the perfusion of hippocampal slices with the aCSF containing AMPA, NMDA and GABA receptor antagonists. Under these conditions, spontaneous epileptiform discharges synchronous between CA3 and CA1 were recorded. Epileptiform discharges were blocked by addition of the calcium-channel blocker Cd2+ and disappeared in CA1 after a surgical cut between CA3 and CA1. Cholinergic antagonist mecamylamine abolished CA3-CA1 synchronous epileptiform discharges, while antagonists of α7 and α4β2 nAChRs, MLA and DhβE, had no effect. Our results suggest that activation of nicotinic acetylcholine receptors can sustain CA3-CA1 synchronous epileptiform activity independently from AMPA, NMDA and GABA transmission. In addition, mecamylamine, but not α7 and α4β2 nAChRs antagonists, reduced bicuculline-induced seizure-like activity. The ability of mecamylamine to decrease hippocampal network synchronization might be associated with its therapeutic effects in a wide variety of CNS disorders including addiction, depression and anxiety.

The loop C hydrophilic residue, threonine 244 lines the orthosteric binding site of ρ1 GABAC receptors was studied by point mutation into serine, alanine and cysteine, and tested with GABA, some representative partial agonists and antagonists. Thr244 has a hydroxyl group essential for GABA activity that is constrained by the threonine methyl group, orienting it toward the binding site. Significant decreases in activation effects of the studied ligands at ρ1 T244S mutant receptors, suggests a critical role for this residue. Results of aliphatic and heteroaromatic partial agonists demonstrate different pharmacological effects at ρ1 T244S mutant receptors when co-applied with GABA EC50 responses. ρ1 T244A and ρ1 T244C mutant receptors have minimal sensitivity to GABA at high mM concentrations, whereas, the ρ1 WT partial agonists, β-alanine and MTSEA demonstrate more efficacy and potency, respectively, than GABA at these mutant receptors. This study explores the role of Thr244 in the binding of agonists as an initial step during channel gating by moving loop C towards the ligand.

GABAA receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of δ subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (α6100Q) allele show that modulators and agonists selective for δ-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that δ subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR δ subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants.

Recent interest in reversal of the hypnotic effects of anesthesia has mainly focused on overcoming a surge in GABA-mediated inhibitory signaling through activation of subcortical arousal circuits or antagonizing GABA receptors. Here we examine the reversal of anesthesia produced from non-GABA agents ketamine/xylazine and the effects of antagonists of adrenoreceptors. These antagonists vary in selectivity and produce temporally unique waking behavior post-anesthesia. We compared two antagonists with differential selectivity for α1- vs. α2-receptors, yohimbine (YOH, 1:40 selectivity) and atipamezole (ATI, 1:8500). Adult mice received intraperitoneal injections of either YOH (4.3 mg/kg), ATI (0.4 mg/kg), or saline after achieving sustained loss of righting following injection of ketamine/xylazine (ketamine: 65.0 mg/kg; xylazine: 9.9 mg/kg). Behaviors indicative of the post-anesthesia, re-animation sequence were carefully monitored and the timing of each behavior relative to anesthesia induction was compared. Both YOH and ATI hastened behaviors indicative of emergence, but ATI was faster than YOH to produce certain behaviors, including whisker movement (YOH: 21.9±1.5 min, ATI: 17.5±0.5 min, p = 0.004) and return of righting reflex (RORR) (YOH: 40.6±8.8 min, ATI: 26.0±1.2 min, p<0.001). Interestingly, although YOH administration hastened early behavioral markers of emergence relative to saline (whisking), the completion of the emergence sequence (time from first marker to appearance of RORR) was delayed with YOH. We attribute this effect to antagonism of α1 receptors by yohimbine. Also notable was the failure of either antagonist to hasten the re-establishment of coordinated motor behavior (e.g., attempts to remove adhesive tape on the forepaw placed during anesthesia) relative to the end of emergence (RORR). In total, our work suggests that in addition to pharmacokinetic effects, re-establishment of normal waking behaviors after anesthesia involves neuronal circuits dependent on time and/or activity.

Lung diseases such as chronic obstructive pulmonary disease (COPD), asthma, and lung infections are major causes of morbidity and mortality among HIV-infected patients even in the era of antiretroviral therapy (ART). Many of these diseases are strongly associated with smoking and smoking is more common among HIV-infected than uninfected people; however, HIV is an independent risk factor for chronic bronchitis, COPD, and asthma. The mechanism by which HIV promotes these diseases is unclear. Excessive airway mucus formation is a characteristic of these diseases and contributes to airway obstruction and lung infections. HIV gp120 plays a critical role in several HIV-related pathologies and we investigated whether HIV gp120 promoted airway mucus formation in normal human bronchial epithelial (NHBE) cells. We found that NHBE cells expressed the HIV-coreceptor CXCR4 but not CCR5 and produced mucus in response to CXCR4-tropic gp120. The gp120-induced mucus formation was blocked by the inhibitors of CXCR4, α7-nicotinic acetylcholine receptor (α7-nAChR), and γ-aminobutyric acid (GABA)AR but not the antagonists of CCR5 and epithelial growth factor receptor (EGFR). These results identify two distinct pathways (α7-nAChR-GABAAR and EGFR) for airway mucus formation and demonstrate for the first time that HIV-gp120 induces and regulates mucus formation in the airway epithelial cells through the CXCR4-α7-nAChR-GABAAR pathway. Interestingly, lung sections from HIV ± ART and simian immunodeficiency virus (SIV) ± ART have significantly more mucus and gp120-immunoreactivity than control lung sections from humans and macaques, respectively. Thus, even after ART, lungs from HIV-infected patients contain significant amounts of gp120 and mucus that may contribute to the higher incidence of obstructive pulmonary diseases in this population.

Repetitive visual training paired with electrical activation of cholinergic projections to the primary visual cortex (V1) induces long-term enhancement of cortical processing in response to the visual training stimulus. To better determine the receptor subtypes mediating this effect the selective pharmacological blockade of V1 nicotinic (nAChR), M1 and M2 muscarinic (mAChR) or GABAergic A (GABAAR) receptors was performed during the training session and visual evoked potentials (VEPs) were recorded before and after training. The training session consisted of the exposure of awake, adult rats to an orientation-specific 0.12 CPD grating paired with an electrical stimulation of the basal forebrain for a duration of 1 week for 10 minutes per day. Pharmacological agents were infused intracortically during this period. The post-training VEP amplitude was significantly increased compared to the pre-training values for the trained spatial frequency and to adjacent spatial frequencies up to 0.3 CPD, suggesting a long-term increase of V1 sensitivity. This increase was totally blocked by the nAChR antagonist as well as by an M2 mAChR subtype and GABAAR antagonist. Moreover, administration of the M2 mAChR antagonist also significantly decreased the amplitude of the control VEPs, suggesting a suppressive effect on cortical responsiveness. However, the M1 mAChR antagonist blocked the increase of the VEP amplitude only for the high spatial frequency (0.3 CPD), suggesting that M1 role was limited to the spread of the enhancement effect to a higher spatial frequency. More generally, all the drugs used did block the VEP increase at 0.3 CPD. Further, use of each of the aforementioned receptor antagonists blocked training-induced changes in gamma and beta band oscillations. These findings demonstrate that visual training coupled with cholinergic stimulation improved perceptual sensitivity by enhancing cortical responsiveness in V1. This enhancement is mainly mediated by nAChRs, M2 mAChRs and GABAARs. The M1 mAChR subtype appears to be involved in spreading the enhancement of V1 cortical responsiveness to adjacent neurons.

Most studies of IR effects on neural cells and tissues in the brain are still focused on loss of neural stem cells. On the other hand, the effects of IR on neuronal differentiation and its implication in IR-induced brain damage are not well defined. To investigate the effects of IR on C17.2 mouse neural stem-like cells and mouse primary neural stem cells, neurite outgrowth and expression of neuronal markers and neuronal function-related genes were examined. To understand this process, the signaling pathways including PI3K, STAT3, metabotrophic glutamate receptor 1 (mGluR1) and p53 were investigated. In C17.2 cells, irradiation significantly increased the neurite outgrowth, a morphological hallmark of neuronal differentiation, in a dose-dependent manner. Also, the expression levels of neuronal marker proteins, β-III tubulin were increased by IR. To investigate whether IR-induced differentiation is normal, the expression of neuronal function-related genes including synaptophysin, a synaptic vesicle forming proteins, synaptotagmin1, a calcium ion sensor, γ-aminobutyric acid (GABA) receptors, inhibitory neurotransmitter receptors and glutamate receptors, excitatory neurotransmitter receptors was examined and compared to that of neurotrophin-stimulated differentiation. IR increased the expression of synaptophysin, synaptotagmin1 and GABA receptors mRNA similarly to normal differentiation by stimulation of neurotrophin. Interestingly, the overall expression of glutamate receptors was significantly higher in irradiated group than normal differentiation group, suggesting that the IR-induced neuronal differentiation may cause altered neuronal function in C17.2 cells. Next, the molecular mechanism of the altered neuronal differentiation induced by IR was studied by investigating signaling pathways including p53, mGluR1, STAT3 and PI3K. Increases of neurite outgrowth, neuronal marker and neuronal function-related gene expressions by IR were abolished by inhibition of p53, mGluR-1, STAT3 or PI3K. The inhibition of PI3K blocked both p53 signaling and STAT3-mGluR1 signaling but inhibition of p53 did not affect STAT3-mGluR1 signaling in irradiated C17.2 cells. Finally, these results of the IR-induced altered differentiation in C17.2 cells were verified in ex vivo experiments using mouse primary neural stem cells. In conclusion, the results of this study demonstrated that IR is able to trigger the altered neuronal differentiation in undifferentiated neural stem-like cells through PI3K-STAT3-mGluR1 and PI3K-p53 signaling. It is suggested that the IR-induced altered neuronal differentiation may play a role in the brain dysfunction caused by IR.

The hypocretin (orexin) system is involved in sleep/wake regulation, and antagonists of both hypocretin receptor type 1 (HCRTR1) and/or HCRTR2 are considered to be potential hypnotic medications. It is currently unclear whether blockade of either or both receptors is more effective for promoting sleep with minimal side effects. Accordingly, we compared the properties of selective HCRTR1 (SB-408124 and SB-334867) and HCRTR2 (EMPA) antagonists with that of the dual HCRTR1/R2 antagonist almorexant in the rat. All 4 antagonists bound to their respective receptors with high affinity and selectivity in vitro. Since in vivo pharmacokinetic experiments revealed poor brain penetration for SB-408124, SB-334867 was selected for subsequent in vivo studies. When injected in the mid-active phase, SB-334867 produced small increases in rapid-eye-movement (REM) and non-REM (NR) sleep. EMPA produced a significant increase in NR only at the highest dose studied. In contrast, almorexant decreased NR latency and increased both NR and REM proportionally throughout the subsequent 6 h without rebound wakefulness. The increased NR was due to a greater number of NR bouts; NR bout duration was unchanged. At the highest dose tested (100 mg/kg), almorexant fragmented sleep architecture by increasing the number of waking and REM bouts. No evidence of cataplexy was observed. HCRTR1 occupancy by almorexant declined 4-6 h post-administration while HCRTR2 occupancy was still elevated after 12 h, revealing a complex relationship between occupancy of HCRT receptors and sleep promotion. We conclude that dual HCRTR1/R2 blockade is more effective in promoting sleep than blockade of either HCRTR alone. In contrast to GABA receptor agonists which induce sleep by generalized inhibition, HCRTR antagonists seem to facilitate sleep by reducing waking "drive".

In in vitro fertilization (IVF) cycles controlled ovarian hyperstimulation (COH) is established by gonadotropins in combination with gonadotropin-releasing hormone (GnRH) agonists or antagonists, to prevent premature luteinizing hormone (LH) surge. The aim of our study was to improve the understanding of gene expression profile of cumulus cells (CC) in terms of ovarian stimulation protocol and oocyte maturity. We applied Affymetrix gene expression profiling in CC of oocytes at different maturation stages using either GnRH agonists or GnRH antagonists. Two analyses were performed: the first involved CC of immature metaphase I (MI) and mature metaphase II (MII) oocytes where 359 genes were differentially expressed, and the second involved the two GnRH analogues where no differentially expressed genes were observed at the entire transcriptome level. A further analysis of 359 differentially genes was performed, focusing on anti-Müllerian hormone receptor 2 (AMHR2), follicle stimulating hormone receptor (FSHR), vascular endothelial growth factor C (VEGFC) and serine protease inhibitor E2 (SERPINE2). Among other differentially expressed genes we observed a marked number of new genes connected to cell adhesion and neurotransmitters such as dopamine, glycine and γ-Aminobutyric acid (GABA). No differential expression in CC between the two GnRH analogues supports the findings of clinical studies where no significant difference in live birth rates between both GnRH analogues has been proven.

A number of antiepileptic medications that modulate GABA(A) mediated synaptic transmission are anxiolytic. The loop diuretics furosemide (Lasix) and bumetanide (Bumex) are thought to have antiepileptic properties. These drugs also modulate GABA(A) mediated signalling through their antagonism of cation-chloride cotransporters. Given that loop diuretics may act as antiepileptic drugs that modulate GABAergic signalling, we sought to investigate whether they also mediate anxiolytic effects. Here we report the first investigation of the anxiolytic effects of these drugs in rat models of anxiety. Furosemide and bumetanide were tested in adult rats for their anxiolytic effects using four standard anxiety models: 1) contextual fear conditioning; 2) fear-potentiated startle; 3) elevated plus maze, and 4) open-field test. Furosemide and bumetanide significantly reduced conditioned anxiety in the contextual fear-conditioning and fear-potentiated startle models. At the tested doses, neither compound had significant anxiolytic effects on unconditioned anxiety in the elevated plus maze and open-field test models. These observations suggest that loop diuretics elicit significant anxiolytic effects in rat models of conditioned anxiety. Since loop diuretics are antagonists of the NKCC1 and KCC2 cotransporters, these results implicate the cation-chloride cotransport system as possible molecular mechanism involved in anxiety, and as novel pharmacological target for the development of anxiolytics. In view of these findings, and since furosemide and bumetanide are safe and well tolerated drugs, the clinical potential of loop diuretics for treating some types of anxiety disorders deserves further investigation.

Understanding how seizures spread throughout the brain is an important problem in the treatment of epilepsy, especially for implantable devices that aim to avert focal seizures before they spread to, and overwhelm, the rest of the brain. This paper presents an analysis of the speed of propagation in a computational model of seizure-like activity in a 2-dimensional recurrent network of integrate-and-fire neurons containing both excitatory and inhibitory populations and having a difference of Gaussians connectivity structure, an approximation to that observed in cerebral cortex. In the same computational model network, alternative mechanisms are explored in order to simulate the range of seizure-like activity propagation speeds (0.1-100 mm/s) observed in two animal-slice-based models of epilepsy: (1) low extracellular [Formula: see text], which creates excess excitation and (2) introduction of gamma-aminobutyric acid (GABA) antagonists, which reduce inhibition. Moreover, two alternative connection topologies are considered: excitation broader than inhibition, and inhibition broader than excitation. It was found that the empirically observed range of propagation velocities can be obtained for both connection topologies. For the case of the GABA antagonist model simulation, consistent with other studies, it was found that there is an effective threshold in the degree of inhibition below which waves begin to propagate. For the case of the low extracellular [Formula: see text] model simulation, it was found that activity-dependent reductions in inhibition provide a potential explanation for the emergence of slowly propagating waves. This was simulated as a depression of inhibitory synapses, but it may also be achieved by other mechanisms. This work provides a localised network understanding of the propagation of seizures in 2-dimensional centre-surround networks that can be tested empirically.

Bmps regulate numerous neural functions with their regulators. We previously identified Brorin, a neural-specific secreted antagonist of Bmp signaling, in humans, mice, and zebrafish. Mouse Brorin has two cysteine-rich domains containing 10 cysteine residues in its core region, and these are located in similar positions to those in the cysteine-rich domains of Chordin family members, which are secreted Bmp antagonists. Zebrafish Brorin had two cysteine-rich domains with high similarity to those of mouse Brorin. We herein examined zebrafish brorin in order to elucidate its in vivo actions. Zebrafish brorin was predominantly expressed in developing neural tissues. The overexpression of brorin led to the inactivation of Bmp signaling. On the other hand, the knockdown of brorin resulted in the activation of Bmp signaling and brorin morphants exhibited defective development of the ventral domain in the forebrain. Furthermore, the knockdown of brorin inhibited the generation of γ-aminobutyric acid (GABA)ergic interneurons and oligodendrocytes and promoted the generation of astrocytes in the forebrain. In addition, brorin was required for axon guidance in the forebrain. The present results suggest that Brorin is a secreted Bmp antagonist predominantly expressed in developing neural tissues and that it plays multiple roles in the development of the zebrafish forebrain.