Γ-aminobutyric Acid Receptor Type Research Articles

Long-Interval Intracortical Inhibition and the Cortical Silent Period in Youth.

The cortical silent period (CSP) and long-interval intracortical inhibition (LICI) are putative markers of γ-aminobutyric acid receptor type B (GABAB)-mediated inhibitory neurotransmission. We aimed to assess the association between LICI and CSP in youths. We analyzed data from three previous studies of youth who underwent CSP and LICI measurements with transcranial magnetic stimulation and electromyography. We assessed CSP and LICI association using Spearman rank correlation tests and multiple linear regression analyses adjusted for demographic and clinical covariates. The sample included 16 healthy participants and 45 participants with depression. The general mean (SD) age was 15.5 (1.7), 14.3 (1.7) for healthy participants, and 15.9 (1.6) years for participants with depression. Measures were nonnormally distributed (Shapiro-Wilk, p < 0.001). CSP and LICI were not correlated at 100-millisecond (ρ = -0.2421, p = 0.06), 150-millisecond (ρ = -0.1612, p = 0.21), or 200-millisecond (ρ = -0.0507, p = 0.70) interstimulus intervals using Spearman rank correlation test. No correlations were found in the multiple regression analysis (p = 0.35). Although previous studies suggest that cortical silent period and long-interval intracortical inhibition measure GABAB receptor-mediated activity, these biomarkers were not associated in our sample of youths. Future studies should focus on the specific physiologic and pharmacodynamic properties assessed by CSP and LICI in younger populations.

Oligomerization of the heteromeric γ-aminobutyric acid receptor GABAB in a eukaryotic cell-free system

Understanding the assembly mechanism and function of membrane proteins is a fundamental problem in biochemical research. Among the membrane proteins, G protein-coupled receptors (GPCRs) represent the largest class in the human body and have long been considered to function as monomers. Nowadays, the oligomeric assembly of GPCRs is widely accepted, although the functional importance and therapeutic intervention remain largely unexplored. This is partly due to difficulties in the heterologous production of membrane proteins. Cell-free protein synthesis (CFPS) with its endogenous endoplasmic reticulum-derived structures has proven as a technique to address this issue. In this study, we investigate for the first time the conceptual CFPS of a heteromeric GPCR, the γ-aminobutyric acid receptor type B (GABAB), from its protomers BR1 and BR2 using a eukaryotic cell-free lysate. Using a fluorescence-based proximity ligation assay, we provide evidence for colocalization and thus suggesting heterodimerization. We prove the heterodimeric assembly by a bioluminescence resonance energy transfer saturation assay providing the manufacturability of a heterodimeric GPCR by CFPS. Additionally, we show the binding of a fluorescent orthosteric antagonist, demonstrating the feasibility of combining the CFPS of GPCRs with pharmacological applications. These results provide a simple and powerful experimental platform for the synthesis of heteromeric GPCRs and open new perspectives for the modelling of protein–protein interactions. Accordingly, the presented technology enables the targeting of protein assemblies as a new interface for pharmacological intervention in disease-relevant dimers.

Prolonging effects of Valeriana fauriei root extract on pentobarbital-induced sleep in caffeine-induced insomnia model mice and the pharmacokinetics of its active ingredients under conditions of glycerol fatty acid ester as emulsifiers

Ethnopharmacological relevanceValeriana plant roots have traditionally been used to treat central nervous system-related disorders in European countries. Among this genus, the Japanese Pharmacopoeia registers the dried roots of V. fauriei Briq. (VF). However, insufficient pharmacological data are available for this species. Aim of the studyWe investigated the sedative effects of VF extract in a murine caffeine-induced insomnia model as well as the active ingredients and their pharmacokinetics to determine its basic pharmacological action mechanisms under conditions glycerol fatty acid ester is used as emulsifiers. Materials and methodsA murine insomnia model was created by caffeine. Samples derived from the ethanol extract of VF were administered per oral (p.o.), and caffeine was injected intraperitoneally (i.p.). Pentobarbital was injected i.p. and the sleep latency and duration were measured. To confirm the mechanism of action of VF, flumazenil, a specific γ-aminobutyric acid receptor type A (GABAA receptor) antagonist, was administered (i.p.) immediately prior to the sample administration. We examined the pharmacokinetic profiles of the active ingredients in the plasma, brain, urine, and feces of mice after the administration (p.o and intravenous (i.v.)) of VF samples. ResultsVF extract (5 g as VF/kg, p.o.) significantly shorten sleep latency and prolonged pentobarbital-induced sleep in caffeine-induced insomnia mice, partially mediated via the GABAergic nervous system, although a higher dose (10 g as VF/kg, p.o.) was required to exhibit the significant effects in normal mice. Kessyl glycol diacetate (KGD), the main constitutive compound in VF, did not shorten sleep latency but exhibited the same sleep prolonged effect at a dose related to VF extract. The concentration of kessyl glycol 8-acetate (KG8) in the plasma was higher than that of KGD in mice treated (p.o.) with VF extract. The profiles of brain concentrations of KGD and KG8 were similar to those in the plasma, and approximately 20% of those in the plasma were distributed throughout the brain. The excretions of KGD and KG8 in urine and feces was slightly detected, and an unknown large peak related to KG8 was detected in the urine of mice administered with VF extract by HPLC-MS/MS analysis. ConclusionsVF exhibits more sedative effects under stressed conditions, such as insomnia, and the major active ingredients are KGD and its metabolite KG8, which are distributed from the blood circulation into the brain by simple diffusion. KG8 is further metabolized into other metabolites that are easily excreted in the urine.

Role of GABAAR in the Transition From Acute to Chronic Pain and the Analgesic Effect of Electroacupuncture on Hyperalgesic Priming Model Rats.

Chronic pain is a costly health problem that impairs health-related quality of life when not effectively treated. Regulating the transition from acute to chronic pain is a new therapeutic strategy for chronic pain that presents a major clinical challenge. The underlying mechanisms of pain transition are not entirely understood, and strategies for preventing this transition are lacking. Here, a hyperalgesic priming model was used to study the potential mechanism by which γ-aminobutyric acid receptor type A (GABAAR) in the dorsal root ganglion (DRG) contributes to pain transition. Furthermore, electroacupuncture (EA), a modern method of acupuncture, was administered to regulate pain transition, and the mechanism underlying EA’s regulatory effect was investigated. Hyperalgesic priming was induced by intraplanar injection of carrageenan (Car)/prostaglandin E2 (PGE2). The decrease in mechanical withdrawal threshold (MWT) induced by PGE2 returned to baseline 4 h after injection in NS + PGE2 group, and still persisted 24 h after injection in Car + PGE2 group. Lower expression of GABAAR in the lumbar DRG was observed in the model rats. Furthermore, activating or blocking GABAAR could reversed the long-lasting hyperalgesia induced by Car/PGE2 injection or produced a persistent hyperalgesia. In addition, GABAAR may be involved in Protein Kinase C epsilon (PKCε) activation in the DRG, a mark molecular of pain transition. EA considerably increased the mechanical pain thresholds of hyperalgesic priming model mammals in both the acute and chronic phases. Furthermore, EA upregulated the expression of GABAAR and inhibited the activation of PKCε in the DRG. In addition, peripheral administration of picrotoxin blocked the analgesic effect of EA on the model rats and abolished the regulatory effect of EA on PKCε activation. These findings suggested that GABAAR plays a key role in both the transition from acute to chronic pain and the analgesic effect of EA on hyperalgesic priming.

Binding activity of Valeriana fauriei root extract on GABAA receptor flunitrazepam sites and distribution of its active ingredients in the brain of mice – A comparison with that of V. officinalis root

Ethnopharmacological relevanceValeriana fauriei root (VF) is a crude drug registered in the Japanese Pharmacopeia 17th Edition and a known substitute for V. officinalis (VO). Although VO has been pharmacologically evaluated for its sedative effects and mechanism of action, data regarding VF remain scarce. Aim of the studyWe compared the binding affinity of VF and VO extracts, as well as examined the active ingredients in the VF extract, on flunitrazepam sites of γ-aminobutyric acid receptor type A (GABAA receptor). Furthermore, we confirmed whether these active ingredients were distributed in the brain of mice orally administered the VF extract. Materials and methodsWe prepared the assay system to evaluate the binding activity of flunitrazepam sites of GABAA receptor using a 96-well plate and assessed the activities of VF and VO extracts. We then analyzed their constituents using HPLC with principal component analysis (PCA) and evaluated active ingredients correlated with their activities. The distribution of active ingredients in the plasma and brain of mice orally administered the VF extract prepared with different emulsifiers were analyzed by LC-MS/MS. ResultsThe ethanol extract of VF exhibited significantly higher activity on flunitrazepam sites of GABAA receptor than VO. For the VF extract, kessyl glycol diacetate (KGD) was markedly associated with the binding activities; however, active ingredients included KGD, kessyl glycol 8-acetate (KG8), α-kessyl acetate (α-KA), and coniferyl isovalerate (CI). For VO, valerenic acid and five other compounds were associated with the binding affinity on flunitrazepam sites of GABAA receptor. On emulsifying the VF extract with a fat-soluble glycerin fatty acid ester, the plasma and brain distributions of KGD tended to be higher, those of KG8 were significantly more than 10-times higher, and those of α-KA was lower than those of the VF extract emulsified with water-soluble gum arabic, after oral administration in mice. ConclusionsBased on the binding activity on flunitrazepam sites of GABAA receptor and brain distribution, KGD, KG8, and α-KA can be considered active ingredients of VF. The addition of a fat-soluble emulsifier promoted the absorption of KGD, the main active ingredient, and KGD was metabolized to KG8 in the body. The present results suggest a possible mechanism underlying the sedative effect for VF, and these three compounds can be used as marker compounds to evaluate the quality of VF products.

Axonal mechanisms mediating γ-aminobutyric acid receptor type A (GABA-A) inhibition of striatal dopamine release.

Axons of dopaminergic neurons innervate the striatum where they contribute to movement and reinforcement learning. Past work has shown that striatal GABA tonically inhibits dopamine release, but whether GABA-A receptors directly modulate transmission or act indirectly through circuit elements is unresolved. Here, we use whole-cell and perforated-patch recordings to test for GABA-A receptors on the main dopaminergic neuron axons and branching processes within the striatum of adult mice. Application of GABA depolarized axons, but also decreased the amplitude of axonal spikes, limited propagation and reduced striatal dopamine release. The mechanism of inhibition involved sodium channel inactivation and shunting. Lastly, we show the positive allosteric modulator diazepam enhanced GABA-A currents on dopaminergic axons and directly inhibited release, but also likely acts by reducing excitation from cholinergic interneurons. Thus, we reveal the mechanisms of GABA-A receptor modulation of dopamine release and provide new insights into the actions of benzodiazepines within the striatum.

A half century of γ-aminobutyric acid.

γ-aminobutyric acid has become one of the most widely known neurotransmitter molecules in the brain over the last 50 years, recognised for its pivotal role in inhibiting neural excitability. It emerged from studies of crustacean muscle and neurons before its significance to the mammalian nervous system was appreciated. Now, after five decades of investigation, we know that most neurons are γ-aminobutyric-acid-sensitive, it is a cornerstone of neural physiology and dysfunction to γ-aminobutyric acid signalling is increasingly documented in a range of neurological diseases. In this review, we briefly chart the neurodevelopment of γ-aminobutyric acid and its two major receptor subtypes: the γ-aminobutyric acidA and γ-aminobutyric acidB receptors, starting from the humble invertebrate origins of being an ‘interesting molecule’ acting at a single γ-aminobutyric acid receptor type, to one of the brain’s most important neurochemical components and vital drug targets for major therapeutic classes of drugs. We document the period of molecular cloning and the explosive influence this had on the field of neuroscience and pharmacology up to the present day and the production of atomic γ-aminobutyric acidA and γ-aminobutyric acidB receptor structures. γ-Aminobutyric acid is no longer a humble molecule but the instigator of rich and powerful signalling processes that are absolutely vital for healthy brain function.

Antagonistic effect of flumazenil on tiletamine-zolazepam-induced anaesthesia in Beagle dogs

Benzodiazepines exert hypnotic/sedative effects through their inhibitory actions on the γ-aminobutyric acid receptor type A. Since flumazenil antagonises these effects through competitive inhibition of the receptor, it has been used to reverse the effect of benzodiazepines. The goal of this study was to characterise the antagonistic effect of flumazenil on anaesthesia induced by tiletamine-zolazepam in dogs. Nine healthy Beagle dogs (four males, five females) were used in this study. The dogs were administered 20 mg/kg of tiletamine-zolazepam intravenously and were then intravenously treated with saline solution (2 ml; control) or flumazenil twenty minutes after tiletamine-zolazepam administration at doses of 0.02, 0.04, 0.06, 0.08 or 0.16 mg/kg. Recovery times after the anaesthesia and cardiorespiratory variation were recorded for each dog. The results of this study indicate that the duration of reversal produced by doses of 0.04 and 0.06 mg/kg flumazenil was more effective than that produced by any of the other doses. In addition, sedation was rapidly reversible at 0.04 and 0.06 mg/kg without resedation. However, at doses of 0.08 and 0.16 mg/kg adverse effects such as shivering, rigidity and opisthotonos were observed. Thus, treatment with flumazenil at doses of 0.04 and 0.06 mg/kg could successfully reverse the anaesthetic effects induced by tiletamine-zolazepam.

γ-Aminobutyric Acid Type A Receptor Potentiation Inhibits Learning in a Computational Network Model.

Propofol produces memory impairment at concentrations well below those abolishing consciousness. Episodic memory, mediated by the hippocampus, is most sensitive. Two potentially overlapping scenarios may explain how γ-aminobutyric acid receptor type A (GABAA) potentiation by propofol disrupts episodic memory-the first mediated by shifting the balance from excitation to inhibition while the second involves disruption of rhythmic oscillations. We use a hippocampal network model to explore these scenarios. The basis for these experiments is the proposal that the brain represents memories as groups of anatomically dispersed strongly connected neurons. A neuronal network with connections modified by synaptic plasticity was exposed to patterned stimuli, after which spiking output demonstrated evidence of stimulus-related neuronal group development analogous to memory formation. The effect of GABAA potentiation on this memory model was studied in 100 unique networks. GABAA potentiation consistent with moderate propofol effects reduced neuronal group size formed in response to a patterned stimulus by around 70%. Concurrently, accuracy of a Bayesian classifier in identifying learned patterns in the network output was reduced. Greater potentiation led to near total failure of group formation. Theta rhythm variations had no effect on group size or classifier accuracy. Memory formation is widely thought to depend on changes in neuronal connection strengths during learning that enable neuronal groups to respond with greater facility to familiar stimuli. This experiment suggests the ability to form such groups is sensitive to alteration in the balance between excitation and inhibition such as that resulting from administration of a γ-aminobutyric acid-mediated anesthetic agent.

Influence of GABA-B Agonist Baclofen on Capsaicin-Induced Excitation of Secondary Peristalsis in Humans.

Objectives:Esophageal instillation of capsaicin enhances secondary peristalsis, but the γ-aminobutyric acid receptor type B (GABA-B) agonist baclofen inhibits secondary peristalsis. This study aimed to investigate whether baclofen could influence heartburn perception and secondary peristalsis subsequent to capsaicin infusion in healthy adults.Methods:Secondary peristalsis was performed by slow and rapid mid-esophagus air injections in 15 healthy subjects. Two different sessions including esophageal infusion of capsaicin-containing red pepper sauce (0.84 mg) following pre-treatment with placebo or baclofen were randomly performed to test the effects on heartburn perception and secondary peristalsis.Results:The intensity of heartburn symptom subsequent to capsaicin infusion was significantly greater after pre-treatment of baclofen as compared with the placebo (P=0.03). Baclofen significantly increased the threshold volume of secondary peristalsis to slow air injections subsequent to esophageal capsaicin infusion (P<0.001). Baclofen significantly increased the threshold volume of secondary peristalsis to rapid air injections subsequent to esophageal capsaicin infusion (P<0.01). The frequency of secondary peristalsis subsequent to capsaicin infusion was significantly decreased with baclofen as compared with the placebo (P<0.002). Baclofen had no effect on any of the peristaltic parameters of secondary peristalsis subsequent to capsaicin infusion.Conclusions:The GABA-B agonist baclofen appears to attenuate the esophagus to capsaicin-induced excitation of secondary peristalsis in healthy adults. Our study suggests the inhibitory modulation for GABA-B receptors on capsaicin-sensitive afferents mediating secondary peristalsis in human esophagus.

Ketamine Increases the Function of γ-Aminobutyric Acid Type A Receptors in Hippocampal and Cortical Neurons.

The "dissociative " general anesthetic ketamine is a well-known N-methyl-D-aspartate receptor antagonist. However, whether ketamine, at clinically relevant concentrations, increases the activity of inhibitory γ-aminobutyric acid (GABA) receptor type A (GABAA) receptors in different brain regions remains controversial. Here, the authors studied the effects of ketamine on synaptic and extrasynaptic GABAA receptors in hippocampal neurons. Ketamine modulation of extrasynaptic GABAA receptors in cortical neurons was also examined. Whole cell currents were recorded from cultured murine neurons. Current evoked by exogenous GABA, miniature inhibitory postsynaptic currents, and currents directly activated by ketamine were studied. Ketamine did not alter the amplitude, frequency, or kinetics of postsynaptic currents but increased a tonic inhibitory current generated by extrasynaptic GABAA receptors in hippocampal neurons. For example, ketamine (100 µM) increased the tonic current by 33.6 ± 6.5% (mean ± SEM; 95% CI, 18.2 to 48.9; n = 8, P < 0.001). Ketamine shifted the GABA concentration-response curve to the left, but only when GABAA receptors were activated by low concentrations of GABA (n = 6). The selective increase in tonic current was attributed to ketamine increasing the apparent potency of GABA at high-affinity extrasynaptic GABAA receptors. Ketamine also increased a tonic current in cortical neurons (n = 11). Ketamine directly gated the opening of GABAA receptors, but only at high concentrations that are unlikely to occur during clinical use. Clinically relevant concentrations of ketamine increased the activity of high-affinity extrasynaptic GABAA receptors in the hippocampus and cortex, an effect that likely contributes to ketamine's neurodepressive properties.

Enhanced Thalamic Spillover Inhibition during Non-rapid-eye-movement Sleep Triggers an Electrocortical Signature of Anesthetic Hypnosis.

Alterations in thalamic γ-aminobutyric acid-mediated signaling are thought to underlie the increased frontal α-β frequency electrocortical activity that signals anesthetic-induced loss of consciousness with γ-aminobutyric acid receptor type A (GABAAR)-targeting general anesthetics. The general anesthetic etomidate elicits phasic extrasynaptic GABAAR activation ("spillover" inhibition) at thalamocortical neurons in vitro. We hypothesize that this action of etomidate at the thalamus is sufficient to trigger an increase in frontal α-β frequency electrocortical activity and that this effect of etomidate is fully recapitulated by enhanced thalamic spillover inhibition in vivo. We recorded electrocortical activity and sleep-wake behavior in freely behaving wild-type (n = 33) and extrasynaptic δ-subunit-containing GABAAR knockout mice (n = 9) during bilateral microperfusion of the thalamus with etomidate and/or other pharmacologic agents that influence GABAAR or T-type Ca channel activity. Microperfusion of etomidate into the thalamus elicited an increase in α-β frequency electrocortical activity that occurred only during non-rapid-eye-movement (REM) sleep (11.0 ± 11.8% and 16.0 ± 14.2% greater 8 to 12- and 12 to 30-Hz power, respectively; mean ± SD; both P < 0.031) and was not affected by blockade of thalamic T-type Ca channels. Etomidate at the thalamus also increased spindle-like oscillations during non-REM sleep (4.5 ± 2.4 spindle per minute with etomidate vs. 3.2 ± 1.7 at baseline; P = 0.002). These effects of etomidate were fully recapitulated by enhanced thalamic extrasynaptic GABAAR-mediated spillover inhibition. These findings identify how a prototypic GABAAR-targeting general anesthetic agent can elicit the characteristic brain wave pattern associated with anesthetic hypnosis when acting at the thalamus by promoting spillover inhibition and the necessity of a preexisting non-REM mode of activity in the thalamus to generate this effect.

Alleviating Bone Cancer-induced Mechanical Hypersensitivity by Inhibiting Neuronal Activity in the Anterior Cingulate Cortex.

The anterior cingulate cortex (ACC) is a brain region that has been critically implicated in the processing of pain perception and modulation. While much evidence has pointed to an increased activity of the ACC under chronic pain states, less is known about whether pain can be alleviated by inhibiting ACC neuronal activity. The authors used pharmacologic, chemogenetic, and optogenetic approaches in concert with viral tracing technique to address this issue in a mouse model of bone cancer-induced mechanical hypersensitivity by intratibia implantation of osteolytic fibrosarcoma cells. Bilateral intra-ACC microinjections of γ-aminobutyric acid receptor type A receptor agonist muscimol decreased mechanical hypersensitivity in tumor-bearing mice (n =10). Using adenoviral-mediated expression of engineered Gi/o-coupled human M4 (hM4Di) receptors, we observed that activation of Gi/o-coupled human M4 receptors with clozapine-N-oxide reduced ACC neuronal activity and mechanical hypersensitivity in tumor-bearing mice (n = 11). In addition, unilateral optogenetic silencing of ACC excitatory neurons with halorhodopsin significantly decreased mechanical hypersensitivity in tumor-bearing mice (n = 4 to 9), and conversely, optogenetic activation of these neurons with channelrhodopsin-2 was sufficient to provoke mechanical hypersensitivity in sham-operated mice (n = 5 to 9). Furthermore, we found that excitatory neurons in the ACC send direct descending projections to the contralateral dorsal horn of the lumbar spinal cord via the dorsal corticospinal tract. The findings of this study indicate that enhanced neuronal activity in the ACC contributes to maintain bone cancer-induced mechanical hypersensitivity and suggest that the ACC may serve as a potential therapeutic target for treating bone cancer pain.

Protein Kinase C γ Interneurons Mediate C-fiber-induced Orofacial Secondary Static Mechanical Allodynia, but Not C-fiber-induced Nociceptive Behavior.

Tissue injury enhances pain sensitivity both at the site of tissue damage and in surrounding uninjured skin (secondary hyperalgesia). Secondary hyperalgesia encompasses several pain symptoms including pain to innocuous punctate stimuli or static mechanical allodynia. How injury-induced barrage from C-fiber nociceptors produces secondary static mechanical allodynia has not been elucidated. Combining behavioral, immunohistochemical, and Western blot analysis, the authors investigated the cell and molecular mechanisms underlying the secondary static mechanical allodynia in the rat medullary dorsal horn (MDH) using the capsaicin model (n = 4 to 5 per group). Intradermal injection of capsaicin (25 μg) into the vibrissa pad produces a spontaneous pain and a secondary static mechanical allodynia. This allodynia is associated with the activation of a neuronal network encompassing lamina I-outer lamina III, including interneurons expressing the γ isoform of protein kinase C (PKCγ) within inner lamina II (IIi) of MDH. PKCγ is concomitantly phosphorylated (+351.4 ± 79.2%, mean ± SD; P = 0.0003). Mechanical allodynia and innocuous punctate stimulus-evoked laminae I to III neuronal activation can be replicated after intracisternally applied γ-aminobutyric acid receptor type A (GABAA) antagonist (bicuculline: 0.05 μg) or reactive oxygen species (ROS) donor (tert-butyl hydroperoxide: 50 to 250 ng). Conversely, intracisternal PKCγ antagonist, GABAA receptor agonist, or ROS scavenger prevent capsaicin-induced static mechanical allodynia and neuronal activation. Sensitization of lamina IIi PKCγ interneurons is required for the manifestation of secondary static mechanical allodynia but not for spontaneous pain. Such sensitization is driven by ROS and GABAAergic disinhibition. ROS released during intense C-fiber nociceptor activation might produce a GABAAergic disinhibition of PKCγ interneurons. Innocuous punctate inputs carried by Aδ low-threshold mechanoreceptors onto PKCγ interneurons can then gain access to the pain transmission circuitry of superficial MDH, producing pain.

Hypothalamic Prolactin Regulation of Luteinizing Hormone Secretion in the Female Rat.

Prolactin (PRL) levels increase in response to long-term antipsychotic treatment that disrupts reproductive function. Recent evidence suggests that activation of central PRL receptors (PRLR) inhibits LH secretion and in ovariectomized rats. However, the mechanisms involved, the mode of LH secretion affected and relevance to hyperprolactinemia remain unknown. We therefore investigated the contribution of central PRL/PRLR signaling to the control of estradiol-induced surges of LH and PRL and pulsatile LH secretion under basal and hyperprolactinemic conditions. First, by subjecting ovariectomized estradiol-primed rats intracerebroventricularly administered with PRL to frequent blood sampling, we demonstrated that acute activation of hypothalamic PRLR disrupts pulsatile LH secretion. Pretreatment (intracerebroventricularly) with the pure PRLR antagonist, Δ1-9-G129R-hPRL, or the γ-aminobutyric acid receptor type A antagonist, bicuculline, blocked this effect. Next, we revealed that sustained blockade of hypothalamic PRLR using Δ1-9-G129R-hPRL augmented the magnitude of LH surges induced by estradiol benzoate and progesterone treatment and suppressed the concomitant surges of PRL. Finally, we determined that acute antagonism of central PRLR is insufficient to normalize the duration of the LH pulse interval prolonged as a result of hyperprolactinemia induced by chronic exposure to the atypical antipsychotic sulpiride. These data serve as the first evidence to suggest that PRL signaling through hypothalamic PRLR inhibits pulsatile secretion of LH in a γ-aminobutyric acid receptor type A-dependent fashion and tonically restrains the magnitude of the LH surge. Furthermore, our results indicate that transient blockade of hypothalamic PRL/PRLR signaling is not an effective strategy for restoring LH pulsatility perturbed by chronic hyperprolactinemia.

Enhancement of GluN2B Subunit-Containing NMDA Receptor Underlies Serotonergic Regulation of Long-Term Potentiation after Critical Period in the Rat Visual Cortex.

Serotonin [5-hydroxytryptamine (5-HT)] regulates synaptic plasticity in the visual cortex. Although the effects of 5-HT on plasticity showed huge diversity depending on the ages of animals and species, it has been unclear how 5-HT can show such diverse effects. In the rat visual cortex, 5-HT suppressed long-term potentiation (LTP) at 5 weeks but enhanced LTP at 8 weeks. We speculated that this difference may originate from differential regulation of neurotransmission by 5-HT between the age groups. Thus, we investigated the effects of 5-HT on apha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-, γ-aminobutyric acid receptor type A (GABAAR)-, and N-methyl-D-aspartic acid receptor (NMDAR)-mediated neurotransmissions and their involvement in the differential regulation of plasticity between 5 and 8 weeks. AMPAR-mediated currents were not affected by 5-HT at both 5 and 8 weeks. GABAAR-mediated currents were enhanced by 5-HT at both age groups. However, 5-HT enhanced NMDAR-mediated currents only at 8 weeks. The enhancement of NMDAR-mediated currents appeared to be mediated by the enhanced function of GluN2B subunit-containing NMDAR. The enhanced GABAAR- and NMDAR-mediated neurotransmissions were responsible for the suppression of LTP at 5 weeks and the facilitation of LTP at 8 weeks, respectively. These results indicate that the effects of 5-HT on neurotransmission change with development, and the changes may underlie the differential regulation of synaptic plasticity between different age groups. Thus, the developmental changes in 5-HT function should be carefully considered while investigating the 5-HT-mediated metaplastic control of the cortical network.

Positron Emission Tomography with [(18)F]FLT Revealed Sevoflurane-Induced Inhibition of Neural Progenitor Cell Expansion in vivo.

Neural progenitor cell expansion is critical for normal brain development and an appropriate response to injury. During the brain growth spurt, exposures to general anesthetics, which either block the N-methyl-d-aspartate receptor or enhance the γ-aminobutyric acid receptor type A can disturb neuronal transduction. This effect can be detrimental to brain development. Until now, the effects of anesthetic exposure on neural progenitor cell expansion in vivo had seldom been reported. Here, minimally invasive micro positron emission tomography (microPET) coupled with 3′-deoxy-3′ [18F] fluoro-l-thymidine ([18F]FLT) was utilized to assess the effects of sevoflurane exposure on neural progenitor cell proliferation. FLT, a thymidine analog, is taken up by proliferating cells and phosphorylated in the cytoplasm, leading to its intracellular trapping. Intracellular retention of [18F]FLT, thus, represents an observable in vivo marker of cell proliferation. Here, postnatal day 7 rats (n = 11/group) were exposed to 2.5% sevoflurane or room air for 9 h. For up to 2 weeks following the exposure, standard uptake values (SUVs) for [18F]-FLT in the hippocampal formation were significantly attenuated in the sevoflurane-exposed rats (p < 0.0001), suggesting decreased uptake and retention of [18F]FLT (decreased proliferation) in these regions. Four weeks following exposure, SUVs for [18F]FLT were comparable in the sevoflurane-exposed rats and in controls. Co-administration of 7-nitroindazole (30 mg/kg, n = 5), a selective inhibitor of neuronal nitric oxide synthase, significantly attenuated the SUVs for [18F]FLT in both the air-exposed (p = 0.00006) and sevoflurane-exposed rats (p = 0.0427) in the first week following the exposure. These findings suggested that microPET in couple with [18F]FLT as cell proliferation marker could be used as a non-invasive modality to monitor the sevoflurane-induced inhibition of neural progenitor cell proliferation in vivo.

Tranexamic Acid Impairs γ-Aminobutyric Acid Receptor Type A–mediated Synaptic Transmission in the Murine Amygdala

Tranexamic acid (TXA) is commonly used to reduce blood loss in cardiac surgery and in trauma patients. High-dose application of TXA is associated with an increased risk of postoperative seizures. The neuronal mechanisms underlying this proconvulsant action of TXA are not fully understood. In this study, the authors investigated the effects of TXA on neuronal excitability and synaptic transmission in the basolateral amygdala. Patch clamp recordings and voltage-sensitive dye imaging were performed in acute murine brain slices. Currents through N-methyl-D-aspartate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and γ-aminobutyric acid receptor type A (GABAA) receptors were recorded. GABAA receptor-mediated currents were evoked upon electrical stimulation or upon photolysis of caged GABA. TXA was applied at different concentrations. Voltage-sensitive dye imaging demonstrates that TXA (1 mM) reversibly enhances propagation of neuronal excitation (mean ± SEM, 129 ± 6% of control; n = 5). TXA at concentrations of 0.1, 0.3, 1, 5, or 10 mM led to a dose-dependent reduction of GABAA receptor-mediated currents in patch clamp recordings. There was no difference in the half-maximal inhibitory concentration for electrically (0.76 mM) and photolytically (0.84 mM) evoked currents (n = 5 to 9 for each concentration), and TXA did not affect the paired-pulse ratio of GABAA receptor-mediated currents. TXA did not impact glutamatergic synaptic transmission. This study clearly demonstrates that TXA enhances neuronal excitation by antagonizing inhibitory GABAergic neurotransmission. The results provide evidence that this effect is mediated via postsynaptic mechanisms. Because GABAA receptor antagonists are known to promote epileptiform activity, this effect might explain the proconvulsant action of TXA.

Chapter 3 - Mutant GABAA receptor subunits in genetic (idiopathic) epilepsy

The γ-aminobutyric acid receptor type A (GABAA receptor) is a ligand-gated chloride channel that mediates major inhibitory functions in the central nervous system. GABAA receptors function mainly as pentamers containing α, β, and either γ or δ subunits. A number of antiepileptic drugs have agonistic effects on GABAA receptors. Hence, dysfunctions of GABAA receptors have been postulated to play important roles in the etiology of epilepsy. In fact, mutations or genetic variations of the genes encoding the α1, α6, β2, β3, γ2, or δ subunits (GABRA1, GABRA6, GABRB2, GABRB3, GABRG2, and GABRD, respectively) have been associated with human epilepsy, both with and without febrile seizures. Epilepsy resulting from mutations is commonly one of following, genetic (idiopathic) generalized epilepsy (e.g., juvenile myoclonic epilepsy), childhood absence epilepsy, genetic epilepsy with febrile seizures, or Dravet syndrome. Recently, mutations of GABRA1, GABRB2, and GABRB3 were associated with infantile spasms and Lennox-Gastaut syndrome. These mutations compromise hyperpolarization through GABAA receptors, which is believed to cause seizures. Interestingly, most of the insufficiencies are not caused by receptor gating abnormalities, but by complex mechanisms, including endoplasmic reticulum (ER)-associated degradation, nonsense-mediated mRNA decay, intracellular trafficking defects, and ER stress. Thus, GABAA receptor subunit mutations are now thought to participate in the pathomechanisms of epilepsy, and an improved understanding of these mutations should facilitate our understanding of epilepsy and the development of new therapies.

Polymorphisms inGABRB3and Oral Clefting in the Brazilian Population

The aim of this study was to evaluate the influence of the γ-aminobutyric acid receptor type A β-3 subunit (GABRB3) polymorphisms in patients with nonsyndromic cleft lip and/or palate (NSCL/P). We carried out a structured case-control analysis of three GABRB3 polymorphisms (rs4477673, rs6576618, and rs981778) in 229 patients with nonsyndromic cleft lip with or without cleft palate (CL±P) and in 314 unaffected controls from Brazil. The polymorphisms were genotyped by the TaqMan 5'-exonuclease allelic discrimination assay, and each sample was independently typed for 40 biallelic short insertion/deletion markers (INDELs) to characterize the genomic ancestry. The genotype distributions of the three polymorphisms were as expected by the Hardy-Weinberg equilibrium test. After adjustment to ancestry contribution, the minor A allele of rs981778 was associated with NSCL/P, but significant results did not persist after Bonferroni correction for multiple tests. Similarly, the haplotype analysis revealed that the CCA haplotype (C allele of rs4477673, C allele of rs6576618, and A allele of rs981778) was correlated with NSCL/P, but this association did not remain statistically significant after Bonferroni correction. With a weak association, our data do not support the hypothesis that the GABRB3 variants are a cause of NSCL/P, but further studies are warranted.