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Severe coronavirus disease-19 (COVID-19) has been associated with fibrin-mediated hypercoagulability and thromboembolic complications. To evaluate potential biomarkers of coagulopathy and disease severity in COVID-19, we measured plasma levels of eight biomarkers potentially associated with coagulation, fibrinolysis, and platelet function in 43 controls and 63 COVID-19 patients, including 47 patients admitted to the intensive care unit (ICU) and 16 non-ICU patients. COVID-19 patients showed significantly elevated levels of fibrinogen, tissue plasminogen activator (t-PA), and its inhibitor plasminogen activation inhibitor 1 (PAI-1), as well as ST2 (the receptor for interleukin 33) and von Willebrand factor (vWF) compared to the control group. We found that higher levels of t-PA, ST2, and vWF at the time of admission were associated with lower survival rates, and that thrombotic events were more frequent in patients with initial higher levels of vWF. These results support a predictive role of specific biomarkers such as t-PA and vWF in the pathophysiology of COVID-19. The data provide support for the case that hypercoagulability in COVID-19 is fibrin-mediated, but also highlights the important role that vWF may play in the genesis of thromboses in the pathophysiology of COVID-19. Interventions designed to enhance fibrinolysis and reduce platelet aggregation might prove to be useful adjuncts in the treatment of coagulopathy in a subset of COVID-19 patients.

Coronavirus disease-2019 (COVID-19) is associated with hypercoagulability that may cause thromobembolic complications. We describe our recent studies investigating the mechanisms of hypercoagulability in patients with severe COVID-19 requiring mechanical ventilation during the COVID-19 crisis in New York City in spring 2020. Using rotational thombelastometry we found that almost all patients with severe COVID-19 had signs of hypercoagulability compared with non-COVID-19 controls. Specifically, the maximal clot firmness in the fibrin-based extrinsically activated test was almost twice the upper limit of normal in COVID patients, indicating a fibrin-mediated cause for hypercoagulability. To better understand the mechanism of this hypercoagulability we measured the components of the fibrinolytic pathways. Fibrinogen, tissue plasminogen activator and plasminogen activator inhibitor-1, but not plasminogen levels were elevated in patients with severe COVID-19. Our studies indicate that hypercoagulability in COVID-19 may be because of decreased fibrinolysis resulting from inhibition of plasmin through high levels of plasminogen activator inhibitor-1. Clinicians creating treatment protocols for anticoagulation in critically ill COVID-19 patients should consider these potential mechanisms of hypercoaguability.

We measured plasma levels of fibrinogen, plasminogen, tissue plasminogen activator (t-PA) and plasminogen activation inhibitor 1 (PAI-1) in blood from 37 patients with severe coronavirus disease-19 (COVID-19) and 23 controls. PAI-1, t-PA and fibrinogen levels were significantly higher in the COVID-19 group. Increased levels of PAI-1 likely result in lower plasmin activity and hence decreased fibrinolysis. These observations provide a partial explanation for the fibrin- mediated increase in blood viscosity and hypercoagulability that has previously been observed in COVID-19. Our data suggest that t-PA administration may be problematic, but that other interventions designed to enhance fibrinolysis might prove useful in the treatment of the coagulopathy that is often associated with severe COVID-19.

The medial prefrontal cortex (mPFC) integrates inputs from multiple subcortical regions including the mediodorsal nucleus of the thalamus (MD) and the ventral hippocampus (vHPC). How the mPFC differentially processes these inputs is not known. One possibility is that these two inputs target discreet populations of mPFC cells. Alternatively, individual prefrontal cells could receive convergent inputs but distinguish between both inputs based on synaptic differences, such as communication frequency. To address this, we utilized a dual wavelength optogenetic approach to stimulate MD and vHPC inputs onto single, genetically defined mPFC neuronal subtypes. Specifically, we compared the convergence and synaptic dynamics of both inputs onto mPFC pyramidal cells, and parvalbumin (PV)- and vasoactive intestinal peptide (VIP)-expressing interneurons. We found that all individual pyramidal neurons in layer 2/3 of the mPFC receive convergent input from both MD and vHPC. In contrast, PV neurons receive input biased from the MD, while VIP cells receive input biased from the vHPC. Independent of the target, MD inputs transferred information more reliably at higher frequencies (20 Hz) than vHPC inputs. Thus, MD and vHPC projections converge functionally onto mPFC pyramidal cells, but both inputs are distinguished by frequency-dependent synaptic dynamics and preferential engagement of discreet interneuron populations.

Key points We report a novel method for the transient expression of SARS‐CoV‐2 envelope (E) protein in intracellular organelles and the plasma membrane of mammalian cells and Xenopus oocytes.Intracellular expression of SARS‐CoV‐2 E protein increases intra‐Golgi pH.By targeting the SARS‐CoV‐2 E protein to the plasma membrane, we show that it forms a cation channel, viroporin, that is modulated by changes of pH.This method for studying the activity of viroporins may facilitate screening for new antiviral drugs to identify novel treatments for COVID‐19. Abstract The envelope (E) protein of coronaviruses such as SARS‐CoV‐1 is proposed to form an ion channel or viroporin that participates in viral propagation and pathogenesis. Here we developed a technique to study the E protein of SARS‐CoV‐2 in mammalian cells by directed targeting using a carboxyl‐terminal fluorescent protein tag, mKate2. The wild‐type SARS‐CoV‐2 E protein can be trafficked to intracellular organelles, notably the endoplasmic reticulum–Golgi intermediate complex, where its expression increases pH inside the organelle. We also succeeded in targeting SARS‐CoV‐2 E to the plasma membrane, which enabled biophysical analysis using whole‐cell patch clamp recording in a mammalian cell line, HEK 293 cells, and two‐electrode voltage clamp electrophysiology in Xenopus oocytes. The results suggest that the E protein forms an ion channel that is permeable to monovalent cations such as Na+, Cs+ and K+. The E current is nearly time‐ and voltage‐independent when E protein is expressed in mammalian cells, and is modulated by changes of pH. At pH 6.0 and 7.4, the E protein current is activated, whereas at pH 8.0 and 9.0, the amplitude of E protein current is reduced, and in oocytes the inward E current fades at pH 9 in a time‐ and voltage‐dependent manner. Using this directed targeting method and electrophysiological recordings, potential inhibitors of the E protein can be screened and subsequently investigated for antiviral activity against SARS‐CoV‐2 in vitro and possible efficacy in treating COVID‐19., Key points We report a novel method for the transient expression of SARS‐CoV‐2 envelope (E) protein in intracellular organelles and the plasma membrane of mammalian cells and Xenopus oocytes.Intracellular expression of SARS‐CoV‐2 E protein increases intra‐Golgi pH.By targeting the SARS‐CoV‐2 E protein to the plasma membrane, we show that it forms a cation channel, viroporin, that is modulated by changes of pH.This method for studying the activity of viroporins may facilitate screening for new antiviral drugs to identify novel treatments for COVID‐19.

Periodic episodes of excessive alcohol consumption ("binge drinking") occur frequently among adolescents, and early binge drinking is associated with an increased risk of alcohol use disorders later in life. The PFC undergoes significant development during adolescence and hence may be especially susceptible to the effects of binge drinking. In humans and in animal models, adolescent alcohol exposure is known to alter PFC neuronal activity and produce deficits in PFC-dependent behaviors, such as decision making, response inhibition, and working memory. Using a voluntary intermittent access to alcohol (IA EtOH) procedure in male mice, we demonstrate that binge-level alcohol consumption during adolescence leads to altered drinking patterns and working memory deficits in young adulthood, two outcomes that suggest medial PFC dysfunction. We recorded from pyramidal neurons (PNs) in the prelimbic subregion of the medial PFC in slices obtained from mice that had IA EtOH and found that they display altered excitability, including a hyperpolarization of the resting membrane potential and reductions in the hyperpolarization-activated cation current (Ih) and in intrinsic persistent activity (a mode of neuronal firing that is dependent on Ih). Many of these effects on intrinsic excitability were sustained following abstinence and observed in mice that showed working memory deficits. In addition, we found that resting membrane potential and the Ih-dependent voltage "sag" in prelimbic PFC PNs are developmentally regulated during adolescence, suggesting that adolescent alcohol exposure may compromise PFC function by arresting the normal developmental trajectory of PN intrinsic excitability.SIGNIFICANCE STATEMENT Binge alcohol drinking during adolescence has negative consequences for the function of the developing PFC. Using a mouse model of voluntary binge drinking during adolescence, we found that this behavior leads to working memory deficits and altered drinking behavior in adulthood. In addition, we found that adolescent drinking is associated with specific changes to the intrinsic excitability of pyramidal neurons in the PFC, reducing the ability of these neurons to generate intrinsic persistent activity, a phenomenon thought to be important for working memory. These findings may help explain why human adolescent binge drinkers show performance deficits on tasks mediated by the PFC.

Emerging evidence indicates that hypnotic anesthetics affect immune function. Many anesthetics potentiate γ-aminobutyric acid A receptor (GABAAR) activation, and these receptors are expressed on multiple subtypes of immune cells, providing a potential mechanistic link. Like immune cells, airway smooth muscle (ASM) cells also express GABAARs, particularly isoforms containing α4-subunits, and activation of these receptors leads to ASM relaxation. We sought to determine if GABAAR signaling modulates the ASM contractile and inflammatory phenotype of a murine allergic asthma model utilizing GABAAR α4-subunit global knockout (KO; Gabra40/0) mice. Wild-type (WT) and Gabra4 KO mice were sensitized with house dust mite (HDM) antigen or exposed to PBS intranasally 5 days/wk for 3 wk. Ex vivo tracheal rings from HDM-sensitized WT and Gabra4 KO mice exhibited similar magnitudes of acetylcholine-induced contractile force and isoproterenol-induced relaxation ( P = not significant; n = 4). In contrast, in vivo airway resistance (flexiVent) was significantly increased in Gabra4 KO mice ( P < 0.05, n = 8). Moreover, the Gabra4 KO mice demonstrated increased eosinophilic lung infiltration ( P < 0.05; n = 4) and increased markers of lung T-cell activation/memory (CD62L low, CD44 high; P < 0.01, n = 4). In vitro, Gabra4 KO CD4+ cells produced increased cytokines and exhibited increased proliferation after stimulation of the T-cell receptor as compared with WT CD4+ cells. These data suggest that the GABAAR α4-subunit plays a role in immune cell function during allergic lung sensitization. Thus GABAAR α4-subunit-specific agonists have the therapeutic potential to treat asthma via two mechanisms: direct ASM relaxation and inhibition of airway inflammation.

The prefrontal cortex (PFC) is involved in executive (“top-down”) control of behavior and its function is especially susceptible to the effects of alcohol, leading to behavioral disinhibition that is associated with alterations in decision making, response inhibition, social anxiety and working memory. The circuitry of the PFC involves a complex interplay between pyramidal neurons (PNs) and several subclasses of inhibitory interneurons (INs), including somatostatin (SST)-expressing INs. Using in vivo calcium imaging, we showed that alcohol dose-dependently altered network activity in layers 2/3 of the prelimbic subregion of the mouse PFC. Low doses of alcohol (1 g/kg, intraperitoneal, i.p.) caused moderate activation of SST INs and weak inhibition of PNs. At moderate to high doses, alcohol (2–3 g/kg) strongly inhibited the activity of SST INs in vivo, and this effect may result in disinhibition, as the activity of a subpopulation of PNs was simultaneously enhanced. In contrast, recordings in brain slices using ex vivo electrophysiology revealed no direct effect of alcohol on the excitability of either SST INs or PNs over a range of concentrations (20 and 50 mM) consistent with the blood alcohol levels reached in the in vivo experiments. This dose-dependent effect of alcohol on SST INs in vivo may reveal a neural basis for the disinhibitory effect of alcohol in the PFC mediated by other neurons within or external to the PFC circuitry.

Enhanced dopamine (DA) neurotransmission from the ventral tegmental area (VTA) to the ventral striatum is thought to drive drug self-administration and mediate positive reinforcement. We examined neuronal firing rates in slices of mouse midbrain following adolescent binge-like alcohol drinking and find that prior alcohol experience greatly enhanced the sensitivity to excitation by ethanol itself (10-50 mM) in a subset of ventral midbrain DA neurons located in the medial VTA. This enhanced response after drinking was not associated with alterations of firing rate or other measures of intrinsic excitability. In addition, the phenomenon appears to be specific to adolescent drinking, as mice that established a drinking preference only after the onset of adulthood showed no change in alcohol sensitivity. Here we demonstrate not only that drinking during adolescence induces enhanced alcohol sensitivity, but also that this DA neuronal response occurs over a range of alcohol concentrations associated with social drinking in humans.

Abnormalities in prefrontal gamma aminobutyric acid (GABA)ergic transmission, particularly in fast-spiking interneurons that express parvalbumin (PV), are hypothesized to contribute to the pathophysiology of multiple psychiatric disorders, including schizophrenia, bipolar disorder, anxiety disorders and depression. While primarily histological abnormalities have been observed in patients and in animal models of psychiatric disease, evidence for abnormalities in functional neurotransmission at the level of specific interneuron populations has been lacking in animal models and is difficult to establish in human patients. Using an animal model of a psychiatric disease risk factor, prenatal maternal immune activation (MIA), we found reduced functional GABAergic transmission in the medial prefrontal cortex (mPFC) of adult MIA offspring. Decreased transmission was selective for interneurons expressing PV, resulted from a decrease in release probability and was not observed in calretinin-expressing neurons. This deficit in PV function in MIA offspring was associated with increased anxiety-like behavior and impairments in attentional set shifting, but did not affect working memory. Furthermore, cell-type specific optogenetic inhibition of mPFC PV interneurons was sufficient to impair attentional set shifting and enhance anxiety levels. Finally, we found that in vivo mPFC gamma oscillations, which are supported by PV interneuron function, were linearly correlated with the degree of anxiety displayed in adult mice, and that this correlation was disrupted in MIA offspring. These results demonstrate a selective functional vulnerability of PV interneurons to MIA, leading to affective and cognitive symptoms that have high relevance for schizophrenia and other psychiatric disorders.

Background and Purpose GABA receptors mediate neuronal inhibition in the brain. They are the primary targets for benzodiazepines, which are widely used to treat neurological disorders including anxiety, epilepsy and insomnia. The mechanism by which benzodiazepines enhance GABA receptor activity has been extensively studied, but there is little mechanistic information on how non-benzodiazepine drugs that bind to the same site exert their effects. Eszopiclone and zolpidem are two non-benzodiazepine drugs for which no mechanism of action has yet been proposed, despite their clinical importance as sleeping aids. Here we investigate how both drugs enhance the activity of α1β2γ2 GABA receptors. Experimental Approach We used rapid ligand application onto macropatches and single-channel kinetic analysis to assess rates of current deactivation. We also studied synaptic currents in primary neuronal cultures and in heterosynapses, whereby native GABAergic nerve terminals form synapses with HEK293 cells expressing α1β2γ2 GABA receptors. Drug binding and modulation was quantified with the aid of an activation mechanism. Key Results At the single-channel level, the drugs prolonged the duration of receptor activation, with similar K values of ∼80-nM. Channel activation was prolonged primarily by increasing the equilibrium constant between two connected shut states that precede channel opening. Conclusions and Implications As the derived mechanism successfully simulated the effects of eszopiclone and zolpidem on ensemble currents, we propose it as the definitive mechanism accounting for the effects of both drugs. Importantly, eszopiclone and zolpidem enhanced GABA receptor currents via a mechanism that differs from that proposed for benzodiazepines.

The clinical need for novel bronchodilators for the treatment of bronchoconstrictive diseases remains a major medical issue. Modulation of airway smooth muscle (ASM) chloride via GABAA receptor activation to achieve relaxation of precontracted ASM represents a potentially beneficial therapeutic option. Since human ASM GABAA receptors express only the α4- and α5-subunits, there is an opportunity to selectively target ASM GABAA receptors to improve drug efficacy and minimize side effects. Recently, a novel compound ( R)-ethyl8-ethynyl-6-(2-fluorophenyl)-4-methyl-4 H-benzo[ f]imidazo[1,5- a][1,4] diazepine-3-carboxylate (SH-053-2′F-R-CH3) with allosteric selectivity for α5-subunit containing GABAA receptors has become available. We questioned whether this novel GABAA α5-selective ligand relaxes ASM and affects intracellular calcium concentration ([Ca2+]i) regulation. Immunohistochemical staining localized the GABAA α5-subunit to human ASM. The selective GABAA α5 ligand SH-053-2′F-R-CH3 relaxes precontracted intact ASM; increases GABA-activated chloride currents in human ASM cells in voltage-clamp electrophysiology studies; and attenuates bradykinin-induced increases in [Ca2+]i, store-operated Ca2+ entry, and methacholine-induced Ca2+ oscillations in peripheral murine lung slices. In conclusion, selective subunit targeting of endogenous α5-subunit containing GABAA receptors on ASM may represent a novel therapeutic option to treat severe bronchospasm.

The mechanisms by which alcohol drinking promotes addiction in humans and self-administration in rodents remain obscure, but it is well known that alcohol can enhance dopamine (DA) neurotransmission from neurons of the ventral tegmental area (VTA) and increase DA levels within the nucleus accumbens and prefrontal cortex. We recorded from identified DA neuronal cell bodies within ventral midbrain slices prepared from a transgenic mouse line (TH-GFP) using long-term stable extracellular recordings in a variety of locations and carefully mapped the responses to applied ethanol (EtOH). We identified a subset of DA neurons in the medial VTA located within the rostral linear and interfascicular nuclei that fired spontaneously and exhibited a concentration-dependent increase of firing frequency in response to EtOH, with some neurons responsive to as little as 20 mM EtOH. Many of these medial VTA DA neurons were also insensitive to the D2 receptor agonist quinpirole. In contrast, DA neurons in the lateral VTA (located within the parabrachial pigmented and paranigral nuclei) were either unresponsive or responded only to 100 mM EtOH. Typically, these lateral VTA DA cells had very slow firing rates, and all exhibited inhibition by quinpirole via D2 “autoreceptors”. VTA non-DA cells did not show any significant response to low levels of EtOH. These findings are consistent with evidence for heterogeneity among midbrain DA neurons and provide an anatomical and pharmacological distinction between DA neuron sub-populations that will facilitate future mechanistic studies on the actions of EtOH in the VTA.

Background Excessive ethanol (EtOH) drinking is associated with white matter loss in the brain at all stages of life. Myelin-forming oligodendrocytes (OLs) are a major component of white matter, but their involvement in EtOH-mediated white matter loss is unclear. Myelination continues throughout the life with highest rates during fetal development and adolescence. However, little is known about the effects of EtOH and its principal metabolite acetaldehyde (ACD) on OLs at the cellular level. Methods We compared the responses to different concentrations of EtOH or ACD by primary OLs in culture. Results EtOH did not cause significant cell death at concentrations lower than 120 mM, even after 24 hours. In comparison, ACD was highly lethal at doses above 50 μM. High concentrations of EtOH (120 mM) and ACD (500 μM) for 24 hours did not reduce myelin in mature OLs. Myelin production and OL differentiation were significantly impaired by 7 days exposure to 500 or 50 μM ACD but not 120 mM EtOH. Conclusions This study shows that OLs are relatively resistant to EtOH, even at a concentration more than 4 times the typical blood EtOH concentrations associated with social drinking (10 to 30 mM). In contrast, OLs are much more sensitive to ACD than EtOH, particularly with long-term exposure. This suggests that part of white matter loss in response to EtOH, especially during high rates of myelin formation, may be due in part to the effects of its principal metabolite ACD.

Brain imaging studies performed in humans have associated low striatal dopamine release and D2R binding with alcohol dependence. Conversely, high striatal D2R binding has been observed in unaffected members of alcoholic families suggesting that high D2R function may protect against alcohol dependence. A possible protective role of increased D2R levels in the striatum is further supported by preclinical studies in non-human primates and rodents. Here, we determined whether there is a causal relationship between D2R levels and alcohol intake. To this end, we upregulated D2R expression levels in the nucleus accumbens of the adult mouse, but selectively restricted the upregulation to the indirect striatal output pathway, which endogenously expresses D2Rs. After overexpression was established, mice were tested in two models of free-choice alcohol drinking: the continuous and intermittent access two-bottle choice models. As anticipated, we found that D2R upregulation leads to hyperactivity in the open field. Contrary to our expectation, D2R upregulation did not reduce alcohol intake during continuous or intermittent access or when alcohol drinking was tested in the context of aversive outcomes. These data argue against a protective role of accumbal indirect pathway D2Rs in alcohol consumption but emphasize their importance in promoting locomotor activity.

Neuronal inhibition can occur via synaptic mechanisms or through tonic activation of extrasynaptic receptors. In spinal cord, glycine mediates synaptic inhibition through the activation of heteromeric glycine receptors (GlyRs) composed primarily of α1 and β subunits. Inhibitory GlyRs are also found throughout the brain, where GlyR α2 and α3 subunit expression exceeds that of α1, particularly in forebrain structures, and coassembly of these α subunits with the β subunit appears to occur to a lesser extent than in spinal cord. Here, we analyzed GlyR currents in several regions of the adolescent mouse forebrain (striatum, prefrontal cortex, hippocampus, amygdala, and bed nucleus of the stria terminalis). Our results show ubiquitous expression of GlyRs that mediate large-amplitude currents in response to exogenously applied glycine in these forebrain structures. Additionally, tonic inward currents were also detected, but only in the striatum, hippocampus, and prefrontal cortex (PFC). These tonic currents were sensitive to both strychnine and picrotoxin, indicating that they are mediated by extrasynaptic homomeric GlyRs. Recordings from mice deficient in the GlyR α3 subunit (Glra3−/−) revealed a lack of tonic GlyR currents in the striatum and the PFC. In Glra2−/Y animals, GlyR tonic currents were preserved; however, the amplitudes of current responses to exogenous glycine were significantly reduced. We conclude that functional α2 and α3 GlyRs are present in various regions of the forebrain and that α3 GlyRs specifically participate in tonic inhibition in the striatum and PFC. Our findings suggest roles for glycine in regulating neuronal excitability in the forebrain.

Emerging evidence indicates that hypnotic anesthetics affect immune function. Many anesthetics potentiate γ-aminobutyric acid A receptor (GABAAR) activation, and these receptors are expressed on multiple subtypes of immune cells, providing a potential mechanistic link. Like immune cells, airway smooth muscle (ASM) cells also express GABAARs, particularly isoforms containing α4-subunits, and activation of these receptors leads to ASM relaxation. We sought to determine if GABAAR signaling modulates the ASM contractile and inflammatory phenotype of a murine allergic asthma model utilizing GABAAR α4-subunit global knockout (KO; Gabra40/0) mice. Wild-type (WT) and Gabra4 KO mice were sensitized with house dust mite (HDM) antigen or exposed to PBS intranasally 5 days/wk for 3 wk. Ex vivo tracheal rings from HDM-sensitized WT and Gabra4 KO mice exhibited similar magnitudes of acetylcholine-induced contractile force and isoproterenol-induced relaxation (P = not significant; n = 4). In contrast, in vivo airway resistance (flexiVent) was significantly increased in Gabra4 KO mice (P < 0.05, n = 8). Moreover, the Gabra4 KO mice demonstrated increased eosinophilic lung infiltration (P < 0.05; n = 4) and increased markers of lung T-cell activation/memory (CD62L low, CD44 high; P < 0.01, n = 4). In vitro, Gabra4 KO CD4+ cells produced increased cytokines and exhibited increased proliferation after stimulation of the T-cell receptor as compared with WT CD4+ cells. These data suggest that the GABAAR α4-subunit plays a role in immune cell function during allergic lung sensitization. Thus GABAAR α4-subunit-specific agonists have the therapeutic potential to treat asthma via two mechanisms: direct ASM relaxation and inhibition of airway inflammation.

Processing of signals within the cerebral cortex requires integration of synaptic inputs and a coordination between excitatory and inhibitory neurotransmission. In addition to the classic form of synaptic inhibition, another important mechanism that can regulate neuronal excitability is tonic inhibition via sustained activation of receptors by ambient levels of inhibitory neurotransmitter, usually GABA. The purpose of this study was to determine whether this occurs in layer II/III pyramidal neurons (PNs) in the prelimbic region of the mouse medial prefrontal cortex (mPFC). We found that these neurons respond to exogenous GABA and to the α4δ-containing GABAA receptor (GABAAR)-selective agonist gaboxadol, consistent with the presence of extrasynaptic GABAAR populations. Spontaneous and miniature synaptic currents were blocked by the GABAAR antagonist gabazine and had fast decay kinetics, consistent with typical synaptic GABAARs. Very few layer II/III neurons showed a baseline current shift in response to gabazine, but almost all showed a current shift (15–25 pA) in response to picrotoxin. In addition to being a noncompetitive antagonist at GABAARs, picrotoxin also blocks homomeric glycine receptors (GlyRs). Application of the GlyR antagonist strychnine caused a modest but consistent shift (∼15 pA) in membrane current, without affecting spontaneous synaptic events, consistent with the tonic activation of GlyRs. Further investigation showed that these neurons respond in a concentration-dependent manner to glycine and taurine. Inhibition of glycine transporter 1 (GlyT1) with sarcosine resulted in an inward current and an increase of the strychnine-sensitive current. Our data demonstrate the existence of functional GlyRs in layer II/III of the mPFC and a role for these receptors in tonic inhibition that can have an important influence on mPFC excitability and signal processing.

Background: The finding that exposure to general anesthetics (GAs) in childhood may increase rates of learning disabilities has raised a concern that anesthetics may interfere with brain development. The generation of neuronal circuits, a complex process in which axons follow guidance cues to dendritic targets, is an unexplored potential target for this type of toxicity. Methods: GA exposures were conducted in developing neocortical neurons in culture and in early postnatal neocortical slices overlaid with fluorescently labeled neurons. Axon targeting, growth cone collapse, and axon branching were measured using quantitative fluorescence microscopy. Results: Isoflurane exposure causes errors in Semaphorin-3A–dependent axon targeting (n = 77 axons) and a disruption of the response of axonal growth cones to Semaphorin-3A (n = 2,358 growth cones). This effect occurs at clinically relevant anesthetic doses of numerous GAs with allosteric activity at γ-aminobutyric acid type A receptors, and it was reproduced with a selective agonist. Isoflurane also inhibits growth cone collapse induced by Netrin-1, but does not interfere branch induction by Netrin-1. Insensitivity to guidance cues caused by isoflurane is seen acutely in growth cones in dissociated culture, and errors in axon targeting in brain slice culture occur at the earliest point at which correct targeting is observed in controls. Conclusions: These results demonstrate a generalized inhibitory effect of GAs on repulsive growth cone guidance in the developing neocortex that may occur via a γ-aminobutyric acid type A receptor mechanism. The finding that GAs interfere with axon guidance, and thus potentially with circuit formation, represents a novel form of anesthesia neurotoxicity in brain development.

Astrocytes are critical for maintaining homeostasis in the central nervous system (CNS), and also participate in the genomic response of the brain to drugs of abuse, including alcohol. In this study, we investigated ethanol regulation of gene expression in astrocytes. A microarray screen revealed that a brief exposure of cortical astrocytes to ethanol increased the expression of a large number of genes. Among the alcohol-responsive genes (ARGs) are glial-specific immune response genes, as well as genes involved in the regulation of transcription, cell proliferation, and differentiation, and genes of the cytoskeleton and extracellular matrix. Genes involved in metabolism were also upregulated by alcohol exposure, including genes associated with oxidoreductase activity, insulin-like growth factor signaling, acetyl-CoA, and lipid metabolism. Previous microarray studies performed on ethanol-treated hepatocyte cultures and mouse liver tissue revealed the induction of almost identical classes of genes to those identified in our microarray experiments, suggesting that alcohol induces similar signaling mechanisms in the brain and liver. We found that acute ethanol exposure activated heat shock factor 1 (HSF1) in astrocytes, as demonstrated by the translocation of this transcription factor to the nucleus and the induction of a family of known HSF1-dependent genes, the heat shock proteins (Hsps). Transfection of a constitutively transcriptionally active Hsf1 construct into astrocytes induced many of the ARGs identified in our microarray study supporting the hypothesis that HSF1 transcriptional activity, as part of the heat shock cascade, may mediate the ethanol induction of these genes. These data indicate that acute ethanol exposure alters gene expression in astrocytes, in part via the activation of HSF1 and the heat shock cascade.

Alcohol has many effects on brain function and hence on human behavior, ranging from anxiolytic and mild disinhibitory effects, sedation and motor incoordination, amnesia, emesis, hypnosis and eventually unconsciousness. In recent years a variety of studies have shown that acute and chronic exposure to alcohol can modulate ion channels that regulate excitability. Modulation of intrinsic excitability provides another way in which alcohol can influence neuronal network activity, in addition to its actions on synaptic inputs. In this review, we review "low dose" effects [between 2 and 20 mM EtOH], and "medium dose"; effects [between 20 and 50 mM], by considering in turn each of the many networks and brain regions affected by alcohol, and thereby attempt to integrate in vitro physiological studies in specific brain regions (e.g. amygdala, ventral tegmental area, prefrontal cortex, thalamus, cerebellum etc.) within the context of alcohol's behavioral actions in vivo (e.g. anxiolysis, euphoria, sedation, motor incoordination). This article is part of the Special Issue entitled "Alcoholism".

Structural plasticity in the adult brain is essential for adaptive behaviors and is thought to contribute to a variety of neurological and psychiatric disorders. Medium spiny neurons of the striatum show a high degree of structural plasticity that is modulated by dopamine through unknown signaling mechanisms. Here, we demonstrate that overexpression of dopamine D2 receptors in medium spiny neurons increases their membrane excitability and decreases the complexity and length of their dendritic arbors. These changes can be reversed in the adult animal after restoring D2 receptors to wild-type levels, demonstrating a remarkable degree of structural plasticity in the adult striatum. Increased excitability and decreased dendritic arborization are associated with downregulation of inward rectifier potassium channels (Kir2.1/2.3). Downregulation of Kir2 function is critical for the neurophysiological and morphological changesin vivobecause virally mediated expression of a dominant-negative Kir2 channel is sufficient to recapitulate the changes in D2 transgenic mice. These findings may have important implications for the understanding of basal ganglia disorders, and more specifically schizophrenia, in which excessive activation of striatal D2 receptors has long been hypothesized to be of pathophysiologic significance.

Embryonic stem cells (ESCs) represent a promising source of midbrain dopaminergic (DA) neurons for applications in Parkinson disease. However, ESC-based transplantation paradigms carry a risk of introducing inappropriate or tumorigenic cells. Cell purification before transplantation may alleviate these concerns and enable identification of the specific DA neuron stage most suitable for cell therapy. Here, we used 3 transgenic mouse ESC reporter lines to mark DA neurons at 3 stages of differentiation (early, middle, and late) following induction of differentiation using Hes5::GFP, Nurr1::GFP, and Pitx3::YFP transgenes, respectively. Transplantation of FACS-purified cells from each line resulted in DA neuron engraftment, with the mid-stage and late-stage neuron grafts being composed almost exclusively of midbrain DA neurons. Mid-stage neuron cell grafts had the greatest amount of DA neuron survival and robustly induced recovery of motor deficits in hemiparkinsonian mice. Our data suggest that the Nurrl(+) stage (middle stage) of neuronal differentiation is particularly suitable for grafting ESC-derived DA neurons. Moreover, global transcriptome analysis of progeny from each of the ESC reporter lines revealed expression of known midbrain DA neuron genes and also uncovered previously uncharacterized midbrain genes. These data demonstrate remarkable fate specificity of ESC-derived DA neurons and outline a sequential stage-specific ESC reporter line paradigm for in vivo gene discovery.

Many synapses within the central nervous system are sensitive to ethanol. Although alcohol is known to affect the probability of neurotransmitter release in specific brain regions, the effects of alcohol on the underlying synaptic vesicle fusion machinery have been little studied. To identify a potential pathway by which ethanol can regulate neurotransmitter release, we investigated the effects of acute alcohol exposure (1–24 h) on the expression of the gene encoding synaptotagmin 1 (Syt1), a synaptic protein that binds calcium to directly trigger vesicle fusion. Syt1 was identified in a microarray screen as a gene that may be sensitive to alcohol and heat shock. We found that Syt1 mRNA and protein expression are rapidly and robustly up-regulated by ethanol in mouse cortical neurons, and that the distribution of Syt1 protein along neuronal processes is also altered. Syt1 mRNA up-regulation is dependent on the activation of the transcription factor heat shock factor 1 (HSF1). The transfection of a constitutively active Hsf1 construct into neurons stimulates Syt1 transcription, while transfection of Hsf1 small interfering RNA (siRNA) or a constitutively inactive Hsf1 construct into neurons attenuates the induction of Syt1 by ethanol. This suggests that the activation of HSF1 can induce Syt1 expression and that this may be a mechanism by which alcohol regulates neurotransmitter release during brief exposures. Further analysis revealed that a subset of the genes encoding the core synaptic vesicle fusion (soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor; SNARE) proteins share this property of induction by ethanol, suggesting that alcohol may trigger a specific coordinated adaptation in synaptic function. This molecular mechanism could explain some of the changes in synaptic function that occur following alcohol administration and may be an important step in the process of neuronal adaptation to alcohol.

The mechanism by which the inhaled anesthetic isoflurane produces amnesia and immobility is not understood. Isoflurane modulates GABA(A) receptors (GABA(A)-Rs) in a manner that makes them plausible targets. We asked whether GABA(A)-R α2 subunits contribute to a site of anesthetic action in vivo. Previous studies demonstrated that Ser270 in the second transmembrane domain is involved in the modulation of GABA(A)-Rs by volatile anesthetics and alcohol, either as a binding site or a critical allosteric residue. We engineered GABA(A)-Rs with two mutations in the α2 subunit, changing Ser270 to His and Leu277 to Ala. Recombinant receptors with these mutations demonstrated normal affinity for GABA, but substantially reduced responses to isoflurane. We then produced mutant (knockin) mice in which this mutated subunit replaced the wild-type α2 subunit. The adult mutant mice were overtly normal, although there was evidence of enhanced neonatal mortality and fear conditioning. Electrophysiological recordings from dentate granule neurons in brain slices confirmed the decreased actions of isoflurane on mutant receptors contributing to inhibitory synaptic currents. The loss of righting reflex EC(50) for isoflurane did not differ between genotypes, but time to regain the righting reflex was increased in N(2) generation knockins. This effect was not observed at the N(4) generation. Isoflurane produced immobility (as measured by tail clamp) and amnesia (as measured by fear conditioning) in both wild-type and mutant mice, and potencies (EC(50)) did not differ between the strains for these actions of isoflurane. Thus, immobility or amnesia does not require isoflurane potentiation of the α2 subunit.

The alpha1beta2gamma2 and alpha3beta3gamma2 are two isoforms of gamma-aminobutyric acid type A (GABAA) receptor that are widely distributed in the brain. Both are found at synapses, for example in the thalamus, where they mediate distinctly different inhibitory postsynaptic current profiles, particularly with respect to decay time. The two isoforms were expressed in HEK293 cells, and single-channel activity was recorded from outside-out patches. The kinetic characteristics of both isoforms were investigated by analyzing single-channel currents over a wide range of GABA concentrations. Alpha1beta2gamma2 channels exhibited briefer active periods than alpha3beta3gamma2 channels over the entire range of agonist concentrations and had lower intraburst open probabilities at subsaturating concentrations. Activation mechanisms were constructed by fitting postulated schemes to data recorded at saturating and subsaturating GABA concentrations simultaneously. Reaction mechanisms were ranked according to log-likelihood values and how accurately they simulated ensemble currents. The highest ranked mechanism for both channels consisted of two sequential binding steps, followed by three conducting and three nonconducting configurations. The equilibrium dissociation constant for GABA at alpha3beta3gamma2 channels was approximately 2.6 microM compared with approximately 19 microM for alpha1beta2gamma2 channels, suggesting that GABA binds to the alpha3beta3gamma2 channels with higher affinity. A notable feature of the mechanism was that two consecutive doubly liganded shut states preceded all three open configurations. The lifetime of the third shut state was briefer for the alpha3beta3gamma2 channels. The longer active periods, higher affinity, and preference for conducting states are consistent with the slower decay of inhibitory currents at synapses that contain alpha3beta3gamma2 channels. The reaction mechanism we describe here may also be appropriate for the analysis of other types of GABAA receptors and provides a framework for rational investigation of the kinetic effects of a variety of therapeutic agents that activate or modulate GABAA receptors and hence influence synaptic and extrasynaptic inhibition in the central nervous system.

Although many people consume alcohol (ethanol), it remains unknown why some become addicted. Elucidating the molecular mechanisms of tolerance and physical dependence (withdrawal) may provide insight into alcohol addiction. While the exact molecular mechanisms of ethanol action are unclear, gamma-aminobutyric acid type A receptors (GABA(A)-Rs) have been extensively implicated in ethanol action. The alpha1 GABA(A)-R subunit is associated with tolerance and physical dependence, but its exact role remains unknown. In this report, we tested the hypothesis that alpha1-GABA(A)-Rs mediate in part these effects of ethanol.Ethanol-induced behavioral responses related to tolerance and physical dependence were investigated in knockin (KI) mice that have ethanol-insensitive alpha1 GABA(A)-Rs and wildtype (WT) controls. Acute functional tolerance (AFT) was assessed using the stationary dowel and loss of righting reflex (LORR) assays. Chronic tolerance was assessed on the LORR, fixed speed rotarod, hypothermia, and radiant tail-flick assays following 10 consecutive days of ethanol exposure. Withdrawal-related hyperexcitability was assessed by handling-induced convulsions following 3 cycles of ethanol vapor exposure/withdrawal. Immunoblots were used to assess alpha1 protein levels.Compared with controls, KI mice displayed decreased AFT and chronic tolerance to ethanol-induced motor ataxia, and also displayed heightened ethanol-withdrawal hyperexcitability. No differences between WT and KI mice were seen in other ethanol-induced behavioral measures. Following chronic exposure to ethanol, control mice displayed reductions in alpha1 protein levels, but KIs did not.We conclude that alpha1-GABA(A)-Rs play a role in tolerance to ethanol-induced motor ataxia and withdrawal-related hyperexcitability. However, other aspects of behavioral tolerance and physical dependence do not rely on alpha1-containing GABA(A)-Rs.

GABAergic neurons in the reticular thalamic nucleus (RTN) synapse onto thalamocortical neurons in the ventrobasal (VB) thalamus, and this reticulo-thalamocortical pathway is considered an anatomic target for general anesthetic-induced unconsciousness. A mutant mouse was engineered to harbor two amino acid substitutions (S270H, L277A) in the GABA(A) receptor (GABA(A)-R) alpha1 subunit; this mutation abolished sensitivity to the volatile anesthetic isoflurane in recombinant GABA(A)-Rs, and reduced in vivo sensitivity to isoflurane in the loss-of-righting-reflex assay. We examined the effects of the double mutation on GABA(A)-R-mediated synaptic currents and isoflurane sensitivity by recording from thalamic neurons in brain slices. The double mutation accelerated the decay, and decreased the (1/2) width of, evoked inhibitory postsynaptic currents (eIPSCs) in VB neurons and attenuated isoflurane-induced prolongation of the eIPSC. The hypnotic zolpidem, a selective modulator of GABA(A)-Rs containing the alpha1 subunit, prolonged eIPSC duration regardless of genotype, indicating that mutant mice incorporate alpha1 subunit-containing GABA(A)-Rs into synapses. In RTN neurons, which lack the alpha1 subunit, eIPSC duration was longer than in VB, regardless of genotype. Isoflurane reduced the efficacy of GABAergic transmission from RTN to VB, independent of genotype, suggesting a presynaptic action in RTN neurons. Consistent with this observation, isoflurane inhibited both tonic action potential and rebound burst firing in the presence of GABA(A)-R blockade. The suppressed excitability in RTN neurons is likely mediated by isoflurane-enhanced Ba(2+)-sensitive, but 4-aminopyridine-insenstive, potassium conductances. We conclude that isoflurane enhances inhibition of thalamic neurons in VB via GABA(A)-R-dependent, but in RTN via GABA(A)-R-independent, mechanisms.

Drinking alcohol is associated with the disturbance of normal sleep rhythms, and insomnia is a major factor in alcoholic relapse. The thalamus is a brain structure that plays a pivotal role in sleep regulation and rhythmicity. A number of studies have implicated GABA(A) receptors (GABA(A)-Rs) in the anxiolytic, amnestic, sedative, and anesthetic effects of ethanol. In the present study, we examined the effects of ethanol on both synaptic and extrasynaptic GABA(A)-Rs of relay neurons in the thalamus. We found that ethanol (or =50 mM) elicits a sustained current in thalamocortical relay neurons from the mouse ventrobasal thalamus, and this current is associated with a decrease in neuronal excitability and firing rate in response to depolarization. The steady current induced by ethanol was totally abolished by gabazine and was absent in relay neurons from GABA(A)-R alpha(4) subunit knockout mice, indicating that the effect of ethanol is to enhance tonic GABA-mediated inhibition. Ethanol (50 mM) enhanced the amplitude of tonic inhibition by nearly 50%. On the other hand, ethanol had no effect on spontaneous or evoked inhibitory postsynaptic currents (IPSCs) at 50 mM but did prolong IPSCs at 100 mM. Ethanol had no effect on the paired-pulse depression ratio, suggesting that the release of GABA from presynaptic terminals is insensitive to ethanol. We conclude that ethanol, at moderate (50 mM) but not low (10 mM) concentrations, can inhibit thalamocortical relay neurons and that this occurs mainly via the actions of ethanol at extrasynaptic GABA(A)-Rs containing GABA(A)-R alpha(4) subunits.

The family of gamma-aminobutyric acid type A receptors (GABA(A)Rs) mediates two types of inhibition in the mammalian brain. Phasic inhibition is mediated by synaptic GABA(A)Rs that are mainly comprised of alpha(1), beta(2), and gamma(2) subunits, whereas tonic inhibition is mediated by extrasynaptic GABA(A)Rs comprised of alpha(4/6), beta(2), and delta subunits. We investigated the activation properties of recombinant alpha(4)beta(2)delta and alpha(1)beta(2)gamma(2S) GABA(A)Rs in response to GABA and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one (THIP) using electrophysiological recordings from outside-out membrane patches. Rapid agonist application experiments indicated that THIP produced faster opening rates at alpha(4)beta(2)delta GABA(A)Rs (beta approximately 1600 s(-1)) than at alpha(1)beta(2)gamma(2S) GABA(A)Rs (beta approximately 460 s(-1)), whereas GABA activated alpha(1)beta(2)gamma(2S) GABA(A)Rs more rapidly (beta approximately 1800 s(-1)) than alpha(4)beta(2)delta GABA(A)Rs (beta440 s(-1)). Single channel recordings of alpha(1)beta(2)gamma(2S) and alpha(4)beta(2)delta GABA(A)Rs showed that both channels open to a main conductance state of approximately 25 pS at -70 mV when activated by GABA and low concentrations of THIP, whereas saturating concentrations of THIP elicited approximately 36 pS openings at both channels. Saturating concentrations of GABA elicited brief (10 ms) openings with low intraburst open probability (P(O) approximately 0.3) at alpha(4)beta(2)delta GABA(A)Rs and at least two "modes" of single channel bursting activity, lasting approximately 100 ms at alpha(1)beta(2)gamma(2S) GABA(A)Rs. The most prevalent bursting mode had a P(O) of approximately 0.7 and was described by a reaction scheme with three open and three shut states, whereas the "high" P(O) mode ( approximately 0.9) was characterized by two shut and three open states. Single channel activity elicited by THIP in alpha(4)beta(2)delta and alpha(1)beta(2)gamma(2S) GABA(A)Rs occurred as a single population of bursts (P(O) approximately 0.4-0.5) of moderate duration (approximately 33 ms) that could be described by schemes containing two shut and two open states for both GABA(A)Rs. Our data identify kinetic properties that are receptor-subtype specific and others that are agonist specific, including unitary conductance.

Volatile anesthetics are used clinically to produce analgesia, amnesia, unconsciousness, blunted autonomic responsiveness, and immobility. Previous work has shown that the volatile anesthetic isoflurane, at concentrations that produce unconsciousness (250-500 microM), enhances fast synaptic inhibition in the brain mediated by GABA(A) receptors (GABA(A)-Rs). In addition, isoflurane causes sedation at concentrations lower than those required to produce unconsciousness or analgesia. In this study, we found that isoflurane, at low concentrations (25-85 microM) associated with its sedative actions, elicits a sustained current associated with a conductance increase in thalamocortical neurons in the mouse ventrobasal (VB) nucleus. These isoflurane-evoked currents reversed polarity close to the Cl(-) equilibrium potential and were totally blocked by the GABA(A)-R antagonist gabazine. Isoflurane (25-250 microM) produced no sustained current in VB neurons from GABA(A)-R alpha(4)-subunit knockout (Gabra4(-/-)) mice, although 250 microM isoflurane enhanced synaptic inhibition in VB neurons from both wild-type and Gabra4(-/-) mice. These data indicate an obligatory requirement for alpha(4)-subunit expression in the generation of the isoflurane-activated current. In addition, isoflurane directly activated alpha(4)beta(2)delta GABA(A)-Rs expressed in human embryonic kidney 293 cells, and it was more potent at alpha(4)beta(2)delta than at alpha(1)beta(2)gamma(2) receptors (the presumptive extrasynaptic and synaptic GABA(A)-R subtypes in VB neurons). We conclude that the extrasynaptic GABA(A)-Rs of thalamocortical neurons are sensitive to low concentrations of isoflurane. In view of the crucial role of the thalamus in sensory processing, sleep, and cognition, the modulation of these extrasynaptic GABA(A)-Rs by isoflurane may contribute to the sedation and hypnosis associated with low doses of this anesthetic agent.

Background gamma-Aminobutyric acid type A receptors (GABA(A)-Rs) have been implicated in mediating some of the behavioral effects of ethanol (EtOH), but the contribution of specific GABA(A)-R subunits is not yet fully understood. The GABA(A)-R alpha 4 subunit often partners with beta2/3 and delta subunits to form extrasynaptic GABA(A)-Rs that mediate tonic inhibition. Several in vitro studies have suggested that these extrasynaptic GABA(A)-Rs may be particularly relevant to the intoxicating effects of low doses of EtOH. In alpha 4 subunit knockout mice, tonic inhibition was greatly reduced, as were the potentiating effects of EtOH. We therefore hypothesized that those behavioral responses to EtOH that are mediated by alpha 4-containing GABA(A)-Rs would be diminished in alpha 4 knockout mice. Methods We investigated behavioral responses to acute administration of moderate/high dose EtOH or pentylenetetrazol in alpha 4 subunit knockout mice. We compared behavioral responses to EtOH in alpha 4 knockout and wild-type littermates in the elevated plus maze (0.0, 1.0 g/kg EtOH), screen test (1.5, 2.0 g/kg), hypothermia (1.5, 2.0 g/kg), fixed speed rotarod (1.5, 2.0, 2.5 g/kg), open field (0.0, 1.0, 2.0 g/kg), radiant tail flick (2.0 g/kg), loss of righting reflex (3.5 g/kg), and EtOH metabolism and clearance assays. Sensitivity to pentylenetetrazol-induced seizures was also analyzed. Results No differences were observed between alpha 4 knockout mice and wild-type controls in terms of the baseline behavior in the absence of EtOH treatment or in the behavioral effects of EtOH in the assays tested. In contrast, alpha 4 knockout mice were significantly more sensitive to pentylenetetrazol-induced seizures. Conclusions We conclude that GABA(A)-Rs containing the alpha 4 subunit are not absolutely required for the acute behavioral responses to moderate/high dose EtOH that were assessed with the elevated plus maze, screen test, hypothermia, fixed speed rotarod, open field, radiant tail flick, and loss of right reflex assays. We further suggest that these findings are complicated by the demonstrated compensatory alterations in synaptic GABA(A)-R EtOH sensitivity and function in alpha 4 knockout mice.

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) has long been implicated in the anxiolytic, amnesic, and sedative behavioral effects of alcohol. A large number of studies have investigated the interactions of alcohol with GABA receptors. Many investigators have reported effects of "high concentrations" (50-100 mM) of alcohol on GABA-mediated synaptic inhibition, but effects of the "low concentrations" (1-30 mM) of alcohol normally associated with mild intoxication have been elusive until recently. A novel form of "tonic inhibition" has been described in the central nervous system (CNS) that is generated by the persistent activation of extrasynaptic gamma-aminobutyric acid type A receptors (GABAA-Rs). These receptors are specific GABAA-R subtypes and distinct from the synaptic subtypes. Tonic inhibition regulates the excitability of individual neurons and the activity and rhythmicity of neural networks. Interestingly, several reports show that tonic inhibition is sensitive to low concentrations of alcohol. The thalamus is a structure that is critically important in the control of sleep and wakefulness. GABAergic inhibition in the thalamus plays a crucial role in the generation of sleep waves. Among the various GABAA-R subunits, the alpha1, alpha4, beta2, and delta subunits are heavily expressed in thalamic relay nuclei. Tonic inhibition has been demonstrated in thalamocortical relay neurons, where it is mediated by alpha4beta2delta GABAA-Rs. These extrasynaptic receptors are highly sensitive to gaboxadol, a novel hypnotic, but insensitive to benzodiazepines. Tonic inhibition is absent in thalamic relay neurons from alpha4 knockout mice, as are the sedative and analgesic effects of gaboxadol. The sedative effects of alcohol can promote sleep. However, alcohol also disrupts the normal sleep pattern and reduces sleep quality. As a result, sleep disturbance caused by alcohol can play a role in the progression of alcoholism. As an important regulator of sleep cycles, inhibition in the thalamus may therefore be involved in both the sedative effects of alcohol and the development of alcoholism. Investigating the effects of alcohol on both synaptic and extrasynaptic GABAA-Rs in the thalamus should help us to understand the mechanisms underlying the interaction between alcohol and sleep.

Background Alcohol abuse and dependence are a serious public health problem. A large number of alcohol-regulated genes, (ARGs) are known to be influenced by alcohol use and withdrawal (AW), and recent evidence suggests that neuroadaptation to alcohol may be due in part to epigenetic changes in the expression of ARGs. Gabra4, which encodes the α4 subunit of GABAA receptors (GABAARs), is one of a number of ARGs that show remarkable plasticity in response to alcohol, being rapidly upregulated by acute alcohol exposure. This study addressed the effects of AW on changes in the expression of Gabra4 and related genes that encode other subunits of GABAARs, and the potential regulation of Gabra4 by microRNAs. Methods We studied gene and microRNAs expression, using RT-PCR and microRNA microarray in cultured cortical neurons treated with alcohol, which was then removed in order to simulate AW in vitro. We also used microRNA mimics or inhibitors, and a promoter-reporter construct carrying the 3′UTR of Gabra4. Results Eleven hours after removal of alcohol, Gabra4 was downregulated, with a modest increase in the expression of Gabrg2, but no change in the expression of Gabra1, Gabrd, or Gabrb2. microRNA profiling in neurons undergoing AW revealed upregulation in the expression of miR-155, miR-186, miR-24, and miR-375 after 8 h of AW. Transfection with molecular mimics of miR-186, miR-24, or miR-375 also downregulated Gabra4 expression, whereas transfection with the corresponding inhibitors of these microRNAs normalized Gabra4 expression in AW neurons to the level measured in control neurons. Promoter-reporter experiments supported the idea that miR-155, miR-186, miR-24, miR-27b, or miR-375 bind to the 3′UTR of Gabra4 and thereby inhibit protein production. Conclusions Our data suggest that AW decreases Gabra4 expression, and that this may be mediated in part by the induction of specific microRNAs in cortical neurons during AW.

The neurotransmitter GABA mediates the majority of rapid inhibition in the CNS. Inhibition can occur via the conventional mechanism, the transient activation of subsynaptic GABA A receptors (GABA A -Rs), or via continuous activation of high-affinity receptors by low concentrations of ambient GABA, leading to “tonic” inhibition that can control levels of excitability and network activity. The GABA A -R α4 subunit is expressed at high levels in the dentate gyrus and thalamus and is suspected to contribute to extrasynaptic GABA A -R-mediated tonic inhibition. Mice were engineered to lack the α4 subunit by targeted disruption of the Gabra4 gene. α4 Subunit knockout mice are viable, breed normally, and are superficially indistinguishable from WT mice. In electrophysiological recordings, these mice show a lack of tonic inhibition in dentate granule cells and thalamic relay neurons. Behaviorally, knockout mice are insensitive to the ataxic, sedative, and analgesic effects of the novel hypnotic drug, gaboxadol. These data demonstrate that tonic inhibition in dentate granule cells and thalamic relay neurons is mediated by extrasynaptic GABA A -Rs containing the α4 subunit and that gaboxadol achieves its effects via the activation of this GABA A -R subtype.

The binding of the neurotransmitter GABA induces conformational changes in the GABAA receptor (GABAAR), leading to the opening of a gate that controls ion permeation through an integral transmembrane pore. A number of structural elements within each subunit, located near the membrane interface, are believed to undergo relative movements during this activation process. In this study, we explored the functional role of the β-10 strand (pre-M1 segment), which connects the extracellular domain to the transmembrane domain. In α1β2γ2s GABAARs, analysis of the 12 residues of the β-10 strand in the α1 subunit proximal to the first transmembrane domain identified two residues, α1V212 and α1K220, in which mutations produced rightward shifts in the GABA concentration–response relationship and also reduced the relative efficacy of the partial agonist, piperidine-4-sulphonic acid. Ultra-fast agonist techniques were applied to mutant α1(K220A)β2γ2s GABAARs and revealed that the macroscopic functional deficit in this mutant could be attributed to a slowing of the opening rate constant, from ∼1500 s−1 in wild-type (WT) channels to ∼730 s−1 in the mutant channels, and a reduction in the time spent in the active state for the mutant. These changes were accompanied by a decrease in agonist affinity, with half-maximal activation rates achieved at 0.77 mm GABA in WT and 1.4 mm GABA in the α1(K220A)β2γ2s channels. The β-10 strand (pre-M1 segment) emerges, from this and other studies, as a key functional component in the activation of the GABAAR.

Despite the pervasiveness of alcohol (ethanol) use, it is unclear how the multiple molecular targets for ethanol contribute to its many behavioral effects. The function of GABA type A receptors (GABA(A)-Rs) is altered by ethanol, but there are multiple subtypes of these receptors, and thus far, individual subunits have not been definitively linked with specific behavioral actions. The alpha1 subunit of the GABA(A)-R is the most abundant alpha subunit in the brain, and the goal of this study was to determine the role of receptors containing this subunit in alcohol action. We designed an alpha1 subunit with serine 270 to histidine and leucine 277 to alanine mutations that was insensitive to potentiation by ethanol yet retained normal GABA sensitivity and constructed knockin mice containing this mutant subunit. Hippocampal slice recordings from these mice indicated that the mutant receptors were less sensitive to ethanol's potentiating effects. Behaviorally, we observed that mutant mice recovered more quickly from the motor-impairing effects of ethanol and etomidate, but not pentobarbital, and showed increased anxiolytic effects of ethanol. No differences were observed in ethanol-induced hypnosis, locomotor stimulation, cognitive impairment, or in ethanol preference and consumption. Overall, these studies demonstrate that the postsynaptic effects of ethanol at GABAergic synapses containing the alpha1 subunit are important for specific ethanol-induced behavioral effects.

Gamma-aminobutyric acid-type A receptors (GABA(A)-Rs) have been proposed as a target for many general anesthetics. We recently created knockin (KI) mice harboring a point mutation (serine 270 to histidine) in the GABA(A)-R alpha1 subunit. This mutation abolishes sensitivity of recombinant GABA(A)-Rs to isoflurane while maintaining normal sensitivity to halothane and increasing the potency of GABA. KI mice showed abnormalities in the EEG baseline, including occasional spike-wave activity and spindle-like bursts. When anesthetized with isoflurane, the KI mice but not the control mice revealed repetitive 4-5 Hz slow wave discharges in the cortical EEG. KI mice did not differ from controls in response to isoflurane or halothane in the standard tail clamp/withdrawal and loss of righting reflex assays. We recorded miniature inhibitory postsynaptic currents (mIPSCs) from hippocampal interneurons and pyramidal cells in brain slices. mIPSCs in neurons from KI mice were of normal amplitude, but decayed more slowly than controls. Hippocampal mIPSCs in control mice were significantly prolonged by 0.4 and 0.9 MAC isoflurane, and by 0.5 MAC halothane. In KI mice, the effect of isoflurane on mIPSC decay was dramatically reduced, while halothane prolonged mIPSCs as for controls. We conclude that the kinetic and pharmacological properties of hippocampal GABA(A)-Rs in the KI mouse recapitulate many features of mutant alpha1beta2gamma2 GABA(A)-Rs observed in vitro. GABA(A)-Rs containing alpha1 subunits do not appear to contribute to the actions of isoflurane in the spinal cord, but both EEG and synaptic recordings provide evidence for effects of isoflurane on these GABA(A)-R isoforms in cortical structures.

Although the depressant effects of the general anesthetic propofol on thalamocortical relay neurons clearly involve gamma-aminobutyric acid (GABA)(A) receptors, other mechanisms may be involved. The hyperpolarization-activated cation current (I(h)) regulates excitability and rhythmic firing in thalamocortical relay neurons in the ventrobasal (VB) complex of the thalamus. Here we investigated the effects of propofol on I(h)-related function in vitro and in vivo. In whole-cell current-clamp recordings from VB neurons in mouse (P23-35) brain slices, propofol markedly reduced the voltage sag and low-threshold rebound excitation that are characteristic of the activation of I(h). In whole-cell voltage-clamp recordings, propofol suppressed the I(h) conductance and slowed the kinetics of activation. The block of I(h) by propofol was associated with decreased regularity and frequency of delta-oscillations in VB neurons. The principal source of the I(h) current in these neurons is the hyperpolarization-activated cyclic nucleotide-gated (HCN) type 2 channel. In human embryonic kidney (HEK)293 cells expressing recombinant mouse HCN2 channels, propofol decreased I(h) and slowed the rate of channel activation. We also investigated whether propofol might have persistent effects on thalamic excitability in the mouse. Three hours following an injection of propofol sufficient to produce loss-of-righting reflex in mice (P35), I(h) was decreased, and this was accompanied by a corresponding decrease in HCN2 and HCN4 immunoreactivity in thalamocortical neurons in vivo. These results suggest that suppression of I(h) may contribute to the inhibition of thalamocortical activity during propofol anesthesia. Longer-term effects represent a novel form of propofol-mediated regulation of I(h).

How general anesthetics change neurotransmitter receptor function is not fully understood. In this short review, we describe the key molecular elements governing GABAA receptor activation and modulation by general anesthetics.

The volatile anesthetic isoflurane both prolongs and reduces the amplitude of GABA-mediated inhibitory postsynaptic currents (IPSCs) recorded in neurons. To explore the latter effect, we investigated isoflurane-induced inhibition of steady-state desensitized GABA currents in Xenopus oocytes expressing wild-type alpha(1)beta(2), alpha(1)beta(2)gamma(2s), mutant alpha(1)(S270H)beta(2) (serine to histidine at residue 270) or alpha(1)(S270H)beta(2)gamma(2s) receptors. The alpha(1) serine 270 site in TM2 (second transmembrane domain of the subunit) is postulated as a binding site for some volatile agents and is critical for positive modulation of sub-maximal GABA responses by isoflurane. For all receptor combinations, at < or =0.6 mM isoflurane (< or =2 minimum alveolar concentration (MAC)) current inhibitions were not pronounced ( approximately 10%) with block reaching half-maximal levels at supraclinical concentrations ( approximately 2 mM isoflurane, 6 MAC). Comparisons with other GABA(A) receptor blockers indicated that isoflurane blocks in a similar manner to picrotoxin, possibly via the pore of the receptor. The extent of isoflurane-induced inhibition was significantly attenuated by inclusion of the gamma(2s)-subunit but was unaffected by introduction of the S270H mutation in the alpha(1)-subunit. In conclusion, isoflurane binds with low affinity and with subunit-specificity to an inhibitory site on the GABA(A) receptor that is distinct from the site that facilitates positive modulation at the extracellular end of the pore.

Combinations of GluR5-GluR7, KA1, and KA2 subunits form kainate receptors, a subtype of excitatory ionotropic glutamate receptors. Isoflurane enhances the action of kainate receptors comprising GluR6 subunits expressed in oocytes. To test whether alterations of the GluR6 subunit gene affect the actions of inhaled anesthetics in vivo, we measured the minimum alveolar concentration of desflurane, isoflurane, and halothane in mice lacking the kainate receptor subunit GluR6 (GluR6 knockout mice) and mice with a dominant negative glutamine/arginine (Q/R) editing mutation in membrane domain 2 of the GluR6 receptor (GluR6 editing mutants), which increases the calcium permeability of kainate receptors containing GluR6Q. We also measured the capacity of isoflurane to interfere with Pavlovian fear conditioning to a tone and to context. Absence of the GluR6 subunit did not change the minimum alveolar concentration of isoflurane, desflurane, or halothane. Possibly, kainate receptors assembled from the remaining kainate receptor subunits compensate for the absent subunits and thereby produce a normal minimum alveolar concentration. A Q/R mutation that dominantly affects kainate receptors containing the GluR6 subunit in mice increased isoflurane minimum alveolar concentration (by 12%; P < 0.01), decreased desflurane minimum alveolar concentration (by 18%; P < 0.001), and did not change halothane minimum alveolar concentration (P = 0.25). These data may indicate that kainate receptors containing GluR6Q subunits differently modulate, directly or indirectly, the mechanism by which inhaled anesthetics cause immobility. The mutations of GluR6 that were studied did not affect the capacity of isoflurane to interfere with fear conditioning.

The gamma-aminobutyric acid type A (GABA(A)) receptor is the target of a structurally diverse group of sedative, hypnotic, and anesthetic drugs, including the volatile anesthetic isoflurane. Previous studies on the GABA(A) receptor have suggested the existence of a cavity located between transmembrane (TM) segments 2 and 3 in both alpha-1 and alpha-2 subunits, within which volatile anesthetics might bind. In this study, we have used site-directed mutagenesis to investigate the involvement of homologous residues of the GABA(A) alpha-3 subunit in allosteric modulation by isoflurane. Mutation of serine residue 294 within the TM2 to histidine or tyrosine increased the potency of GABA and decreased positive modulation by isoflurane. Mutation of alanine residue 315 within the TM3 to tryptophan increased the potency of GABA and abolished isoflurane modulation. The activity of the intravenous anesthetic propofol was unaltered from wild-type at these mutant receptors. These findings are consistent with the action of isoflurane on a critical site within the transmembrane domains of the receptor and suggest a degree of functional homology between the GABA(A) alpha-1, -2, and -3 subunits.