On the participation of glycine receptors in the reconsolidation of spatial long-term memory in male rats.

The effects of intra-hippocampal manipulation of glycine receptors on the reconsolidation of recent and late long-term spatial memory were evaluated and assessed in the Morris water maze. The results obtained from the intra-hippocampal infusion of glycine and taurine demonstrated that taurine at a 100 nmol/side dose impaired the reconsolidation of recent and late long-term spatial memory. In comparison, at a dose of 10 nmol/side, it only affected the reconsolidation of late long-term spatial memory, reinforcing that there are differences between molecular mechanisms underlying recent and late long-term memory reconsolidation. On the other hand, glycine impaired the reconsolidation of early and late spatial memory when infused at a dose of 10 nmol/side, but not at a dose of 100 nmol/side, unless it is co-infused with an allosteric site antagonist of the NMDA receptor. Altogether these results show that glycine acting in situ in the hippocampal CA1 region exerts a pharmacological effect on U-curve, which can be explained by its concomitant action on its ionotropic receptor GlyR and on its NMDA receptor co-agonist site.

Antiviral Drug Ivermectin at Nanomolar Concentrations Inhibits Glycine-Induced Chloride Current in Rat Hippocampal Neurons.

Ivermectin (IVM) belongs to the class of macrocyclic lactones, which is used as an antiparasitic agent. At present, the researchers focus on possibility to use IVM in treatment of certain forms of cancer and viral diseases such as COVID-19. The mechanisms of IVM action are not clear. It is assumed that IVM affects chloride channels and increases cytoplasmic concentration of chloride. This study examines the effect of IVM on chloride currents induced by glycine (IGly). Experiments were carried out on isolated pyramidal neurons of the rat hippocampus with whole-cell patch clamp. A short-term (600 msec) application of IVM in a concentration of 10 µM induced a slow inward current, which persisted after washing the neurons. The low concentrations (0.1-1000 nM) of IVM did not induce any novel current, but it rapidly and reversibly reduced the peak amplitude and accelerated desensitization of IGly in a dose-dependent manner. The threshold concentrations of IVM sufficient to reduce peak amplitude of IGly and to accelerate desensitization of IGly were 100 nM and 0.1 nM, respectively. The study revealed a high sensitivity of neuronal glycine receptors to IVM.

Recognition of Potential COVID-19 Drug Treatments through the Study of Existing Protein-Drug and Protein-Protein Structures: An Analysis of Kinetically Active Residues.

We report the results of our in silico study of approved drugs as potential treatments for COVID-19. The study is based on the analysis of normal modes of proteins. The drugs studied include chloroquine, ivermectin, remdesivir, sofosbuvir, boceprevir, and α-difluoromethylornithine (DMFO). We applied the tools we developed and standard tools used in the structural biology community. Our results indicate that small molecules selectively bind to stable, kinetically active residues and residues adjoining them on the surface of proteins and inside protein pockets, and that some prefer hydrophobic sites over other active sites. Our approach is not restricted to viruses and can facilitate rational drug design, as well as improve our understanding of molecular interactions, in general.

Comparative Pro-cognitive and Neurochemical Profiles of Glycine Modulatory Site Agonists and Glycine Reuptake Inhibitors in the Rat: Potential Relevance to Cognitive Dysfunction and Its Management.

Frontocortical NMDA receptors are pivotal in regulating cognition and mood, are hypofunctional in schizophrenia, and may contribute to autistic spectrum disorders. Despite extensive interest in agents potentiating activity at the co-agonist glycine modulatory site, few comparative functional studies exist. This study systematically compared the actions of the glycine reuptake inhibitors, sarcosine (40-200 mg/kg) and ORG24598 (0.63-5 mg/kg), the agonists, glycine (40-800 mg/kg), and D-serine (10-160 mg/kg) and the partial agonists, S18841 (2.5 mg/kg s.c.) and D-cycloserine (2.5-40 mg/kg) that all dose-dependently prevented scopolamine disruption of social recognition in adult rats. Over similar dose ranges, they also prevented a delay-induced impairment of novel object recognition (NOR). Glycine reuptake inhibitors specifically elevated glycine but not D-serine levels in rat prefrontal cortical (PFC) microdialysates, while glycine and D-serine markedly increased levels of glycine and D-serine, respectively. D-Cycloserine slightly elevated D-serine levels. Conversely, S18841 exerted no influence on glycine, D-serine, other amino acids, monamines, or acetylcholine. Reversal of NOR deficits by systemic S18841 was prevented by the NMDA receptor antagonist, CPP (20 mg/kg), and the glycine modulatory site antagonist, L701,324 (10 mg/kg). S18841 blocked deficits in NOR following microinjection into the PFC (2.5-10 µg/side) but not the striatum. Finally, in rats socially isolated from weaning (a neurodevelopmental model of schizophrenia), S18841 (2.5 and 10 mg/kg s.c.) reversed impairment of NOR and contextual fear-motivated learning without altering isolation-induced hyperactivity. In conclusion, despite contrasting neurochemical profiles, partial glycine site agonists and glycine reuptake inhibitors exhibit comparable pro-cognitive effects in rats of potential relevance to treatment of schizophrenia and other brain disorders where cognitive performance is impaired.

Influence of nonsynaptic α1 glycine receptors on ethanol consumption and place preference.

Here, we used knock-in (KI) mice that have ethanol-insensitive alpha 1 glycine receptors (GlyRs) (KK385/386AA) to examine how alpha 1 GlyRs might affect binge drinking and conditioned place preference. Data show that tonic alpha 1 GlyR-mediated currents were exclusively sensitive to ethanol only in wild-type mice. Behavioral studies showed that the KI mice have a higher intake of ethanol upon first exposure to drinking and greater conditioned place preference to ethanol. This study suggests that nonsynaptic alpha 1-containing GlyRs have a role in motivational and early reinforcing effects of ethanol.

Alcohol abuse leads to great medical, social, and economic burdens throughout the world. It is believed that the rewarding actions of alcohol are mediated by alterations in the mesolimbic dopaminergic system leading to increased levels of dopamine in the nucleus accumbens (NAc). Little is known about the role that ligand-gated ion channels (LGICs), such as glycine receptors (GlyRs), have in regulating levels of ethanol intake and place preference. In this study, we used knock-in (KI) mice that have ethanol-insensitive α1 GlyRs (KK385/386AA) and a combination of electrophysiological and behavioral approaches to examine how expression of ethanol-resistant α1 GlyRs in brain neurons might affect binge drinking and conditioned place preference. Data show that tonic α1 GlyR-mediated currents that modulate accumbal excitability were exclusively sensitive to ethanol only in wild-type (WT) mice. Behavioral studies showed that the KI mice have a higher intake of ethanol upon first exposure to drinking and greater conditioned place preference to ethanol, suggesting that α1 GlyRs in the brain have a protective role against abuse. This study suggests that nonsynaptic α1-containing GlyRs have a role in motivational and early reinforcing effects of ethanol and open a novel opportunity for pharmacotherapy development to treat alcohol use disorders.

Thyrotropin-Releasing-Hormone-Synthesizing Neurons of the Hypothalamic Paraventricular Nucleus Are Inhibited by Glycinergic Inputs.

Background: Glycine is a classical neurotransmitter that has role in both inhibitory and excitatory synapses. To understand whether glycinergic inputs are involved in the regulation of the hypophysiotropic thyrotropin-releasing hormone (TRH) neurons, the central controllers of the hypothalamic-pituitary-thyroid axis, the glycinergic innervation of the TRH neurons was studied in the hypothalamic paraventricular nucleus (PVN). Methods: Double-labeling immunocytochemistry and patch-clamp electrophysiology were used to determine the role of glycinergic neurons in the regulation of TRH neurons in the PVN. Anterograde and retrograde tracing methods were used to determine the sources of the glycinergic input of TRH neurons. Results: Glycine transporter-2 (GLYT2), a marker of glycinergic neurons, containing axons were found to establish symmetric type of synapses on TRH neurons in the PVN. Furthermore, glycine receptor immunoreactivity was observed in these TRH neurons. The raphe magnus (RMg) and the ventrolateral periaqueductal gray (VLPAG) were found to be the exclusive sources of the glycinergic innervation of the TRH neurons within the PVN. Patch-clamp electrophysiology using sections of TRH-IRES-tdTomato mice showed that glycine hyperpolarized the TRH neurons and completely blocked the firing of these neurons. Glycine also markedly hyperpolarized the TRH neurons in the presence of tetrodotoxin demonstrating the direct effect of glycine. In more than 60% of the TRH neurons, spontaneous inhibitory postsynaptic currents (sIPSCs) were observed, even after the pharmacological inhibition of glutamatergic and GABAergic neuronal transmission. The glycine antagonist, strychnine, almost completely abolished these sIPSCs, demonstrating the inhibitory nature of the glycinergic input of TRH neurons. Conclusions: These data demonstrate that TRH neurons in the PVN receive glycinergic inputs from the RMg and the VLPAG. The symmetric type of synaptic connection and the results of the electrophysiological experiments demonstrate the inhibitory nature of these inputs.

High ethanol sensitive glycine receptors regulate firing in D1 medium spiny neurons in the nucleus accumbens.

Inhibitory glycine receptors (GlyRs) are widely expressed in spinal cord and brain stem. They are also expressed in the nucleus Accumbens (nAc) where they have been implicated in the release of dopamine from the ventral tegmental area to the nAc in the presence of ethanol. One of the major types of neurons in the nAc are the Dopamine 1 receptor-expressing (D1+) medium spiny neurons (MSNs) that are activated when addictive drugs, like ethanol, are administrated. Thus, D1(+) MSNs are a relevant target for the study of ethanol effects. Here, using electrophysiological recordings, we report that GlyRs in D1(+) MSNs are highly sensitive to ethanol, with potentiation starting at 5 mM (26 ± 5%). Single channel recordings in D1(+) MSNs showed that 10 mM ethanol increased the open probability of the channel (0.22 ± 0.05 versus 0.66 ± 0.16), but did not affect channel conductance (~40 pS). A glycinergic mediated tonic current in D1(+) MSNs was potentiated by 10 and 50 mM ethanol causing a reduction in the excitability of these cells. A 34 ± 7% reduction in action potential firing was observed in these neurons in the presence of 50 mM ethanol. Interestingly, no effects of ethanol were detected in the presence of strychnine or in D1(-) MSNs in the nAc. These results indicate that GlyRs present in D1(+) MSNs are sensitive to low concentrations of ethanol, and that potentiation of this inhibitory current regulates the activation of nAc, acting as a homeostatic signal that would prevent over-activation of the reward system when drugs like ethanol are consumed.

Kavalactones from Kava (Piper methysticum) root extract as modulators of recombinant human glycine receptors.

Roots of kava (Piper methysticum) plant are used in almost all Pacific Ocean cultures to prepare a drink with sedative, anesthetic and euphoric properties. One of the main active ingredients of the extract are kava lactones. Here, kava root CO2 extract and three kavalactones, DL-kavain, dihydrokavain and yangonin (isolated from whole extract by column chromatography) were tested for their inhibitory action on recombinant homomeric human α1 glycine receptors expressed in HEK293 cells. Kava CO2 root extract, as well as the individual components DL-kavain, dihydrokavain and yangonin inhibited glycine receptor activity in a dose-dependent manner. DL-kavain was the most potent inhibitor (IC50 = 0.077 ± 0.002 mm), followed by yangonin (IC50 = 0.31 ± 0.04 mm) and dihydrokavain (IC50 = 3.23 ± 0.10 mm) which were 4- and 40-fold less active than DL-kavain, respectively. Application of kava root extract did not reduce maximum currents, but increased EC50 of glycine. Simultaneous application of kava extract and strychnine showed additive inhibition, suggesting that binding of kavalactones and strychnine on the receptor is mutually exclusive. Overall, kavalactones exert a moderate inhibitory effect on the human α1 glycine receptor with DL-kavain being the most potent constituent.

Ubiquitination and inhibition of glycine receptor by HUWE1 in spinal cord dorsal horn.

Glycine receptors (GlyRs) are pentameric proteins that consist of α (α1-α4) subunits and/or ß subunit. In the spinal cord of adult animals, the majority of inhibitory glycinergic neurotransmission is mediated by α1 subunit-containing GlyRs. The reduced glycinergic inhibition (disinhibition) is proposed to increase the excitabilities and spontaneous activities of spinal nociceptive neurons during pathological pain. However, the molecular mechanisms by which peripheral lesions impair GlyRs-α1-mediated synaptic inhibition remain largely unknown. Here we found that activity-dependent ubiquitination of GlyRs-α1 subunit might contribute to glycinergic disinhibition after peripheral inflammation. Our data showed that HUWE1 (HECT, UBA, WWE domain containing 1), an E3 ubiquitin ligase, located at spinal synapses and specifically interacted with GlyRs-α1 subunit. By ubiquitinating GlyRs-α1, HUWE1 reduced the surface expression of GlyRs-α1 through endocytic pathway. In the dorsal horn of Complete Freund's Adjuvant-injected mice, shRNA-mediated knockdown of HUWE1 blunted GlyRs-α1 ubiquitination, potentiated glycinergic synaptic transmission and attenuated inflammatory pain. These data implicated that ubiquitin modification of GlyRs-α1 represented an important way for peripheral inflammation to reduce spinal glycinergic inhibition and that interference with HUWE1 activity generated analgesic action by resuming GlyRs-α1-mediated synaptic transmission.

Modulation of Glycine-Mediated Spinal Neurotransmission for the Treatment of Chronic Pain.

Chronic pain constitutes a significant and expanding worldwide health crisis. Currently available analgesics poorly serve individuals suffering from chronic pain, and new therapeutic agents that are more effective, safer, and devoid of abuse liabilities are desperately needed. Among the myriad of cellular and molecular processes contributing to chronic pain, spinal disinhibition of pain signaling to higher cortical centers plays a critical role. Accumulating evidence shows that glycinergic inhibitory neurotransmission in the spinal cord dorsal horn gates nociceptive signaling, is essential in maintaining physiological pain sensitivity, and is diminished in pathological pain states. Thus, it is hypothesized that agents capable of enhancing glycinergic tone within the dorsal horn could obtund nociceptor signaling to the brain and serve as analgesics for persistent pain. This Perspective highlights the potential that pharmacotherapies capable of increasing inhibitory spinal glycinergic neurotransmission hold in providing new and transformative analgesic therapies for the treatment of chronic pain.

Identification of Positive Allosteric Modulators of Glycine Receptors from a High-Throughput Screen Using a Fluorescent Membrane Potential Assay.

Glycine receptor 3 (GlyRα3) is a ligand-gated ion channel of the cys-loop family that plays a key role in mediating inhibitory neurotransmission and regulation of pain signaling in the dorsal horn. Potentiation of GlyRα3 function is therefore of interest as a putative analgesic mechanism with which to target new therapeutics. However, to date, positive allosteric modulators (PAMs) of this receptor with sufficient selectivity to enable target validation studies have not been described. To address this lack of pharmacological tools, we developed a suite of in vitro assays comprising a high-throughput fluorescent membrane potential screen and a medium-throughput electrophysiology assay using IonFlux HT together with conventional manual patch clamp. Using these assays, we conducted a primary screening campaign and report the structures of hit compounds identified as GlyR PAMs. Our functional characterization data reveal a hit compound with high efficacy relative to current known potentiators and selectivity over GABAAR, another major class of inhibitory neurotransmission receptors of importance to pain. These small-molecule GlyR PAMs have high potential both as early tool compounds to enable pharmacological studies of GlyR inhibitory neurotransmission and as a starting point for the development of potent, selective GlyRα3 PAMs as novel analgesics.

Activation of Strychnine-Sensitive Glycine Receptors by Shilajit on Preoptic Hypothalamic Neurons of Juvenile Mice.

Shilajit, a mineral pitch, has been used in Ayurveda and Siddha system of medicine to treat many human ailments, and is reported to contain at least 85 minerals in ionic form. This study examined the possible mechanism of Shilajit action on preoptic hypothalamic neurons using juvenile mice. The hypothalamic neurons are the key regulator of many hormonal systems. In voltage clamp mode at a holding potential of -60 mV, and under a high chloride pipette solution, Shilajit induced dose-dependent inward current. Shilajit-induced inward currents were reproducible and persisted in the presence of 0.5 µM tetrodotoxin (TTX) suggesting a postsynaptic action of Shilajit on hypothalamic neurons. The currents induced by Shilajit were almost completely blocked by 2 µM strychnine (Stry), a glycine receptor antagonist. In addition, Shilajit-induced inward currents were partially blocked by bicuculline. Under a gramicidin-perforated patch clamp mode, Shilajit induced membrane depolarization on juvenile neurons. These results show that Shilajit affects hypothalamic neuronal activities by activating the Stry-sensitive glycine receptor with α2/α2ß subunit. Taken together, these results suggest that Shilajit contains some ingredients with possible glycine mimetic activities and might influence hypothalamic neurophysiology through activation of Stry-sensitive glycine receptor-mediated responses on hypothalamic neurons postsynaptically.

Nonsynaptic glycine release is involved in the early KCC2 expression.

The cation-chloride co-transporters are important regulators of the cellular Cl(-) homeostasis. Among them the Na(+) -K(+) -2Cl(-) co-transporter (NKCC1) is responsible for intracellular chloride accumulation in most immature brain structures, whereas the K(+) -Cl(-) co-transporter (KCC2) extrudes chloride from mature neurons, ensuring chloride-mediated inhibitory effects of GABA/glycine. We have shown that both KCC2 and NKCC1 are expressed at early embryonic stages (E11.5) in the ventral spinal cord (SC). The mechanisms by which KCC2 is prematurely expressed are unknown. In this study, we found that chronically blocking glycine receptors (GlyR) by strychnine led to a loss of KCC2 expression, without affecting NKCC1 level. This effect was not dependent on the firing of Na(+) action potentials but was mimicked by a Ca(2+) -dependent PKC blocker. Blocking the vesicular release of neurotransmitters did not impinge on strychnine effect whereas blocking volume-sensitive outwardly rectifying (VSOR) chloride channels reproduced the GlyR blockade, suggesting that KCC2 is controlled by a glycine release from progenitor radial cells in immature ventral spinal networks. Finally, we showed that the strychnine treatment prevented the maturation of rhythmic spontaneous activity. Thereby, the GlyR-activation is a necessary developmental process for the expression of functional spinal motor networks. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 764-779, 2016.

Advances in the pharmacology of lGICs auxiliary subunits.

Ligand-gated ion channels (LGICs) are cell surface integral proteins that mediate the fast neurotransmission in the nervous system. LGICs require auxiliary subunits for their trafficking, assembly and pharmacological modulation. Auxiliary subunits do not form functional homomeric receptors, but are reported to assemble with the principal subunits in order to modulate their pharmacological profiles. For example, nACh receptors are built at least by co-assemble of α and ß subunits, and the neuronal auxiliary subunits ß3 and α5 and muscle type ß, δ, γ, and ϵ determine the agonist affinity of these receptors. Serotonergic 5-HT3B, 5-HT3C, 5-HT3D and 5-HT3E are reported to assemble with the 5-HT3A subunit to modulate its pharmacological profile. Functional studies evaluating the role of γ2 and δ auxiliary subunits of GABAA receptors have made important advances in the understanding of the action of benzodiazepines, ethanol and neurosteroids. Glycine receptors are composed principally by α1-3 subunits and the auxiliary subunit ß determines their synaptic location and their pharmacological response to propofol and ethanol. NMDA receptors appear to be functional as heterotetrameric channels. So far, the existence of NMDA auxiliary subunits is controversial. On the other hand, Kainate receptors are modulated by NETO 1 and 2. AMPA receptors are modulated by TARPs, Shisa 9, CKAMP44, CNIH2-3 auxiliary proteins reported that controls their trafficking, conductance and gating of channels. P2X receptors are able to associate with auxiliary Pannexin-1 protein to modulate P2X7 receptors. Considering the pharmacological relevance of different LGICs auxiliary subunits in the present work we will highlight the therapeutic potential of these modulator proteins.

Ethanol effects on glycinergic transmission: From molecular pharmacology to behavior responses.

It is well accepted that ethanol is able to produce major health and economic problems associated to its abuse. Because of its intoxicating and addictive properties, it is necessary to analyze its effect in the central nervous system. However, we are only now learning about the mechanisms controlling the modification of important membrane proteins such as ligand-activated ion channels by ethanol. Furthermore, only recently are these effects being correlated to behavioral changes. Current studies show that the glycine receptor (GlyR) is a susceptible target for low concentrations of ethanol (5-40mM). GlyRs are relevant for the effects of ethanol because they are found in the spinal cord and brain stem where they primarily express the α1 subunit. More recently, the presence of GlyRs was described in higher regions, such as the hippocampus and nucleus accumbens, with a prevalence of α2/α3 subunits. Here, we review data on the following aspects of ethanol effects on GlyRs: (1) direct interaction of ethanol with amino acids in the extracellular or transmembrane domains, and indirect mechanisms through the activation of signal transduction pathways; (2) analysis of α2 and α3 subunits having different sensitivities to ethanol which allows the identification of structural requirements for ethanol modulation present in the intracellular domain and C-terminal region; (3) Genetically modified knock-in mice for α1 GlyRs that have an impaired interaction with G protein and demonstrate reduced ethanol sensitivity without changes in glycinergic transmission; and (4) GlyRs as potential therapeutic targets.

Alterations in ethanol-induced accumbal transmission after acute and long-term zinc depletion.

Alcoholism is subject to extensive research, but the role of changes in metabolism caused by alcohol consumption has been poorly investigated. Zinc (Zn(2+) ) deficiency is a common metabolic aberration among alcoholics and Zn(2+) influences the function of ligand-gated ion channels, known pharmacological targets of ethanol (EtOH). Here, we investigate whether manipulation of extracellular levels of Zn(2+) modulates EtOH-induced increases of dopamine (DA) output, as measured by in vivo microdialysis in the rat, and whether voluntary EtOH consumption is altered by Zn(2+) deficiency. Our findings show that the Zn(2+) -chelating agent tricine slowly raises DA levels when perfused in the nucleus accumbens (nAc), whereas the more potent Zn(2+) chelator TPEN reduces DA levels. We also show that pre-treatment with either tricine or TPEN blocks the EtOH-induced DA elevation. Chronic Zn(2+) deficiency induced by a Zn(2+) -free diet did not affect EtOH consumption, but excitatory transmission, assessed by striatal field-potential recordings in the nAc shell, was significantly modulated both by Zn(2+) -free diet and by EtOH consumption, as compared with the EtOH naïve controls. The present study indicates that Zn(2+) influences EtOH's interaction with the brain reward system, possibly by interfering with glycine receptor and GABAA receptor function. This also implies that Zn(2+) deficiency among alcoholics may be important to correct in order to normalize important aspects of brain function.

Glycine and GABA(A) ultra-sensitive ethanol receptors as novel tools for alcohol and brain research.

A critical obstacle to developing effective medications to prevent and/or treat alcohol use disorders is the lack of specific knowledge regarding the plethora of molecular targets and mechanisms underlying alcohol (ethanol) action in the brain. To identify the role of individual receptor subunits in ethanol-induced behaviors, we developed a novel class of ultra-sensitive ethanol receptors (USERs) that allow activation of a single receptor subunit population sensitized to extremely low ethanol concentrations. USERs were created by mutating as few as four residues in the extracellular loop 2 region of glycine receptors (GlyRs) or γ-aminobutyric acid type A receptors (GABA(A)Rs), which are implicated in causing many behavioral effects linked to ethanol abuse. USERs, expressed in Xenopus oocytes and tested using two-electrode voltage clamp, demonstrated an increase in ethanol sensitivity of 100-fold over wild-type receptors by significantly decreasing the threshold and increasing the magnitude of ethanol response, without altering general receptor properties including sensitivity to the neurosteroid, allopregnanolone. These profound changes in ethanol sensitivity were observed across multiple subunits of GlyRs and GABA(A)Rs. Collectively, our studies set the stage for using USER technology in genetically engineered animals as a unique tool to increase understanding of the neurobiological basis of the behavioral effects of ethanol.

4-bromopropofol decreases action potential generation in spinal neurons by inducing a glycine receptor-mediated tonic conductance.

BACKGROUND AND PURPOSE: Impaired function of spinal strychnine-sensitive glycine receptors gives rise to chronic pain states and movement disorders. Therefore, increased activity of glycine receptors should help to treat such disorders. Although compounds targeting glycine receptors with a high selectivity are lacking, halogenated analogues of propofol have recently been considered as potential candidates. Therefore we asked whether 4-bromopropofol attenuated the excitability of spinal neurons by promoting glycine receptor-dependent inhibition. EXPERIMENTAL APPROACH: The actions of sub-anaesthetic concentrations of propofol and 4-bromopropofol were investigated in spinal tissue cultures prepared from mice. Drug-induced alterations in action potential firing were monitored by extracellular multi-unit recordings. The effects on GABAA and glycine receptor-mediated inhibition were quantified by whole-cell voltage-clamp recordings. KEY RESULTS: Low concentrations of 4-bromopropofol (50 nM) reduced action potential activity of ventral horn neurons by about 30%, compared with sham-treated slices. This effect was completely abolished by strychnine (1 µM). In voltage-clamped neurons, 4-bromopropofol activated glycine receptors, generating a tonic current of 65 ± 10 pA, while GABAA - and glycine receptor-mediated synaptic transmission remained unaffected. CONCLUSIONS AND IMPLICATIONS: The highest glycine levels in the CNS are found in the ventral horn of the spinal cord, a region mediating pain-induced motor reflexes and participating in the control of muscle tone. 4-Bromopropofol may serve as a starting point for the development of non-sedative, non-addictive, muscle relaxants and analgesics to be used to treat low back pain.

Contaminating levels of zinc found in commonly-used labware and buffers affect glycine receptor currents.

Zinc is an allosteric modulator of glycine receptor function, enhancing the effects of glycine at nM to low µM concentrations, and inhibiting its effects at higher concentrations. Because of zinc's high potency at the glycine receptor, there exists a possibility that effects attributed solely to exogenously-applied glycine in fact contain an undetected contribution of zinc acting as an allosteric modulator. We found that glycine solutions made up in standard buffers and using deionized distilled water produced effects that could be decreased by the zinc chelator tricine. This phenomenon was observed in three different vials tested and persisted even if vials were extensively washed, suggesting the zinc was probably present in the buffer constituents. In addition, polystyrene, but not glass, pipets bore a contaminant that enhanced glycine receptor function and that could also be antagonized by tricine. Our findings suggest that without checking for this effect using a chelator such as tricine, one cannot assume that responses elicited by glycine applied alone are not necessarily also partially due to some level of allosteric modulation by zinc.

Molecular and genetic determinants of alcohol dependence.

Alcohol dependence is a complex disorder affecting all social and ethnic groups. Although the scientific understanding of the mechanism governing this multifactorial disease is still in its infancy, understanding its biological bases, including the potential contribution of genetic factors, is key to characterizing individual's risk and developing efficacious therapeutic target to combat the disease. This review provides an overview of different approaches that are being increasingly integrated to extend our knowledge of the genetic underpinnings of alcohol dependence.