Search

Aims: Glycine receptors (GlyRs) are potentiated by physiologically relevant concentrations of ethanol, and mutations in the intracellular loop of α1 and α2 subunits reduced the effect of the drug. Knock-in (KI) mice having these individual mutations revealed that α1 and α2 subunits played a role in ethanol-induced sedation and ethanol intake. In this study, we wanted to examine if the effects of stacking both mutations in a 2xKI mouse model (α1/α2) generated by a selective breeding strategy further impacted cellular and behavioral responses to ethanol., Main Methods: We used electrophysiological recordings to examine ethanol's effect on GlyRs and evaluated ethanol-induced neuronal activation using c-Fos immunoreactivity and the genetically encoded calcium indicator GCaMP6s in the nucleus accumbens (nAc). We also examined ethanol-induced behavior using open field, loss of the righting response, and drinking in the dark (DID) paradigm., Key Findings: Ethanol did not potentiate GlyRs nor affect neuronal excitability in the nAc from 2xKI. Moreover, ethanol decreased the Ca 2+ signal in WT mice, whereas there were no changes in the signal in 2xKI mice. Interestingly, there was an increase in c-Fos baseline in the 2xKI mice in the absence of ethanol. Behavioral assays showed that 2xKI mice recovered faster from a sedative dose of ethanol and had higher ethanol intake on the first test day of the DID test than WT mice. Interestingly, an open-field assay showed that 2xKI mice displayed less anxiety-like behavior than WT mice., Significance: The results indicate that α1 and α2 subunits are biologically relevant targets for regulating sedative effects and ethanol consumption., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Previous studies have shown the presence of several subunits of the inhibitory glycine receptor (GlyR) in the reward system, specifically in medium spiny neurons (MSNs) of the nucleus Accumbens (nAc). It was suggested that GlyR α1 subunits regulate nAc excitability and ethanol consumption. However, little is known about the role of the α2 subunit in the adult brain since it is a subunit highly expressed during early brain development. In this study, we used genetically modified mice with a mutation (KR389-390AA) in the intracellular loop of the GlyR α2 subunit which results in a heteromeric α2β receptor that is insensitive to ethanol. Using this mouse model denoted knock-in α2 (KI α2), our electrophysiological studies showed that neurons in the adult nAc expressed functional KI GlyRs that were rather insensitive to ethanol when compared with WT GlyRs. In behavioral tests, the KI α2 mice did not show any difference in basal motor coordination, locomotor activity, or conditioned place preference compared with WT littermate controls. In terms of ethanol response, KI α2 male mice recovered faster from the administration of ataxic and sedative doses of ethanol. Furthermore, KI α2 mice consumed higher amounts of ethanol in the first days of the drinking in the dark protocol, as compared with WT mice. These results show that the α2 subunit is important for the potentiation of GlyRs in the adult brain and this might result in reduced sedation and increased ethanol consumption.

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., (© 2019 Society for the Study of Addiction.)

Some cannabinoids have been shown to suppress chronic pain by targeting glycine receptors (GlyRs). Although cannabinoid potentiation of α3 GlyRs is thought to contribute to cannabinoid-induced analgesia, the role of cannabinoid potentiation of α1 GlyRs in cannabinoid suppression of chronic pain remains unclear. Here we report that dehydroxylcannabidiol (DH-CBD), a nonpsychoactive cannabinoid, significantly suppresses chronic inflammatory pain caused by noxious heat stimulation. This effect may involve spinal α1 GlyRs since the expression level of α1 subunits in the spinal cord is positively correlated with CFA-induced inflammatory pain and the GlyRs antagonist strychnine blocks the DH-CBD-induced analgesia. A point-mutation of S296A in TM3 of α1 GlyRs significantly inhibits DH-CBD potentiation of glycine currents (I Gly ) in HEK-293 cells and neurons in lamina I-II of spinal cord slices. To explore the in vivo consequence of DH-CBD potentiation of α1 GlyRs, we generated a GlyRα1 S296A knock-in mouse line. We observed that DH-CBD-induced potentiation of I Gly and analgesia for inflammatory pain was absent in GlyRα1 S296A knock-in mice. These findings suggest that spinal α1 GlyR is a potential target for cannabinoid analgesia in chronic inflammatory pain., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We recently developed ultra-sensitive ethanol receptors (USERs) as a novel tool for investigation of single receptor subunit populations sensitized to extremely low ethanol concentrations that do not affect other receptors in the nervous system. To this end, we found that mutations within the extracellular Loop 2 region of glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABAARs) can significantly increase receptor sensitivity to micro-molar concentrations of ethanol resulting in up to a 100-fold increase in ethanol sensitivity relative to wild-type (WT) receptors. The current study investigated: (1) Whether structural manipulations of Loop 2 in α1 GlyRs could similarly increase receptor sensitivity to other anesthetics; and (2) If mutations exclusive to the C-terminal end of Loop 2 are sufficient to impart these changes. We expressed α1 GlyR USERs in Xenopus oocytes and tested the effects of three classes of anesthetics, isoflurane (volatile), propofol (intravenous), and lidocaine (local), known to enhance glycine-induced chloride currents using two-electrode voltage clamp electrophysiology. Loop 2 mutations produced a significant 10-fold increase in isoflurane and lidocaine sensitivity, but no increase in propofol sensitivity compared to WT α1 GlyRs. Interestingly, we also found that structural manipulations in the C-terminal end of Loop 2 were sufficient and selective for α1 GlyR modulation by ethanol, isoflurane, and lidocaine. These studies are the first to report the extracellular region of α1 GlyRs as a site of lidocaine action. Overall, the findings suggest that Loop 2 of α1 GlyRs is a key region that mediates isoflurane and lidocaine modulation. Moreover, the results identify important amino acids in Loop 2 that regulate isoflurane, lidocaine, and ethanol action. Collectively, these data indicate the commonality of the sites for isoflurane, lidocaine, and ethanol action, and the structural requirements for allosteric modulation on α1 GlyRs within the extracellular Loop 2 region., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Alcohol abuse and alcoholism are major health problems and one of the leading preventable causes of death. Before achieving better treatments for alcoholism, it is necessary to understand the critical actions of alcohol on membrane proteins that regulate fundamental functions in the central nervous system. After generating a genetically modified knock-in (KI) mouse having a glycine receptor (GlyR) with phenotypical silent mutations at KK385/386AA, we studied its cellular and in vivo ethanol sensitivity. Analyses with western blotting and immunocytochemistry indicated that the expression of α1 GlyRs in nervous tissues and spinal cord neurons (SCNs) were similar between WT and KI mice. The analysis of synaptic currents recorded from KI mice showed that the glycinergic synaptic transmission had normal properties, but the sensitivity to ethanol was significantly reduced. Furthermore, the glycine-evoked current in SCNs from KI was resistant to ethanol and G-protein activation by GTP-γ-S. In behavioral studies, KI mice did not display the foot-clasping behavior upon lifting by the tail and lacked an enhanced startle reflex response that are characteristic of other glycine KI mouse lines with markedly impaired glycine receptor function. The most notable characteristic of the KI mice was their significant lower sensitivity to ethanol (∼40%), expressed by shorter times in loss of righting reflex (LORR) in response to a sedative dose of ethanol (3.5 g/Kg). These data provide the first evidence to link a molecular site in the GlyR with the sedative effects produced by intoxicating doses of ethanol.

Although postsynaptic glycine receptors (GlyRs) as αβ heteromers attract considerable research attention, little is known about the role of presynaptic GlyRs, likely α homomers, in diseases. Here, we demonstrate that dehydroxylcannabidiol (DH-CBD), a nonpsychoactive cannabinoid, can rescue GlyR functional deficiency and exaggerated acoustic and tactile startle responses in mice bearing point mutations in α1 GlyRs that are responsible for a hereditary startle-hyperekplexia disease. The GlyRs expressed as α1 homomers either in HEK-293 cells or at presynaptic terminals of the calyceal synapses in the auditory brainstem are more vulnerable than heteromers to hyperekplexia mutation-induced impairment. Homomeric mutants are more sensitive to DH-CBD than are heteromers, suggesting presynaptic GlyRs as a primary target. Consistent with this idea, DH-CBD selectively rescues impaired presynaptic GlyR activity and diminished glycine release in the brainstem and spinal cord of hyperekplexic mutant mice. Thus, presynaptic α1 GlyRs emerge as a potential therapeutic target for dominant hyperekplexia disease and other diseases with GlyR deficiency.

Background: Volatile anesthetics (VAs) alter the function of key central nervous system proteins but it is not clear which, if any, of these targets mediates the immobility produced by VAs in the face of noxious stimulation. A leading candidate is the glycine receptor, a ligand-gated ion channel important for spinal physiology. VAs variously enhance such function, and blockade of spinal glycine receptors with strychnine affects the minimal alveolar concentration (an anesthetic EC50) in proportion to the degree of enhancement., Methods: We produced single amino acid mutations into the glycine receptor α1 subunit that increased (M287L, third transmembrane region) or decreased (Q266I, second transmembrane region) sensitivity to isoflurane in recombinant receptors, and introduced such receptors into mice. The resulting knockin mice presented impaired glycinergic transmission, but heterozygous animals survived to adulthood, and we determined the effect of isoflurane on glycine-evoked responses of brainstem neurons from the knockin mice, and the minimal alveolar concentration for isoflurane and other VAs in the immature and mature knockin mice., Results: Studies of glycine-evoked currents in brainstem neurons from knockin mice confirmed the changes seen with recombinant receptors. No increases in the minimal alveolar concentration were found in knockin mice, but the minimal alveolar concentration for isoflurane and enflurane (but not halothane) decreased in 2-week-old Q266I mice. This change is opposite to the one expected for a mutation that decreases the sensitivity to volatile anesthetics., Conclusion: Taken together, these results indicate that glycine receptors containing the α1 subunit are not likely to be crucial for the action of isoflurane and other VAs.

Glycine receptors (GlyRs) are inhibitory ligand-gated ion channels. Ethanol potentiates glycine activation of the GlyR, and putative binding sites for alcohol are located in the transmembrane (TM) domains between and within subunits. To alter alcohol sensitivity of GlyR, we introduced two mutations in the GlyR α1 subunit, M287L (TM3) and Q266I (TM2). After expression in Xenopus laevis oocytes, both mutants showed a reduction in glycine sensitivity and glycine-induced maximal currents. Activation by taurine, another endogenous agonist, was almost abolished in the M287L GlyR. The ethanol potentiation of glycine currents was reduced in the M287L GlyR and eliminated in Q266I. Physiological levels of zinc (100 nM) potentiate glycine responses in wild-type GlyR and also enhance the ethanol potentiation of glycine responses. Although zinc potentiation of glycine responses was unchanged in both mutants, zinc enhancement of ethanol potentiation of glycine responses was absent in M287L GlyRs. The Q266I mutation decreased conductance but increased mean open time (effects not seen in M287L). Two lines of knockin mice bearing these mutations were developed. Survival of homozygous knockin mice was impaired, probably as a consequence of impaired glycinergic transmission. Glycine showed a decreased capacity for displacing strychnine binding in heterozygous knockin mice. Electrophysiology in isolated neurons of brain stem showed decreased glycine-mediated currents and decreased ethanol potentiation in homozygous knockin mice. Molecular models of the wild-type and mutant GlyRs show a smaller water-filled cavity within the TM domains of the Q266I α1 subunit. The behavioral characterization of these knockin mice is presented in a companion article (J Pharmacol Exp Ther 340:317-329, 2012).

We used behavioral pharmacology to characterize heterozygous knockin mice with mutations (Q266I or M287L) in the α1 subunit of the glycine receptor (GlyR) (J Pharmacol Exp Ther 340:304-316, 2012). These mutations were designed to reduce (M287L) or eliminate (Q266I) ethanol potentiation of GlyR function. We asked which behavioral effects of ethanol would be reduced more in the Q266I mutant than the M287L and found rotarod ataxia to be the behavior that fulfilled this criterion. Compared with controls, the mutant mice also differed in ethanol consumption, ethanol-stimulated startle response, signs of acute physical dependence, and duration of loss of righting response produced by ethanol, butanol, ketamine, pentobarbital, and flurazepam. Some of these behavioral changes were mimicked in wild-type mice by acute injections of low, subconvulsive doses of strychnine. Both mutants showed increased acoustic startle response and increased sensitivity to strychnine seizures. Thus, in addition to reducing ethanol action on the GlyRs, these mutations reduced glycinergic inhibition, which may also alter sensitivity to GABAergic drugs.

Strychnine-sensitive glycine receptors (GlyRs) inhibit neurotransmission in the spinal cord and brainstem. To better define the function of this receptor in vivo, we constructed a point mutation that impairs receptor function in the alpha1-subunit and compared these knock-in mice to oscillator (spdot) mice lacking functional GlyR alpha1-subunits. Mutation of the serine residue at amino acid 267 to glutamine (alpha1S267Q) results in a GlyR with normal glycine potency but decreased maximal currents, as shown by electrophysiological recordings using Xenopus oocytes. In addition, single-channel recordings using human embryonic kidney 293 cells indicated profoundly altered properties of the mutated GlyR. We produced knock-in mice bearing the GlyR alpha1 S267Q mutation to assess the in vivo consequences of selectively decreasing GlyR efficacy. Chloride uptake into brain synaptoneurosomes from knock-in mice revealed decreased responses to maximally effective glycine concentrations, although wild-type levels of GlyR expression were observed using 3H-strychnine binding and immunoblotting. A profound increase in the acoustic startle response was observed in knock-in mice as well as a "limb clenching" phenotype. In contrast, no changes in coordination or pain perception were observed using the rotarod or hot-plate tests, and there was no change in GABA(A)-receptor-mediated chloride uptake. Homozygous S267Q knock-in mice, like homozygous spdot mice, exhibited seizures and died within 3 weeks of birth. In heterozygous spdot mice, both decreased 3H-strychnine binding and chloride flux were observed; however, neither enhanced acoustic startle responses nor limb clenching were seen. These data demonstrate that a dominant-negative point mutation in GlyR disrupting normal function can produce a more dramatic phenotype than the corresponding recessive null mutation, and provides a new animal model to evaluate GlyR function in vivo.

Unlabelled: We used two mouse lines with glycine receptor mutations to determine whether glycine receptors might play an important role in anesthetic responses in vivo. Spastic (spA) mutants were slightly more sensitive (P = 0.02) to enflurane in the loss-of-righting reflex assay (50% effective concentration [EC(50)] = 1.17 +/- 0.06 atm for controls versus 0.97 +/- 0.06 atm for spA) but were also substantially more resistant (P = 0.01) to enflurane in the tail clamp assay (EC(50) = 1.96 +/- 0.10 atm for controls versus 2.58 +/- 0.25 atm for spA). spA mice were also more sensitive to halothane (P < 0.001) in the loss-of-righting reflex assay (EC(50) = 0.81 +/- 0.03 atm for controls versus 0.57 +/- 0.04 atm for spA), but the responses of mutant and control mice to tail clamp in the presence of halothane were similar. Spasmodic control and mutant mice did not differ in their responses to the two drugs. Sleep time was substantially longer in both mutant mouse lines after injection of three hypnotics (midazolam, pentobarbital, and ethanol). Our results suggest a complex involvement of glycinergic pathways in mediating anesthetic responses. Greater sensitivity to the hypnotic effect of enflurane, halothane, midazolam, pentobarbital, and ethanol in mutant mice with diminished glycinergic capacity suggests that glycinergic activity is inversely related to hypnosis, whereas resistance to enflurane in the tail clamp assay suggests that glycinergic activity potentiates the minimum alveolar anesthetic concentration response. Halothane seems to share some, but not all, of enflurane's mechanisms, indicating that not all volatile anesthetics modulate glycinergic pathways equally., Implications: We tested two mouse lines with glycine receptor mutations to determine whether glycine receptors might play an important role in anesthetic responses in vivo. Both sensitivity and resistance to common anesthetics were observed in mutant mice, depending on the behavioral end-point evaluated.