The glycine receptor is an important inhibitory receptor for both spinal and supraspinal function... more The glycine receptor is an important inhibitory receptor for both spinal and supraspinal functions, and mutations in receptor subunits are responsible for neurological disorders in several species, including humans. However, functional measurements of glycine receptors have generally been restricted to electrophysiological analysis of immature, cultured neurons. We developed a 36Cl- flux assay to measure glycine receptor function using membrane vesicles from spinal cord and brainstem of adult mice. The uptake of 36Cl- stimulated by glycine was characterized by a glycine EC50 of 22 microM for the major component and an EC50 of 0.5 microM for a minor component. Strychnine inhibited the glycine-stimulated uptake with an IC50 of 0.4 microM. The uptake was not affected by picrotoxin, bicuculline, or pentobarbital. Glycine-stimulated uptake reached a maximum by 10 s. This technique should prove useful for genetic and pharmacological analysis of the function of glycine receptors.
Behavioral studies on genetically diverse mice have proven powerful for determining relationships... more Behavioral studies on genetically diverse mice have proven powerful for determining relationships between phenotypes and have been widely used in alcohol research. Most of these studies rely on naturally occurring genetic polymorphisms among inbred strains and selected lines. Another approach is to introduce variation by engineering single-gene mutations in mice. We have tested 37 different mutant mice and their wildtype controls for a variety (31) of behaviors and have mined this data set by K-means clustering and analysis of correlations. We found a correlation between a stress-related response (activity in a novel environment) and alcohol consumption and preference for saccharin. We confirmed several relationships detected in earlier genetic studies, including positive correlation of alcohol consumption with saccharin consumption and negative correlations with conditioned taste aversion and alcohol withdrawal severity. Introduction of single-gene mutations either eliminated or greatly diminished these correlations. The three tests of alcohol consumption used (continuous two-bottle choice and two limited access tests: drinking in the dark and sustained high alcohol consumption) share a relationship with saccharin consumption, but differ from each other in their correlation networks. We suggest that alcohol consumption is controlled by multiple physiological systems where single-gene mutations can disrupt the networks of such systems.
Alcoholism: Clinical and Experimental Research, May 1, 2001
This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Jap... more This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were R. Adron Harris and Susumu Ueno. The presentations were (1) Protein kinase C-regulated sensitivity of ␥-aminobutyric acid type A (GABA A
Astrocytes play critical roles in central nervous system (CNS) homeostasis and are implicated in ... more Astrocytes play critical roles in central nervous system (CNS) homeostasis and are implicated in the pathogenesis of neurological and psychiatric conditions, including drug dependence. Little is known about the effects of chronic ethanol consumption on astrocyte gene expression. To address this gap in knowledge, we performed transcriptome-wide RNA sequencing of astrocytes isolated from the prefrontal cortex (PFC) of mice following chronic ethanol consumption. Differential expression analysis revealed ethanol-induced changes unique to astrocytes that were not identified in total homogenate preparations. Astrocyte-specific gene expression revealed calcium-related signaling and regulation of extracellular matrix genes as responses to chronic ethanol use. These findings emphasize the importance of investigating expression changes in specific cellular populations to define molecular consequences of chronic ethanol consumption in mammalian brain. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
The International Journal of Neuropsychopharmacology, Apr 1, 2008
Recent studies have demonstrated that metabotropic glutamate receptor 5 (mGluR5) antagonists decr... more Recent studies have demonstrated that metabotropic glutamate receptor 5 (mGluR5) antagonists decrease alcohol self-administration and suggest that the anti-craving medication, acamprosate, may also act to decrease mGluR5 function. To address the role of mGluR5 in behavioural actions of ethanol and acamprosate, we compared mutant mice with deletion of the mGluR5 gene and mice treated with a mGluR5 antagonist (MPEP) or acamprosate. Lack of mGluR5 or administration of MPEP reduced the severity of alcohol-induced withdrawal (AW), increased the sedative effect of alcohol (duration of loss of righting reflex ; LORR), and increased basal motor activity. The motor stimulation produced by ethanol was blocked by deletion of mGluR5, but not by injection of MPEP. Both acamprosate and MPEP increased ethanol-induced LORR and reduced AW. Importantly, the protective effects of both MPEP and acamprosate on AW were found when the drugs were injected before, but not after, injection of ethanol. This indicates that the drugs prevented development of dependence rather than merely producing an anticonvulsant action. No effects of acamprosate or MPEP on ethanol-induced LORR and AW were found in mGluR5 knockout mice, demonstrating that mGluR5 is required for these actions. mGluR5 null mutant mice showed decreased alcohol consumption in some, but not all, tests. These data show the importance of mGluR5 for several actions of alcohol and support the hypothesis that some effects of acamprosate require mGluR5 signalling.
Pharmacological studies implicate toll-like receptor 3 (TLR3) signaling in alcohol drinking. We e... more Pharmacological studies implicate toll-like receptor 3 (TLR3) signaling in alcohol drinking. We examined the role of TLR3 in behavioral responses to alcohol and GABAergic drugs by studying Tlr3 −/− mice. Because of opposing signaling between TLR3 and MyD88 pathways, we also evaluated Myd88 −/− mice. Ethanol consumption and preference decreased in male but not female Tlr3 −/− mice during two-bottle choice every-other-day (2BC-EOD) drinking. There were no genotype differences in either sex during continuous or limited-access drinking. Null mutations in Tlr3 or Myd88 did not alter conditioned taste aversion to alcohol and had small or no effects on conditioned place preference. The Tlr3 null mutation did not alter acute alcohol withdrawal. Male, but not female, Tlr3 −/− mice took longer than wild-type littermates to recover from ataxia by ethanol or diazepam and longer to recover from sedative-hypnotic effects of ethanol or gaboxadol, indicating regulation of GABAergic signaling by TLR3. Acute functional tolerance (AFT) to alcohol-induced ataxia was decreased in Tlr3 −/− mice but was increased in Myd88 −/− mice. Thus, MyD88 and TLR3 pathways coordinately regulate alcohol consumption and tolerance to intoxicating doses of alcohol and GABAergic drugs. Despite similar alcohol metabolism and similar amounts of total alcohol consumed during 2BC and 2BC-EOD procedures in C57BL/6J mice, only 2BC-EOD drinking induced tolerance to alcohol-induced ataxia. Ataxia recovery was inversely correlated with level of drinking in wild-type and Tlr3 −/− littermates. Thus, deleting Tlr3 reduces alcohol consumption by reducing AFT to alcohol and not by altering tolerance induced by 2BC-EOD drinking.
The glycine receptor is an important inhibitory receptor for both spinal and supraspinal function... more The glycine receptor is an important inhibitory receptor for both spinal and supraspinal functions, and mutations in receptor subunits are responsible for neurological disorders in several species, including humans. However, functional measurements of glycine receptors have generally been restricted to electrophysiological analysis of immature, cultured neurons. We developed a 36Cl- flux assay to measure glycine receptor function using membrane vesicles from spinal cord and brainstem of adult mice. The uptake of 36Cl- stimulated by glycine was characterized by a glycine EC50 of 22 microM for the major component and an EC50 of 0.5 microM for a minor component. Strychnine inhibited the glycine-stimulated uptake with an IC50 of 0.4 microM. The uptake was not affected by picrotoxin, bicuculline, or pentobarbital. Glycine-stimulated uptake reached a maximum by 10 s. This technique should prove useful for genetic and pharmacological analysis of the function of glycine receptors.
Behavioral studies on genetically diverse mice have proven powerful for determining relationships... more Behavioral studies on genetically diverse mice have proven powerful for determining relationships between phenotypes and have been widely used in alcohol research. Most of these studies rely on naturally occurring genetic polymorphisms among inbred strains and selected lines. Another approach is to introduce variation by engineering single-gene mutations in mice. We have tested 37 different mutant mice and their wildtype controls for a variety (31) of behaviors and have mined this data set by K-means clustering and analysis of correlations. We found a correlation between a stress-related response (activity in a novel environment) and alcohol consumption and preference for saccharin. We confirmed several relationships detected in earlier genetic studies, including positive correlation of alcohol consumption with saccharin consumption and negative correlations with conditioned taste aversion and alcohol withdrawal severity. Introduction of single-gene mutations either eliminated or greatly diminished these correlations. The three tests of alcohol consumption used (continuous two-bottle choice and two limited access tests: drinking in the dark and sustained high alcohol consumption) share a relationship with saccharin consumption, but differ from each other in their correlation networks. We suggest that alcohol consumption is controlled by multiple physiological systems where single-gene mutations can disrupt the networks of such systems.
Alcoholism: Clinical and Experimental Research, May 1, 2001
This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Jap... more This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were R. Adron Harris and Susumu Ueno. The presentations were (1) Protein kinase C-regulated sensitivity of ␥-aminobutyric acid type A (GABA A
Astrocytes play critical roles in central nervous system (CNS) homeostasis and are implicated in ... more Astrocytes play critical roles in central nervous system (CNS) homeostasis and are implicated in the pathogenesis of neurological and psychiatric conditions, including drug dependence. Little is known about the effects of chronic ethanol consumption on astrocyte gene expression. To address this gap in knowledge, we performed transcriptome-wide RNA sequencing of astrocytes isolated from the prefrontal cortex (PFC) of mice following chronic ethanol consumption. Differential expression analysis revealed ethanol-induced changes unique to astrocytes that were not identified in total homogenate preparations. Astrocyte-specific gene expression revealed calcium-related signaling and regulation of extracellular matrix genes as responses to chronic ethanol use. These findings emphasize the importance of investigating expression changes in specific cellular populations to define molecular consequences of chronic ethanol consumption in mammalian brain. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
The International Journal of Neuropsychopharmacology, Apr 1, 2008
Recent studies have demonstrated that metabotropic glutamate receptor 5 (mGluR5) antagonists decr... more Recent studies have demonstrated that metabotropic glutamate receptor 5 (mGluR5) antagonists decrease alcohol self-administration and suggest that the anti-craving medication, acamprosate, may also act to decrease mGluR5 function. To address the role of mGluR5 in behavioural actions of ethanol and acamprosate, we compared mutant mice with deletion of the mGluR5 gene and mice treated with a mGluR5 antagonist (MPEP) or acamprosate. Lack of mGluR5 or administration of MPEP reduced the severity of alcohol-induced withdrawal (AW), increased the sedative effect of alcohol (duration of loss of righting reflex ; LORR), and increased basal motor activity. The motor stimulation produced by ethanol was blocked by deletion of mGluR5, but not by injection of MPEP. Both acamprosate and MPEP increased ethanol-induced LORR and reduced AW. Importantly, the protective effects of both MPEP and acamprosate on AW were found when the drugs were injected before, but not after, injection of ethanol. This indicates that the drugs prevented development of dependence rather than merely producing an anticonvulsant action. No effects of acamprosate or MPEP on ethanol-induced LORR and AW were found in mGluR5 knockout mice, demonstrating that mGluR5 is required for these actions. mGluR5 null mutant mice showed decreased alcohol consumption in some, but not all, tests. These data show the importance of mGluR5 for several actions of alcohol and support the hypothesis that some effects of acamprosate require mGluR5 signalling.
Pharmacological studies implicate toll-like receptor 3 (TLR3) signaling in alcohol drinking. We e... more Pharmacological studies implicate toll-like receptor 3 (TLR3) signaling in alcohol drinking. We examined the role of TLR3 in behavioral responses to alcohol and GABAergic drugs by studying Tlr3 −/− mice. Because of opposing signaling between TLR3 and MyD88 pathways, we also evaluated Myd88 −/− mice. Ethanol consumption and preference decreased in male but not female Tlr3 −/− mice during two-bottle choice every-other-day (2BC-EOD) drinking. There were no genotype differences in either sex during continuous or limited-access drinking. Null mutations in Tlr3 or Myd88 did not alter conditioned taste aversion to alcohol and had small or no effects on conditioned place preference. The Tlr3 null mutation did not alter acute alcohol withdrawal. Male, but not female, Tlr3 −/− mice took longer than wild-type littermates to recover from ataxia by ethanol or diazepam and longer to recover from sedative-hypnotic effects of ethanol or gaboxadol, indicating regulation of GABAergic signaling by TLR3. Acute functional tolerance (AFT) to alcohol-induced ataxia was decreased in Tlr3 −/− mice but was increased in Myd88 −/− mice. Thus, MyD88 and TLR3 pathways coordinately regulate alcohol consumption and tolerance to intoxicating doses of alcohol and GABAergic drugs. Despite similar alcohol metabolism and similar amounts of total alcohol consumed during 2BC and 2BC-EOD procedures in C57BL/6J mice, only 2BC-EOD drinking induced tolerance to alcohol-induced ataxia. Ataxia recovery was inversely correlated with level of drinking in wild-type and Tlr3 −/− littermates. Thus, deleting Tlr3 reduces alcohol consumption by reducing AFT to alcohol and not by altering tolerance induced by 2BC-EOD drinking.
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Papers by Yuri Blednov