NMDA receptor subunit mRNA and protein expression in ethanol-withdrawal seizure-prone and -resistant mice

John N. Mason, Amy J. Eshleman, John Belknap, John Jr Crabbe, Jennifer Loftis, Tara A. Macey, Aaron Janowsky

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Background: Ethanol withdrawal seizure-prone (WSP) and -resistant (WSR) mice have been genetically selected for differences in handling-induced convulsion severity during withdrawal from chronic ethanol administration. Importantly, drug-naive mice from these selected lines also differ in handling-induced convulsion severity. Different N-methyl-D-aspartate (NMDA) receptor subunit and splice variant associations confer varying sensitivities to ethanol, and may play a role in the different behavioral responses of the WSP and WSR mice. Methods: In situ hybridization of riboprobes was used to characterize NMDA receptor subunit and splice variant mRNA expression in cortex and hippocampus from WSP and WSR mice. In addition, immunoblotting and immunohistochemistry were used to examine the expression of specific NMDA receptor subunits and splice variants in hippocampus and cortex from the selected mouse lines. Results: In situ hybridization of riboprobes indicated that, in brain sections from both WSP and WSR mice, there was a differential regional distribution of mRNA for the mouse NR1, NR2A, NR2B, and NR2C NMDA receptor subunits. However, there were no differences between the selected lines in the hybridization of riboprobes to hippocampal subfields or cortical layers. In addition, hybridization of the probe for a 63-base N1-terminal cassette of ethanol-sensitive NR1 splice variants labeled both cortex and hippocampus. The level of hybridization did not differ across subfields of the hippocampus. Results from Western blot and immunohistochemical experiments also indicated that there were no differences between selected lines in NMDA receptor subunit protein expression. However, there was a correlation between mRNA and protein expression in hippocampus and cortex for each NMDA receptor subunit that was examined. Conclusions: The data suggest that at the level of both mRNA and protein, NMDA receptor subunit and splice variant expression can be uncoupled from convulsion severity in mice that have been selectively bred for symptoms of ethanol withdrawal.

Original languageEnglish (US)
Pages (from-to)651-660
Number of pages10
JournalAlcoholism: Clinical and Experimental Research
Volume25
Issue number5
StatePublished - May 2001

Fingerprint

Protein Subunits
N-Methyl-D-Aspartate Receptors
Seizures
Ethanol
Messenger RNA
Hippocampus
Proteins
In Situ Hybridization
Substance Withdrawal Syndrome
Immunoblotting
Brain
Western Blotting
Immunohistochemistry
Pharmaceutical Preparations

Keywords

  • Alcohol
  • Cortex
  • Glutamate
  • Hippocampus
  • In Situ Hybridization

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • Toxicology

Cite this

NMDA receptor subunit mRNA and protein expression in ethanol-withdrawal seizure-prone and -resistant mice. / Mason, John N.; Eshleman, Amy J.; Belknap, John; Crabbe, John Jr; Loftis, Jennifer; Macey, Tara A.; Janowsky, Aaron.

In: Alcoholism: Clinical and Experimental Research, Vol. 25, No. 5, 05.2001, p. 651-660.

Research output: Contribution to journalArticle

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abstract = "Background: Ethanol withdrawal seizure-prone (WSP) and -resistant (WSR) mice have been genetically selected for differences in handling-induced convulsion severity during withdrawal from chronic ethanol administration. Importantly, drug-naive mice from these selected lines also differ in handling-induced convulsion severity. Different N-methyl-D-aspartate (NMDA) receptor subunit and splice variant associations confer varying sensitivities to ethanol, and may play a role in the different behavioral responses of the WSP and WSR mice. Methods: In situ hybridization of riboprobes was used to characterize NMDA receptor subunit and splice variant mRNA expression in cortex and hippocampus from WSP and WSR mice. In addition, immunoblotting and immunohistochemistry were used to examine the expression of specific NMDA receptor subunits and splice variants in hippocampus and cortex from the selected mouse lines. Results: In situ hybridization of riboprobes indicated that, in brain sections from both WSP and WSR mice, there was a differential regional distribution of mRNA for the mouse NR1, NR2A, NR2B, and NR2C NMDA receptor subunits. However, there were no differences between the selected lines in the hybridization of riboprobes to hippocampal subfields or cortical layers. In addition, hybridization of the probe for a 63-base N1-terminal cassette of ethanol-sensitive NR1 splice variants labeled both cortex and hippocampus. The level of hybridization did not differ across subfields of the hippocampus. Results from Western blot and immunohistochemical experiments also indicated that there were no differences between selected lines in NMDA receptor subunit protein expression. However, there was a correlation between mRNA and protein expression in hippocampus and cortex for each NMDA receptor subunit that was examined. Conclusions: The data suggest that at the level of both mRNA and protein, NMDA receptor subunit and splice variant expression can be uncoupled from convulsion severity in mice that have been selectively bred for symptoms of ethanol withdrawal.",
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T1 - NMDA receptor subunit mRNA and protein expression in ethanol-withdrawal seizure-prone and -resistant mice

AU - Mason, John N.

AU - Eshleman, Amy J.

AU - Belknap, John

AU - Crabbe, John Jr

AU - Loftis, Jennifer

AU - Macey, Tara A.

AU - Janowsky, Aaron

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N2 - Background: Ethanol withdrawal seizure-prone (WSP) and -resistant (WSR) mice have been genetically selected for differences in handling-induced convulsion severity during withdrawal from chronic ethanol administration. Importantly, drug-naive mice from these selected lines also differ in handling-induced convulsion severity. Different N-methyl-D-aspartate (NMDA) receptor subunit and splice variant associations confer varying sensitivities to ethanol, and may play a role in the different behavioral responses of the WSP and WSR mice. Methods: In situ hybridization of riboprobes was used to characterize NMDA receptor subunit and splice variant mRNA expression in cortex and hippocampus from WSP and WSR mice. In addition, immunoblotting and immunohistochemistry were used to examine the expression of specific NMDA receptor subunits and splice variants in hippocampus and cortex from the selected mouse lines. Results: In situ hybridization of riboprobes indicated that, in brain sections from both WSP and WSR mice, there was a differential regional distribution of mRNA for the mouse NR1, NR2A, NR2B, and NR2C NMDA receptor subunits. However, there were no differences between the selected lines in the hybridization of riboprobes to hippocampal subfields or cortical layers. In addition, hybridization of the probe for a 63-base N1-terminal cassette of ethanol-sensitive NR1 splice variants labeled both cortex and hippocampus. The level of hybridization did not differ across subfields of the hippocampus. Results from Western blot and immunohistochemical experiments also indicated that there were no differences between selected lines in NMDA receptor subunit protein expression. However, there was a correlation between mRNA and protein expression in hippocampus and cortex for each NMDA receptor subunit that was examined. Conclusions: The data suggest that at the level of both mRNA and protein, NMDA receptor subunit and splice variant expression can be uncoupled from convulsion severity in mice that have been selectively bred for symptoms of ethanol withdrawal.

AB - Background: Ethanol withdrawal seizure-prone (WSP) and -resistant (WSR) mice have been genetically selected for differences in handling-induced convulsion severity during withdrawal from chronic ethanol administration. Importantly, drug-naive mice from these selected lines also differ in handling-induced convulsion severity. Different N-methyl-D-aspartate (NMDA) receptor subunit and splice variant associations confer varying sensitivities to ethanol, and may play a role in the different behavioral responses of the WSP and WSR mice. Methods: In situ hybridization of riboprobes was used to characterize NMDA receptor subunit and splice variant mRNA expression in cortex and hippocampus from WSP and WSR mice. In addition, immunoblotting and immunohistochemistry were used to examine the expression of specific NMDA receptor subunits and splice variants in hippocampus and cortex from the selected mouse lines. Results: In situ hybridization of riboprobes indicated that, in brain sections from both WSP and WSR mice, there was a differential regional distribution of mRNA for the mouse NR1, NR2A, NR2B, and NR2C NMDA receptor subunits. However, there were no differences between the selected lines in the hybridization of riboprobes to hippocampal subfields or cortical layers. In addition, hybridization of the probe for a 63-base N1-terminal cassette of ethanol-sensitive NR1 splice variants labeled both cortex and hippocampus. The level of hybridization did not differ across subfields of the hippocampus. Results from Western blot and immunohistochemical experiments also indicated that there were no differences between selected lines in NMDA receptor subunit protein expression. However, there was a correlation between mRNA and protein expression in hippocampus and cortex for each NMDA receptor subunit that was examined. Conclusions: The data suggest that at the level of both mRNA and protein, NMDA receptor subunit and splice variant expression can be uncoupled from convulsion severity in mice that have been selectively bred for symptoms of ethanol withdrawal.

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