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Voluntary Ethanol Consumption Induced by Social Isolation Reverses the Increase of α(4)/δ GABA(A) Receptor Gene Expression and Function in the Hippocampus of C57BL/6J Mice.

Sanna E, Talani G, Obili N, Mascia MP, Mostallino MC, Secci PP, Pisu MG, Biggio F, Utzeri C, Olla P, Biggio G, Follesa P - Front Neurosci (2011)

Bottom Line: SI stress induced a marked reduction in hippocampal 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG) and was associated with molecular and functional changes of the GABA(A)R.Nevertheless, the increase in EtOH consumption did not restore the reduction in hippocampal 3α,5α-TH PROG induced by SI.We conclude that self-administration of EtOH selectively abolishes the increase of α(4) subunit but not other neurochemical, molecular, and functional modifications induced by SI prolonged mild stress.

View Article: PubMed Central - PubMed

Affiliation: Section of Neuroscience, Department of Experimental Biology, Center of Excellence for the Neurobiology of Dependence, University of Cagliari Monserrato, Cagliari, Italy.

ABSTRACT
Post-weaning social isolation (SI) is a model of prolonged mild stress characterized by behavioral and neurochemical alterations. We used SI in C57BL/6J mice to investigate the effects of ethanol (EtOH) in the free-choice drinking paradigm on gene expression and function of γ-aminobutyric acid type A receptors (GABA(A)Rs) and the role of neuroactive steroids in the actions of EtOH in the hippocampus. SI stress induced a marked reduction in hippocampal 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG) and was associated with molecular and functional changes of the GABA(A)R. The gene expression of the α(4) and δ subunits was increased in the hippocampus of SI C57BL/6J mice; the expression of the γ(2) subunit was decreased whereas that of the α(1) did not change. Patch-clamp recordings in dentate gyrus (DG) granule cells obtained from SI C57BL/6J mice revealed a greater enhancement of tonic currents induced by α-(4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridin-3-ol (THIP) compared to that in control C57BL/6J mice. These neurochemical, molecular and functional changes observed in SI C57BL/6J mice were associated with an increased EtOH intake and EtOH preference. Nevertheless, the increase in EtOH consumption did not restore the reduction in hippocampal 3α,5α-TH PROG induced by SI. EtOH self-administration blocked the changes in gene expression of the α(4) subunit but not those of the δ and γ(2) subunits induced by SI. In addition, EtOH self-administration did not block the SI-induced changes in GABA(A)R-mediated tonic inhibition in hippocampal granule cells but increased the frequency of basal GABAergic sIPSCs in DG granule cells. We conclude that self-administration of EtOH selectively abolishes the increase of α(4) subunit but not other neurochemical, molecular, and functional modifications induced by SI prolonged mild stress.

No MeSH data available.


Related in: MedlinePlus

Representative immunohistochemical (A,B) and RNase protection assay (C) analysis images. Distribution of the α4 (A) and δ (B) subunits in molecular layer of DG of the hippocampus slices from GH (a), SI (b), GH with free access for 2 h a day to EtOH (c), and SI mice with free access for 2 h a day to EtOH (d). Scale bar, 50 μm. (C) Autoradiograph on a urea/polyacrylamide electrophoresis gel showing protected fragments of the mRNAs encoding for δ, α1, α4 subunits of GABAAR and cyclophilin (internal standard). On each band, 25 μg of total RNA extracted from the whole hippocampus of individual mice of the indicated experimental groups. Pd = digested probe; P = probe. (D) Semi-quantitative measurement of image (C) of the indicated subunits. Data are expressed as percentage change in optical density of the corresponding bands relative to GH.
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Figure 5: Representative immunohistochemical (A,B) and RNase protection assay (C) analysis images. Distribution of the α4 (A) and δ (B) subunits in molecular layer of DG of the hippocampus slices from GH (a), SI (b), GH with free access for 2 h a day to EtOH (c), and SI mice with free access for 2 h a day to EtOH (d). Scale bar, 50 μm. (C) Autoradiograph on a urea/polyacrylamide electrophoresis gel showing protected fragments of the mRNAs encoding for δ, α1, α4 subunits of GABAAR and cyclophilin (internal standard). On each band, 25 μg of total RNA extracted from the whole hippocampus of individual mice of the indicated experimental groups. Pd = digested probe; P = probe. (D) Semi-quantitative measurement of image (C) of the indicated subunits. Data are expressed as percentage change in optical density of the corresponding bands relative to GH.

Mentions: In order to assay whether SI stress is associated with changes in GABAAR gene expression in the hippocampus and whether the increased drinking behavior in the SI mice could reverse these changes we measured both the abundance of mRNA and peptide, in the whole hippocampus or DG respectively, of discrete GABAAR subunits. RNase protection assay and immunohistochemistry measurements (Figure 5) revealed that SI-induced a marked and significant increase in the abundance of both the δ and α4 subunit (Figures 6 and 7A,B; p < 0.05). Moreover, SI did not alter the mRNA levels of the α1 subunit (Figure 8A) but significantly decreased the γ2 subunit peptide levels (Figure 8B; p < 0.05). In hippocampus of mice that had free access to EtOH the gene expression of the GABAAR subunits was also altered. Similarly to our recent finding in the rat (Pisu et al., 2010) EtOH intake induced changes in gene expression in the GH mice namely a marked and statistically significant increase of the δ subunit (Figures 6A,B; p < 0.05) and a significant decrease of the γ2 subunit peptide (Figure 8B; p < 0.05). On the other hand, in mice the intake of EtOH was able to completely block the increase in the abundance of the α4 subunit induced by SI stress in a selective manner (Figures 7A,B; p < 0.05). In fact, EtOH intake did not abolish the effects of SI on the gene expression of neither δ nor γ2 subunit which remained still up-regulated (Figures 6A,B) and down-regulated, respectively (Figure 8B). Moreover, EtOH intake did not have any significant effect on the gene expression of the α1 subunit in the hippocampus of mice from both GH and SI experimental groups (Figure 8A).


Voluntary Ethanol Consumption Induced by Social Isolation Reverses the Increase of α(4)/δ GABA(A) Receptor Gene Expression and Function in the Hippocampus of C57BL/6J Mice.

Sanna E, Talani G, Obili N, Mascia MP, Mostallino MC, Secci PP, Pisu MG, Biggio F, Utzeri C, Olla P, Biggio G, Follesa P - Front Neurosci (2011)

Representative immunohistochemical (A,B) and RNase protection assay (C) analysis images. Distribution of the α4 (A) and δ (B) subunits in molecular layer of DG of the hippocampus slices from GH (a), SI (b), GH with free access for 2 h a day to EtOH (c), and SI mice with free access for 2 h a day to EtOH (d). Scale bar, 50 μm. (C) Autoradiograph on a urea/polyacrylamide electrophoresis gel showing protected fragments of the mRNAs encoding for δ, α1, α4 subunits of GABAAR and cyclophilin (internal standard). On each band, 25 μg of total RNA extracted from the whole hippocampus of individual mice of the indicated experimental groups. Pd = digested probe; P = probe. (D) Semi-quantitative measurement of image (C) of the indicated subunits. Data are expressed as percentage change in optical density of the corresponding bands relative to GH.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3039156&req=5

Figure 5: Representative immunohistochemical (A,B) and RNase protection assay (C) analysis images. Distribution of the α4 (A) and δ (B) subunits in molecular layer of DG of the hippocampus slices from GH (a), SI (b), GH with free access for 2 h a day to EtOH (c), and SI mice with free access for 2 h a day to EtOH (d). Scale bar, 50 μm. (C) Autoradiograph on a urea/polyacrylamide electrophoresis gel showing protected fragments of the mRNAs encoding for δ, α1, α4 subunits of GABAAR and cyclophilin (internal standard). On each band, 25 μg of total RNA extracted from the whole hippocampus of individual mice of the indicated experimental groups. Pd = digested probe; P = probe. (D) Semi-quantitative measurement of image (C) of the indicated subunits. Data are expressed as percentage change in optical density of the corresponding bands relative to GH.
Mentions: In order to assay whether SI stress is associated with changes in GABAAR gene expression in the hippocampus and whether the increased drinking behavior in the SI mice could reverse these changes we measured both the abundance of mRNA and peptide, in the whole hippocampus or DG respectively, of discrete GABAAR subunits. RNase protection assay and immunohistochemistry measurements (Figure 5) revealed that SI-induced a marked and significant increase in the abundance of both the δ and α4 subunit (Figures 6 and 7A,B; p < 0.05). Moreover, SI did not alter the mRNA levels of the α1 subunit (Figure 8A) but significantly decreased the γ2 subunit peptide levels (Figure 8B; p < 0.05). In hippocampus of mice that had free access to EtOH the gene expression of the GABAAR subunits was also altered. Similarly to our recent finding in the rat (Pisu et al., 2010) EtOH intake induced changes in gene expression in the GH mice namely a marked and statistically significant increase of the δ subunit (Figures 6A,B; p < 0.05) and a significant decrease of the γ2 subunit peptide (Figure 8B; p < 0.05). On the other hand, in mice the intake of EtOH was able to completely block the increase in the abundance of the α4 subunit induced by SI stress in a selective manner (Figures 7A,B; p < 0.05). In fact, EtOH intake did not abolish the effects of SI on the gene expression of neither δ nor γ2 subunit which remained still up-regulated (Figures 6A,B) and down-regulated, respectively (Figure 8B). Moreover, EtOH intake did not have any significant effect on the gene expression of the α1 subunit in the hippocampus of mice from both GH and SI experimental groups (Figure 8A).

Bottom Line: SI stress induced a marked reduction in hippocampal 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG) and was associated with molecular and functional changes of the GABA(A)R.Nevertheless, the increase in EtOH consumption did not restore the reduction in hippocampal 3α,5α-TH PROG induced by SI.We conclude that self-administration of EtOH selectively abolishes the increase of α(4) subunit but not other neurochemical, molecular, and functional modifications induced by SI prolonged mild stress.

View Article: PubMed Central - PubMed

Affiliation: Section of Neuroscience, Department of Experimental Biology, Center of Excellence for the Neurobiology of Dependence, University of Cagliari Monserrato, Cagliari, Italy.

ABSTRACT
Post-weaning social isolation (SI) is a model of prolonged mild stress characterized by behavioral and neurochemical alterations. We used SI in C57BL/6J mice to investigate the effects of ethanol (EtOH) in the free-choice drinking paradigm on gene expression and function of γ-aminobutyric acid type A receptors (GABA(A)Rs) and the role of neuroactive steroids in the actions of EtOH in the hippocampus. SI stress induced a marked reduction in hippocampal 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG) and was associated with molecular and functional changes of the GABA(A)R. The gene expression of the α(4) and δ subunits was increased in the hippocampus of SI C57BL/6J mice; the expression of the γ(2) subunit was decreased whereas that of the α(1) did not change. Patch-clamp recordings in dentate gyrus (DG) granule cells obtained from SI C57BL/6J mice revealed a greater enhancement of tonic currents induced by α-(4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridin-3-ol (THIP) compared to that in control C57BL/6J mice. These neurochemical, molecular and functional changes observed in SI C57BL/6J mice were associated with an increased EtOH intake and EtOH preference. Nevertheless, the increase in EtOH consumption did not restore the reduction in hippocampal 3α,5α-TH PROG induced by SI. EtOH self-administration blocked the changes in gene expression of the α(4) subunit but not those of the δ and γ(2) subunits induced by SI. In addition, EtOH self-administration did not block the SI-induced changes in GABA(A)R-mediated tonic inhibition in hippocampal granule cells but increased the frequency of basal GABAergic sIPSCs in DG granule cells. We conclude that self-administration of EtOH selectively abolishes the increase of α(4) subunit but not other neurochemical, molecular, and functional modifications induced by SI prolonged mild stress.

No MeSH data available.


Related in: MedlinePlus