Functional regulation of PI3K-associated signaling in the accumbens by binge alcohol drinking in male but not female mice - PubMed (original) (raw)
Functional regulation of PI3K-associated signaling in the accumbens by binge alcohol drinking in male but not female mice
Debra K Cozzoli et al. Neuropharmacology. 2016 Jun.
Abstract
It is well established that binge alcohol consumption produces alterations in Group 1 metabotropic glutamate receptors (mGlus) and related signaling cascades in the nucleus accumbens (NAC) of adult male mice, but female and adolescent mice have not been examined. Thus, the first set of studies determined whether repeated binge alcohol consumption produced similar alterations in protein and mRNA levels of Group 1 mGlu-associated signaling molecules in the NAC of male and female adult and adolescent mice. The adult (9 weeks) and adolescent (4 weeks) C57BL/6J mice were exposed to 7 binge alcohol sessions every 3rd day while controls drank water. Repeated binge alcohol consumption produced sexually divergent changes in protein levels and mRNA expression for Group 1 mGlus and downstream signaling molecules in the NAC, but there was no effect of age. Binge alcohol intake decreased mGlu5 levels in females, whereas it decreased indices of phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), 4E-binding protein 1, and p70 ribosomal protein S6 kinase in males. Expression of genes encoding mGlu1, mGlu5, the NR2A subunit of the NMDA receptor, and Homer2 were all decreased by binge alcohol consumption in males, while females were relatively resistant (only phosphoinositide-dependent protein kinase 1 was decreased). The functional implication of these differences was investigated in a separate study by inhibiting mTOR in the NAC (via infusions of rapamycin) before binge drinking sessions. Rapamycin (50 and 100 ng/side) significantly decreased binge alcohol consumption in males, while consumption in females was unaffected. Altogether these results highlight that mTOR signaling in the NAC was necessary to maintain binge alcohol consumption only in male mice and that binge drinking recruits sexually divergent signaling cascades downstream of PI3K and presumably, Group 1 mGlus. Importantly, these findings emphasize that sex should be considered in the development of potential pharmacotherapeutic targets.
Keywords: Group 1 metabotropic glutamate receptor; Homer2; Nucleus accumbens; Rapamycin; SHAC procedure; Sex differences.
Published by Elsevier Ltd.
Figures
Figure 1. Withdrawal from repeated binge alcohol consumption significantly altered protein levels of phosphoinositide 3-kinase (PI3K) and downstream signaling molecules without altering metabotropic glutamate receptor 1 or 5 (mGlu1 or mGlu5) in the nucleus accumbens (NAC) of male mice
Withdrawal (24 hr) from repeated binge alcohol consumption (SHAC) did not alter (A) mGlu1 or mGlu5 or (C) phosphoinositide-dependent protein kinase 1 (PDK1), but it significantly decreased the phosphorylation or ratio (index of activation level) of (B) PI3K, (D) mammalian target of rapamycin (mTOR), (E) 4E-binding protein 1 (4EBP1), and (F) p70 ribosomal protein S6 kinase (p70s6K), when compared with water drinking control animals (Water). Values are collapsed across age and represent the mean ± SEM for each group (Water: n=15-16; SHAC: n=17-18). All levels were initially normalized to α-tubulin. Fold regulation was then determined by normalizing all values to the mean of the relative expression of the Water group. Representative immunoblots for each protein are included beneath each graph. +p<0.09, *p<0.05 vs. water.
Figure 2. Withdrawal from repeated binge alcohol consumption significantly altered protein levels of metabotropic glutamate receptor 5 (mGlu5) without altering phosphoinositide 3-kinase (PI3K) or downstream signaling molecules in the nucleus accumbens (NAC) of female mice
Withdrawal (24 hr) from repeated binge alcohol consumption (SHAC) significantly decreased (A) mGlu5, but not mGlu1, and it did not alter indices of (B) PI3K, (C) phosphoinositide-dependent protein kinase 1 (PDK1), (D) mammalian target of rapamycin (mTOR), (E) 4E-binding protein 1 (4EBP1), or (F) p70 ribosomal protein S6 kinase (p70s6K), when compared with water drinking control animals (Water). Values are collapsed across age and represent the mean ± SEM for each group (Water: n=16-18; SHAC: n=16-17; except for p-4EBP1, 4EBP1, and mGlu5 where Water: n=9-14 and SHAC: n=8-13). All levels were initially normalized to α-tubulin. Fold regulation was then determined by normalizing all values to the mean of the relative expression of the Water group. Representative immunoblots for each protein are included beneath each graph. *p<0.05 vs. water.
Figure 3. Sex differences in the effect of withdrawal from repeated binge alcohol intake on RNA expression of glutamatergic genes in the nucleus accumbens (NAC)
Withdrawal (24 hr) from repeated binge alcohol consumption (SHAC) significantly decreased the expression of (A) Grm1 & Grm5 (mGlu1 and 5 gene), (B) Grin2A (NR2A subunit of the NMDA receptor gene), and (C) Homer2 (homer homolog 2 gene) without altering expression of (B) Grin2B (NR2B subunit of the NMDA receptor gene) or (C) Pdkp1 (3-phosphoinositide dependent protein kinase 1 gene) in the NAC of male mice, when compared with controls (Water). Conversely, withdrawal from repeated binge alcohol intake (SHAC) did not alter (D) Grm1 & Grm5, (E) Grin2A or (F) Homer2, but it decreased the expression of (F) Pdpk1 in the NAC of female mice, when compared with controls (Water). Values are collapsed across age and represent the mean ± SEM for each group (Water: n=11-14; SHAC: n=7-14). Fold regulation was determined by normalizing all values to the mean of the relative expression of the Water group. +p<0.09, *p<0.05 vs. water. N.D. = not detectable.
Figure 4. Inhibition of mammalian target of rapamycin (mTOR) in the nucleus accumbens (NAC) decreases binge alcohol consumption in male but not female mice without altering water consumption
(A) Rapamycin dose-dependently decreased alcohol intake during a 30-min binge alcohol session in male but not in female mice. (B) Summary of the effects of the intra-NAC doses of rapamycin on the subsequent 3.5 hr water access period as well as (C) the total intake (in mLs) from the entire 4 hr fluid access period. (D) Intra-NAC infusion of vehicle or 100 ng/side rapamycin did not alter 30 min water intake or (E) 4 hr total water intake on days when animals only had access to water, demonstrating selectively to reduce binge alcohol intake in male mice. (F) Schematic representation of microinjection placements in the NAC for the male (black circles) and female (gray circles) animals included in the study. Values represent the mean ± SEM of each group (Males: n=8; Females: n=6-7). *p<0.05, **p<0.01 vs. respective vehicle infusion;
**
p<0.01 ANOVA.
References
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