Glucocorticoid receptors regulate central amygdala GABAergic synapses in Marchigian-Sardinian alcohol-preferring rats

doi:10.1016/j.ynstr.2023.100547

. 2023 Jun 4:25:100547.

doi: 10.1016/j.ynstr.2023.100547. eCollection 2023 Jul.

Glucocorticoid receptors regulate central amygdala GABAergic synapses in Marchigian-Sardinian alcohol-preferring rats

Sophia Khom^{1

2}, Vittoria Borgonetti¹, Valentina Vozella¹, Dean Kirson^{1

3}, Larry Rodriguez¹, Pauravi Gandhi¹, Paula Cristina Bianchi^{1

4}, Angela Snyder¹, Roman Vlkolinsky¹, Michal Bajo¹, Christopher S Oleata¹, Roberto Ciccocioppo⁵, Marisa Roberto^{1

6}

Affiliations

¹ Department of Molecular Medicine, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.
² Department of Pharmaceutical Sciences, University of Vienna, Josef-Holaubek-Platz 2, Vienna, A 1090, Austria.
³ Department of Pharmacology, Addiction Science, and Toxicology, The University of Tennessee Health Science Center, Memphis, TN 38163, USA.
⁴ Department of Pharmacology, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP 04024-002, Brazil.
⁵ School of Pharmacy, Center of Neuroscience, University of Camerino, Italy.
⁶ Department of Neuroscience, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.

PMID: 37547774
PMCID: PMC10401345
DOI: 10.1016/j.ynstr.2023.100547

Glucocorticoid receptors regulate central amygdala GABAergic synapses in Marchigian-Sardinian alcohol-preferring rats

Sophia Khom et al. Neurobiol Stress. 2023.

. 2023 Jun 4:25:100547.

doi: 10.1016/j.ynstr.2023.100547. eCollection 2023 Jul.

Authors

Affiliations

¹ Department of Molecular Medicine, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.
² Department of Pharmaceutical Sciences, University of Vienna, Josef-Holaubek-Platz 2, Vienna, A 1090, Austria.
³ Department of Pharmacology, Addiction Science, and Toxicology, The University of Tennessee Health Science Center, Memphis, TN 38163, USA.
⁴ Department of Pharmacology, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP 04024-002, Brazil.
⁵ School of Pharmacy, Center of Neuroscience, University of Camerino, Italy.
⁶ Department of Neuroscience, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.

PMID: 37547774
PMCID: PMC10401345
DOI: 10.1016/j.ynstr.2023.100547

Abstract

Impairments in the function of the hypothalamic-pituitary-adrenal (HPA) axis and enhanced glucocorticoid receptor (GR) activity in the central amygdala (CeA) are critical mechanisms in the pathogenesis of alcohol use disorder (AUD). The GR antagonist mifepristone attenuates craving in AUD patients, alcohol consumption in AUD models, and decreases CeA γ-aminobutyric acid (GABA) transmission in alcohol-dependent rats. Previous studies suggest elevated GR activity in the CeA of male alcohol-preferring Marchigian-Sardinian (msP) rats, but its contribution to heightened CeA GABA transmission driving their characteristic post-dependent phenotype is largely unknown. We determined Nr3c1 (the gene encoding GR) gene transcription in the CeA in male and female msP and Wistar rats using in situ hybridization and studied acute effects of mifepristone (10 μM) and its interaction with ethanol (44 mM) on pharmacologically isolated spontaneous inhibitory postsynaptic currents (sIPSCs) and electrically evoked inhibitory postsynaptic potentials (eIPSPs) in the CeA using ex vivo slice electrophysiology. Female rats of both genotypes expressed more CeA GRs than males, suggesting a sexually dimorphic GR regulation of CeA activity. Mifepristone reduced sIPSC frequencies (GABA release) and eIPSP amplitudes in msP rats of both sexes, but not in their Wistar counterparts; however, it did not prevent acute ethanol-induced increase in CeA GABA transmission in male rats. In msP rats, GR regulates CeA GABAergic signaling under basal conditions, indicative of intrinsically active GR. Thus, enhanced GR function in the CeA represents a key mechanism contributing to maladaptive behaviors associated with AUD.

PubMed Disclaimer

Conflict of interest statement

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.

Figures

**Fig. 1**
Elevated GR expression in the female CeA in both msP and Wistar rats. (A) Representative images of the medial subdivision of the CeA after *in situ* hybridization for *Gad2* (*yellow signal*) as a marker of GABAergic neurons, *Slc17a7* (*red signal*) as a marker of glutamatergic neurons, *Nr3c1* (*green signal*) for expression of GR, and DAPI to label cell bodies. The data are expressed as the mean ± SEM of normalized cell counts of *GAD2*-positive (B), *Slc17a7*-positive (C), and *Nr3c1*-positive (D) neurons in the CeA in rats of the indicated sexes and genotypes. The data are expressed as the mean ± SEM of the number of neurons that co-expressed either *Nr3c1* and *GAD2* (E) or *Nr3c1* and *SLc17a7* (F). Main effects of sex and genotype and a sex × genotype interaction were determined by two-way ANOVA (^$p < 0.05, main effect of sex; ^%p < 0.05, main effect of genotype). Two sections from 3 animals for each sex and genotype were imaged and analyzed. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

**Fig. 2**
Glucocorticoid receptors regulate CeA GABA activity in msP rats, but not in Wistar rats. (A) Representative sIPSC traces from CeA neurons from the indicated genotype and sex before (control, left panel) and during 10 μM mifepristone application (right panel). Color-coded scaled sIPSC averages of each trace (black line: control conditions; red line: in the presence of mifepristone) are shown in the adjacent boxes. (B) Cumulative frequency plots for the indicated animal groups. Scatter dot plots depict means ± SEM of sIPSC (C) frequencies, (D) amplitudes, (E) rise times, and (F) decay times in the presence of mifepristone from 9 to 19 different neurons. Differences between sexes and genotypes were determined using two-way ANOVA (^$p = 0.05). Simple drug effects (i.e., differences from baseline) were calculated using one-sample t-tests (*p = 0.05, **p = 0.01). Means ± SEM were derived from at least four different animals per group (9 male and 5 female msP rats; 9 male and 4 female Wistar rats). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

**Fig. 3**
Mifepristone decreases evoked CeA IPSP amplitudes in msP rats, but not in Wistar rats. (A) Representative CeA eIPSP traces during baseline control (left side) and during 10 μM mifepristone superfusion in the indicated animal groups. Scatter dot plots illustrate means ± SEM of the effects of mifepristone on (B) eIPSP amplitudes and (C) the PPF compared with baseline control (indicated as dashed line). Differences between groups were determined using two-way ANOVA (main effects: ^%%p < 0.01). Differences from baseline control (dashed line) were determined using one-sample t-tests (*p < 0.05, **p < 0.01, ***p < 0.001). Means ± SEM were derived from 8 to 14 individual cells from 12 male, 8 female msP rats, 8 male and 7 female Wistar rats, respectively.

**Fig. 4**
Acute ethanol enhances CeA GABA transmission only in male rats. (A) Representative traces of CeA sIPSCs from the indicated genotypes and sexes during baseline control (left trace) and during 44 mM ethanol application (right trace). Bar graphs summarize effects of ethanol on sIPSC (B) frequencies and (C) amplitudes and (D) rise and (E) decay times. The data are expressed as the mean ± SEM from 7 to 10 cells, derived from at least three different animals per group (6 male and 6 female msP rats; 3 male and 3 female Wistar rats). Differences from the baseline control (dashed line) were calculated using one-sample t-tests (*p = 0.05, **p = 0.01). Differences between groups were determined using two-way ANOVA.

**Fig. 5**
Mifepristone does not block acute effects of ethanol on CeA GABA transmission in male rats. (A) Representative sIPSC recordings from CeA neurons during baseline control conditions (left panel), during 10 μM mifepristone (middle panel), and during 44 mM ethanol in the continued presence of mifepristone (right panel) from the indicated rat groups. Effects of ethanol were normalized to the last 3 min of mifepristone application (indicated as dashed line). Scatter dot plots depict means ± SEM of effects of ethanol in the presence of mifepristone from 8 to 9 individual cells on sIPSC (B) frequencies and (C) amplitudes and (D) rise and (E) decay times. Differences between groups were calculated using two-way ANOVA (^$p > 0.05). Differences from pre-ethanol baseline were calculated using one-sample t-tests (*p < 0.05). Data were derived from at least four different animals per group (4 male and 5 female msP rats; 5 male and 4 female Wistar rats).

**Fig. 6**
Similar regulation of CeA GABA transmission in male alcohol-dependent Wistar and msP rats. (A) Representative CeA sIPSC traces from a male, alcohol-dependent Wistar rat during baseline control conditions (upper trace) and in the presence of mifepristone (lower trace). Bars represent means ± SEM of sIPSC (B) frequencies and (C) postsynaptic measures in the presence of mifepristone from the indicated treatment group compared with baseline control conditions (dashed line) from 6 and 18 cells, respectively. (D) Representative CeA eIPSP traces from a male, alcohol-dependent Wistar rat before and during mifepristone application. Bars represent means ± SEM of (E) eIPSP amplitudes and (F) PPF during mifepristone application compared with baseline control (dashed line) from 11 to 16 cells. All data for dependent Wistar rats were derived from 8 animals. Differences from baseline control were determined using one-sample t-tests, and differences between groups were determined using two-tailed unpaired t-tests (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). All data for msP rats in these graphs were taken from Fig. 2.

See this image and copyright information in PMC

Cited by

Withdrawal from chronic alcohol impairs the serotonin-mediated modulation of GABAergic transmission in the infralimbic cortex in male rats.
Vlkolinsky R, Khom S, Vozella V, Bajo M, Roberto M. Vlkolinsky R, et al. Neurobiol Dis. 2024 Sep;199:106590. doi: 10.1016/j.nbd.2024.106590. Epub 2024 Jul 10. Neurobiol Dis. 2024. PMID: 38996987
Chemogenetic inhibition of central amygdala CRF-expressing neurons decreases alcohol intake but not trauma-related behaviors in a rat model of post-traumatic stress and alcohol use disorder.
Cruz B, Vozella V, Borgonetti V, Bullard R, Bianchi PC, Kirson D, Bertotto LB, Bajo M, Vlkolinsky R, Messing RO, Zorrilla EP, Roberto M. Cruz B, et al. Mol Psychiatry. 2024 Sep;29(9):2611-2621. doi: 10.1038/s41380-024-02514-8. Epub 2024 Mar 21. Mol Psychiatry. 2024. PMID: 38509197 Free PMC article.
Early social isolation differentially affects the glucocorticoid receptor system and alcohol-seeking behavior in male and female Marchigian Sardinian alcohol-preferring rats.
Benvenuti F, De Carlo S, Rullo L, Caffino L, Losapio LM, Morosini C, Ubaldi M, Soverchia L, Cannella N, Domi E, Candeletti S, Mottarlini F, Fattore L, Romualdi P, Fumagalli F, Trezza V, Roberto M, Ciccocioppo R. Benvenuti F, et al. Neurobiol Stress. 2023 Dec 7;28:100598. doi: 10.1016/j.ynstr.2023.100598. eCollection 2024 Jan. Neurobiol Stress. 2023. PMID: 38115888 Free PMC article.
IL-18 Signaling in the Rat Central Amygdala Is Disrupted in a Comorbid Model of Post-Traumatic Stress and Alcohol Use Disorder.
Borgonetti V, Cruz B, Vozella V, Khom S, Steinman MQ, Bullard R, D'Ambrosio S, Oleata CS, Vlkolinsky R, Bajo M, Zorrilla EP, Kirson D, Roberto M. Borgonetti V, et al. Cells. 2023 Jul 27;12(15):1943. doi: 10.3390/cells12151943. Cells. 2023. PMID: 37566022 Free PMC article.

References

1. Augier E., Barbier E., Dulman R.S., Licheri V., Augier G., Domi E., Barchiesi R., Farris S., Nätt D., Mayfield R.D., Adermark L., Heilig M. A molecular mechanism for choosing alcohol over an alternative reward. Science. 2018;360:1321–1326. doi: 10.1126/science.aao1157. - DOI - PubMed
1. Benvenuti F., Cannella N., Stopponi S., Soverchia L., Ubaldi M., Lunerti V., Vozella V., Cruz B., Roberto M., Ciccocioppo R. Effect of glucocorticoid receptor antagonism on alcohol self-administration in genetically-selected Marchigian Sardinian alcohol-preferring and non-preferring Wistar rats. Int. J. Mol. Sci. 2021;22:4184. doi: 10.3390/ijms22084184. - DOI - PMC - PubMed
1. Borruto A.M., Stopponi S., Li H., Weiss F., Roberto M., Ciccocioppo R. Genetically selected alcohol-preferring msP rats to study alcohol use disorder: anything lost in translation? Neuropharmacology. 2021;186 doi: 10.1016/j.neuropharm.2020.108446. - DOI - PMC - PubMed
1. Burnette E.M., Nieto S.J., Grodin E.N., Meredith L.R., Hurley B., Miotto K., Gillis A.J., Ray L.A. Novel agents for the pharmacological treatment of alcohol use disorder. Drugs. 2022;82:251–274. doi: 10.1007/s40265-021-01670-3. - DOI - PMC - PubMed
1. Carmack S.A., Vendruscolo J.C.M., Adrienne McGinn M., Miranda-Barrientos J., Repunte-Canonigo V., Bosse G.D., Mercatelli D., Giorgi F.M., Fu Y., Hinrich A.J., Jodelka F.M., Ling K., Messing R.O., Peterson R.T., Rigo F., Edwards S., Sanna P.P., Morales M., Hastings M.L., Koob G.F., Vendruscolo L.F. Corticosteroid sensitization drives opioid addiction. Mol. Psychiatr. 2022;27:2492–2501. doi: 10.1038/s41380-022-01501-1. - DOI - PMC - PubMed

Grants and funding

LinkOut - more resources

Full Text Sources
Research Materials
- NCI CPTC Antibody Characterization Program

[1] Augier E., Barbier E., Dulman R.S., Licheri V., Augier G., Domi E., Barchiesi R., Farris S., Nätt D., Mayfield R.D., Adermark L., Heilig M. A molecular mechanism for choosing alcohol over an alternative reward. Science. 2018;360:1321–1326. doi: 10.1126/science.aao1157. - DOI - PubMed

[2] Augier E., Barbier E., Dulman R.S., Licheri V., Augier G., Domi E., Barchiesi R., Farris S., Nätt D., Mayfield R.D., Adermark L., Heilig M. A molecular mechanism for choosing alcohol over an alternative reward. Science. 2018;360:1321–1326. doi: 10.1126/science.aao1157. - DOI - PubMed

[3] Benvenuti F., Cannella N., Stopponi S., Soverchia L., Ubaldi M., Lunerti V., Vozella V., Cruz B., Roberto M., Ciccocioppo R. Effect of glucocorticoid receptor antagonism on alcohol self-administration in genetically-selected Marchigian Sardinian alcohol-preferring and non-preferring Wistar rats. Int. J. Mol. Sci. 2021;22:4184. doi: 10.3390/ijms22084184. - DOI - PMC - PubMed

[4] Benvenuti F., Cannella N., Stopponi S., Soverchia L., Ubaldi M., Lunerti V., Vozella V., Cruz B., Roberto M., Ciccocioppo R. Effect of glucocorticoid receptor antagonism on alcohol self-administration in genetically-selected Marchigian Sardinian alcohol-preferring and non-preferring Wistar rats. Int. J. Mol. Sci. 2021;22:4184. doi: 10.3390/ijms22084184. - DOI - PMC - PubMed

[5] Borruto A.M., Stopponi S., Li H., Weiss F., Roberto M., Ciccocioppo R. Genetically selected alcohol-preferring msP rats to study alcohol use disorder: anything lost in translation? Neuropharmacology. 2021;186 doi: 10.1016/j.neuropharm.2020.108446. - DOI - PMC - PubMed

[6] Borruto A.M., Stopponi S., Li H., Weiss F., Roberto M., Ciccocioppo R. Genetically selected alcohol-preferring msP rats to study alcohol use disorder: anything lost in translation? Neuropharmacology. 2021;186 doi: 10.1016/j.neuropharm.2020.108446. - DOI - PMC - PubMed

[7] Burnette E.M., Nieto S.J., Grodin E.N., Meredith L.R., Hurley B., Miotto K., Gillis A.J., Ray L.A. Novel agents for the pharmacological treatment of alcohol use disorder. Drugs. 2022;82:251–274. doi: 10.1007/s40265-021-01670-3. - DOI - PMC - PubMed

[8] Burnette E.M., Nieto S.J., Grodin E.N., Meredith L.R., Hurley B., Miotto K., Gillis A.J., Ray L.A. Novel agents for the pharmacological treatment of alcohol use disorder. Drugs. 2022;82:251–274. doi: 10.1007/s40265-021-01670-3. - DOI - PMC - PubMed

[9] Carmack S.A., Vendruscolo J.C.M., Adrienne McGinn M., Miranda-Barrientos J., Repunte-Canonigo V., Bosse G.D., Mercatelli D., Giorgi F.M., Fu Y., Hinrich A.J., Jodelka F.M., Ling K., Messing R.O., Peterson R.T., Rigo F., Edwards S., Sanna P.P., Morales M., Hastings M.L., Koob G.F., Vendruscolo L.F. Corticosteroid sensitization drives opioid addiction. Mol. Psychiatr. 2022;27:2492–2501. doi: 10.1038/s41380-022-01501-1. - DOI - PMC - PubMed

[10] Carmack S.A., Vendruscolo J.C.M., Adrienne McGinn M., Miranda-Barrientos J., Repunte-Canonigo V., Bosse G.D., Mercatelli D., Giorgi F.M., Fu Y., Hinrich A.J., Jodelka F.M., Ling K., Messing R.O., Peterson R.T., Rigo F., Edwards S., Sanna P.P., Morales M., Hastings M.L., Koob G.F., Vendruscolo L.F. Corticosteroid sensitization drives opioid addiction. Mol. Psychiatr. 2022;27:2492–2501. doi: 10.1038/s41380-022-01501-1. - DOI - PMC - PubMed

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Glucocorticoid receptors regulate central amygdala GABAergic synapses in Marchigian-Sardinian alcohol-preferring rats

Affiliations

Glucocorticoid receptors regulate central amygdala GABAergic synapses in Marchigian-Sardinian alcohol-preferring rats

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

Grants and funding

LinkOut - more resources

Full Text Sources

Research Materials

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

Related information

Grants and funding

LinkOut - more resources

Full Text Sources

Research Materials