The Amygdala Noradrenergic System Is Compromised With Alcohol Use Disorder

Abstract

Background: Alcohol use disorder (AUD) is a leading preventable cause of death. The central amygdala (CeA) is a hub for stress and AUD, while dysfunction of the noradrenaline stress system is implicated in AUD relapse.

Methods: Here, we investigated whether alcohol (ethanol) dependence and protracted withdrawal alter noradrenergic regulation of the amygdala in rodents and humans. Male adult rats were housed under control conditions, subjected to chronic intermittent ethanol vapor exposure to induce dependence, or withdrawn from chronic intermittent ethanol vapor exposure for 2 weeks, and ex vivo electrophysiology, biochemistry (catecholamine quantification by high-performance liquid chromatography), in situ hybridization, and behavioral brain-site specific pharmacology studies were performed. We also used real-time quantitative polymerase chain reaction to assess gene expression of α_1B, β₁, and β₂ adrenergic receptors in human postmortem brain tissue from men diagnosed with AUD and matched control subjects.

Results: We found that α₁ receptors potentiate CeA GABAergic (gamma-aminobutyric acidergic) transmission and drive moderate alcohol intake in control rats. In dependent rats, β receptors disinhibit a subpopulation of CeA neurons, contributing to their excessive drinking. Withdrawal produces CeA functional recovery with no change in local noradrenaline tissue concentrations, although there are some long-lasting differences in the cellular patterns of adrenergic receptor messenger RNA expression. In addition, postmortem brain analyses reveal increased α_1B receptor messenger RNA in the amygdala of humans with AUD.

Conclusions: CeA adrenergic receptors are key neural substrates of AUD. Identification of these novel mechanisms that drive alcohol drinking, particularly during the alcohol-dependent state, supports ongoing new medication development for AUD.

Keywords: Adrenergic receptor; Ethanol; Norepinephrine/noradrenaline; Prazosin; Propranolol; Translation.

Figure 1.

NA increases CeA GABA release via α1 receptors in naïve rats. A: Noradrenaline (1 μM NA for 15 min) decreased action potential firing in all neurons (n=8 cells from 5 rats). B: Representative firing traces and graph demonstrating that NA decreased the neuronal firing rate, with recovery after a 15 min ACSF wash period. C: Pharmacological blockade of GABA transmission prevented the NA-induced decrease in neuronal firing (n=7 cells from 5 rats, though 1 cell did not complete washout). D: NA increased the sIPSC frequency in 9/15 CeA neurons and had no effect in the remaining neurons (N=14 rats). E: Representative sIPSC traces and graph demonstrating that NA increased the sIPSC frequency in the majority of CeA neurons, with no effect on sIPSC amplitude or kinetics. F: NA did not alter the sIPSC properties of the remaining neurons. G: Representative mIPSC traces and graph demonstrating that NA had no effect on mIPSC characteristics (n=7 cells from 4 rats). H: Pretreatment with the α1 receptor inverse agonist (10 μM prazosin) prevented NA’s actions on the sIPSC frequency in 6/8 neurons (N=6 rats), indicating that NA-induced GABA release is mediated by the α1 receptor. I: After pretreatment with the β receptor inverse agonist (20 μM propranolol), NA was still able to increase the sIPSC frequency (n=6 cells from 4 rats). All data are normalized to a pre-drug baseline and presented as mean±SEM. **p<.01, ***p<.001 by one-sample t-test; ^###p<.001 by paired t-test.

Figure 2.

Acute alcohol does not interact with the CeA noradrenergic system. A: Pretreatment with acute ethanol (44 mM EtOH) increased the sIPSC frequency, and noradrenaline (1 μM NA) further potentiated the sIPSC frequency in 4/7 neurons compared to EtOH alone (N=5 rats). B: NA pretreatment either increased or had no effect on the sIPSC frequency, and EtOH co-application furthered increased it compared to NA alone (n=10 cells from 8 rats). C: The magnitude of EtOH’s effect on the sIPSC frequency in neurons pretreated with NA was similar to EtOH alone (cells from panel A and B). D: The magnitude of NA’s effect on the sIPSC frequency in neurons pretreated with EtOH was similar to NA alone (cells from panel A and B). All data are normalized to a pre-drug baseline and presented as mean±SEM. *p<.05, **p<.01 by one-sample t-test; ^#p<.05 by paired t-test.

Figure 3.

Alcohol dependence recruits tonic α1 and NA-induced β receptor signaling. A: Noradrenaline (1 μM NA) decreased action potential firing in 10/17 neurons, increased it in 4/17 neurons, and had no effect in the remaining neurons (N=14 rats). B: Representative firing traces and graphs demonstrating that NA either decreased or increased the neuronal firing rate. C: NA increased the sIPSC frequency in 8/18 neurons, decreased it in 9/18 neurons, and had no effect in the remaining neuron (N=14 rats). D: Representative sIPSC traces and graphs indicating that NA either increased or decreased the sIPSC frequency. E: 10 μM prazosin decreased sIPSC frequency, indicating tonic α1 receptor activity. After prazosin pretreatment, NA either decreased or had no effect on the sIPSC frequency (n=9 cells from 7 rats). F: After 20 μM propranolol pretreatment, NA increased or had no effect on the sIPSC frequency, revealing β receptor recruitment in dependence (n=8 cells from 5 rats). All data are normalized to a pre-drug baseline and presented as mean±SEM. *p<.05, **p<.01, ***p<.001 by one-sample t-test; ^#p<.05, ^##p<.01 by paired t-test.

Figure 4.

Noradrenergic regulation of CeA activity recovers with protracted withdrawal. A: Noradrenaline (1 μM NA) decreased action potential firing in 10/11 neurons and had no effect in the remaining neuron (N=9 rats). B: Representative firing traces and graph demonstrating that NA decreased the firing rate in most neurons. C: NA increased the sIPSC frequency in 9/11 CeA neurons and had no effect in the remaining neurons (N=10 rats). D: Representative sIPSC traces and graph demonstrating that NA increased the sIPSC frequency in most neurons. E: After 10 μM prazosin pretreatment, NA either decreased or had no effect on the sIPSC frequency in 7/8 neurons (N=6 rats). F: After 20 μM propranolol pretreatment, NA increased the sIPSC frequency (n=8 cells from 6 rats). All data are normalized to a pre-drug baseline and presented as mean±SEM. *p<.05, ***p<.001 by one-sample t-test; ^###p<.001 by paired t-test.

Figure 5.

Dependence and withdrawal do not alter CeA NA concentration. Tissue concentrations (pmol/mg) of A: noradrenaline (NA) or B: dopamine (DA) in the CeA of naïve, alcohol-dependent (Dep), or withdrawn (WD) rats. All data are presented as mean±SEM. N=5–7 rats/group.

Figure 6.

Withdrawal alters α1 and β mRNA expression patterns in the CeA. A: Representative images of Adra1a (red), Adrb1 (green), Adrb2 (yellow) and DAPI (blue) for naive, dependent (Dep), and withdrawal (WD) rats. Scale bar = 10μm. Summary bar graphs indicate the change in the percent of nuclei expressing B: Adra1a, C: Adrb1, D: Adrb2 and E: co-expressing Adrb1+Adrb2 in the medial CeA of Dep and WD rats relative to the Naïve group. All data are presented as mean±SEM. ^p<.05 by Tukey’s post hoc test; ^$p<.05 by one-way ANOVA; n=13–15 images from 4–6 rats/group.

Figure 7.

CeA α1 and β receptor signaling drive alcohol intake in non-dependent and dependent rats. A: Intra-CeA administration of prazosin decreased alcohol intake only in the non-dependent rats. B: Intra-CeA propranolol decreased alcohol intake only in dependent rats. with no effect on the inactive lever across both groups. C and D: Responses at the inactive lever were unaffected. E: Histological reconstruction showing CeA microinfusion sites (drawing from the atlas of Paxinos and Watson). All data are presented as mean±SEM. **p<.01 vs. basal intake by Sidak post hoc test; ^p<.05, ^^^p<.001 vs. vehicle by Sidak post hoc test; ^#p<.05, ^##p<.01 vs. dependent rats by Sidak post hoc test. N=14 rats/group. Bsl, baseline during training before CIE vapor exposure; Veh, vehicle.

Figure 8.

Noradrenergic receptor expression in post-mortem human brain. A: α1_B receptor mRNA expression level (2^−ΔΔCt) is higher in the amygdala in humans with AUD compared to controls, but not in the hippocampus or prefrontal cortex (PFC). There was no α1_B receptor mRNA expression in the VTA and NA and these brain regions were excluded in the analysis. B and C: There was no significant difference in the β1 and β2 receptor mRNA expression level, however, there was a trend for β1 overexpression toward the same direction of the α1_B mRNA only in the amygdala in humans with AUD compared to controls. All data presented as mean±SEM. ^$p<.05 by GLM; *p<.05 by post hoc t-test.