The role of anterior insula-brainstem projections and alpha-1 noradrenergic receptors for compulsion-like and alcohol-only drinking

Abstract

Compulsion-like alcohol drinking (CLAD), where consumption continues despite negative consequences, is a major obstacle to treating alcohol use disorder. The locus coeruleus area in the brainstem and norepinephrine receptor (NER) signaling in forebrain cortical regions have been implicated in adaptive responding under stress, which is conceptually similar to compulsion-like responding (adaptive responding despite the presence of stress or conflict). Thus, we examined whether anterior insula (aINS)-to-brainstem connections and alpha-1 NERs regulated compulsion-like intake and alcohol-only drinking (AOD). Halorhodopsin inhibition of aINS-brainstem significantly reduced CLAD, with no effect on alcohol-only or saccharin intake, suggesting a specific aINS-brainstem role in aversion-resistant drinking. In contrast, prazosin inhibition of alpha-1 NERs systemically reduced both CLAD and AOD. Similar to systemic inhibition, intra-aINS alpha-1-NER antagonism reduced both CLAD and AOD. Global aINS inhibition with GABAR agonists also strongly reduced both CLAD and AOD, without impacting saccharin intake or locomotion, while aINS inhibition of calcium-permeable AMPARs (with NASPM) reduced CLAD without impacting AOD. Finally, prazosin inhibition of CLAD and AOD was not correlated with each other, systemically or within aINS, suggesting the possibility that different aINS pathways regulate CLAD versus AOD, which will require further study to definitively address. Together, our results provide important new information showing that some aINS pathways (aINS-brainstem and NASPM-sensitive) specifically regulate compulsion-like alcohol consumption, while aINS more generally may contain parallel pathways promoting CLAD versus AOD. These findings also support the importance of the adaptive stress response system for multiple forms of alcohol drinking.

Fig. 1. aINS inputs to brainstem regulated CLAD but not AOD or saccharin intake.

(A) Schematic of an alcohol-drinking study: 3mo intermittent intake, then 5d/week 20-min access; test sessions for “Opto” (optogenetics) involved “laser” or “no-las” (no laser) tests during alcohol-only (AOD) and compulsion-like alcohol-drinking (CLAD) sessions; “Pharm” (pharmacology) experiments were similar except with “drug” or “veh” (vehicle) (details in “Materials and Methods”). (B) Cartoon of optogenetic methods targeting AINS to LC Area (LCA). (C) Horizontal section showing aINS inputs (green) overlap LC (red for TH + staining) but are primarily lateral to LC. (D, E) Examples of fiber-optic placement in (F) eYFP rat and (G) halorhodopsin rat (TH + LC is torn in E). Note that eYFP expression is less strong in brainstem relative to halorhodopsin–eYFP, which likely reflects that the latter has targeting to the surface membrane. (F) 488 nanometer laser light in brainstem significantly reduced CLAD in halorhodopsin- but not eYFP-expressing rats. * indicates p < 0.05 difference between CLAD/Halorh/Laser and both CLAD/eYFP/Laser and AOD/Halorh/Laser. For the 3-way ANOVA, there was a significant interaction for drinkXlaser (F_1,52 = 8.982, p = 0.004) but no interaction for drinkXvirus (F_1,52 = 2.306, p = 0.135) or laserXvirus (F_1,52 = 0.011, p = 0.917), and significant main effect of drink (F_1,52 = 7.766, p = 0.007) but not laser (F_1,52 = 3.538, p = 0.066) or virus (F_1,52 = 0.171, p = 0.681). Two-way ANOVAs showed a significant effect of laser stimulation with halorhodopsin [F_{(drinking-condition;1,6)} = 8.653, p = 0.007; F_(laser;1,6) = 1.841, p = 0.187; F_{(interaction;1,6)} = 11.811, p = 0.002] but not eYFP [F_{(drinking-condition;1,7)} = 1.952, p = 0.205; F_(laser;1,7) = 4.559, p = 0.070; F_{(interaction;1,7)} = 1.060, p = 0.337]. Two-way ANOVAs determined post hocs. (G) No effect of halorhodopsin inhibition of aINS–brainstem on saccharin intake. Scale bars indicate 1, 2, and 0.6 mm in (C), (D), and (E). PB parabrachium, TH tyrosine hydroxylase.

Fig. 2. Systemic prazosin reduced alcohol drinking.

Effects of systemic (i.p.) 0.75 mg/kg (A) or 1.5 mg/kg (B) prazosin on CLAD and AOD. **,***: p < 0.01, p < 0.005.

Fig. 3. Relationships among systemic prazosin effects on intake.

Changes in intake were determined by taking a log transformation of drug intake/vehicle intake (see “Materials and methods”); dotted lines indicate when log of change in intake equals 2, indicating no change in drinking [log(100)]. Neither 0.75 (A) nor 1.5 (B) mg/kg prazosin changes in AOD were correlated with changes in CLAD. 0.75 mg/kg (C) but not 1.5-mg/kg (D) prazosin changes in CLAD were correlated with basal CLAD. Neither 0.75 (E) nor 1.5 (F) mg/kg prazosin changes in AOD were correlated with basal AOD. A-O alcohol-only (AOD), Praz prazosin, Q-A quinine–alcohol (CLAD). *p < 0.05.

Fig. 4. Intra-aINS prazosin reduced both AOD and CLAD.

Prazosin (0.3 µg/side) in aINS reduced AOD and CLAD drinking (A) but not saccharin intake (B). (C) A lower dose of prazosin (0.1 µg/side) in aINS had no effect on AOD or CLAD drinking compared with vehicle (F_{(treatment;1,10)} = 0.319, p = 0.585; F_{(drinking-condition;1,10)} = 5.865, p = 0.036; F_{(interaction;1,10)} = 0.588, p = 0.461), although there was small (~21.5%) but significant lower CLAD vs. AOD intake. (D) For 0.3-µg/side aINS prazosin, changes in AOD were not correlated with changes in CLAD. **,***: p < 0.01, p < 0.005.

Fig. 5. More global aINS inhibition suppressed aspects of alcohol drinking.

Inhibition of aINS with M/B strongly reduced AOD and CLAD (A) but not saccharin intake (B) or locomotion (C). (D) OFC M/B did not reduce CLAD. (E) Intra-aINS M/B changes in AOD were not correlated with changes in CLAD. (F) Intra-aINS NASPM reduced CLAD but not AOD. Bacl baclofen, Musc muscimol, Quin quinine. *,***: p < 0.05, p < 0.005.