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. 2021 Aug 18;7(34):eabg9045.
doi: 10.1126/sciadv.abg9045. Print 2021 Aug.

A neural substrate of compulsive alcohol use

Esi Domi  1 , Li Xu  2   3 , Sanne Toivainen  2 , Anton Nordeman  2 , Francesco Gobbo  4 , Marco Venniro  5 , Yavin Shaham  6 , Robert O Messing  7 , Esther Visser  8 , Michel C van den Oever  8 , Lovisa Holm  2 , Estelle Barbier  2 , Eric Augier  2 , Markus Heilig  2
Affiliations

A neural substrate of compulsive alcohol use

Esi Domi et al. Sci Adv. .

Abstract

Alcohol intake remains controlled in a majority of users but becomes "compulsive," i.e., continues despite adverse consequences, in a minority who develop alcohol addiction. Here, using a footshock-punished alcohol self-administration procedure, we screened a large population of outbred rats to identify those showing compulsivity operationalized as punishment-resistant self-administration. Using unsupervised clustering, we found that this behavior emerged as a stable trait in a subpopulation of rats and was associated with activity of a brain network that included central nucleus of the amygdala (CeA). Activity of PKCδ+ inhibitory neurons in the lateral subdivision of CeA (CeL) accounted for ~75% of variance in punishment-resistant alcohol taking. Activity-dependent tagging, followed by chemogenetic inhibition of neurons activated during punishment-resistant self-administration, suppressed alcohol taking, as did a virally mediated shRNA knockdown of PKCδ in CeA. These findings identify a previously unknown mechanism for a core element of alcohol addiction and point to a novel candidate therapeutic target.

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Figures

Fig. 1
Fig. 1. Punishment-resistant alcohol self-administration emerges in a minority of outbred rats.
(A and B) Schematic representation of the footshock punishment procedure and resistance score distribution of punished alcohol self-administration in the total cohort of outbred rats screened (n = 301) over 14 days (color-coded). Insets show individual distribution for days 1 to 3 and 12 to 14 of punished alcohol self-administration. (C) Resistance score distribution of punished saccharin self-administration across 14 days (color-coded). (D) Bimodal distribution of the population of punished alcohol self-administration; 38% (n = 114) of rats were punishment resistant, while 62% (187) were punishment sensitive. (E) Mean resistance score (±SEM) during a 30-min punished self-administration session of 20% EtOH (FR2). *P < 0.001. (F) Mean break points (±SEM) reached during a progressive ratio session of 20% alcohol in punishment-resistant (n = 10) and punishment-sensitive (n = 9) rats. *P < 0.01. (G) Mean reinforcers (±SEM) earned during a 30-min self-administration session of punished 20% EtOH (FR2) and unpunished alcohol self-administration in punishment-resistant (n = 7) and punishment-sensitive (n = 9) rats. Punishment-resistant rats obtained a significantly higher number of punished alcohol reinforcers during the last 3 days of baseline punished self-administration (left, *P < 0.01) and when footshock punishment was reintroduced. *P < 0.001. (H) Aversion-resistant alcohol drinking in punishment-resistant (n = 7) and punishment-sensitive rats (n = 9), shown by resistance to quinine adulteration of the alcohol solution (resistance score ± SEM for quinine-adulterated alcohol drinking, #P < 0.001, *P < 0.05). SA, self-administration; FR, fixed ratio; d, day; BL, baseline.
Fig. 2
Fig. 2. A brain network associated with punishment resistance.
(A) Factor analysis using principal component extraction followed by varimax normalized rotation of Fos immunoreactivity data identified two networks that showed highly correlated within-network activity. Network 1 consisted of CeA, PAG, NAcC, and NAcSh; network 2 consisted of OFC, PrL, IL, and BLA. (B and C) Activity of network 1, but not network 2, was increased in punishment-resistant rats (*P < 0.001) and correlated with punishment-resistant self-administration. (D) Representative images of Fos immunohistochemistry in CeA in shock-resistant and shock-sensitive rats. Scale bar, 50 μm. (E) Mean number of Fos-immunoreactive neurons/mm2 (±SEM) in shock-resistant (n = 9), shock-sensitive (n = 9), and yoked rats (n = 8). *P < 0.001 versus the punishment-sensitive group; #P < 0.001 versus the yoked group. (F) Activity of CeA was particularly highly correlated with the resistance score. CeA, central nucleus of amygdala; PAG, periaqueductal gray; NAcC, nucleus accumbens core; NAcSh, NAc shell; OFC, orbitofrontal cortex; PrL, prelimbic cortex; IL, infralimbic cortex; BLA, basolateral amygdala.
Fig. 3
Fig. 3. Activity of CeA ensembles is necessary for punishment-resistant self-administration.
(A) Schematic overview of the experimental design. (B) Virus injection site and cannula placement (scale bar, 2 mm). (C) Schematic representation of the TRAP approach. (D and E) Representative images of CeA photomicrographs showing Fos immunoreactivity (green) and mCherry colabeling in control (n = 8) and hM4Di (n = 9). Scale bar, 50 μm. Mean number of Fos-immunoreactive neurons (±SEM), mean number of mCherry immunoreactive neurons (±SEM), and mean number of Fos+ mCherry-immunoreactive double-labeled neurons/mm2. *P < 0.001, *P < 0.05. (F) Mean number of alcohol-reinforced lever presses (±SEM) during the 30-min test in control (n = 11) and hM4Di (n = 12) punishment-resistant CNO injected rats. *P < 0.001, #P < 0.01. (G) Mean resistance score (±SEM). *P < 0.01. BL, baseline; 4TM, 4-hydroxytamoxifen; CNO, clozapine N-oxide.
Fig. 4
Fig. 4. Punishment resistance is driven by activity of PKCδ+ inhibitory neurons in CeA.
(A) Representative images of CeA photomicrographs showing Fos (green) and PKCδ (red) immunoreactivity colabeling [scale bars, 200 μm (left) and 50 μm (right)] in punishment-resistant (n = 10) and punishment-sensitive (n = 9) rats. (B to D) Mean number of cells (±SEM) positive for Fos, PKCδ, and double-labeled cells/mm2. *P < 0.001, *P < 0.05, *P < 0.01. (E) Mean fold change in Prkcd mRNA levels in punishment-resistant (n = 8) and punishment-sensitive (n = 6) rats, measured by qPCR. *P < 0.01.
Fig. 5
Fig. 5. A mechanistic role of CeA-PKCδ in punishment-resistant alcohol self-administration.
(A) Experimental design. (B) Virus injection site (scale bar, 2 mm) and mean fold change in Prkcd mRNA levels following a viral-mediated knockdown (n = 8 per group). *P < 0.05. (C) Expression of Prkcd in the CeA measured by RNAscope. Brown dots represent the expression of Prkcd [scale bars, 2 mm (left), 50 μm (right), and 5 μm (inset)] (n = 8 per group). *P < 0.05. (D) Representative images of CeA photomicrographs (scale bar, 50 μm) showing Fos (blue), PKCδ (magenta) immunoreactivity, and yellow fluorescent protein (YFP) colabeling. Mean number of cells positive (±SEM) for Fos, PKCδ, and double-labeled cells in punishment-resistant (n = 6 per group) and punishment-sensitive (n = 5 per group) rats/mm2. #P < 0.001, *P < 0.001, *P < 0.05, *P < 0.01. (E) Mean number of alcohol-reinforced lever presses (±SEM) during the 30-min punishment session control in punishment-resistant (n = 19) and punishment-sensitive rats (n = 19) receiving shCtrl, or shRNA knockdown of PKCδ (n = 18; n = 19). #P < 0.001. (F) Mean resistance score (±SEM). #P < 0.001.
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