Characterization in the rat of the individual tendency to rely on alcohol to cope with distress and the ensuing vulnerability to drink compulsively

Abstract

Only some vulnerable individuals who recreationally drink alcohol eventually develop the compulsive drinking pattern that characterizes alcohol use disorder. A new frontier in biomedical research lies in understanding the neurobehavioural mechanisms of this individual vulnerability, a necessary step towards developing novel effective therapeutic strategies. Translational research has been hindered by the lack of valid, reliable and robust approaches that enable the study of the influence of the reliance on alcohol to cope with stress or self-medicate negative emotional states on the subsequent transition to alcohol use disorder. We have therefore developed a behavioural task in the rat that enables the investigation of the neural and cellular basis of the exacerbation of the vulnerability to develop compulsive alcohol drinking by the use of alcohol to develop an adjunctive, anxiolytic, polydipsic drinking behaviour in a schedule-induced polydipsia procedure. Hence, in our task, alcohol is introduced in the schedule-induced polydipsia context after several weeks of training with water so that rats are exposed to alcohol for the first time in a distressing context and learn to drink alcohol as a coping strategy. Capitalizing on this protocol, we have consistently been able to identify a subpopulation of rats that were unable to learn to cope with negative states by drinking water and relied on alcohol to do so. This maladaptive reliance on alcohol drinking to cope with distress has been shown to be associated with an exacerbation of the subsequent transition to compulsive drinking. Furthermore, these vulnerable rats reached blood alcohol levels comparable to that of intoxication in humans, thereby developing two key features of alcohol use disorder, namely excessive alcohol intake and compulsive drinking. Altogether, this behavioural task provides a novel and unique tool for the investigation of the neurobehavioural mechanisms underlying the exacerbation of the individual vulnerability to developing compulsive alcohol drinking by the use of alcohol as a strategy to cope with distress, and for the evaluation of the efficacy of potential therapeutic strategies in a personalized medicine approach. This procedure, which focuses on an understudied but key factor of the development of alcohol use disorder, may become widely used as it benefits the fields of alcohol, emotion regulation and stress, the interest in which has substantially increased since the evidence of a profound exacerbation of alcohol use and alcohol-related negative consequences by the distress and social isolation engendered by the various measures implemented worldwide in response to the COVID-19 pandemic.

Keywords: alcohol; alcohol use disorder; individual vulnerability; schedule-induced polydipsia; self-medication.

Graphical Abstract

Figure 1

Individual differences in the development of adjunctive behaviour under schedule-induced polydipsia. While, at the population level, individuals acquire a coping strategy expressed as an adjunctive polydipsic water drinking response under a fixed time 60-s schedule of food delivery [two-way repeated-measures ANOVA with Greenhouse–Geisser correction, main effect of time: F(4.4,176.3) = 21.31, P < 0.001, ηp2 = 0.35], some vulnerable individuals (High Drinkers) lose control over their coping strategy over time and develop hyperdipsia, an excessive polydipsic water intake. Other individuals (Low Drinkers) do not acquire a polydipsic coping response with water [two-way repeated-measures ANOVA, main effect of group: F(1,18) = 18.67, P < 0.001, ηp2 = 0.51; time: F(20,360) = 12.22, P < 0.001, ηp2 = 0.40 and group × time interaction: F(20,360) = 6.97, P < 0.001, ηp2 = 0.28]. Data are presented as mean ± SEM. #P < 0.001 group × time interaction, *P < 0.001 main effect of time. All data were analysed with a two-way repeated-measures ANOVA with time as within-subject factor and group as between-subject factor, and Greenhouse–Geisser correction when needed. B, baseline session; min, minutes; mL: millilitres.

Figure 2

The reliance on alcohol to develop a coping response facilitates the development of compulsive alcohol drinking (reproduced from Fouyssac et al.). (A) While some individuals exposed to a schedule-induced polydipsia procedure progressively developed a polydipsic water drinking response (water copers), others did not [two-way repeated-measures ANOVA, main effect of group: F(1,10) = 33.33, P < 0.001, ηp2 = 0.77; time: F(20,200) = 11.74, P < 0.001, ηp2 = 0.54; group × time interaction: F(20,200) = 7.96, P < 0.001, ηp2 = 0.44] unless they were given access to alcohol (alcohol copers). Thus, the introduction of the opportunity to drink alcohol as a means to cope resulted in the fast development of polydipsic alcohol drinking in these alcohol copers so that alcohol and water copers did not differ in their alcohol drinking behaviour [two-way repeated-measures ANOVA, main effect of group: F(1,10) = 1.28, P = 0.28; time: F(19,190) = 1.23, P = 0.23; group × time interaction: F(19,190) = 1.44, P = 0.11]. (B) Water and alcohol coper rats did not differ in their levels of alcohol intoxication [one-way ANOVA, main effect of group: F(1,9) = 1.59, P = 0.24]. (C) However, as compared to the former, the latter persisted in drinking alcohol despite adulteration with quinine [one-way ANOVA, main effect of group: F(1,10) = 6.77, P = 0.03, ηp2 = 0.40]. Data are presented as mean ± SEM or box plots [medians ± 25% (percentiles) and Min/Max as whiskers]. #P < 0.001 group × time interaction, *P < 0.05 alcohol coper different from water coper rats, ns: no significant. All data were analysed with a two-way repeated-measures ANOVA with time as within-subject factor and group as between-subject factor or one-way ANOVA with group as between-subject factor. B, baseline session; min, minutes; mL, millilitres; mg, milligram.

Figure 3

Timeline of the experimental procedure. After reception of rats from the supplier or when those bred in-house have reached the required age/body weight, house them in groups with ad libitum access to water and chow and allow them to habituate to the vivarium and the experimenter for at least one week (steps 1–3). Following the habituation, restrict the daily amount of food provided until rats reach 80–85% of their theoretical free-feeding body weight, and house them individually. These conditions are to be maintained for the duration of the experiment (steps 4–7). Once the desired body weight has been stable for at least 3 days, give rats some reward food pellets in their home cage the day before starting the training in order to prevent hyponeophagia (step 8). Next day, start the training in the schedule-induced polydipsia (SIP) procedure using either the 60- or 30-min sessions. Start the training with one habituation (steps 9–16 + steps 17–19) and one magazine training session (steps 9–16 + steps 20–21). The total amount of water that each rat drinks over these sessions will provide you with the volume of water ingested to meet their homoeostatic needs while eating 60 reward pellets over the duration of the session. It will be considered as baseline intake. Twenty-four hours after the magazine training session, start the SIP water training stage that consists of at least 20 daily sessions during which rats have free access to a water bottle while 60 reward pellets are delivered under a fixed time (FT) 60- or 30-s schedule over 60- or 30-min sessions, respectively. After each session, calculate the total amount of water consumed by each rat (steps 9–16 + steps 22–24). Twenty-four hours after the last SIP water session, replace the water with 10% ethanol and train the rats in the SIP alcohol procedure for at least 20 sessions. In this stage, rats have free access to the alcohol bottle while 60 reward pellets are delivered under a FT 60- or 30-s schedule over 60- or 30-min sessions, respectively. At the end of each session, calculate the amount of alcohol consumed by each rat (steps 9–16 + steps 25–28). The average of water and/or alcohol consumed over the last two to four sessions will be used to identify different subpopulations of rats (see ‘Overview of the procedure section’ for a detailed description). Pellet hab, pellet habituation; Mag Train, magazine training.

Figure 4

Identification of individual differences in the development of a coping strategy with water. (A, C) At the population level, food-restricted rats exposed to a fixed time 60- or 30-s schedule of food delivery progressively developed adjunctive polydipsic water drinking over 20 sessions [two-way repeated-measures ANOVA with Greenhouse–Geisser correction, main effect of time, 60-min sessions: F(3.8,177.8) = 32.82, P < 0.001, ηp2 = 0.41; 30-min sessions: F(5.3,244.5) = 46.76, P < 0.001, ηp2 = 0.50]. Polydipsic drinking became different from baseline drinking from session 3 onwards in both experiments. The average water intake over the last days of training (dashed-line rectangle) was used to subsequently identify differences in the development of a coping polydipsic response with water. (B, D) Marked individual differences in the propensity to lose control over adjunctive water drinking behaviour as a coping strategy emerged over the 20 sessions of training with High water Drinker (HDw) rats, selected in the upper quartile of the population, developing hyperdipsia, whereas Low water Drinker (LDw) rats, selected in the lower quartile of the population, did not develop a coping strategy with water. The other Intermediate water Drinker (Intw) individuals developed a controlled, stable, adjunctive polydipsic adjunctive drinking [two-way repeated-measures ANOVA with Greenhouse–Geisser correction, 60-min session: main effect of group: F(2,45) = 49.16, P < 0.001, ηp2 = 0.68; time: F(20,900) = 45.51, P < 0.001, ηp2 = 0.50; group × time interaction: F(15.8,355) = 11.66, P < 0.001, ηp2 = 0.34; 30-min session: main effect of group: F(2,44) = 70.08, P < 0.001, ηp2 = 0.76; time: F(20,880) = 58.58, P < 0.001, ηp2 = 0.57; group × time interaction: F(16,352.03) = 9.43, P < 0.001, ηp2 = 0.30]. Data are presented as mean ± SEM. #P < 0.001 group × time interaction, *P < 0.05 different from baseline intake. B, baseline session; min, minutes; mL, millilitres. A and B are reproduced from Fouyssac et al. All data were analysed with a two-way repeated-measures ANOVA with time as within-subject factor and group as between-subject factor and Greenhouse–Geisser correction when needed.

Figure 5

Identification of individual differences in the tendency to rely on alcohol to develop an adjunctive response. (A, D) Rats in the upper and lower quartiles of a population exposed to the schedule-induced polydipsia (SIP) procedure were characterized as High water Drinkers (HDw) and Low water Drinkers (LDw), respectively, based on their water consumption averaged across the last sessions of SIP with water [one-way ANOVA, main effect of group, 60-min sessions: F(1,22) = 94.77, P < 0.001, ηp2 = 0.81; 30-min sessions: F(1,22) = 170.91, P < 0.001, ηp2 = 0.89]. HDw rats developed hyperdipsia whereas LDw rats failed to develop a coping polydipsic response with water [two-way repeated-measures ANOVA without or with Greenhouse–Geisser correction, 60-min sessions: main effect of group: F(1,22) = 53.97, P < 0.001, ηp2 = 0.71; time: F(20,440) = 23.96, P < 0.001, ηp2 = 0.52; group × time interaction: F(20,440) = 16.03, P < 0.001, ηp2 = 0.42; 30-min sessions: main effect of group: F(1,22) = 133.05, P < 0.001, ηp2 = 0.86; time: F(20,440) = 34.89, P < 0.001, ηp2 = 0.61; group × time interaction: F(5.83,128.35) = 18.68, P < 0.001, ηp2 = 0.46]. (B, E) The introduction of alcohol resulted in significant changes in coping behaviour. While HDw rats maintained overall a steady level of polydipsic drinking, now of alcohol, over time some LDw rats acquired a coping response with alcohol and eventually developed polydipsic drinking. While analysis of the average alcohol intake over the last sessions of alcohol training revealed that HDw and LDw rats differed in their consumption of alcohol [one-way ANOVA, main effect of group, 60-min sessions: F(1,22) = 6.86, P = 0.015, ηp2 = 0.24; 30-min sessions: F(1,22) = 7.92, P = 0.010, ηp2 = 0.26], (C, F, bottom panel) a median split applied to each group identified LDw rats that relied on alcohol to acquire a polydipsic coping strategy (Low Drinker water–High Drinker alcohol, LDwHDa) in contrast with others (Low Drinker water–Low Drinker alcohol, LDwLDa) that continued not to show any adjunctive response [one-way ANOVA, main effect of group, 60-min sessions: F(1,10) = 21.22, P < 0.001, ηp2 = 0.68; 30-min sessions: F(1,10) = 22.84, P < 0.001, ηp2 = 0.70]. (C, F, top panel) Similarly, half the HDw rats were revealed to have a level of polydipsic alcohol drinking similar to that they used to show with water (High Drinker water–High Drinker alcohol, HDwHDa), while others drank less alcohol than they did water (High Drinker water–Low Drinker alcohol, HDwLDa) [one-way ANOVA, main effect of group, 60-min sessions: F(1,10) = 24.47, P < 0.001, ηp2 = 0.71; 30-min sessions: F(1,10) = 28.83, P < 0.001, ηp2 = 0.74]. HDwLDa and LDwHDa are referred to as water copers (WC) and alcohol copers (AC), respectively. Data are presented as mean ± SEM. #P < 0.001 group × time interaction, **P < 0.001 and *P < 0.05 main effect of group. B, baseline session; min, minutes; mL, millilitres. A and B are reproduced from Fouyssac et al. All data were analysed with a two-way repeated-measures ANOVA with time as within-subject factor and group as between-subject factor and Greenhouse–Geisser correction when needed or one-way ANOVA with group as between-subject factor.

Figure 6

Carrying box for rats. Carrying box used in our laboratory.

Figure 7

Schedule-induced polydipsia apparatus. Description of a standard modular chamber for schedule-induced polydipsia in rats (L × W × H: 32 × 27 × 25 cm) made of stainless steel (side walls) and clear polycarbonate (top, door and rear panels) with a stainless-steel grid floor (1) and stainless-steel waste pan (2), placed on a white polypropylene base (3). The bottle with a curved sipper tube (4) is fitted with the dedicated screw (5) on a magazine that has an open access (6) through which the curved sipper tube (7) protrudes inside the chamber. This bottle magazine, equipped with an infra-red photobeam (8, manufacturer)/contact (9, home-made) lickometer system, is placed at the bottom of the left wall (5 cm above the base). Each chamber is illuminated by a house light (3 W) (10) located at the top of the centre panel of the left wall (22 cm above the base), opposite the food magazine (11) equipped with an infra-red photobeam detection system (5.5 cm above the base) (12). The pellet dispenser (13) is located outside the chamber on the right and delivers the reward pellets to the food magazine (11) through a plastic tube (14). Inputs and outputs are connected to a SmartCtrl panel (15). The apparatus is enclosed within a sound-attenuating cubicle (16) equipped with a fan (17).

Figure 8

Rat identification system. Tail labelling system used in our laboratory to identify rats.