Psychopharmacology DOI 10.1007/s00213-015-3910-5
ORIGINAL INVESTIGATION
Differential involvement of anxiety and novelty preference levels on oral ethanol consumption in rats Yann Pelloux & Jean Costentin & Dominique Duterte-Boucher
Received: 16 September 2014 / Accepted: 23 February 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Rationale Drug addiction is defined as a recurring cycle of intoxication, abstinence and relapse. The behavioural trait of novelty seeking is frequently observed in alcohol abusers. Moreover, converging evidence indicates that anxious individuals are also predisposed to alcohol abuse. Objectives We have analyzed the respective implication of those two behavioural factors on vulnerability to ethanol intake on rats in situations designed to reflect drug intoxication and relapse phases in humans. Methods In a general population of Wistar rats, animals were tested in both the light/dark box and the novelty preference tests. Ethanol consumption was measured in a two-bottle freechoice procedure across three successive procedures. Animals were first exposed to increasing concentrations of ethanol (2, 4, 6, 8, 10, 12 % for 8 days at each concentration). Then, the concentration of the solution was diminished from 12 to 6 %.
Finally, all rats were re-exposed to 6 % ethanol after 12 days of ethanol deprivation. Results Novelty preference predicted the amount of ethanol consumed across all phases. In contrast, anxiety was associated with a quicker recovery of ethanol consumption after the concentration drop and a greater increase in ethanol consumption after deprivation. Conclusions Novelty seeking and anxiety are both but differentially implicated in predisposition to ethanol abuse. Whereas novelty seeking is related to the amount of ethanol consumed, anxiety is associated to higher ethanol consumption when ethanol concentration is decreased or after ethanol deprivation. Keywords Ethanol . Oral consumption . Anxiety . Novelty seeking . Individual differences . Rat
Electronic supplementary material The online version of this article (doi:10.1007/s00213-015-3910-5) contains supplementary material, which is available to authorized users. Y. Pelloux : J. Costentin : D. Duterte-Boucher Unité de Neuropsychopharmacologie Expérimentale, EA 4359, Institut de Recherche et d’Innovation Biomédicale (IRIB), Faculté de Médecine et de Pharmacie de Rouen, 22, Bld Gambetta, 76183 Rouen Cedex, France Present Address: D. Duterte-Boucher (*) Equipe Facteurs Neurotrophiques et Différenciation Neuronale, INSERM U982, Différenciation et Communication Neuronale et Neuroendocrine (DC2N), IRIB, Faculté de Médecine et de Pharmacie de Rouen, 22, Bld Gambetta, 76183 Rouen Cedex, France e-mail:
[email protected] Present Address: Y. Pelloux Institut de Neuroscience de la Timone, CNRS UMR 7289, Université de Aix Marseille, Campus Santé Timone, 27, Bd Jean Moulin, 13385 Marseille Cedex 05, France
Introduction Understanding drug addiction requires taking into account the interrelationship between the substance, the individual and the environment (Zinberg 1986). Large individual differences exist regarding susceptibility to drug abuse in humans and laboratory animals. Several personality factors, including anxiety and sensation seeking, have been shown to be directly associated with an increased risk of using alcohol and other drugs of abuse (Woicik et al. 2009). Considerable evidence has implicated sensation/novelty seeking as a major behavioural feature in drug addicts (Zuckerman 1996) which could preexist (Cloninger et al. 1993) or result from drug use (Ersche et al. 2010). Experiments using laboratory animals have demonstrated the importance of novelty preference in free-choice situations (an
Psychopharmacology
index of novelty seeking) as a predisposition factor relevant to different responses induced by several drugs of abuse (Bardo et al. 2013 for review). In rats, novelty preference measured prior to any drug use has been shown to be positively correlated with levels of oral consumption of amphetamine (Pelloux et al. 2004) and morphine (Pelloux et al. 2006). Moreover, high novelty seekers (HNS) show greater conditioned place preference induced by morphine (Zheng et al. 2003; Pelloux et al. 2006), amphetamine (Robinet et al. 1998; Klebaur and Bardo 1999) or cocaine (Vidal-Infer et al. 2012) and are more likely to maintain cocaine self-administration despite adverse consequences, a hallmark of drug addiction (Belin et al. 2011). However, it has been argued that depressant and psychostimulant addictions are behaviourally and neurobiologically distinct (Badiani et al. 2011). Preclinical studies investigating the relationship between alcohol intake and preexisting high levels of novelty seeking as measured by increased locomotion in a new environment have yielded inconsistent outcomes (Bisaga and Kostowski 1993; Fahlke et al. 1995; Gingras and Cools 1995; Koros et al. 1998; Bienkowski et al. 2001; Nadal et al. 2002; Hayton et al. 2012; Manzo et al. 2014). The locomotor response to inescapable novelty may depend, at least in part, on escape behaviour due to novelty-induced stress (Exner and Clark 1993). In contrast, allowing rats a free choice between novel and familiar compartments favours the assessment of novelty seeking in a less stressful situation (Misslin et al. 1982). Novelty preference assessed by the novel object test does not predict individual differences in operant oral ethanol self-administration (Bienkowski et al. 2001). However, a positive relationship between ethanol preference and novelty seeking in the Y-maze test has been observed in high- and lowavoidance Roman inbred strains (Manzo et al. 2014). To date, no studies have assessed the relationship between novelty- induced place preference and ethanol intake in outbred rats. In parallel, clinical studies have reported a strong comorbidity between several anxiety-related disorders and drug abuse. In addition to the role of withdrawal-induced anxiety in the maintenance of drug use (Koob and Le Moal 1997), several epidemiological studies support the idea of a predisposition to drugs of abuse in individuals suffering from psychoemotional disorders such as anxiety and depression that preexisted any drug consumption (Rounsaville et al. 1991, Merikangas et al. 1998, Marquenie et al. 2007). In rodents, the same relationship between preexisting anxiety and predisposition to consumption of drugs of abuse has been observed. We previously found that, in rats regarded as anxious (A) and non-anxious (NA) on the basis of their scores on both the elevated plus-maze and light-dark box tests, anxious animals exhibit increased place conditioning induced by cocaine (Pelloux et al. 2009). High anxiety also predicts higher oral cocaine consumption (Walker et al. 2009). High levels of grooming expressed in the elevated plus-maze, thought to represent an anxious behaviour, are associated with a higher
motivation for cocaine as assessed by the break point reached under a progressive ratio schedule of reinforcement (Homberg et al. 2002). Finally, anxiety expressed in the elevated plusmaze predicts the loss of control over cocaine intake (Dilleen et al. 2012). Within general populations of rats and mice, anxiety as assessed in the elevated plus-maze not only predicts initiation of ethanol consumption (Spanagel et al. 1995), but also that carried out during limited or continuous access to alcohol (Hayton et al. 2012; Bahi 2013). Furthermore, only rats selected as anxious show conditioned place preference induced by alcohol (Blatt and Takahashi 1999). Notably, drug addiction as a chronic relapsing disorder is associated with recurring cycles of drug intoxication and withdrawal phases (Leshner 1997). These phases are thought to mobilize separate brain substrates (Koob 2009), suggesting that addicted individuals may exhibit distinct responsiveness to excess and lack of drug, thereby explaining the heterogeneity observed among addicts and, particularly, alcoholdependent patients (Cloninger 1987). In fact, high level of anxiety and novelty seeking are both strong predictors for relapse in detoxified alcohol-dependent patients (Meszaros et al. 1999; Willinger et al. 2002). On one side, withdrawalinduced negative state such as anxiety and depression resulting from disruption of the stress system comprising the extended amygdala seems to play a crucial role in prompting individuals to relapse. On the other side, exacerbated drug consumption and drug seeking as seen in alcoholic patients also involve an alteration of the rewarding system in the nucleus accumbens, including a decrease in dopaminergic neurotransmission (Koob 2013). Now, the incentive value of novelty is also mediated, at least in part, by activation of the mesocorticolimbic dopamine circuitry, and exaggerated responsiveness of this system seems to be linked to a high level of novelty seeking ((Bardo et al. 1996, 2013). Consequently, one might expect HNS to be especially sensitive to a decrease in brain reward function. We therefore hypothesized that various behavioural traits could maintain drug use by differentially acting on the distinct phases of the addiction cycle. Determination of such interactions could serve to greatly improve treatment for addiction through individual-based therapeutic strategies. To test the hypothesis, we analyzed, in a first experiment, the respective implications of preexisting anxiety and novelty seeking on voluntary alcohol consumption in situations aiming to reflect drug intoxication and withdrawal phases in humans. Exaggerated intake was induced by progressively increasing the concentration of ethanol for the purpose of contrasting high and low drinkers. We expected that rats exposed to different concentrations of ethanol would adapt their preference to maintain a stable consumption of ethanol, and since we previously reported that novelty seeking is associated with increased consumption of amphetamine (Pelloux et al. 2004) and morphine (Pelloux et al. 2006), we expected the same
Psychopharmacology
relationship with ethanol. In parallel, we predicted that during a concentration drop, animals would re-increase their consumption to maintain an optimal level of stimulation and that after a protracted period of abstinence, animals would increase their consumption, the so-called alcohol deprivation effect that can serve as a measure of relapse behaviour (Spanagel 2000). Since we previously observed an increased preference in anxious animals for cocaine in a drug-free state (Pelloux et al. 2009) and as ethanol deprivation is anxiogenic, we expected increased ethanol consumption in anxious rats during the reduction of the alcohol concentration or after an extended lack of ethanol. Finally, to verify whether the influence of levels of anxiety or novelty preference was specific to ethanol and therefore did not extend to other type of reward, we further investigated, in a second experiment, the relationships between anxiety or novelty preference and sucrose consumption. Considering our previous data (Pelloux et al. 2006), we did not expect differences between HNS and low novelty seekers (LNS) in sucrose consumption. Now, considering that drugs acting on anxiety affect the frustrative reduction in sucrose consumption during a negative contrast, that is when rats are shifted from a more preferred sucrose solution to a less preferred solution (Becker and Flaherty 1983; Becker and Hale 1991), we tested whether the level of anxiety would modulate sucrose consumption during negative contrast.
Materials and methods Animals One hundred and four male outbred Wistar rats (180–200 g on arrival) were purchased from Charles River/IFFA CREDO (Saint Germain sur l’Arbresle, France). Four rats were housed together in large Makrolon cages (L=40 cm, W=25 cm, H= 18 cm). They were maintained on a 12-h/12-h light/dark cycle (lights on at 07:00 a.m.), at a constant temperature (21±1 °C), with laboratory chow (SDS, Peypin, France) and water ad libitum. The experiments were carried out between 09:00 a.m. and 7:00 p.m. They were conducted in accordance with the guidelines for the use of animals in research (French Decree no. 87.848) and were approved by the ethical committee for animal experimentation in Normandy.
apparatus consisted of a wooden box (L=50 cm; W=45 cm; H=23 cm), divided in two equal compartments (L=45 cm; W=24 cm; H=23 cm), connected by a sliding door (L= 10 cm; H=8 cm). One compartment was painted white and dimly lit by a halogen lamp (100 lux), while the other was painted in black (illumination 40 lux). The degree of luminosity in both compartments was optimized to evaluate individual differences in anxiety level. The animal was confined for 30 s in the white compartment. Afterwards, the door was opened allowing free access to the black compartment, and the time spent in each compartment and the number of entries into the white compartment were measured for a 5-min test period using a video analysis system (see below). The third of the population that spent the least amount of time in the white compartment was regarded as A, while the third of the population that spent the most time in the white compartment was designated NA. Novelty preference test The novelty preference test has been described previously (Pelloux et al. 2004). The apparatus consisted of a twocompartment wooden chamber (L=35, W=35, H=30 cm) connected by a sliding door (10×10 cm). The floor was black. One compartment had grey walls, and the other had nonpainted wooden walls. Rats were confined to the grey compartment for 30 min, for two consecutive days. On the third day, the sliding door was removed and the rats were placed in the Bfamiliar^ grey compartment, from which they could freely access the novel compartment. The time spent and the number of entries into the novel compartment were measured for 15 min using a video analysis system (see below). The third of the population that spent the least amount of time in the novel compartment was regarded as LNS, while the third of the population that spent the longest duration in the novel compartment was designated HNS. Image analysis system
Ethanol was purchased from SDS and diluted in tap water.
The image analysis system (Videotrack 512, Viewpoint, Lyon, France) consisted of video cameras positioned above the apparatus, a video interface and a microcomputer. It converted the video input signal into binary images so that each animal corresponded to a white spot against a black background. During experimentation, the movements of the spots were translated into the time (s) spent, the distance (cm) and the entries in each compartment by the centre of gravity of the spots.
Light/dark box test
Ethanol oral consumption
The light/dark box test used in the present study was similar to the one previously described (Pelloux et al. 2009). The
Ethanol oral consumption tests were performed in a two-bottle free-choice paradigm. After assessment of novelty preference
Drugs
Psychopharmacology
and anxiety levels, 50 animals were isolated in individual cages (L=35, W=35, H=30 cm) and first habituated to the procedure by allowing them free access to two drinking bottles filled with water for 4 days. Bottles with conical top were hung upside down with an angle of approximately 45°. They were secured to the cage grid with metal strings. Liquid loss due to spillage, measured by using separate empty cages, revealed negligible loss compared to average oral consumption (less than 1.5 %). To further minimize overestimation of oral consumption by occasional spillage, data were averaged across four or eventually across two consecutive days. After habituation, rats were allowed to choose between water and an ethanol solution, the concentration (v/v) of which was progressively increased every 8 days, from 2 to 12 %. The concentration was then decreased from 12 to 6 % and maintained at 6 % for 20 days. Finally, ethanol (6 %) was presented to rats over 4 days following 12 days of ethanol deprivation. Each day, the consumption of the water and ethanol solutions was measured and the position of the bottles changed in order to avoid a position preference during both habituation and the free-choice period. Animals were weighed every 4 days. Average ethanol intake (g/kg/day) and the Bethanol preference ratio,^ representing ethanol consumption (ml/day) as a percentage of total fluid consumption, were calculated for each ethanol concentration.
Statistical analysis Behavioural scores (means±SEM) in rats selected as A and NA or as HNS and LNS were first tested for normality and homogeneity of variance. As some behavioural data did not fit a normal distribution, behavioural scores were compared using a Mann-Whitney U test. In oral consumption experiments, evolutions were analyzed within the entire experimental population by repeatedmeasures one-way analysis of variance (ANOVA), with concentrations or time periods as main factors, followed by a Fisher’s least significant difference (LSD) test for post hoc analysis. The relationship between behavioural performance and ethanol consumption or ratio was then evaluated using Pearson’s coefficient product (r) or Spearman’s rank correlation (ρ), depending whether the normality of population data was fulfilled or not. Furthermore, within rats selected as A or NA and as HNS or LNS, repeated measures two-way ANOVAs were performed with the anxiety or novelty seeking level and time period as main factors.
Results First experimental procedure Behavioural scores
First experimental procedure Rats were first tested in the novelty preference test and 3 days later in the light/dark box test. Two days later, all rats were isolated in individual cages in order to assess ethanol oral consumption during the different experimental phases as described above. Second experimental procedure: negative contrast for sucrose To test whether differences in ethanol intake did not extend to other type of reward, we investigated the relationships between anxiety and novelty and sucrose consumption using a similar but simplified procedure as that employed for ethanol. Rats were selected from a separate group of 54 rats according to their level of novelty preference and anxiety as previously described. Two days later, they were tested in a negative contrast procedure. For this purpose, rats were first habituated to the procedure by allowing them free access to two drinking bottles filled with water for 4 days. They were then given water in one bottle and sucrose in the other one. The first 4 days, animals were exposed to 4 % sucrose solution, then to 16 % for 4 days and back to 4 % for the last 4 days. Average intake (ml/kg/day) was calculated for each sucrose concentration.
The behaviours of rats selected as A and NA or as HNS and LNS in the light-dark box and in the novelty preference test are illustrated in Table 1. HNS spent more time in the novel compartment than LNS, this being the basis of group selection. This was due to HNS presenting a greater average duration per visit than LNS, as the groups did not differ in the number of visits to the novel compartment. No differences between HNS and LNS appeared in the light-dark box test. Similarly, while the differences between A and NA in the duration spent in the white compartment extended to all behaviours expressed in the light-dark box test, A and NA did not differ in any of the behavioural indices assessed in the novelty preference test. Ethanol consumption during the different experimental phases Preliminary analysis within the total population of 50 rats exposed to ethanol revealed significant changes in consumption between each experimental phase. Firstly, as the ethanol concentration was gradually raised, animals increased their ethanol intake and decreased their Bpreference ratio^ for this drug [main effect of concentration: F(5,245)= 21.64 and 63.24, respectively, for intake and preference; p
