Frustrative nonreward and emotional self-medication:Factors modulating alcohol consumption following reward downshift in rats.

Life experience involving unexpected incentive loss (e.g., loss of job or a significant other) may result in negative emotional reactions (frustration) and promote alcohol drinking. Similarly, animals exposed to a frustrative 32-to-4% sucrose downshift increase their preference for alcohol (2%) vs. water. This result was interpreted as reflecting emotional self-medication-the consumption of substances that reduce negative emotions. We conducted three experiments examining parametric manipulations of the animal model: (1) effects of a severe reward downshift (32-to-4% sucrose) on consumption of various alcohol concentrations (Experiment 1); (2) effects of different magnitudes of reward downshifts on consumption of 32% alcohol (Experiment 2); and (3) effects of partial reinforcement (an intervention that increases resistance to frustration) on 2% alcohol intake induced by a 32-to-4% sucrose downshift (Experiment 3). The results show that (1) a 32-to-4% sucrose downshift leads to an increase in alcohol intake over a wide range of alcohol concentrations; (2) the greater the reward downshift, the higher the relative increase in alcohol consumption; and (3) a treatment that increases resistance to frustration (partial reinforcement) also attenuates alcohol consumption after a sucrose downshift. These data are discussed in relation to the role of frustrative nonreward in alcohol consumption.

Effects of alcohol consumption induced by reward loss on behavior in the hole-board test.

Rats exposed to reward downshift (from 32 to 4% sucrose) increase 2% alcohol intake in a 2-h, free-choice preference test which also offered water. This effect was accompanied by augmented general activity in the elevated plus maze (Donaire et al., 2018, Behav Proc, 150, 59-65). In the present study we analyzed the effect of alcohol consumption induced by reward downshift on anxiety behaviors registered in the hole-board (HB) test. Sixteen food-deprived female Wistar rats received 32% sucrose for ten 5-min daily sessions and were then downshifted to 4% sucrose for two 5-min daily sessions (postshift). Sessions also involved testing animals in a 2-h, 2-bottle preference task with 2% alcohol vs. water (Group A), or water vs. water (Group W). On postshift sessions, animals were exposed to a 6-min HB test after the preference task. Reward devaluation significantly reduced sucrose intake in Groups A and W, and increased alcohol consumption in Group A, but had no effect on water consumption in Group W. Increased alcohol consumption was followed by higher head-dipping frequency in the HB test compared with Group W. The results are discussed in terms of the impact of reward loss on anxiety behaviors in the HB test and the anxiolytic effects of alcohol in situations involving negative affect.

Lateral habenula lesions disrupt appetitive extinction, but do not affect voluntary alcohol consumption.

This study analyzed the effects of LHb lesions on appetitive extinction and alcohol consumption. Eighteen male Wistar rats received neurochemical lesions of the LHb (quinolinic acid) and 12 received a vehicle infusion (PBS). In a runway instrumental task, rats received acquisition (12 pellets/trial, 6 trials/session, 10 sessions) and extinction training (5 sessions). In a consummatory task, rats had daily access to 32% sucrose (5 min, 10 sessions) followed by access to water (5 sessions). Then, animals received 2 h preference tests with escalating alcohol concentrations (2%-24%), followed by two 24 h preference tests with 24% alcohol. Relative to Shams, LHb lesions delayed extinction, as indicated by lower response latencies (instrumental task) and higher fluid consumption (consummatory task). LHb lesions did not affect alcohol consumption regardless of alcohol concentration or test duration. The LHb modulates appetitive extinction and needs to be considered as part of the brain circuit underlying reward loss.

Augmented voluntary consumption of ethanol induced by reward downshift increases locomotor activity of male Wistar rats in the elevated plus maze.

Rats exposed to unexpected reward loss increase voluntary oral consumption of ethanol. Such consumption has been assumed to attenuate loss-induced negative affect (called emotional self-medication). To test this assumption, food-deprived male Wistar rats were exposed to 10 sessions of access to 32% sucrose followed by 5 sessions of access to 4% sucrose (reward downshift). A two-bottle preference test was initiated immediately after each consummatory session to assess ethanol intake. The experimental group received access to 2% ethanol and water, whereas the control group received access to two water bottles. On sessions 11, 12, and 15, immediately after the preference test, animals were tested in the elevated plus maze (EPM) for signs of anxiety. Sucrose consumption was reduced after the 32-to-4% sucrose downshift on sessions 11 and 12, but behavior recovered by session 15. Consummatory suppression was followed by increased ethanol intake in the preference test after sessions 11 and 12, but intake was reduced to preshift levels by session 15; no changes were observed in water controls. Finally, general activity (closed-arm entries and total arm entries) in the EPM increased in the ethanol group on session 12, but not on session 15, relative to water controls. The increase in ethanol consumption induced by reward downshift had measurable effects on activity as assessed in the EPM. These results show that voluntary oral 2% ethanol consumption after reward downshift can affect subsequent behavior, but fall short of providing unambiguous evidence that such ethanol consumption reduces negative affect.

Exploration of a novel object in late adolescence predicts novelty-seeking behavior in adulthood: Associations among behavioral responses in four novelty-seeking tests.

UNLABELLED: The sensation/novelty seeking behavioral trait refers to the exploration/preference for a novel environment. Novelty seeking increases during late adolescence and it has been associated with several neurobehavioral disorders. In this experiment, we asked whether inbred Roman high- and low-avoidance (RHA-I, RLA-I) rats (1) differ in novelty seeking in late adolescence and (2) whether late adolescent novelty seeking predicts this trait in adulthood. Thirty six male RHA-I and 36 RLA-I rats were exposed to a novel object exploration (NOE) test during late adolescence (pnd: 52-59; DEPENDENT VARIABLES: contact latency, contact time, contact frequency). Head-dipping (hole-board, HB), time and visits to a novel-arm (Y-maze), and latency-in and emergence latency (emergence test) were registered in adulthood (pnd: 83-105). The results showed strain differences in all these tests (RHA-I>RLA-I). Factor analysis (RHA-I+RLA-I) revealed two clusters. The first one grouped HB and emergence test measures. The second one grouped NOE and Y-maze variables. Time exploring a novel object (NOE) was a significant predictor of novel arm time (RHA-I+RLA, RHA-I); contact latency was a significant predictor of novel arm frequency (RLA-I). Present results show consistent behavioral associations across four novelty-seeking tests and suggest that late adolescent novelty seeking predicts this genetically-influenced temperamental trait in adult Roman rats.

Anti-anxiety self-medication in rats: oral consumption of chlordiazepoxide and ethanol after reward devaluation.

Rats increased preference for ethanol after sessions of appetitive extinction, but not after acquisition (reinforced) sessions (Manzo et al., 2014). Drinking was not influenced by appetitive extinction in control groups with postsession access to water, rather than ethanol. Because ethanol has anxiolytic properties in tasks involving reward loss, these results were interpreted as anti-anxiety self-medication. The present experiment tested the potential for self-medication with the prescription anxiolytic chlordiazepoxide, a benzodiazepine with an addictive profile used in the treatment of anxiety disorders. To test this hypothesis, Wistar rats exposed to a 32-to-4% sucrose devaluation received a two-bottle, 2-h preference test immediately after consummatory training. One bottle contained 1 mg/kg of chlordiazepoxide, 2% ethanol, or water for different groups (the second bottle contained water for all groups). Three additional groups received the same postsession preference tests, but were exposed to 4% sucrose during consummatory training. Rats showed suppression of consummatory behavior after reward devaluation relative to unshifted controls. This effect was accompanied by a selective increase in preference for chlordiazepoxide and ethanol. Downshifted animals with access to water or unshifted controls with access to the anxiolytics failed to exhibit postsession changes in preference. Similar results were observed in terms of absolute consumption and consumption relative to body weight. This study shows for the first time that a prescription anxiolytic supports enhanced voluntary consumption during periods of emotional distress triggered by reward loss. Such anti-anxiety self-medication provides insights into the early stages of addictive behavior.

Relationship between ethanol preference and sensation/novelty seeking.

High- and low-avoidance Roman inbred rat strains (RHA-I, RLA-I) were selected for extreme differences in two-way active avoidance. RHA-I rats also express less anxiety than RLA-I rats. This study compared male Roman rats in ethanol preference and sensation/novelty seeking. Rats were first exposed in counterbalanced order to the hole-board test (forced exposure to novelty) and the Y-maze and emergence tests (free choice between novel and familiar locations). Then, rats were tested in 24-h, two-bottle preference tests with water in one bottle and ethanol (2, 4, 6, 8, or 10% in successive days). Compared to RLA-I rats, RHA-I rats showed (1) higher frequency and time in head dipping, (2) higher activity, and (3) lower frequency of rearing and grooming in the hole-board test, and (4) remained in the novel arm longer in the Y-maze test. No strain differences were observed in the emergence test. RHA-I rats exhibited higher preference for and consumed more ethanol than RLA-I rats at all concentrations. However, both strains preferred ethanol over water for 2-4% concentrations, but water over ethanol for 6-10% concentrations. Factorial analysis with all the rats pooled identified a two-factor solution, one grouping preferred ethanol concentrations (2-4%) with head dipping and grooming in the hole board, and another factor grouping the nonpreferred ethanol concentrations (6-10%) with activity in the hole board and novel-arm time in the Y-maze test. These results show that preference for ethanol is associated with different aspects of behavior measured in sensation/novelty-seeking tests.

Gene expression in hippocampus as a function of differential trait anxiety levels in genetically heterogeneous NIH-HS rats.

To identify genes involved in the development/expression of anxiety/fear, we analyzed the gene expression profile in the hippocampus of genetically heterogeneous NIH-HS rats. The NIH-HS rat stock is a unique genetic resource for the fine mapping of quantitative trait loci (QTLs) to very small genomic regions, due to the high amount of genetic recombinants accumulated along more than 50 breeding generations, and for the same reason it can be expected that those genetically heterogeneous rats should be especially useful for studying differential gene expression as a function of anxiety, fearfulness or other complex traits. We selected high- and low-anxious NIH-HS rats according to the number of avoidance responses they performed in a single 50-trial session of the two-way active avoidance task. Rats were also tested in unconditioned anxiety/fearfulness tests, i.e. the elevated zero-maze and a "novel-cage activity" test. Three weeks after behavioral testing, the hippocampus was dissected and prepared for the microarray study. There appeared 29 down-regulated and 37 up-regulated SNC-related genes (fold-change>|2.19|, FDR<0.05) in the "Low-anxious" vs. the "High-anxious" group. Regression analyses (stepwise) revealed that differential expression of some genes could be predictive of anxiety/fear responses. Among those genes for which the present results suggest a link with individual differences in trait anxiety, nine relevant genes (Avpr1b, Accn3, Cd74, Ltb, Nrg2, Oprdl1, Slc10a4, Slc5a7 and RT1-EC12), tested for validation through qRT-PCR, have either neuroendocrinological or neuroinmunological/inflammation-related functions, or have been related with the hippocampal cholinergic system, while some of them have also been involved in the modulation of anxiety or stress-related (neurobiological and behavioral) responses (i.e. Avpr1b, Oprdl1). The present work confirms the usefulness of NIH-HS rats as a good animal model for research on the neurogenetic basis or mechanisms involved in anxiety and/or fear, and suggest that some MHC-(neuroinmunological/inflammation)-related pathways, as well as the cholinergic system within the hippocampus, may play a role in shaping individual differences in trait anxiety.

Incentive loss and hippocampal gene expression in inbred Roman high- (RHA-I) and Roman low- (RLA-I) avoidance rats.

Two recent microarray and qRT-PCR studies showed that inbred Roman high- (RHA-I, low anxiety and frustration vulnerability) and low-avoidance (RLA-I, high anxiety and frustration vulnerability) rats, psychogenetically selected on the basis of their divergence in two-way avoidance performance, differed in basal whole-brain and hippocampal expression of genes related to neurotransmission, emotion, stress, aversive learning, and drug seeking behavior. We have extended these studies by analyzing strain differences in hippocampal gene expression following a frustrative experience involving reward downshift, i.e. instrumental successive negative contrast (iSNC), a phenomenon in which the sudden reduction of an expected reward induces frustration/anxiety. Food-deprived male Roman rats were exposed to a reduction in the amount of solid food presented in the goal of a straight alley (from 12 pellets in "training" trials - i.e. preshift trials- to 2 pellets in "frustration testing" trials - i.e. postshift trials-). The iSNC effect, as measured by response latencies in the "postshift" trials, appeared only in RLA-I rats (i.e. higher response latencies in the 12-2 RLA-I group as compared to the 2-2 RLA-I control group in postshift trials). Two and a half hours after the "postshift" behavioral test, hippocampi were removed and stored (-80°C) until analysis. Microarray analysis of these hippocampi showed that four differentially-expressed, and qRT-PCR-validated genes (TAAR2, THAP1, PKD2L1, NANOS), have relevance for brain function and behavior, including schizophrenia, depression, anxiety, and drug addiction, thus showing the usefulness of Roman strains as a genetic model for research on the neurogenetic basis of frustration.

Gene expression in amygdala as a function of differential trait anxiety levels in genetically heterogeneous NIH-HS rats.

To identify genes involved in anxiety/fear traits, we analyzed the gene expression profile in the amygdala of genetically heterogeneous NIH-HS rats. The NIH-HS rat stock has revealed to be a unique genetic resource for the fine mapping of Quantitative Trait Loci (QTLs) to very small genomic regions, due to the high amount of genetic recombinants accumulated along more than 50 breeding generations, and for the same reason it can be expected that those genetically heterogeneous rats should be especially useful for studying differential gene expression as a function of anxiety-(or other)-related traits. We selected high- and low-anxious NIH-HS rats differing in their number of avoidances in a single 50-trial session of the two-way active avoidance task. Rats were also tested in unconditioned anxiety tests (e.g., elevated zero-maze). Three weeks after behavioural testing, the amygdala was dissected and prepared for the microarray study. There appeared 6 significantly down-regulated and 28 up-regulated genes (fold-change >|2|, FDR<0.05) between the low- and high-anxious groups, with central nervous system-related functions. Regression analyses (stepwise) revealed that differential expression of some genes could be predictive of anxiety/fear responses. Among those genes for which the present results suggest a link with individual differences in trait anxiety, six relevant genes were examined with qRT-PCR, four of which (Ucn3, Tacr3, H2-M9 and Arr3) were validated. Remarkably, some of them are characterized by sharing known functions related with hormonal HPA-axis responses to (and/or modulation of) stress, anxiety or fear, and putative involvement in related neurobehavioural functions. The results confirm the usefulness of NIH-HS rats as a good animal model for research on the neurogenetic basis of anxiety and fear, while suggesting the involvement of some neuropeptide/neuroendocrine pathways on the development of differential anxiety profiles.