Flexible Behavioral Adjustment to Frustrative Nonreward in Anticipatory Behavior, but Not in Consummatory Behavior, Requires the Dorsal Hippocampus.

The hippocampus (HC) is recognized for its pivotal role in memory-related plasticity and facilitating adaptive behavioral responses to reward shifts. However, the nature of its involvement in the response to reward downshifts remains to be determined. To bridge this knowledge gap, we explored the HC's function through a series of experiments in various tasks involving reward downshifts and using several neural manipulations in rats. In Experiment 1, complete excitotoxic lesions of the HC impaired choice performance in a modified T-maze after reducing the quantity of sugar pellet rewards. In Experiment 2, chemogenetic inhibition of the dorsal HC (dHC) disrupted anticipatory behavior following a food-pellet reward reduction. Experiments 3-5 impaired HC function by using peripheral lipopolysaccharide (LPS) administration. This treatment, which induces peripheral inflammation affecting HC function, significantly increased cytokine levels in the dHC (Experiment 3) and impaired anticipatory choice behavior (Experiment 4). None of these dorsal hippocampal manipulations affected consummatory responses in animals experiencing sucrose downshifts. Accordingly, we found no evidence of increased neural activation in either the dorsal or ventral HC, as measured by c-Fos expression, after a sucrose downshift task involving consummatory suppression (Experiment 6). The results highlight the HC's pivotal role in adaptively modulating anticipatory behavior in response to a variety of situations involving frustrative nonreward, while having no effect on adjustments on consummatory behavior. The data supporting this conclusion were obtained under heterogeneous experimental conditions derived from a multi-laboratory collaboration, ensuring the robustness and high reproducibility of our findings. Spatial orientation, memory update, choice of reward signals of different values, and anticipatory versus consummatory adjustments to reward downshift are discussed as potential mechanisms that could account for the specific effects observed from HC manipulations.

Memory deficits and hippocampal cytokine expression in a rat model of ADHD.

Attention-deficit/hyperactivity disorder (ADHD) is a complex behavioral disorder characterized by hyperactivity, impulsivity, inattention, and deficits in working memory and time perception. While animal models have advanced our neurobiological understanding of this condition, there are limited and inconsistent data on working and elapsed time memory function. Inflammatory signaling has been identified as a key factor in memory and cognitive impairments, but its role in ADHD remains unclear. Additionally, the disproportionate investigation of male subjects in ADHD research has contributed to a poor understanding of the disorder in females. This study sought to investigate the potential connections between memory, neuroimmunology, and ADHD in both male and female animals. Specifically, we utilized the spontaneously hypertensive rat (SHR), one of the most extensively studied animal models of ADHD. Compared to their control, the Wistar-Kyoto (WKY) rat, male SHR are reported to exhibit several behavioral phenotypes associated with ADHD, including hyperactivity, impulsivity, and poor sustained attention, along with impairments in learning and memory. As the hippocampus is a key brain region for learning and memory, we examined the behavior of male and female SHR and WKY rats in two hippocampal-dependent memory tasks. Our findings revealed that SHR have delay-dependent working memory deficits that were similar to, albeit less severe than, those seen in hippocampal-lesioned rats. We also observed impairments in elapsed time processing in female SHR, particularly in the discrimination of longer time durations. To investigate the impact of inflammatory signaling on memory in these rats, we analyzed the levels of several cytokines in the dorsal and ventral hippocampus of SHR and WKY. Although we found some sex and genotype differences, concentrations were generally similar between groups. Taken together, our results indicate that SHR exhibit deficits in spatial working memory and memory for elapsed time, as well as some differences in hippocampal cytokine concentrations. These findings contribute to a better understanding of the neurobiological basis of ADHD in both sexes and may inform future research aimed at developing effective treatments for the disorder. Nonetheless, the potential mediating role of neuroinflammation in the memory symptomatology of SHR requires further investigation.

Medial entorhinal cortex lesions produce delay-dependent disruptions in memory for elapsed time.

Our memory for time is a fundamental ability that we use to judge the duration of events, put our experiences into a temporal context, and decide when to initiate actions. The medial entorhinal cortex (MEC), with its direct projections to the hippocampus, has been proposed to be the key source of temporal information for hippocampal time cells. However, the behavioral relevance of such temporal firing patterns remains unclear, as most of the paradigms used for the study of temporal processing and time cells are either spatial tasks or tasks for which MEC function is not required. In this study, we asked whether the MEC is necessary for rats to perform a time duration discrimination task (TDD), in which rats were trained to discriminate between 10-s and 20-s delay intervals. After reaching a 90% performance criterion, the rats were assigned to receive an excitotoxic MEC-lesion or sham-lesion surgery. We found that after recovering from surgery, rats with MEC lesions were impaired on the TDD task in comparison to rats with sham lesions, failing to return to criterion performance. Their impairment, however, was specific to the longer, 20-s delay trials. These results indicate that time processing is dependent on MEC neural computations only for delays that exceed 10 s, perhaps because long-term memory resources are needed to keep track of longer time intervals.

A Time Duration Discrimination Task for the Study of Elapsed Time Processing in Rats.

Space and time are both essential features of episodic memory. However, while spatial tasks have been used effectively to study the behavioral relevance of place cells, the behavioral paradigms utilized for the study of time cells have not used time duration as a variable that animals need to be aware of to solve the task. In order to evaluate how time flow is coded into memory, time duration needs to be a variable that animals use to solve the behavioral task. This protocol describes a novel behavioral paradigm, the time duration discrimination (TDD) task, which is designed to directly investigate the neurological mechanisms that underlie temporal processing. During the TDD task, rats navigate around a Figure-8 Maze, which contains a rectangular track with a central arm and a delay box at the end of the central arm. While confined to the delay box, rats experience a 10- or 20-second time delay, during which a tone will play for the duration of the 10- or 20-second delay. When the delay box opens, the rat will choose whether to turn left or right out of the delay box and receive a reward for the correct choice (e.g., 10 seconds = left turn; 20 seconds = right turn). By directly manipulating elapsed time, we can better explore the behavioral relevance of hippocampal time cells and whether the time-dependent activity seen in physiological recordings of hippocampal neurons reflects a neuronal representation of time flow that can be used by the animal for learning and storing memories. Graphic abstract: Elapsed time duration discrimination in rats.

In the temporal organization of episodic memory, the hippocampus supports the experience of elapsed time.

Space and time are both essential features of episodic memory, for which the hippocampus is critical (Howard & Eichenbaum, 2015). Spatial tasks have been used effectively to study the behavioral relevance of place cells. However, the behavioral paradigms utilized for the study of time cells have not used time duration as a variable that animals need to be aware of to solve the task. Therefore, the behavioral relevance of this cell firing is unclear. In order to directly study the role of the hippocampus in processing elapsed time, we created a novel time duration discrimination task. Rats learned to make a decision to turn left or right depending on the preceding tone duration (10 s, left turn; 20 s, right turn). Once the rats reached criterion performance of 90% correct on two out of three consecutive days, they received either an excitotoxic hippocampal lesion or a sham-lesion surgery. After recovery, rats were tested to determine hippocampal involvement in discriminating time duration. Rats with hippocampal lesions performed at chance level on their first testing day postlesion, and they were impaired relative to the sham-lesioned rats. Although the hippocampal-lesioned rats began discriminating at above chance level, their performance never returned to criterion even with 50 days of postoperative testing. Furthermore, while sham rats showed no difference in the number of errors they made on 10- versus 20-s delay trials, hippocampal lesion rats similarly improved their performance under the 10-s delay condition, but not under the 20-s delay condition. Results indicate that hippocampal lesions resulted in a selective impairment in discriminating elapsed time only during the longer delay trials. The implications of these results are discussed in relation to the limits of working-memory capacity and to the role of sustained hippocampal time cell activity in memory performance depending on the perceived relevance of the delay period.

Delayed Alternation Task for the Study of Spatial Working and Long Term Memory in Rats.

Memory systems can hold previously presented information for several seconds, bridging gaps between discontinuous events. It has been previously demonstrated that the hippocampus and the medial entorhinal cortex (mEC) are necessary for memory retention over delay intervals in alternation tasks. Here we describe the delayed alternation task, a spatial working memory (WM) task in which animals need to alternate between left and right sides of a figure-8 maze on a trial-by-trial basis to receive a reward. On each trial of this task, the rat has to remember the last episode and turn in the opposite direction compared to the previous trial. We manipulated the WM load by introducing delays of various lengths (10 s and 60 s) between trials. While other alternation task protocols use short delay intervals between trials, our protocol introduces a longer delay condition that requires animals to use long-term memory resources that are not necessarily supported by sequential neuronal firing patterns (i.e., time cells) as are seen with shorter delay intervals.

Time Cells in the Hippocampus Are Neither Dependent on Medial Entorhinal Cortex Inputs nor Necessary for Spatial Working Memory.

A key function of the hippocampus and entorhinal cortex is to bridge events that are discontinuous in time, and it has been proposed that medial entorhinal cortex (mEC) supports memory retention by sustaining the sequential activity of hippocampal time cells. Therefore, we recorded hippocampal neuronal activity during spatial working memory and asked whether time cells depend on mEC inputs. Working memory was impaired in rats with mEC lesions, but the occurrence of time cells and of trajectory-coding cells in the stem did not differ from controls. Rather, the main effect of mEC lesions was an extensive spatial coding deficit of CA1 cells, which included inconsistency over time and reduced firing differences between positions on the maze. Therefore, mEC is critical for providing stable and distinct spatial information to hippocampus, while working memory (WM) maintenance is likely supported either by local synaptic plasticity in hippocampus or by activity patterns elsewhere in the brain.

Hippocampal CA1 replay becomes less prominent but more rigid without inputs from medial entorhinal cortex.

The hippocampus is an essential brain area for learning and memory. However, the network mechanisms underlying memory storage, consolidation and retrieval remain incompletely understood. Place cell sequences during theta oscillations are thought to be replayed during non-theta states to support consolidation and route planning. In animals with medial entorhinal cortex (MEC) lesions, the temporal organization of theta-related hippocampal activity is disrupted, which allows us to test whether replay is also compromised. Two different analyses-comparison of co-activation patterns between running and rest epochs and analysis of the recurrence of place cell sequences-reveal that the enhancement of replay by behavior is reduced in MEC-lesioned versus control rats. In contrast, the degree of intrinsic network structure prior and subsequent to behavior remains unaffected by MEC lesions. The MEC-dependent temporal coordination during theta states therefore appears to facilitate behavior-related plasticity, but does not disrupt pre-existing functional connectivity.

Active avoidance learning differentially activates ERK phosphorylation in the primary auditory and visual cortices of Roman high- and low-avoidance rats.

Understanding the mechanisms underlying conditioned avoidance is a critical step toward the development of novel treatments of anxiety. In this context, the two-way active avoidance (2WAA) task is a validated paradigm to investigate uncontrolled avoidance, a hallmark of anxiety disorders. The outbred Roman high- (RHA) and low-avoidance (RLA) rat lines are selected for respectively rapid vs. poor acquisition of active avoidant behavior, and emotional reactivity appears to be the most prominent behavioral difference between the two lines, with RLA rats being more fearful/anxious than their RHA counterparts. This study was aimed at assessing the relationship between the different performance of RHA and RLA rats in the 2WAA task and the number of phosphorylated ERK positive (pERK+) neurons in the primary auditory and visual cortices, in three sub-nuclei of the amygdala, as well as in the nucleus accumbens, and the prefrontal cortex. The results indicate that: (1) RHA rats, but not their RLA counterparts, learn very rapidly to avoid mild electric foot-shocks by crossing to the opposite compartment of the shuttle-box during the presentation of the conditioned stimulus and (2) the different behavior of the Roman lines during active avoidance training is associated with differential changes in the number of pERK+ neurons in the primary auditory and visual cortices (where the proactive coping of RHA rats is associated with increased ERK phosphorylation), but not in the other brain areas examined. These results are consistent with the hypothesis that the activation of the ERK signaling cascade in the auditory and visual cortices may be involved in the acquisition of aversive learning in RHA rats.

Effects of morphine on place conditioning and ERK1/2 phosphorylation in the nucleus accumbens of psychogenetically selected Roman low- and high-avoidance rats.

RATIONALE: Extracellular signal-regulated kinase (ERK1/2) phosphorylation is critical for neuronal and behavioural functions; in particular, phosphorylated ERK1/2 (pERK1/2) expression in the nucleus accumbens (Acb) of the rat is stimulated by addictive drugs with the exception of morphine, which decreases accumbal ERK1/2 phosphorylation in the Sprague-Dawley and Wistar rats. The psychogenetically selected Roman low- (RLA) and high-avoidance (RHA) rats differ behaviourally and neurochemically in many responses to addictive drugs. In particular, morphine elicits a greater increment in locomotor activity and in dopamine transmission in the Acb of RHA vs RLA rats. However, the effects of morphine on place conditioning (conditioned place preference (CPP)) and ERK1/2 phosphorylation in the Roman lines remain unknown. OBJECTIVES AND METHODS: To characterize in the Roman lines the reinforcing properties of morphine (i.e. morphine-elicited CPP acquisition) and the relationship between these properties and its effects on ERK1/2 phosphorylation in the Acb, the behavioural effects of morphine were evaluated in a place-conditioning apparatus and ERK1/2 phosphorylation was assessed by immunohistochemistry in the shell and core subregions of the Acb of rats both acutely administered with morphine or undergoing conditioning. RESULTS: Morphine elicited CPP in both Roman lines and decreased pERK1/2 expression in the Acb of RLA but not RHA rats. Such decrease was prevented by conditioning. CONCLUSIONS: These findings indicate that the selective breeding of the Roman lines has generated a divergence, in terms of morphine-elicited pERK1/2 expression but not of morphine-elicited CPP, between RLA and RHA rats and sustain the observation that changes in pERK1/2 expression in the Acb are not a requisite for the reinforcing effects of morphine.

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.

Differential effects of cocaine on extracellular signal-regulated kinase phosphorylation in nuclei of the extended amygdala and prefrontal cortex of psychogenetically selected Roman high- and low-avoidance rats.

Roman high (RHA)- and low (RLA)-avoidance rats are selectively bred for rapid vs. poor acquisition of active avoidance, respectively, and differ markedly in emotional reactivity, coping style, and behavioral and neurochemical responses to morphine and psychostimulants. Accordingly, acute cocaine induces more robust increments in locomotion and dopamine output in the nucleus accumbens shell (AcbSh) of RHA than of RLA rats. Cocaine induces short- and long-term neuronal plasticity via activation of the extracellular signal-regulated kinase (ERK) pathway. This study compares the effects of acute cocaine on ERK phosphorylation (pERK) in limbic brain areas of Roman rats. In RHA but not RLA rats, cocaine (5 mg/kg) increased pERK in the infralimbic prefrontal cortex and AcbSh, two areas involved in its acute effects, but did not modify pERK in the prelimbic prefrontal cortex and Acb core, which mediate the chronic effects of cocaine. Moreover, cocaine failed to affect pERK immunolabeling in the bed nucleus of stria terminalis pars lateralis and central amygdala of either line but increased it in the basolateral amygdala of RLA rats. These results extend to pERK expression previous findings on the greater sensitivity to acute cocaine of RHA vs. RLA rats and confirm the notion that genetic factors influence the differential responses of the Roman lines to addictive drugs. Moreover, they support the view that the Roman lines are a useful tool to investigate the molecular underpinnings of individual vulnerability to drug addiction.

Differential sensitivity of ethanol-elicited ERK phosphorylation in nucleus accumbens of Sardinian alcohol-preferring and -non preferring rats.

Sardinian alcohol-preferring (sP) and -non preferring (sNP) rats have been selectively bred for opposite ethanol preference and consumption; sP rats represent a validated experimental tool to model several aspects of excessive ethanol drinking in humans. Phosphorylated Extracellular signal-Regulated Kinase (pERK) in dopamine-rich terminal areas plays a critical role in several psychopharmacological effects of addictive drugs, including ethanol. This study was aimed at investigating whether ethanol-elicited ERK activation may differ in key brain areas of ethanol-naïve sP and sNP rats. To this end, the effects of ethanol (0, 0.5, 1, and 2 g/kg, administered intra-gastrically [i.g.]) on ERK phosphorylation were assessed by pERK immunohistochemistry in the shell (AcbSh) and core (AcbC) of the nucleus accumbens (Acb) as well as in the prelimbic (PrL) and infralimbic (IL) prefrontal cortex (PFCx), in the bed nucleus of stria terminalis (BSTL) and in the central nucleus of the amygdala (CeA). Ethanol (1 g/kg) significantly increased pERK immunoreactivity in AcbSh and AcbC of sP but not sNP rats. Conversely, ethanol failed to affect pERK expression in PrL and IL PFCx as well as in BSTL and CeA of both sP and sNP rats. These results suggest that selective breeding of these rat lines results in differential effects of acute ethanol on ERK phosphorylation in brain regions critical for the psychopharmacological effects of ethanol.

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.

Effects of L-cysteine on reinstatement of ethanol-seeking behavior and on reinstatement-elicited extracellular signal-regulated kinase phosphorylation in the rat nucleus accumbens shell.

BACKGROUND: Alcoholism is a neuroadaptive disorder, and the understanding of the mechanisms of the high rates of relapse, which characterize it, represents one of the most demanding challenges in alcoholism and addiction research. The extracellular signal-regulated kinase (ERK) is an intracellular kinase, critical for neuroplasticity in the adult brain that is suggested to play a fundamental role in the molecular mechanisms underlying drug addiction and relapse. We previously observed that a nonessential amino acid, L-cysteine, significantly decreases oral ethanol (EtOH) self-administration, reinstatement of EtOH-drinking behavior, and EtOH self-administration break point. METHODS: Here, we tested whether L-cysteine can affect the ability of EtOH priming to induce reinstatement of EtOH-seeking behavior. In addition, we determined the ability of EtOH priming to induce ERK phosphorylation as well as the ability of L-cysteine to affect reinstatement-elicited ERK activation. To these purposes, Wistar rats were trained to nose-poke for a 10% v/v EtOH solution. After stable drug-taking behavior was obtained, nose-poking for EtOH was extinguished, and reinstatement of drug seeking, as well as reinstatement-elicited pERK, was determined after an oral, noncontingent, priming of EtOH (0.08 g/kg). Rats were pretreated with either saline or L-cysteine (80 to 120 mg/kg) 30 minutes before testing for reinstatement. RESULTS: The findings of this study confirm that the noncontingent delivery of a nonpharmacologically active dose of EtOH to rats, whose previous self-administration behavior had been extinguished, results in significant reinstatement into EtOH-seeking behavior. In addition, the results indicate that reinstatement selectively activates ERK phosphorylation in the shell of the nucleus accumbens (Acb) and that pretreatment with L-cysteine reduces either reinstatement of EtOH seeking and reinstatement-elicited pERK in the AcbSh. CONCLUSIONS: Altogether, these results indicate that L-cysteine could be an effective pharmacological agent for the prevention of behavioral and molecular correlates of EtOH-primed reinstatement of EtOH seeking and that the shell of the Acb represents a critical neural substrate for priming-elicited reinstatement mechanisms involving ERK phosphorylation.