Male Long-Evans Rats Research Articles

Heightened Exploratory Behavior Following Chronic Excessive Ethanol Drinking: Mediation by Neurotensin Receptor Type 2 in the Anterior Paraventricular Thalamus.

Chronic, excessive alcohol drinkers, even without dependence, can exhibit changes in behavior and neurochemical systems. Identifying these changes and their relationship with one another could provide novel avenues for the prevention and treatment of alcohol use disorder. We recently demonstrated, in rats, that neurotensin (NTS) in the paraventricular thalamus (PVT) regulates excessive ethanol (EtOH) drinking. Here, we investigate the effects of chronic EtOH drinking on the PVT-NTS system and its contribution to EtOH-induced behavioral changes. We gave adult male Long-Evans rats 20% EtOH under the intermittent access 2-bottle-choice paradigm or maintained them on chow and water for up to 11weeks. Prior to EtOH exposure and following several weeks of access, during acute abstinence, we tested these groups for multiple behaviors. In the 12th week, during acute abstinence, we examined gene expression and peptide levels of NTS and its receptors in the anterior and posterior subregions of the PVT. Finally, in chronic EtOH drinkers, during acute abstinence, we microinjected the NTS receptor type 2 (NTS2R) agonist, JMV-431, in the anterior PVT (aPVT) and examined subsequent EtOH intake and behavior. Following chronic intermittent EtOH access, rats were classified by cluster analysis as high or low EtOH drinkers. High EtOH drinkers spent more time in the light chamber of a light-dark box and open arms of an elevated plus maze and entered fewer familiar holes in a hole-board apparatus. These differences were absent prior to EtOH exposure but were detectable as early as 4weeks into drinking. Time in the light chamber following chronic drinking also predicted level of subsequent drinking. High EtOH drinkers also showed elevated protein levels of NTS2R in the aPVT, and pharmacological stimulation of aPVT NTS2R in low drinkers mimicked the increased time spent in the light chamber that was observed in high drinkers. Our findings suggest that chronic, excessive, but not lower level, EtOH drinking induces heightened or flexible exploratory behavior, which predicts future EtOH drinking and is partly mediated by elevated NTS2R signaling in the aPVT. These EtOH-induced alterations represent adaptations that could perpetuate excessive drinking and lead to the development of EtOH dependence.

Decreased motor impulsivity following chronic lithium treatment in male rats is associated with reduced levels of pro-inflammatory cytokines in the orbitofrontal cortex

Lithium’s efficacy in reducing both symptom severity in bipolar disorder (BD) and suicide risk across clinical populations may reflect its ability to reduce impulsivity. Changes in immune markers are associated with BD and suicidality yet their exact role in symptom expression remains unknown. Evidence also suggests that lithium may decrease levels of pro-inflammatory cytokines in the periphery and central nervous system, and that such changes are related to its therapeutic efficacy. However, issues of cause and effect are hard to infer from clinical data alone. Here, we investigated the effects of chronic dietary lithium treatment on rats’ performance of the 5-Choice Serial Reaction Time Task (5CSRTT), a well-validated operant behavioural task measuring aspects of impulsivity, attention and motivation. Male Long-Evans rats received a diet supplemented with 0.3% LiCl (n = 13), or the equivalent control diet (n = 16), during behavioural testing. Blood and brain tissue samples were assayed for a wide range of cytokines once any changes in impulsivity became significant. After 12 weeks, chronic lithium treatment reduced levels of motor impulsivity, as indexed by premature responses in the 5CSRTT; measures of sustained attention and motivation were unaffected. Plasma levels of IL-1β, IL-10 and RANTES (CCL-5) were reduced in lithium-treated rats at this time point. IL-1β, IL-6 and RANTES were also reduced selectively within the orbitofrontal cortex of lithium-treated rats, whereas cytokine levels in the medial prefrontal cortex and nucleus accumbens were comparable with control subjects. These results are consistent with the hypothesis that lithium may improve impulse control deficits in clinical populations by minimising the effects of pro-inflammatory signalling on neuronal activity, particularly within the orbitofrontal cortex.

Chronic sleep deprivation prolongs the reduction of sexual behaviour associated with daily sexual encounter in male rats

Chronic sleep deprivation (CSD) is common in many societies. Consecutive sleep loss increases allostatic load, which is known to negatively affect health outcomes. We investigated the impact of CSD on male sexual behaviour. Sexually-experienced male Long-Evans rats (singly housed under 14:10 light:dark) were either subjected to CSD or no CSD for 7 days, followed by a 7-day sleep recovery (SR) period. Their sexual behaviours were tested daily during both periods. CSD was performed by a ‘gentle-handling’ protocol for 4 hours per day, at the end of the light phase. Daily sexual behaviour tests led to a change in sexual behaviour over time. Intromission and ejaculation frequencies declined with repeated testing, but the reduction in these behaviours lasted for a longer period in rats that were previously subjected to CSD. Ejaculation latency was significantly longer towards the end of the recovery period in rats that had undergone CSD, but not in the control group. Post-ejaculatory interval increased and mounting behaviour did not change with daily mating tests, regardless of sleep deprivation protocol. CSD prolongs the decline in sexual behaviours associated with daily sexual encounters in male rats, and thus the return to baseline for these parameters requires days.

Chronic intermittent ethanol and lipopolysaccharide exposure differentially alter Iba1-derived microglia morphology in the prelimbic cortex and nucleus accumbens core of male Long-Evans rats.

Accumulating evidence has linked pathological changes associated with chronic alcohol exposure to neuroimmune signaling mediated by microglia. Prior characterization of the microglial structure-function relationship demonstrates that alterations in activity states occur concomitantly with reorganization of cellular architecture. Accordingly, gaining a better understanding of microglial morphological changes associated with ethanol exposure will provide valuable insight into how neuroimmune signaling may contribute to ethanol-induced reshaping of neuronal function. Here we have used Iba1-staining combined with high-resolution confocal imaging and 3D reconstruction to examine microglial structure in the prelimbic (PL) cortex and nucleus accumbens (NAc) in male Long-Evans rats. Rats were either sacrificed at peak withdrawal following 15days of exposure to chronic intermittent ethanol (CIE) or 24hr after two consecutive injections of the immune activator lipopolysaccharide (LPS), each separated by 24hr. LPS exposure resulted in dramatic structural reorganization of microglia in the PL cortex, including increased soma volume, overall cellular volume, and branching complexity. In comparison, CIE exposure was associated with a subtle increase in somatic volume and differential effects on microglia processes, which were largely absent in the NAc. These data reveal that microglial activation following a neuroimmune challenge with LPS or exposure to chronic alcohol exhibits distinct morphometric profiles and brain region-dependent specificity.

Early Physiological Changes To The Vastus Lateralis After Non-invasive Anterior Cruciate Ligament Injury

Insufficient recovery of quadriceps muscle strength is commonly reported after anterior cruciate ligament (ACL) injury. Although weakness is secondary to a complex manifestation of inhibition, the extent and time course of the morphological changes in muscle are largely unknown. Using a novel, translational animal model of ACL injury, a longitudinal study was performed to illuminate the mechanisms underlying acute muscle atrophy. PURPOSE: To investigate the role of atrophic pathways after non-invasive ACL rupture. METHODS: Male Long-Evans rats were randomly assigned to 8 groups (n=8 per group): 1 control group and 7 ACL injury groups (6, 12, 24, 48-hours, and 1, 2, 4-weeks). The right hindlimbs of ACL injury rats were exposed to a single impulse, longitudinal tibial compression, to induce a non-invasive ACL rupture, followed by normal cage activity. After which rats were euthanized as per assigned group. Right and left vastus lateralis muscles (VL) were harvested, weighed and flash frozen in liquid nitrogen. The VL were immunoreacted for dystrophin to quantify fiber cross sectional area (CSA), and RNA was isolated to measure the abundance of MuRF-1, MAFbx (markers of protein degradation) and 45s (marker of translational capacity). rRNA expression was determined using RT-PCR. One-way ANOVAs with Bonferroni post-hoc were used to determine differences between groups, and paired t-tests were used to detect VL differences between limbs (P < 0.05). RESULTS: ACL injury resulted in a decrease in muscle wet weight (p=0.0003) and a trend toward reduced CSA (p=0.06) at 1-week post injury, compared to control, 2-and 4-week time points. CSA of the ACL injured limb VL was smaller than the VL of the contralateral limb at 1-week only (p= 0.01). MAFbx abundance was significantly increased at 48-hours post-ACL injury (p=0.0001), with no differences for 45s rRNA, total RNA concentration or MuRF-1. CONCLUSIONS: Results indicate that ACL injury induces atrophy which is transient and not related to a decrease in ribosome biogenesis but likely due to increased protein degradation. Future studies should focus on a comprehensive analysis of atrophic pathways after ACL injury, to establish key therapeutic windows for targeting therapy-resistant quadriceps weakness after ACL injury. Supported by K01AR071503.

Reinforcement Learning during Adolescence in Rats.

The most dynamic period of postnatal brain development occurs during adolescence, the period between childhood and adulthood. Neuroimaging studies have observed morphologic and functional changes during adolescence, and it is believed that these changes serve to improve the functions of circuits that underlie decision-making. Direct evidence in support of this hypothesis, however, has been limited because most preclinical decision-making paradigms are not readily translated to humans. Here, we developed a reversal-learning protocol for the rapid assessment of adaptive choice behavior in dynamic environments in rats as young as postnatal day 30. A computational framework was used to elucidate the reinforcement-learning mechanisms that change in adolescence and into adulthood. Using a cross-sectional and longitudinal design, we provide the first evidence that value-based choice behavior in a reversal-learning task improves during adolescence in male and female Long-Evans rats and demonstrate that the increase in reversal performance is due to alterations in value updating for positive outcomes. Furthermore, we report that reversal-learning trajectories in adolescence reliably predicted reversal performance in adulthood. This novel behavioral protocol provides a unique platform for conducting biological and systems-level analyses of the neurodevelopmental mechanisms of decision-making.SIGNIFICANCE STATEMENT The neurodevelopmental adaptations that occur during adolescence are hypothesized to underlie age-related improvements in decision-making, but evidence to support this hypothesis has been limited. Here, we describe a novel behavioral protocol for rapidly assessing adaptive choice behavior in adolescent rats with a reversal-learning paradigm. Using a computational approach, we demonstrate that age-related changes in reversal-learning performance in male and female Long-Evans rats are linked to specific reinforcement-learning mechanisms and are predictive of reversal-learning performance in adulthood. Our behavioral protocol provides a unique platform for elucidating key components of adolescent brain function.

Modified limited bedding and nesting is a model of early-life stress that affects reproductive physiology and behavior in female and male Long-Evans rats

We used a modification of the limited bedding and nesting (LBN) model to evaluate the effects of early-life stress (ELS) on female and male reproductive physiology and behavior in Long-Evans rats. On postnatal day (PD) 2, dams and pups were transferred to a cage containing 100 mL of bedding (LBN condition) or to a cage containing 500 mL of bedding (control condition); bedding conditions remained until PD 10. In female rats, we measured vaginal opening, estrous cyclicity, female sexual behavior and motivation, and anxiety-like behavior. In male rats, we measured preputial separation, the development of male copulatory behavior, sexual motivation, and anxiety-like behavior. We found that relative to controls, female rats reared with LBN experienced precocious puberty and enhanced sexual motivation, but normal estrous cyclicity. Relative to controls, male rats reared with LBN experienced delayed puberty and enhanced sexual motivation, but normal development of copulatory behavior. Anxiety-like behavior was not affected by LBN in either female or male rats. In summary, the ELS of being reared with LBN affected the onset of puberty in the opposite direction in females and males, but enhanced sexual motivation in both. The current study is the first to examine the effects of ELS on sexual motivation using the LBN model. These findings further support the hypothesis that maternal care affects the development of sexual maturation and sexual motivation.

Prefrontal Cortex Representation of Learning of Punishment Probability During Reward-Motivated Actions.

Actions executed toward obtaining a reward are frequently associated with the probability of harm occurring during action execution. Learning this probability allows for appropriate computation of future harm to guide action selection. Impaired learning of this probability may be critical for the pathogenesis of anxiety or reckless and impulsive behavior. Here we designed a task for punishment probability learning during reward-guided actions to begin to understand the neuronal basis of this form of learning, and the biological or environmental variables that influence action selection after learning. Male and female Long-Evans rats were trained in a seek-take behavioral paradigm where the seek action was associated with varying probability of punishment. The take action remained safe and was followed by reward delivery. Learning was evident as subjects selectively adapted seek action behavior as a function of punishment probability. Recording of neural activity in the mPFC during learning revealed changes in phasic mPFC neuronal activity during risky-seek actions but not during the safe take actions or reward delivery, revealing that this region is involved in learning of probabilistic punishment. After learning, the variables that influenced behavior included reinforcer and punisher value, pretreatment with the anxiolytic diazepam, and biological sex. In particular, females were more sensitive to probabilistic punishment than males. These data demonstrate that flexible encoding of risky actions by mPFC is involved in probabilistic punishment learning and provide a novel behavioral approach for studying the pathogenesis of anxiety and impulsivity with inclusion of sex as a biological variable.SIGNIFICANCE STATEMENT Actions we choose to execute toward obtaining a reward are often associated with the probability of harm occurring. Impaired learning of this probability may be critical for the pathogenesis of anxiety or reckless behavior and impulsivity. We developed a behavioral model to assess this mode of learning. This procedure allowed us to determine biological and environmental factors that influence the resistance of reward seeking to probabilistic punishment and to identify the mPFC as a region that flexibly adapts its response to risky actions as contingencies are learned.

Effects of SCH‐23390 and Alpha‐Methyl‐Tyrosine on the Discriminative Stimulus Effects of Mephedrone in Male Rats

The illicit synthetic cathinone mephedrone (4‐methylmethcathinone) often termed a “bath salt” produces discriminative stimulus effects that are similar, but not identical, to those for central nervous system stimulants such as cocaine and methamphetamine. This may be due to its capacity to bind atypically at both dopamine and serotonin transporters compared to those drugs. The purpose of the present study was to further elucidate the mechanism of action underlying mephedrone’s discriminative stimulus effects by administering it with the dopamine type‐1 receptor antagonist SCH‐23390 (n=11) or the catecholamine synthesis inhibitor alpha‐methyl‐tyrosine (alpha‐MT; n=8). Male Long‐Evans rats were trained to discriminate intraperitoneal injections of mephedrone (3.2 mg/kg) from saline under a fixed‐ratio 20 (FR‐20) schedule of food presentation. Mephedrone dose‐effect curves (0.32 – 10 mg/kg) were then established by administering increasing cumulative doses of mephedrone on test days, and comparing those effects with multiple (4–5) injections of saline. The interaction of SCH‐23390 (0.032 – 0.1 mg/kg) and alpha‐MT (200 mg/kg) with mephedrone was examined by administering an acute injection of these drugs prior to increasing cumulative doses of mephedrone. As shown previously by our laboratory, saline administration produced almost exclusively saline‐lever responding while increasing cumulative doses of mephedrone dose‐dependently increased mephedrone‐lever responding. When SCH‐23390 preceded cumulative doses of mephedrone, neither dose alone produced mephedrone‐lever responding and only the larger of the two doses tested (0.1 mg/kg) shifted the mephedrone dose‐effect curve rightward; however, this shift was still less than 2‐fold and produced rate‐decreasing effects when administered alone. Acute administration of alpha‐MT alone also did not produce mephedronelever responding nor decrease rate, and in combination did not produce a marked rightward shift in the mephedrone dose‐effect curve. Taken together, these data indicate that neither SCH‐23390 nor alpha‐MT effectively antagonize the discriminative stimulus effects of mephedrone, and the alpha‐MT data in particular suggest that mephedrone’s discriminative stimulus effects do not depend on newly synthesized catecholamines.

Pharmacokinetics, mass balance, tissue distribution, metabolism, and excretion of praliciguat, a clinical-stage soluble guanylate cyclase stimulator in rats.

The pharmacokinetics (PK), metabolism, excretion, mass balance, and tissue distribution of [14C]praliciguat were evaluated following oral administration of a 3‐mg/kg dose in Sprague‐Dawley rats and in a quantitative whole‐body autoradiography (QWBA) study conducted in male Long‐Evans rats. Plasma T max was 1 hour and the t 1/2 of total plasma radioactivity was 23.7 hours. Unchanged praliciguat accounted for 87.4%, and a minor metabolite (N‐dealkylated‐praliciguat) accounted for 7.6% of the total radioactivity in plasma through 48 hours (AUC0‐48). Tissues with the highest exposure ratios relative to plasma were liver, intestines, adrenal gland, and adipose, and those with the lowest values were seminal vesicle, blood, CNS tissues, lens of the eye, and bone. Most of the [14C]praliciguat‐derived radioactivity was excreted within 48 hours after oral administration. Mean cumulative recovery of the administered radioactivity in urine and feces over 168 hours was 3.7% and 95.7%, respectively. Unchanged praliciguat was not quantifiable in urine or bile of cannulated rats; however, based on the total radioactivity in these fluids, a minimum of approximately 82% of the orally administered dose was absorbed. [14C]Praliciguat was metabolized via oxidative and glucuronidation pathways and the most abundant metabolites recovered in bile were praliciguat‐glucuronide and hydroxy‐praliciguat‐glucuronide. These results indicate that praliciguat had rapid absorption, high bioavailability, extensive tissue distribution, and elimination primarily via hepatic metabolism.

Impairment of Pattern Separation of Ambiguous Scenes by Single Units in the CA3 in the Absence of the Dentate Gyrus.

Theoretical models and experimental evidence have suggested that connections from the dentate gyrus (DG) to CA3 play important roles in representing orthogonal information (i.e., pattern separation) in the hippocampus. However, the effects of eliminating the DG on neural firing patterns in the CA3 have rarely been tested in a goal-directed memory task that requires both the DG and CA3. In this study, selective lesions in the DG were made using colchicine in male Long-Evans rats, and single units from the CA3 were recorded as the rats performed visual scene memory tasks. The original scenes used in training were altered during testing by blurring to varying degrees or by using visual masks, resulting in maximal recruitment of the DG-CA3 circuits. Compared with controls, the performance of rats with DG lesions was particularly impaired when blurred scenes were used in the task. In addition, the firing rate modulation associated with visual scenes in these rats was significantly reduced in the single units recorded from the CA3 when ambiguous scenes were presented, largely because DG-deprived CA3 cells did not show stepwise, categorical rate changes across varying degrees of scene ambiguity compared with controls. These findings suggest that the DG plays key roles not only during the acquisition of scene memories but also during retrieval when modified visual scenes are processed in conjunction with the CA3 by making the CA3 network respond orthogonally to ambiguous scenes.SIGNIFICANCE STATEMENT Despite the behavioral evidence supporting the role of the dentate gyrus in pattern separation in the hippocampus, the underlying neural mechanisms are largely unknown. By recording single units from the CA3 in DG-lesioned rats performing a visual scene memory task, we report that the scene-related modulation of neural firing was significantly reduced in the DG-lesion rats compared with controls, especially when the original scene stimuli were ambiguously altered. Our findings suggest that the dentate gyrus plays an essential role during memory retrieval and performs a critical computation to make categorical rate modulation occur in the CA3 between different scenes, especially when ambiguity is present in the environment.

Morphology and Anabolic Response of Skeletal Muscles Subjected to Eccentrically or Concentrically Biased Exercise.

Long-term eccentric exercise is known to promote muscle growth better than concentric exercise, but its acute effect on muscle is not well understood because of misinterpreted modeling and in situ and in vitro stretch protocols. Knowing if the initial bout of eccentric exercise promotes muscle growth and limits damage is critical to understanding the effect of this mode of exercise. To directly evaluate the immediate effects of eccentric and concentric exercises on untrained muscle when fiber strains were physiological and exercise doses were comparable. Controlled laboratory study. Laboratory. A total of 40 skeletally mature male Long-Evans rats (age = 16 weeks, mass = 452.1 ± 35.2 g) were randomly assigned to an eccentric exercise (downhill walking, n = 16), concentric exercise (uphill walking, n = 16), or control (no exercise, n = 8) group. Rats were exposed to a single 15-minute bout of eccentric or concentric exercise on a motorized treadmill and then were euthanized at 6 or 24 hours postexercise. We harvested the vastus lateralis muscle bilaterally. The percentage increase or decrease in protein abundance in exercised animals relative to that in unexercised control animals was evaluated as elevated phosphorylated p70S6k relative to total p70S6k. Fiber damage was quantified using immunoglobulin G permeability staining. One-way analysis of variance and post hoc Tukey tests were performed. Rats exposed to eccentric exercise and euthanized at 24 hours had higher percentage response protein synthesis rates than rats exposed to eccentric exercise and euthanized at 6 hours (P = .02) or to concentric exercise and euthanized at 6 (P = .03) or 24 (P = .03) hours. We assessed 9446 fibers for damage and found only 1 fiber was infiltrated (in the concentric exercise group euthanized at 6 hours). Furthermore, no between-groups differences in immunoglobulin G fluorescent intensity were detected (P = .94). Incorporating eccentric exercise is a simple, universally available therapeutic intervention for promoting muscle recovery. A single 15-minute dose of eccentric exercise to a novice muscle can better exert an anabolic effect than a comparable dose of concentric exercise, with very limited evidence of fiber damage.

Adolescent methylmercury exposure alters short-term remembering, but not sustained attention, in male Long-Evans rats

Methylmercury is an environmental neurotoxicant found in fish that produces behavioral deficits following early developmental exposure. The impact of adolescent exposure to this developmental neurotoxicant is only recently being explored in animal models. Here, short-term memory and sustained attention were examined using a rodent model of adolescent methylmercury exposure. Rats were exposed to 0, 0.5, or 5 ppm methylmercury throughout the adolescent period and tested on a two-choice visual signal detection task in adulthood. Methylmercury improved short-term remembering in this procedure but the dose-effect curve was nonmonotonic, as has been reported previously: effects on memory were observed in animals exposed to 0.5 ppm methylmercury, but not 5 ppm. Methylmercury did not significantly alter sustained attention, which is in contrast to effects following gestational exposure in human populations. The results may suggest that attention is not involved with previously reported effects of methylmercury during adolescence, but certain procedural issues remain unresolved.

Ventral Hippocampal Input to the Prelimbic Cortex Dissociates the Context from the Cue Association in Trace Fear Memory.

The PFC, through its high degree of interconnectivity with cortical and subcortical brain areas, mediates cognitive and emotional processes in support of adaptive behaviors. This includes the formation of fear memories when the anticipation of threat demands learning about temporal or contextual cues, as in trace fear conditioning. In this variant of fear learning, the association of a cue and shock across an empty trace interval of several seconds requires sustained cue-elicited firing in the prelimbic cortex (PL). However, it is unknown how and when distinct PL afferents contribute to different associative components of memory. Among the prominent inputs to PL, the hippocampus shares with PL a role in both working memory and contextual processing. Here we tested the necessity of direct hippocampal input to the PL for the acquisition of trace-cued fear memory and the simultaneously acquired contextual fear association. Optogenetic silencing of ventral hippocampal (VH) terminals in the PL of adult male Long-Evans rats selectively during paired trials revealed that direct communication between the VH and PL during training is necessary for contextual fear memory, but not for trace-cued fear acquisition. The pattern of the contextual memory deficit and the disruption of local PL firing during optogenetic silencing of VH-PL suggest that the VH continuously updates the PL with the current contextual state of the animal, which, when disrupted during memory acquisition, is detrimental to the subsequent rapid retrieval of aversive contextual associations.SIGNIFICANCE STATEMENT Learning to anticipate threat from available contextual and discrete cues is crucial for survival. The prelimbic cortex is required for forming fear memories when temporal or contextual complexity is involved, as in trace fear conditioning. However, the respective contribution of distinct prelimbic afferents to the temporal and contextual components of memory is not known. We report that direct input from the ventral hippocampus enables the formation of the contextual, but not trace-cued, fear memory necessary for the subsequent rapid expression of a fear response. This finding dissociates the contextual and working-memory contributions of prelimbic cortex to the formation of a fear memory and demonstrates the crucial role for hippocampal input in contextual fear learning.

Selective impact of lateral orbitofrontal cortex inactivation on reinstatement of alcohol seeking in male Long-Evans rats

The orbitofrontal cortex (OFC) plays a fundamental role in motivated behavior and decision-making. In humans, OFC structure and function is significantly disrupted in drug using and dependent individuals, including those exhibiting chronic alcohol use and alcoholism. In animal models, the OFC has been shown to significantly influence the seeking of non-alcohol drugs of abuse. However direct investigations of the OFC during alcohol seeking and use have been more limited. In the studies reported here, we inactivated lateral (lOFC) or medial OFC (mOFC) subregions in rats during multiple stages of alcohol seeking. After one month of intermittent access to homecage 20% ethanol (EtOH), rats were trained to self-administer EtOH under an FR3 schedule and implanted with cannulae directed to lOFC or mOFC. We inactivated OFC subregions with baclofen/muscimol during EtOH self-administration, extinction, cue-induced reinstatement, and progressive ratio testing to broadly characterize the influence of these subregions on alcohol seeking. There were no significant effects of mOFC or lOFC inactivation during FR3 self-administration, extinction, or progressive ratio self-administration. However, lOFC, and not mOFC, inactivation significantly decreased cue-induced reinstatement of EtOH seeking. These findings contribute new information to the specific impact of OFC manipulation on operant alcohol seeking, support previous studies investigating the role of OFC in seeking and consumption of alcohol and other drugs of abuse, and indicate a specific role for lOFC vs. mOFC in reinstatement.

Female and male rats readily consume and prefer oxycodone to water in a chronic, continuous access, two-bottle oral voluntary paradigm

The increasing abuse of opioids - such as oxycodone - poses major challenges for health and socioeconomic systems. Human prescription opioid abuse is marked by chronic, voluntary, oral intake and sex differences. To develop interventions, the field would benefit from a preclinical paradigm that similarly provides rodents with chronic, continuous, oral, voluntary and free-choice access to oxycodone. Here we show female and male rats voluntarily ingest and choose oxycodone over water and show both dependence and motivation to take oxycodone during a chronic oral voluntary, two-bottle choice, continuous access paradigm. Adult female and male Long-Evans rats were given unlimited, continuous homecage access to two bottles containing water (Control) or one bottle of water and one bottle of oxycodone dissolved in water (Experimental). Virtually all experimental rats voluntarily drank oxycodone (~10 mg/kg/day) and escalated their intake over 22 weeks. Females self-administered twice as much oxycodone by body weight (leading to higher blood levels of oxycodone) and engaged in more gnawing behavior of wooden blocks relative to males. Precipitated withdrawal revealed high levels of dependence in both sexes. Reflecting motivation to drink oxycodone, ascending concentrations of citric acid suppressed the intake of oxycodone (Experimental) and the intake of water (Control); however, Experimental rats returned to pre-citric acid preference levels whereas Controls rats did not. Pre-screening behaviors of rats on open field exploration predicted oxycodone intake. Thus, rats consumed and preferred oxycodone over time in this chronic two-bottle oral choice paradigm and both sexes displayed many features of human oxycodone abuse.

P.010 Dose increase of s-adenosyl-methionine and escitalopram in a randomized clinical trial for major depressive disorder

The mechanisms behind relapse to ethanol intake in recovering alcoholics are still unclear. The negative reinforcing effects contributing to ethanol addiction, including relapse, are considered to be partly driven by the κ-opioidergic system. As the κ-opioidergic system interacts with the mesolimbic reward pathway, the aim of the study was to clarify the role of nucleus accumbens shell κ-opioidergic mechanisms in relapse to ethanol intake by using the alcohol deprivation effect (ADE) paradigm. The ADE is defined as a transient increase in voluntary ethanol intake after a forced period of abstinence. Male Long-Evans rats were trained to voluntarily consume 10% (v/v) ethanol solution. Ethanol access and deprivation cycles were initiated after stable ethanol intake baselines had been reached and bilateral guide cannulas had been implanted above the nucleus accumbens shell. One cycle consisted of 10 days of 90 min access to ethanol followed by 6 days of ethanol deprivation. The ADE was measured in the beginning of a new cycle. Rats received JDTic, a selective κ-antagonist, either subcutaneously (10 mg/kg) or intra-accumbally (15 µg/site) or, as a reference substance, systemic naltrexone (0.3 mg/kg) before ethanol re-access, and the effects on the ADE were evaluated. Systemic and intra-accumbal JDTic significantly attenuated the ADE on the first day of ethanol re-access, as did systemic naltrexone. Additionally, naltrexone decreased ethanol intake levels. These results suggest that nucleus accumbens shell κ-opioidergic mechanisms may have a role in mediating relapse to ethanol intake. Additionally, κ-antagonism could be a valuable adjunct in ethanol relapse prevention.

Abstract A114: Quantitative mass spectrometry imaging: A game changer in the pharmaceutical industry

Abstract Developing a drug requires to demonstrate its efficacy in preclinical and clinical studies but also to characterize its pharmacokinetics properties. If Quantitative Whole-Body Autoradiography (QWBA) is still a gold standard to support the design of radiolabelled clinical studies, its lack of molecular specificity limits the understanding of the distribution of the drug and its metabolites, with no establishing of separate quantitative results for each target tissue of interest. On the contrary, the use of high resolution mass spectrometers such as FT-ICR MS allows a quantitative characterization of the metabolic profile of the drug at the tissue level in order to further understand its behaviour in the organism. A comparative analysis between QWBA and QMSI was applied as a complementary approach to follow chloroquine and one of its metabolites (desethyl-chloroquine) at different time-points after a single administration of a radiolabeled dose to Long-Evans male rats (30 mg/kg). 1H-Chloroquine and 1H-desethyl chloroquine were well detected by MALDI-FTICR in the eye including; the uveal tract, the vitreous humor, the lens and the Harderian gland. In the mid whole-body region, both compounds were also detected in various organs from the unique prepared T4h sections. Interestingly the two compounds were co-localized into the tissue sections and their distributions matched the zones obtained by QWBA. In QWBA additional regions containing the radiolabeled moiety could be clearly identified because of the high sensitivity of the technique. The advantage brought by MALDI QMSI was the ability to discriminate between parent drug 1H-choloroquine and its metabolite 1H-desethyl chloroquine so that each compound had its own distribution image and thus its own quantification data directly in one tissue section. The used labeled forms of both compounds during the matrix deposit allowed normalizing the data for each position targeted with the MALDI onto the section of interest and the calibration range of both 1H-choloroquine and 1H-desethyl chloroquine and quantifying each compound into the organs of interest with the ILC approach. Finally the QMSI was able to demonstrate the disappearance of the drug and its metabolite with time to better understand differential pharmacokinetics analysis demonstrating the additional input of the technology compared to QWBA. Citation Format: Rima Ait-Belkacem, Guillaume Hochart, Joseph Marini, Aurore Tomezyk, p Mantefeul, B. Jung, David Bonnel, Don Mc Kenzie, Jonathan Stauber. Quantitative mass spectrometry imaging: A game changer in the pharmaceutical industry [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A114. doi:10.1158/1535-7163.TARG-19-A114

Anxiety during alcohol withdrawal involves 5-HT2C receptors and M-channels in the lateral habenula

Anxiety disorders often co-occur with alcohol use disorders, but the mechanisms underlying this comorbidity remain elusive. Previously, we reported that rats withdrawn from chronic alcohol consumption (Post-EtOH rats) exhibited robust anxiety-like behaviors (AB), which were accompanied by neuronal hyperexcitability, and the downregulation of M-type potassium channels (M-channels) in the lateral habenula (LHb); and that serotonin (5-HT) stimulated LHb neurons via type 2C receptors (5-HT2CRs). Also, 5-HT2CR activation is known to inhibit M-current in mouse hypothalamic neurons. The present study investigated whether LHb 5-HT2CRs and M-channels contribute to AB in adult male Long-Evans rats. We used the intermittent-access to 20% ethanol two-bottle free-choice drinking paradigm to induce dependence. We measured AB with the elevated plus-maze, open-field, and marble-burying tests at 24 h withdrawal. We found that intra-LHb infusion of SB242084, a selective 5-HT2CR antagonist alleviated AB and reduced the elevated c-Fos expression in the LHb of Post-EtOH rats. By contrast, intra-LHb infusion of the selective 5-HT2CR agonist WAY161503 induced AB and increased c-Fos expression in the LHb in alcohol-naive but not Post-EtOH rats. Also, intra-LHb SB242084 significantly reduced self-administration of alcohol intake in the operant chambers. Furthermore, both 5-HT2CR protein levels and 5-HIAA/5-HT ratio was increased in the LHb of Post-EtOH rats. Finally, intra-LHb SB242084 increased LHb KCNQ2/3 membrane protein expression in Post-EtOH rats. Collectively, these results suggest that enhanced LHb 5-HT2CR signaling that interacted with M-channels triggers AB in Post-EtOH rats and that 5-HT2CRs may be a promising target for treating comorbid anxiety disorders in alcoholics.

Effects of negative incentive shifts in food reward on rats' consumption of concurrent ethanol solutions.

Frustration stress, typically operationalized as the unexpected loss of reinforcement, has been shown to engender substance use. Abrupt reductions in reinforcer magnitude likely also function as frustration stressors. These negative incentive shifts were previously shown to produce tap- and sweetened-water drinking in rats. The purpose of this study was to investigate whether these shifts in food reward would occasion oral ethanol self-administration. Nine male Long-Evans rats operated on a two-component multiple fixed-ratio schedule with signaled components producing either a large (4 pellets) or small (1pellet) reinforcer. Components were pseudorandomly arranged to present 4 transitions between past and upcoming reinforcer magnitudes: small-to-large, small-to-small, large-to-large, and large-to-small (negative incentive shift). Experiment 1 investigated the effects of negative incentive shifts on consumption of concurrent, freely available 10% sucrose, 10% sucrose plus 10% ethanol, and following sucrose fading, 10% ethanol. Experiment 2 entailed continuation of schedule contingencies with a dose manipulation of 4 ethanol concentrations (0, 5, 10, and 20%) to assess dose-dependent differences in transition-type control and consumption. A lever-press extinction condition was then conducted with 10% ethanol availability. In this novel model of frustration stress, negative incentive shifts prompted ethanol self-administration at each dose investigated, whereas the other transitions did not.