Flow Cytometry of Synaptoneurosomes (FCS) Reveals Increased Ribosomal S6 and Calcineurin Proteins in Activated Medial Prefrontal Cortex to Nucleus Accumbens Synapses.

Learning and behavior activate cue-specific patterns of sparsely distributed cells and synapses called ensembles that undergo memory-encoding engram alterations. While Fos is often used to label selectively activated cell bodies and identify neuronal ensembles, there is no comparable endogenous marker to label activated synapses and identify synaptic ensembles. For the purpose of identifying candidate synaptic activity markers, we optimized a flow cytometry of synaptoneurosome (FCS) procedure for assessing protein alterations in activated synapses from male and female rats. After injecting yellow fluorescent protein (YFP)-expressing adeno-associated virus into medial prefrontal cortex (mPFC) to label terminals in nucleus accumbens (NAc) of rats, we injected 20 mg/kg cocaine in a novel context (cocaine+novelty) to activate synapses, and prepared NAc synaptoneurosomes 0-60 min following injections. For FCS, we used commercially available antibodies to label presynaptic and postsynaptic markers synaptophysin and PSD-95 as well as candidate markers of synaptic activity [activity-regulated cytoskeleton protein (Arc), CaMKII and phospho-CaMKII, ribosomal protein S6 (S6) and phospho-S6, and calcineurin and phospho-calcineurin] in YFP-labeled synaptoneurosomes. Cocaine+novelty increased the percentage of S6-positive synaptoneurosomes at 5-60 min and calcineurin-positive synaptoneurosomes at 5-10 min. Electron microscopy verified that S6 and calcineurin levels in synaptoneurosomes were increased 10 min after cocaine+novelty. Pretreatment with the anesthetic chloral hydrate blocked cocaine+novelty-induced S6 and calcineurin increases in synaptoneurosomes, and novel context exposure alone (without cocaine) increased S6, both of which indicate that these increases were due to neural activity per se. Overall, FCS can be used to study protein alterations in activated synapses coming from specifically labeled mPFC projections to NAc.SIGNIFICANCE STATEMENT Memories are formed during learning and are stored in the brain by long-lasting molecular and cellular alterations called engrams formed within specific patterns of cue-activated neurons called neuronal ensembles. While Fos has been used to identify activated ensemble neurons and the engrams within them, we have not had a similar marker for activated synapses that can be used to identify synaptic engrams. Here we developed a procedure for high-throughput in-line analysis of flow cytometry of synaptoneurosome (FCS) and found that ribosomal S6 protein and calcineurin were increased in activated mPFC-NAc synapses. FCS can be used to study protein alterations in activated synapses within specifically labeled circuits.

New technologies for examining the role of neuronal ensembles in drug addiction and fear.

Correlational data suggest that learned associations are encoded within neuronal ensembles. However, it has been difficult to prove that neuronal ensembles mediate learned behaviours because traditional pharmacological and lesion methods, and even newer cell type-specific methods, affect both activated and non-activated neurons. In addition, previous studies on synaptic and molecular alterations induced by learning did not distinguish between behaviourally activated and non-activated neurons. Here, we describe three new approaches--Daun02 inactivation, FACS sorting of activated neurons and Fos-GFP transgenic rats--that have been used to selectively target and study activated neuronal ensembles in models of conditioned drug effects and relapse. We also describe two new tools--Fos-tTA transgenic mice and inactivation of CREB-overexpressing neurons--that have been used to study the role of neuronal ensembles in conditioned fear.

Distinct Fos-Expressing Neuronal Ensembles in the Ventromedial Prefrontal Cortex Mediate Food Reward and Extinction Memories.

UNLABELLED: In operant learning, initial reward-associated memories are thought to be distinct from subsequent extinction-associated memories. Memories formed during operant learning are thought to be stored in "neuronal ensembles." Thus, we hypothesize that different neuronal ensembles encode reward- and extinction-associated memories. Here, we examined prefrontal cortex neuronal ensembles involved in the recall of reward and extinction memories of food self-administration. We first trained rats to lever press for palatable food pellets for 7 d (1 h/d) and then exposed them to 0, 2, or 7 daily extinction sessions in which lever presses were not reinforced. Twenty-four hours after the last training or extinction session, we exposed the rats to either a short 15 min extinction test session or left them in their homecage (a control condition). We found maximal Fos (a neuronal activity marker) immunoreactivity in the ventral medial prefrontal cortex of rats that previously received 2 extinction sessions, suggesting that neuronal ensembles in this area encode extinction memories. We then used the Daun02 inactivation procedure to selectively disrupt ventral medial prefrontal cortex neuronal ensembles that were activated during the 15 min extinction session following 0 (no extinction) or 2 prior extinction sessions to determine the effects of inactivating the putative food reward and extinction ensembles, respectively, on subsequent nonreinforced food seeking 2 d later. Inactivation of the food reward ensembles decreased food seeking, whereas inactivation of the extinction ensembles increased food seeking. Our results indicate that distinct neuronal ensembles encoding operant reward and extinction memories intermingle within the same cortical area. SIGNIFICANCE STATEMENT: A current popular hypothesis is that neuronal ensembles in different prefrontal cortex areas control reward-associated versus extinction-associated memories: the dorsal medial prefrontal cortex (mPFC) promotes reward seeking, whereas the ventral mPFC inhibits reward seeking. In this paper, we use the Daun02 chemogenetic inactivation procedure to demonstrate that Fos-expressing neuronal ensembles mediating both food reward and extinction memories intermingle within the same ventral mPFC area.

Chronic nicotine activates stress/reward-related brain regions and facilitates the transition to compulsive alcohol drinking.

Alcohol and nicotine are the two most co-abused drugs in the world. Previous studies have shown that nicotine can increase alcohol drinking in nondependent rats, yet it is unknown whether nicotine facilitates the transition to alcohol dependence. We tested the hypothesis that chronic nicotine will speed up the escalation of alcohol drinking in rats and that this effect will be accompanied by activation of sparsely distributed neurons (neuronal ensembles) throughout the brain that are specifically recruited by the combination of nicotine and alcohol. Rats were trained to respond for alcohol and made dependent using chronic, intermittent exposure to alcohol vapor, while receiving daily nicotine (0.8 mg/kg) injections. Identification of neuronal ensembles was performed after the last operant session, using immunohistochemistry. Nicotine produced an early escalation of alcohol drinking associated with compulsive alcohol drinking in dependent, but not in nondependent rats (air exposed), as measured by increased progressive-ratio responding and increased responding despite adverse consequences. The combination of nicotine and alcohol produced the recruitment of discrete and phenotype-specific neuronal ensembles (∼4-13% of total neuronal population) in the nucleus accumbens core, dorsomedial prefrontal cortex, central nucleus of the amygdala, bed nucleus of stria terminalis, and posterior ventral tegmental area. Blockade of nicotinic receptors using mecamylamine (1 mg/kg) prevented both the behavioral and neuronal effects of nicotine in dependent rats. These results demonstrate that nicotine and activation of nicotinic receptors are critical factors in the development of alcohol dependence through the dysregulation of a set of interconnected neuronal ensembles throughout the brain.

Context-induced reinstatement of methamphetamine seeking is associated with unique molecular alterations in Fos-expressing dorsolateral striatum neurons.

Context-induced reinstatement of drug seeking is a well established animal model for assessing the neural mechanisms underlying context-induced drug relapse, a major factor in human drug addiction. Neural activity in striatum has previously been shown to contribute to context-induced reinstatement of heroin, cocaine, and alcohol seeking, but not yet for methamphetamine seeking. In this study, we found that context-induced reinstatement of methamphetamine seeking increased expression of the neural activity marker Fos in dorsal but not ventral striatum. Reversible inactivation of neural activity in dorsolateral but not dorsomedial striatum using the GABA agonists muscimol and baclofen decreased context-induced reinstatement. Based on our previous findings that Fos-expressing neurons play a critical role in conditioned drug effects, we assessed whether context-induced reinstatement was associated with molecular alterations selectively induced within context-activated Fos-expressing neurons. We used fluorescence-activated cell sorting to isolate reinstatement-activated Fos-positive neurons from Fos-negative neurons in dorsal striatum and used quantitative PCR to assess gene expression within these two populations of neurons. Context-induced reinstatement was associated with increased expression of the immediate early genes Fos and FosB and the NMDA receptor subunit gene Grin2a in only Fos-positive neurons. RNAscope in situ hybridization confirmed that Grin2a, as well as Grin2b, expression were increased in only Fos-positive neurons from dorsolateral, but not dorsomedial, striatum. Our results demonstrate an important role of dorsolateral striatum in context-induced reinstatement of methamphetamine seeking and that this reinstatement is associated with unique gene alterations in Fos-expressing neurons.

Role of nucleus accumbens shell neuronal ensembles in context-induced reinstatement of cocaine-seeking.

Environmental contexts previously associated with drug use provoke relapse to drug use in humans and reinstatement of drug seeking in animal models of drug relapse. We examined whether context-induced reinstatement of cocaine seeking is mediated by activation of context-selected nucleus accumbens neurons. We trained rats to self-administer cocaine in Context A and extinguished their lever-pressing in a distinct Context B. On test day, reexposure to the cocaine-associated Context A reinstated cocaine seeking and increased expression of the neural activity marker Fos in 3.3% of accumbens shell and 1.6% of accumbens core neurons. To assess a causal role for these activated neurons, we used the Daun02 inactivation procedure to selectively inactivate these neurons. We trained c-fos-lacZ transgenic rats to self-administer cocaine in Context A and extinguished their lever-pressing in Context B. On induction day, we exposed rats to either Context A or a novel Context C for 30 min and injected Daun02 or vehicle into accumbens shell or core 60 min later. On test day, 3 d after induction day, the ability of Context A to reinstate cocaine seeking and increase neuronal activity in accumbens shell was attenuated when Daun02 was previously injected after exposure to Context A. Daun02 injections after exposure to the novel Context C had no effect on context-induced reinstatement of cocaine seeking despite much greater numbers of Fos-expressing neurons induced by Context C. Daun02 injections in accumbens core had no effect. Our data suggest that context-induced reinstatement of cocaine seeking is mediated by activation of context-selected accumbens shell but not core neuronal ensembles.

Stress vulnerability during adolescence: comparison of chronic stressors in adolescent and adult rats.

OBJECTIVE: This study investigated the physiological and somatic changes evoked by daily exposure to the same type of stressor (homotypic) or different aversive stressor stimuli (heterotypic) in adolescent and adult rats, with a focus on cardiovascular function. The long-term effects of stress exposure during adolescence were also investigated longitudinally. METHODS: Male Wistar rats were exposed to repeated restraint stress (RRS, homotypic) or chronic variable stress (CVS, heterotypic). RESULTS: Adrenal hypertrophy, thymus involution, and elevated plasma glucocorticoid were observed only in adolescent animals, whereas reduction in body weight was caused by both stress regimens in adults. CVS increased mean arterial pressure (adolescent: p = .001; adult: p = .005) and heart rate (HR; adolescent: p = .020; adult: p = .011) regardless of the age, whereas RRS increased blood pressure selectively in adults (p = .001). Rest tachycardia evoked by CVS was associated with increased cardiac sympathetic activity in adults, whereas a decreased cardiac parasympathetic activity was observed in adolescent animals. Changes in cardiovascular function and cardiac autonomic activity evoked by both CVS and RRS were followed by alterations in baroreflex activity and vascular reactivity to vasoconstrictor and vasodilator agents in adolescent adult animals. Except for the circulating glucocorticoid change, all alterations observed during adolescence were reversed in adulthood. CONCLUSIONS: These findings suggest a stress vulnerability of adolescents to somatic and neuroendocrine effects regardless of stress regimen. Our results indicated an age-stress type-specific influence in stress-evoked cardiovascular/autonomic changes. Data suggest minimal consequences in adulthood of stress during adolescence.

Long-lasting mechanical hypersensitivity and CRF receptor type-1 neuron activation in the BNST following adolescent ethanol exposure.

BACKGROUND: Adolescent alcohol use can produce long-lasting alterations in brain function, potentially leading to adverse health outcomes in adulthood. Emerging evidence suggests that chronic alcohol use can increase pain sensitivity or exacerbate existing pain conditions, but the potential neural mechanisms underlying these effects require further investigation. Here, we evaluate the impact of chronic ethanol vapor on mechanical sensitivity over the course of acute and protracted withdrawal in adolescent and adult male and female mice, and its potential association with alterations in corticotropin-releasing factor (CRF) signaling within the bed nucleus of the stria terminalis (BNST). METHODS: Adolescent and adult male and female mice underwent intermittent ethanol vapor exposure on 4 days/week for 2 weeks. Mechanical thresholds were evaluated 5 h and 7, 14, 21, and 28 d after cessation of ethanol exposure using the von Frey test. For mice with a history of adolescent ethanol exposure, brains were collected for in situ RNAscope processing after the final test. Messenger RNA expression of c-Fos, Crfr1, and Crf in the BNST subregions was examined. RESULTS: Exposure to intermittent ethanol vapor induced persistent mechanical hypersensitivity during withdrawal in both adolescent and adult mice. Notably, the effect was more transient in mice exposed to ethanol during adulthood, resolving by day 28 after ethanol exposure. Furthermore, both male and female mice with a history of adolescent ethanol exposure exhibited increased activation of CRF receptor type 1 (CRFR1) neurons within the dorsolateral BNST. CONCLUSIONS: Our results support the conclusion that intermittent ethanol exposure can induce mechanical hypersensitivity, potentially through the activation of BNST CRFR1 neurons. These findings provide a basis for future studies aimed at evaluating specific subpopulations of BNST neurons and their contribution to pain in individuals with a history of alcohol use.

Biperiden reverses the increase in 50-kHz ultrasonic vocalizations but not the increase in locomotor activity induced by cocaine.

Cocaine use disorder (CUD) is a worldwide public health problem, associated with severe psychosocial and economic impacts. Currently, no FDA-approved treatment is available for CUD. However, an emerging body of evidence from clinical and preclinical studies suggests that biperiden, an M1 muscarinic receptor antagonist, presents potential therapeutic use for CUD. These studies have suggested that biperiden may reduce the reinforcing effects of cocaine. It is well established that rodents emit 50-kHz ultrasonic vocalizations (USV) in response to natural rewards and stimulant drugs, including cocaine. Nonetheless, the effects of biperiden on the cocaine-induced increase of 50-kHz USV remains unknown. Here, we hypothesized that biperiden could antagonize the acute effects of cocaine administration on rat 50-kHz USV. To test this hypothesis, adult male Wistar rats were divided into four experimental groups: saline, 5 mg/kg biperiden, 10 mg/kg cocaine, and biperiden/cocaine (5 and 10 mg/kg, i.p., respectively). USV and locomotor activity were recorded in baseline and test sessions. As expected, cocaine administration significantly increased the number of 50-kHz USV. Biperiden administration effectively antagonized the increase in 50-kHz USV induced by cocaine. Cocaine administration also increased the emission of trill and mixed 50 kHz USV subtypes and this effect was antagonized by biperiden. Additionally, we showed that biperiden did not affect the cocaine-induced increase in locomotor activity, although biperiden administration per se increased locomotor activity. In conclusion, our findings indicate that administering biperiden acutely reduces the positive affective effects of cocaine, as demonstrated by its ability to inhibit the increase in 50-kHz USV.

The Matricellular Protein Hevin Is Involved in Alcohol Use Disorder.

Astrocytic-secreted matricellular proteins have been shown to influence various aspects of synaptic function. More recently, they have been found altered in animal models of psychiatric disorders such as drug addiction. Hevin (also known as Sparc-like 1) is a matricellular protein highly expressed in the adult brain that has been implicated in resilience to stress, suggesting a role in motivated behaviors. To address the possible role of hevin in drug addiction, we quantified its expression in human postmortem brains and in animal models of alcohol abuse. Hevin mRNA and protein expression were analyzed in the postmortem human brain of subjects with an antemortem diagnosis of alcohol use disorder (AUD, n = 25) and controls (n = 25). All the studied brain regions (prefrontal cortex, hippocampus, caudate nucleus and cerebellum) in AUD subjects showed an increase in hevin levels either at mRNA or/and protein levels. To test if this alteration was the result of alcohol exposure or indicative of a susceptibility factor to alcohol consumption, mice were exposed to different regimens of intraperitoneal alcohol administration. Hevin protein expression was increased in the nucleus accumbens after withdrawal followed by a ethanol challenge. The role of hevin in AUD was determined using an RNA interference strategy to downregulate hevin expression in nucleus accumbens astrocytes, which led to increased ethanol consumption. Additionally, ethanol challenge after withdrawal increased hevin levels in blood plasma. Altogether, these results support a novel role for hevin in the neurobiology of AUD.

Stress-induced cross-sensitization to amphetamine is related to changes in the dopaminergic system.

Repeated stress engenders behavioral sensitization. The mesolimbic dopamine system is critically involved in drug-induced behavioral sensitization. In the present study we examined the differences between adolescent and adult rats in stress-induced behavioral sensitization to amphetamine and changes in dopamine (DA) and its metabolite levels in the mesolimbic system. Adolescent or adult rats were restrained for 2 h, once a day, for 7 days. Three days after the last exposure to stress, the animals were challenged with saline or amphetamine (1.0 mg/kg i.p.) and amphetamine-induced locomotion was recorded for 40 min. Immediately after the behavioral tests, rats were decapitated and the nucleus accumbens (NAcc), ventral tegmental area (VTA) and amygdala (AM) were removed to measure tissue levels of DA and its metabolites by HPLC. Exposure to repeated restraint stress promoted behavioral sensitization to amphetamine in both adult and adolescent rats. In adult rats, amphetamine administration increased DA levels in both the stress and control groups in the NAcc and VTA. In adolescent rats, amphetamine increased DA levels in the NAcc in rats exposed to stress. Furthermore, in the AM of adolescent rats in the control group, amphetamine increased the DA levels; however, amphetamine reduced this neurotransmitter in the rats that were exposed to stress. No alteration was observed in the dopamine metabolite levels. Therefore, stress promoted behavioral sensitization to amphetamine and this may be related to changes in DA levels in the mesolimbic system. These changes appear to be dependent on ontogeny.

Effect of the single or combined administration of cocaine and testosterone on cardiovascular function and baroreflex activity in unanesthetized rats.

Abuse of cocaine and androgenic-anabolic steroids has become a serious public health problem. Despite reports of an increase in the incidence of simultaneous illicit use of these substances, potential toxic interactions between cocaine and androgenic-anabolic steroids in the cardiovascular system are unknown. In the present study, we investigated the effect of single or combined administration of testosterone and cocaine for 1 or 10 consecutive days on basal cardiovascular parameters, baroreflex activity, and hemodynamic responses to vasoactive agents in unanesthetized rats. Ten-day combined administration of testosterone and cocaine increased baseline arterial pressure. Changes in arterial pressure were associated with altered baroreflex activity and impairment of both hypotensive response to intravenous sodium nitroprusside and pressor effect of intravenous phenylephrine. Chronic single administration of either testosterone or cocaine did not affect baseline arterial pressure. However, testosterone-treated animals presented rest bradycardia, cardiac hypertrophy, alterations in baroreflex activity, and enhanced response to sodium nitroprusside. Repeated administration of cocaine affected baroreflex activity and impaired vascular responsiveness to both sodium nitroprusside and phenylephrine. One-day single or combined administration of the drugs did not affect any parameter investigated. In conclusion, the present results suggest a potential interaction between toxic effects of cocaine and testosterone on the cardiovascular activity. Changes in baseline arterial pressure after combined administration of these 2 drugs may result from alterations in baroreflex activity and impairment of vascular responsiveness to vasoactive agents.

Neuroinflammation in early, late and recovery stages in a progressive parkinsonism model in rats.

Parkinson's disease (PD) is characterized by motor and non-motor signs, which are accompanied by progressive degeneration of dopaminergic neurons in the substantia nigra. Although the exact causes are unknown, evidence links this neuronal loss with neuroinflammation and oxidative stress. Repeated treatment with a low dose of reserpine-inhibitor of VMAT2-has been proposed as a progressive pharmacological model of PD. The aim of this study was to investigate whether this model replicates the neuroinflammation characteristic of this disease. Six-month-old Wistar rats received repeated subcutaneous injections of reserpine (0.1 mg/kg) or vehicle on alternate days. Animals were euthanized after 5, 10, or 15 injections, or 20 days after the 15th injection. Catalepsy tests (motor assessment) were conducted across treatment. Brains were collected at the end of each treatment period for immunohistochemical and RT-PCR analyzes. Reserpine induced a significant progressive increase in catalepsy duration. We also found decreased immunostaining for tyrosine hydroxylase (TH) in the substantia nigra pars compacta (SNpc) and increased GFAP + cells in the SNpc and dorsal striatum after 10 and 15 reserpine injections. Phenotyping microglial M1 and M2 markers showed increased number of CD11b + cells and percentage of CD11b + /iNOS + cells in reserpine-treated animals after 15 injections, which is compatible with tissue damage and production of cytotoxic factors. In addition, increased CD11b + /ArgI + cells were found 20 days after the last reserpine injection, together with an increment in IL-10 gene expression in the dorsal striatum, which is indicative of tissue repair or regeneration. Reserpine also induced increases in striatal interleukin TNF-alpha mRNA levels in early stages. In view of these results, we conclude that reserpine-induced progressive parkinsonism model leads to neuroinflammation in regions involved in the pathophysiology of PD, which is reversed 20 days after the last injection. These findings reveal that withdrawal period, together with the shift of microglial phenotypes from the pro-inflammatory to the anti-inflammatory stage, may be important for the study of the mechanisms involved in reversing this condition, with potential clinical applicability.

Cocaine-induced increases in motivation require 2-arachidonoylglycerol mobilization and CB1 receptor activation in the ventral tegmental area.

A wide body of evidence supports an integral role for mesolimbic dopamine (DA) in motivated behavior. In brief, drugs that increase DA in mesolimbic terminal regions, like cocaine, enhance motivation, while drugs that decrease DA concentration reduce motivation. Data from our laboratory and others shows that phasic activation of mesolimbic DA requires signaling at cannabinoid type-1 (CB1) receptors in the ventral tegmental area (VTA), and systemic delivery of CB1 receptor antagonists reduces DA cell activity and attenuates motivated behaviors. Recent findings demonstrate that cocaine mobilizes the endocannabinoid 2-arachidonoylglycerol (2-AG) in the VTA to cause phasic activation of DA neurons and terminal DA release. It remains unclear, however, if cocaine-induced midbrain 2-AG signaling contributes to the motivation-enhancing effects of cocaine. To examine this, we trained male and female rats on a progressive ratio (PR) task for a food reinforcer. Each rat underwent a series of tests in which they were pretreated with cocaine alone or in combination with systemic or intra-VTA administration of the CB1 receptor antagonist rimonabant or the 2-AG synthesis inhibitor tetrahydrolipstatin (THL). Cocaine increased motivation, measured by augmented PR breakpoints, while rimonabant dose-dependently decreased motivation. Importantly, intra-VTA administration of rimonabant or THL, at doses that did not decrease breakpoints on their own, blocked systemic cocaine administration from increasing breakpoints in male and female rats. These data suggest that cocaine-induced increases in motivation require 2-AG signaling at CB1 receptors in the VTA and may provide critical insight into cannabinoid-based pharmacotherapeutic targets for the successful treatment of substance abuse.

CB1 and CB2 receptors in the bed nucleus of the stria terminalis differently modulate anxiety-like behaviors in rats.

The endocannabinoid system is implicated in anxiety, but the brain sites involved are not completely understood. The bed nucleus of the stria terminalis (BNST) has been related to anxiety and responses to aversive threats. Besides, endocannabinoid neurotransmission acting via CB1 receptors was identified in the BNST. However, the presence of CB2 receptors and the role of BNST endocannabinoid system in anxiety-like behaviors have never been reported. Therefore, this study investigated the presence of CB1 and CB2 receptors in the BNST and their role in anxiety-like behaviors. For this, gene expression of the endocannabinoid receptors was evaluated in samples from anterior and posterior BNST. Besides, behaviors were evaluated in the elevated plus-maze (EPM) in unstressed rats (trait anxiety-like behavior) and after exposure to restraint stress (restraint-evoked anxiety-like behavior) in rats treated with either the CB1 receptor antagonist AM251 or the CB2 receptor antagonist JTE907 into the anterior BNST. The presence of CB1 and CB2 receptors gene expression was identified in anterior and posterior divisions of the BNST. Bilateral microinjection of AM251 into the anterior BNST dose-dependently increased EPM open arms exploration in unstressed animals and inhibited the anxiety-like behavior in the EPM evoked by restraint. Conversely, intra-BNST microinjection of JTE907 decreased EPM open arms exploration in a dose-dependent manner and inhibited restraint-evoked behavioral changes in the EPM. Taken together, these results indicate that CB1 and CB2 receptors present in the BNST are involved in control of anxiety-like behaviors, and control by the latter is affected by previous stress experience.

Ethanol-induced locomotor sensitization: Neuronal activation in the nucleus accumbens and medial prefrontal cortex.

Ethanol consumption may promote neuroplasticity and alterations in synapses, resulting in modifications in neuronal activity. Here, we treated male Swiss mice with ethanol (2.2 g/kg) or saline once per day for 21 consecutive days. Nine days after the last ethanol administration, they received a challenge injection of ethanol or saline, and we assessed locomotor activity. After the behavioral analysis, we evaluated neuronal activation in the medial Prefrontal Cortex (Cingulate, Prelimbic, and Infralimbic) and the Nucleus Accumbens (Shell and Core) using Fos/DAB immunohistochemistry. In another group of animals, we performed the quantitative analysis of the ARC and PSD-95 protein levels by Western blotting in the medial prefrontal cortex and nucleus accumbens brain areas. Repeated ethanol administration produced locomotor sensitization, accompanied by an increase in the nucleus accumbens shell's activation but not core. Furthermore, the ethanol pretreatment reduced ARC expression in the nucleus accumbens and medial prefrontal cortex. Our results suggest the participation of the nucleus accumbens shell in ethanol behavioral sensitization and add new pieces of evidence that neuroplasticity in synapses may contribute to the mechanism underlying this behavior.

Chronic ethanol vapor exposure potentiates cardiovascular responses to acute stress in male but not in female rats.

BACKGROUND: Ethanol use is related to a wide variety of negative health outcomes, including cardiovascular diseases. Stress is also involved in numerous pathologies, such as cardiovascular diseases and psychiatric disorders. Sexual dimorphism is an important factor affecting cardiovascular response and has been proposed as a potential risk factor for sex-specific health problems in humans. Here, we evaluated the effect of prolonged ethanol vapor inhalation on arterial pressure, heart rate, and tail skin temperature responses to acute restraint stress, investigating differences between male and female rats. METHODS: We exposed male and female Long-Evans rats to ethanol vapor for 14 h, followed by ethanol withdrawal for 10 h, for 30 consecutive days, or to room air (control groups). The animals underwent surgical implantation of a cannula into the femoral artery for assessment of arterial pressure and heart rate values. The tail skin temperature was measured as an indirect measurement of sympathetic vasomotor response. RESULTS: Chronic ethanol vapor inhalation reduced basal heart rate in both female and male rats. Sex-related difference was observed in the decrease of tail cutaneous temperature evoked by stress, but not in the pressor and tachycardiac responses. Furthermore, prolonged ethanol inhalation enhanced the blood pressure and heart rate increase caused by acute restraint stress in male, but not in female rats. However, no effect of chronic ethanol vapor was observed in the tail cutaneous temperature response to restraint in either sex. CONCLUSION: Chronic ethanol vapor exposure increased the cardiovascular reactivity to stress in male, but not in female rats.

Aripiprazole and topiramate, alone or in combination, block the expression of ethanol-induced conditioned place preference in mice.

BACKGROUND: Ethanol is the most largely consumed drug in the world. Because of its complex mechanisms of action, studies suggest that the combination of drugs with distinct pharmacological effects may be a promising alternative for treating ethanol use disorder. In the present study, we aimed to investigate the effects of topiramate, alone and in combination with aripiprazole, on ethanol-induced conditioned place preference (CPP). METHODS: Adult male mice were conditioned with ethanol (1.8 g/kg, i.p.) in the conditioned place preference (CPP) apparatus. Animals were then treated with vehicle, topiramate (2.5, 5 or 10 mg/kg, i.p.), aripiprazole (0.025, 0.05, 0.075 or 0.1 mg/kg, i.p.) or a combination of subthreshold doses of topiramate and aripiprazole (5 and 0.075 mg/kg, respectively) in the ethanol-paired compartment for 8 consecutive days. The expression of ethanol-induced CPP was then evaluated during a drug-free test performed 24 h after a re-exposure to ethanol in the ethanol-paired compartment. RESULTS: Treatment with 10 mg/kg topiramate or 0.1 mg/kg aripiprazole blocked the expression of ethanol-induced CPP. Combined treatment with 5 mg/kg topiramate and 0.075 mg/kg aripiprazole, doses that alone were not effective, also blocked the expression of CPP to ethanol. CONCLUSIONS: Topiramate and aripiprazole, alone or in combination, blocked the expression of ethanol-induced CPP. By showing that a combination of lower, subthreshold doses or topiramate and aripiprazole was effective in blocking the conditioned reinforcing properties of the ethanol-paired environment in mice, our current findings provide important insights into the therapeutic use of these drugs in ethanol use disorder.

Ibogaine Blocks Cue- and Drug-Induced Reinstatement of Conditioned Place Preference to Ethanol in Male Mice.

Ibogaine is a psychedelic extracted from the plant Tabernanthe iboga Baill. (Apocynaceae), natural from Africa, and has been proposed as a potential treatment for substance use disorders. In animal models, ibogaine reduces ethanol self-administration. However, no study to date has investigated the effects of ibogaine on ethanol-induced conditioned place preference (CPP). The present study aimed to investigate the effects of repeated treatment with ibogaine on the reinstatement of CPP to ethanol in male mice. The rewarding effects of ethanol (1.8 g/kg, i. p.) or ibogaine (10 or 30 mg/kg, p. o.) were investigated using the CPP model. Furthermore, we evaluated the effects of repeated treatment with ibogaine (10 or 30 mg/kg, p. o.) on the reinstatement of ethanol-induced CPP. Reinstatement was evaluated under two conditions: 1) during a priming injection re-exposure test in which animals received a priming injection of ethanol and had free access to the CPP apparatus; 2) during a drug-free test conducted 24 h after a context-paired re-exposure, in which subjects received an injection of ethanol and were confined to the compartment previously conditioned to ethanol. Our results show that ethanol, but not ibogaine, induced CPP in mice. Treatment with ibogaine after conditioning with ethanol blocked the reinstatement of ethanol-induced CPP, both during a drug priming reinstatement test and during a drug-free test conducted after re-exposure to ethanol in the ethanol-paired compartment. Our findings add to the literature suggesting that psychedelics, in particular ibogaine, may have therapeutic properties for the treatment of alcohol use disorder at doses that do not have rewarding effects per se.

Multiparameter Optimization of Trypanocidal Cruzain Inhibitors With In Vivo Activity and Favorable Pharmacokinetics.

Cruzain, the main cysteine protease of Trypanosoma cruzi, plays key roles in all stages of the parasite's life cycle, including nutrition acquisition, differentiation, evasion of the host immune system, and invasion of host cells. Thus, inhibition of this validated target may lead to the development of novel drugs for the treatment of Chagas disease. In this study, a multiparameter optimization (MPO) approach, molecular modeling, and structure-activity relationships (SARs) were employed for the identification of new benzimidazole derivatives as potent competitive inhibitors of cruzain with trypanocidal activity and suitable pharmacokinetics. Extensive pharmacokinetic studies enabled the identification of metabolically stable and permeable compounds with high selectivity indices. CYP3A4 was found to be involved in the main metabolic pathway, and the identification of metabolic soft spots provided insights into molecular optimization. Compound 28, which showed a promising trade-off between pharmacodynamics and pharmacokinetics, caused no acute toxicity and reduced parasite burden both in vitro and in vivo.