Molecular, Circuit, and Stress Response Characterization of Ventral Pallidum Npas1-Neurons.

Altered activity of the ventral pallidum (VP) underlies disrupted motivation in stress and drug exposure. The VP is a very heterogeneous structure composed of many neuron types with distinct physiological properties and projections. Neuronal PAS 1-positive (Npas1+) VP neurons are thought to send projections to brain regions critical for motivational behavior. While Npas1+ neurons have been characterized in the globus pallidus external, there is limited information on these neurons in the VP. To address this limitation, we evaluated the projection targets of the VP Npas1+ neurons and performed RNA-sequencing on ribosome-associated mRNA from VP Npas1+ neurons to determine their molecular identity. Finally, we used a chemogenetic approach to manipulate VP Npas1+ neurons during social defeat stress (SDS) and behavioral tasks related to anxiety and motivation in Npas1-Cre mice. We used a similar approach in females using the chronic witness defeat stress (CWDS). We identified VP Npas1+ projections to the nucleus accumbens, ventral tegmental area, medial and lateral habenula, lateral hypothalamus, thalamus, medial and lateral septum, and periaqueductal gray area. VP Npas1+ neurons displayed distinct translatome representing distinct biological processes. Chemogenetic activation of hM3D(Gq) receptors in VP Npas1+ neurons increased susceptibility to a subthreshold SDS and anxiety-like behavior in the elevated plus maze and open field while the activation of hM4D(Gi) receptors in VP Npas1+ neurons enhanced resilience to chronic SDS and CWDS. Thus, the activity of VP Npas1+ neurons modulates susceptibility to social stressors and anxiety-like behavior. Our studies provide new information on VP Npas1+ neuron circuitry, molecular identity, and their role in stress response.SIGNIFICANCE STATEMENT The ventral pallidum (VP) is a structure connected to both reward-related and aversive brain centers. It is a key brain area that signals the hedonic value of natural rewards. Disruption in the VP underlies altered motivation in stress and substance use disorder. However, VP is a very heterogeneous area with multiple neuron subtypes. This study characterized the projection pattern and molecular signatures of VP Neuronal PAS 1-positive (Npas1+) neurons. We further used tools to alter receptor signaling in VP Npas1+ neurons in stress to demonstrate a role for these neurons in stress behavioral outcomes. Our studies have implications for understanding brain cell type identities and their role in brain disorders, such as depression, a serious disorder that is precipitated by stressful events.

Effects of N-acetylcysteine treatment on ethanol's rewarding properties and dopaminergic alterations in mesocorticolimbic and nigrostriatal pathways.

Recent reports have shown that N-acetylcysteine (N-AC) has beneficial effects in the treatment of cocaine and nicotine abuse. Considering the similar neurobiologic mechanisms involved in the development of addiction to different drugs, N-AC treatment could be useful in the treatment of ethanol abuse. The rewarding properties of the drugs of abuse plays an important role in the development of addiction and can be studied using the conditioned place preference (CPP) paradigm. Thus, to study the effects of N-AC treatment in the rewarding effects of ethanol, we investigated the effects of N-AC administration in the ethanol-induced CPP and neurochemical alterations within the mesocorticolimbic and the nigrostriatal dopaminergic pathways. Adult male Swiss mice were pretreated with N-AC (60 or 120 mg/kg intraperitoneal) and tested for the development, expression, or extinction of the ethanol-induced CPP. Another cohort of animals received N-AC (60 or 120 mg/kg intraperitoneal) 2-h before an acute administration of ethanol and had their brains removed for dopamine and its metabolites quantification in the mesocorticolimbic and nigrostriatal pathways. Pretreatment with N-AC (120 mg/kg) blocked the development of ethanol-induced CPP. On the other hand, N-AC at both doses did not alter the expression nor the extinction of ethanol-induced CPP. N-AC increased 3,4-dihydroxyphenylacetic acid content in the medial prefrontal cortex and dopaminergic turnover within the substantia nigra. Besides that, there was an increase in dopamine content in the nucleus accumbens of ethanol-treated animals. In summary, N-AC treatment blocked the development of ethanol CPP, without altering ethanol effects on dopaminergic neurotransmission.

Short and prolonged maternal separation impacts on ethanol-related behaviors in rats: sex and age differences.

Maternal separation (MS) is an animal model widely used to evaluate the influence of early-life stress exposure on ethanol consumption and dependence. The goal of this study was to evaluate the effects of brief and prolonged MS on the pattern of consumption and ethanol conditioned place preference (CPP) in male and female rats during adolescence and adulthood. Wistar rat pups were separated daily from their dams for 15 or 180 minutes during the 2 to 10 postnatal days (PND). In adolescence, half of the litter from each group was evaluated in the ethanol consumption test using the three-bottle test choice paradigm. In addition, using biased procedure, ethanol-conditioned place preference was also evaluated. In adulthood, the other half of the litter was evaluated on the same tests. Our results showed that there are differences in consumption pattern and in alcohol reinforcement between males and females, adolescents and adults. While prolonged MS had no effect on total ethanol consumption in adolescents of both sexes, it induced CPP in these animals. In turn, in adults, previous exposure to prolonged MS increased ethanol consumption without altering ethanol-CPP.Lay summaryGiving the importance of the mother-children (dam-pups when talking about rodents) relationship to proper brain development, the separation of pups from their dam is broadly used as an animal model to study the impact of early-life stress exposure. Here, we used a protocol of brief or prolonged maternal separation to study the impact of early-life stress exposure in the alcohol consumption and conditioned place preference in rats, and how age and sex influence it. We showed that, overall, the prolonged maternal separation increased alcohol consumption in both males and females, but only when animals were tested during the adulthood. In the other hand, prolonged maternal separation increased ethanol conditioned place preference in adolescent rats, both male and female.

Stress induces behavioral sensitization, increases nicotine-seeking behavior and leads to a decrease of CREB in the nucleus accumbens.

Experimental evidence shows that exposure to stress engenders behavioral sensitization and increases drug-seeking and leads to intense drug taking. However the molecular mechanisms involved in these processes is not well known yet. The present experiments examined the effects of exposure to variable stress on nicotine-induced locomotor activation, cAMP-response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK) activity and nicotine intravenous self-administration in rats. Male Wistar rats were exposed to variable stress that consisted of the exposure to different stressors twice a day in random order for 10 days. During this period the control group was left undisturbed except for cage cleaning. Ten days after the last stress episode, rats were challenged with either saline or nicotine (0.4 mg/kgs.c.) and the locomotor activity was recorded for 20 min. Immediately after behavioral recordings rats were sacrificed and their brains were removed to posterior western blotting analysis of CREB, phosphoCREB, ERK and phosphoERK in the nucleus accumbens. An independent set of control and stressed animals were subjected to an intravenous nicotine self-administration protocol. The break point during a progressive ratio schedule and nicotine intake patterns during a 24-hour binge was analyzed. Repeated variable stress caused a sensitized motor response to a single challenge of nicotine and decreased CREB in the nucleus accumbens. Furthermore, in the self-administration experiments previous stress exposure caused an increase in the break point and nicotine intake.

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.

Ontogênese, estresse e dependência de substâncias psicoativas / Ortogenesis, stress and psychoactive substances addiction

O estudo da dependência de substâncias psicoativas apresentou grandes avanços conceituais nas últimas décadas. A evolução dos conceitos foi paralela às evidências científicas que têm revelado os aspectos comportamentais e os mecanismos neurais envolvidos nesse fenômeno. Contudo, um grande desafio que permanece na pesquisa sobre a dependência de substâncias psicoativas é a identificação de quais fatores são responsáveis pela transição do uso controlado para o uso compulsivo. Está demonstrado que muitas variáveis interagem para influenciar a probabilidade de que qualquer indivíduo inicie o uso abusivo de substâncias psicoativas ou se torne dependente. Nos últimos anos, o estresse tem sido destacado como um fator importante na iniciação, manutenção e recaída da utilização de substâncias psicoativas. Neste trabalho analisamos os conceitos e teorias da farmacodependência e as principais evidências comportamentais pré-clínicas que demonstram a relação entre estresse e a vulnerabilidade ao abuso e dependência de psicoestimulantes.

The investigation of the mechanisms of drug abuse and addiction showed great advances in the last decades. New concepts emerged from the scientific evidences on behavioral and neural aspects of this phenomenon. However, the biggest challenge for the future is the identification of which risk factors are implicated in the transition from controlled to compulsive drug use. Stress has been pointed as an important factor related to initiation, maintenance and relapse to drug use. In the present paper we discuss the concepts and theories of drug addiction, and the main behavioral pre-clinical evidences showing the relationship between stress and psychostimulant addiction.