Brief and long maternal separation in C57Bl6J mice: behavioral consequences for the dam and the offspring.

Introduction: Animal models, especially rodents, have become instrumental to experimentally investigate the effects of an adverse post-natal environment on the developing brain. For this purpose, maternal separation (MS) paradigms have been widely used in the last decades. Nonetheless, how MS affects maternal behavior and, ultimately, the offspring depend on multiple variables. Methods: To gain further insights into the consequences of MS, we decided to thoroughly measure and compare the effects of short (15 min, 3 times/day) vs. long (3 h, 1 time/day) separation on multiple maternally-associated behaviors and across the entire post-natal period. Results: Compared to unhandled control litters, our results confirmed previous studies and indicated that SMS enhanced the time and variety of maternal care whereas LMS resulted in poor caregiving. We also showed that SMS-accrued caregiving persisted during the whole post-natal period. In contrast, LMS effects on maternal behavior were restricted to the early life (P2-P10). Finally, we also analyzed the behavioral consequences of these different rearing social environments on the offspring. We found that MS has profound effects in social tasks. We showed that affiliative touch, a type of prosocial behavior that provides comfort to others, is particularly sensitive to the modification of maternal caregiving. Discussion: Our results provide further support to the contention that interactions during the early post-natal period critically contribute to emotional processing and brain co-construction.

Effects of heroin self-administration and forced withdrawal on the expression of genes related to the mTOR network in the basolateral complex of the amygdala of male Lewis rats.

RATIONALE: The development of substance use disorders involves long-lasting adaptations in specific brain areas that result in an elevated risk of relapse. Some of these adaptations are regulated by the mTOR network, a signalling system that integrates extracellular and intracellular stimuli and modulates several processes related to plasticity. While the role of the mTOR network in cocaine- and alcohol-related disorders is well established, little is known about its participation in opiate use disorders. OBJECTIVES: To use a heroin self-administration and a withdrawal protocol that induce incubation of heroin-seeking in male rats and study the associated effects on the expression of several genes related to the mTOR system and, in the specific case of Rictor, its respective translated protein and phosphorylation. RESULTS: We found that heroin self-administration elicited an increase in the expression of the genes Igf1r, Igf2r, Akt2 and Gsk3a in the basolateral complex of the amygdala, which was not as evident at 30 days of withdrawal. We also found an increase in the expression of Rictor (a protein of the mTOR complex 2) after heroin self-administration compared to the saline group, which was occluded at the 30-day withdrawal period. The activation levels of Rictor, measured by the phosphorylation rate, were also reduced after heroin self-administration, an effect that seemed more apparent in the protracted withdrawal group. CONCLUSIONS: These results suggest that heroin self-administration under extended access conditions modifies the expression profile of activators and components of the mTOR complexes and show a putative irresponsive mTOR complex 2 after withdrawal from heroin use.

The interactions of alcohol and cocaine regulate the expression of genes involved in the GABAergic, glutamatergic and endocannabinoid systems of male and female rats.

Although the pharmacological and behavioural interactions between cocaine and alcohol are well established, less is known about how polyconsumption of these drugs affects the neurotransmitter systems involved in their psychoactive effects and in particular, in the process of addiction. Here, rats of both sexes at two stages of development were studied under a chronic regime of intravenous cocaine and/or alcohol administration. Brain samples from the medial prefrontal cortex, nucleus accumbens, hippocampus and amygdala were extracted to analyse the mRNA expression of genes encoding subunits of the GABA, NMDA and AMPA receptors, as well as the expression of the CB1 receptor, and that of enzymes related to the biosynthesis and degradation of endocannabinoids. Moreover, two synaptic scaffold proteins related to GABA and NMDA receptors, gephyrin and PSD-95, were quantified in Western blots. Significant interactions between cocaine and alcohol were common, affecting the GABAergic and endocannabinoid systems in the medial prefrontal cortex and amygdala of young adults, whereas such interactions were evident in the glutamatergic and endocannabinoid systems in adults, as well as a more pronounced sex effect. Significant interactions between these drugs affecting the scaffold proteins were evident in the medial prefrontal cortex and nucleus accumbens of young adults, and in the nucleus accumbens and amygdala of adults, but not in the hippocampus. These results highlight the importance of considering the interactions between cocaine and alcohol on neurotransmitter systems in the context of polyconsumption, specifically when treating problems of abuse of these two substances.

Ex vivo 1H-MRS brain metabolic profiling in a two-hit model of neurodevelopmental disorders: Prenatal immune activation and peripubertal stress.

Prenatal infections are environmental risk factors for neurodevelopmental disorders. In addition, traumatic experiences during adolescence in individuals exposed to infections during gestation could increase the risk of schizophrenia. It is of the most crucial importance to discover potential markers of the disease in its early stages or before its onset, so that therapeutic strategies may be implemented. In the present study, we combined a proposed two-hit model of schizophrenia-related symptoms with proton magnetic resonance spectroscopy (1H-MRS) to discover potential biomarkers. To this end, we i.p. injected 100 µg/kg/ml of lipopolysaccharide (LPS) or saline on gestational days 15 and 16 to pregnant rats. Their male offspring were then subjected to five episodes of stress or handling on alternate days during postnatal days (PND) 28-38. Once the animals reached adulthood (PND70), we evaluated prepulse inhibition (PPI). At PND90, we performed an ex vivo 1H-MRS study in the cortex and striatum. While we did not detect alterations in PPI at the age tested, we found neurochemical disturbances induced by LPS, stress or (more interestingly) their interaction. LPS decreased glucose levels in the cortex and striatum and altered glutamate, glutamine and N-acetylaspartate levels. Glutamate and glutamine levels in the left (but not right) striatum were differentially affected by prenatal LPS exposure in a manner that depended on stress experiences. These results suggest that alterations in the glutamate cycle in the striatum could be used as early markers of developmental disorders.

Δ 9-Tetrahydrocannabinol During Adolescence Reprograms the Nucleus Accumbens Transcriptome, Affecting Reward Processing, Impulsivity, and Specific Aspects of Cocaine Addiction-Like Behavior in a Sex-Dependent Manner.

BACKGROUND: Cannabis exposure during adolescence is associated with emotional and motivational alterations that may entail an enhanced risk of developing psychiatric disorders. In rodent models, exposure to cannabinoids during adolescence leads to increased self-administration of opiates and cocaine, however, the psychological and neural mechanisms and the sex-specificity of this phenomenon are largely unknown. METHODS: We exposed male and female adolescent rats to Δ9-tetrahydrocannabinol (THC) and studied at adulthood the effects of such treatment on psychological processes related to reward, such as Pavlovian conditioned approach, Pavlovian to instrumental transfer, habit formation and waiting impulsivity. In the light of these data and given the involvement of the nucleus accumbens in the processes examined, we performed an RNASeq transcriptomic study and assessed cocaine addiction-like behavior. RESULTS: THC exposure increased goal-tracking (in males and females) and enhanced Pavlovian to instrumental transfer (especially in males) but did not affect habit formation. THC-exposed rats exhibited subtle, state-dependent changes in premature responding in the 2-CSRTT task. RNASeq data showed gene expression alterations in a marked sex-specific manner. While no effects were found on the acquisition of cocaine self-administration or punished drug-seeking, rats exposed to THC self-administered more cocaine under a progressive ratio schedule (males), had a higher rebound upon returning to continuous access to the drug (females) and showed reduced drug-seeking after 30 days of withdrawal (females). CONCLUSIONS: Adolescent THC affects specific aspects of reward- (and cocaine-) guided behavior and the function of a key brain region mediating these effects, in a remarkable sex-specific manner.

Cocaine-induced Fos expression in the rat brain: Modulation by prior Δ9-tetrahydrocannabinol exposure during adolescence and sex-specific effects.

It has been suggested that cannabis consumption during adolescence may be an initial step to cocaine use in adulthood. Indeed, previous preclinical data show that adolescent exposure to cannabinoids (both natural and synthetic) potentiates cocaine self-administration in rats. Here we aimed at gaining a deeper understanding of the cellular activation patterns induced by cocaine as revealed by Fos imaging and how these patterns may change due to adolescent exposure to THC. Male and female Wistar rats were administered every other day THC (3 mg/kg i.p.) or vehicle from postnatal day 28-44. At adulthood (PND90) they were given an injection of cocaine (20 mg/kg i.p.) or saline and sacrificed 90 min later. Cocaine-induced Fos activation was measured by immunohistochemistry as an index of cellular activation. We found that cocaine-induced activation in the motor cortex was stronger in THC-exposed rats. Moreover, there was significant sex-dependent interaction between cocaine and adolescent THC exposure in the dorsal hypothalamus, suggesting that cocaine induced a more robust cellular activation in THC-exposed females but not in THC-treated males. Other THC- and cocaine-induced effects were also evident. These results add to the previous literature suggesting that the behavioral, cellular, molecular, and brain-activating actions of cocaine are modulated by early experience with cannabinoids and provide additional knowledge that may explain the enhanced actions of cocaine in rats exposed to cannabinoids during their adolescence.

Comparative analysis of the modulation of perineuronal nets in the prefrontal cortex of rats during protracted withdrawal from cocaine, heroin and sucrose self-administration.

Relapse into drug use is a significant problem for people recovering from addiction. The ability that conditioned cues have to reinstate and reinvigorate drug-seeking is potentiated over time (incubation of seeking), posing an additional difficulty for maintaining abstinence. While the prefrontal cortex has been involved in the incubation phenomenon and the extracellular matrix, perineuronal nets (PNNs) in particular, may play a vital role in brain plasticity associated to drug relapse, there are no comparative analyses between different drug classes and natural reinforcers. Here, we compare the effects of early (1 day) and protracted (30 days) withdrawal from to cocaine, heroin and sucrose self-administration on the total density and density per intensity range of PNNs of different territories of the prefrontal cortex of male Lewis rats. Our results show that cocaine self-administration increases the density of PNNs in the dorsal prelimbic, infralimbic and ventral orbitofrontal cortices, while protracted withdrawal reversesthis effect in the dorsal prelimbic cortex. Also, heroin self-administration increases the density of PNNs in the infralimbic cortex and ventral orbitofrontal cortices, but this effect is lost after 30 days of withdrawal in the infralimbic cortex. Finally, the self-administration of sucrose-sweetened water or the protracted withdrawal from this powerful reinforcer does not affect any of the PNN parameters analysed. Our results show that two different drugs of abuse (but not a natural reward) with specific pharmacological and physiological actions, differentially modulate PNNs in specific areas of the rodent prefrontal cortex with potential implications for the incubation of seeking phenomenon.

The role of the mTOR pathway in models of drug-induced reward and the behavioural constituents of addiction.

BACKGROUND: Exposure to drugs of abuse induces neuroadaptations in critical nodes of the so-called reward systems that are thought to mediate the transition from controlled drug use to the compulsive drug-seeking that characterizes addictive disorders. These neural adaptations are likely to require protein synthesis, which is regulated, among others, by the mechanistic target of the rapamycin kinase (mTOR) signalling cascade. METHODS: We have performed a narrative review of the literature available in PubMed about the involvement of the mTOR pathway in drug-reward and addiction-related phenomena. AIMS: The aim of this study was to review the underlying architecture of this complex intracellular network and to discuss the alterations of its components that are evident after exposure to drugs of abuse. The aim was also to delineate the effects that manipulations of the mTOR network have on models of drug reward and on paradigms that recapitulate some of the psychological components of addiction. RESULTS: There is evidence for the involvement of the mTOR pathway in the acute and rewarding effects of drugs of abuse, especially psychostimulants. However, the data regarding opiates are scarce. There is a need to use sophisticated animal models of addiction to ascertain the real role of the mTOR pathway in this pathology and not just in drug-mediated reward. The involvement of this pathway in behavioural addictions and impulsivity should also be studied in detail in the future. CONCLUSIONS: Although there is a plethora of data about the modulation of mTOR by drugs of abuse, the involvement of this signalling pathway in addictive disorders requires further research.

Central nucleus of the amygdala as a common substrate of the incubation of drug and natural reinforcer seeking.

Relapse into drug use is a major problem faced by recovering addicts. In humans, an intensification of the desire for the drug induced by environmental cues-incubation of drug craving-has been observed. In rodents, this phenomenon has been modeled by studying drug seeking under extinction after different times of drug withdrawal (or using a natural reinforcer). Although much progress has been made, an integrated approach simultaneously studying different drug classes and natural reward and examining different brain regions is lacking. Lewis rats were used to study the effects of cocaine, heroin, and sucrose seeking incubation on six key brain regions: the nucleus accumbens shell/core, central/basolateral amygdala, and dorsomedial/ventromedial prefrontal cortex. We analyzed PSD95 and gephyrin protein levels, gene expression of glutamatergic, GABAergic and endocannabinoid elements, and amino acid transmitter levels. The relationships between the areas studied were examined by Structural Equation Modelling. Pathways from medial prefrontal cortex and basolateral complex of the amygdala to central nucleus of the amygdala, but not to the nucleus accumbens, were identified as common elements involved in the incubation phenomenon for different substances. These results suggest a key role for the central nucleus of amygdala and its cortical and amygdalar afferences in the incubation phenomenon, and we suggest that by virtue of its regulatory effects on glutamatergic and GABAergic dynamics within amygdalar circuits, the endocannabinoid system might be a potential target to develop medications that are effective in the context of relapse.

Impulsive Action and Impulsive Choice Are Differentially Associated With Gene Expression Variations of the GABAA Receptor Alfa 1 Subunit and the CB1 Receptor in the Lateral and Medial Orbitofrontal Cortices.

The orbitofrontal cortex (OFC) is a key brain region for decision-making, action control and impulsivity. Quite notably, previous research has identified a double dissociation regarding the role of this cortical territory in impulsive choice. While medial orbitofrontal lesions increase preference for a large but delayed reward, lateral orbitofrontal lesions have the opposite effect. However, there are no data regarding this anatomical dissociation in impulsive action. The neurochemical basis of impulsivity is still being elucidated, however, in recent years a role for the endocannabinoids and the related glutamatergic and GABAergic neurotransmitter systems has been suggested. Here, we submitted male Wistar rats to a delay-discounting task (DDT) or a two-choice serial reaction time task (2-CSRTT) and classified them as high impulsive or low impulsive in either task using cluster analysis. We then examined the gene expression of several elements of the endocannabinoid system or different subunits of certain glutamatergic or GABAergic ionotropic receptors (AMPA, NMDA, or GABAA) in the lateral or medial divisions of their orbitofrontal cortices. Our results confirm, at the gene expression level, the dissociation in the participation of the medial, and lateral divisions of the orbitofrontal cortex in impulsivity. While in the 2-CSRTT (inhibitory control) we found that high impulsive animals exhibited lower gene expression levels of the α1 GABAA receptor subunit in the lateral OFC, no such differences were evident in the medial OFC. When we analyzed DDT performance, we found that high impulsive animals displayed lower levels of CB1 gene expression in the medial but not in the lateral OFC. We propose that GABAergic dynamics in the lateral OFC might contribute to the inhibitory control mechanisms that are altered in impulsive behavior while endocannabinoid receptor gene transcription in the medial OFC may subserve the delay-discounting processes that participate in certain types of impulsiveness.

Morphine self-administration alters the expression of translational machinery genes in the amygdala of male Lewis rats.

BACKGROUND: Addiction is a chronic disorder with a high risk of relapse. The neural mechanisms mediating addictions require protein synthesis, which could be relevant for the development of more effective treatments. The mTOR signaling pathway regulates protein synthesis processes that have recently been linked to the development of drug addiction. AIMS: To assess the effects of morphine self-administration and its subsequent extinction on the expression of several genes that act in this pathway, and on the levels of specific phosphoproteins (Akt, Gsk3α/ß, mTOR, PDK1 and p70 S6 kinase) in the amygdala, nucleus accumbens, and the prefrontal cortex. METHODS: Male Lewis rats underwent morphine self-administration (1 mg/kg) for 19 days. They subsequently were submitted to extinction training for 15 days. Rats were killed either after self-administration or extinction, their brains extracted, and gene expression or phosphoprotein levels were assessed. RESULTS: We found an increase in Raptor and Eif4ebp2 expression in the amygdala of rats that self-administered morphine, even after extinction. The expression of Insr in the amygdala of control animals decreased over time while the opposite effect was seen in the rats that self-administered morphine. CONCLUSIONS: Our results suggest that morphine self-administration affects the gene expression of some elements of the translational machinery in the amygdala.

Pyrethroid insecticide lambda-cyhalothrin induces hepatic cytochrome P450 enzymes, oxidative stress and apoptosis in rats.

This study aimed to examine in rats the effects of the Type II pyrethroid lambda-cyhalothrin on hepatic microsomal cytochrome P450 (CYP) isoform activities, oxidative stress markers, gene expression of proinflammatory, oxidative stress and apoptosis mediators, and CYP isoform gene expression and metabolism phase I enzyme PCR array analysis. Lambda-cyhalothrin, at oral doses of 1, 2, 4 and 8mg/kg bw for 6days, increased, in a dose-dependent manner, hepatic activities of ethoxyresorufin O-deethylase (CYP1A1), methoxyresorufin O-demethylase (CYP1A2), pentoxyresorufin O-depentylase (CYP2B1/2), testosterone 7α- (CYP2A1), 16ß- (CYP2B1), and 6ß-hydroxylase (CYP3A1/2), and lauric acid 11- and 12-hydroxylase (CYP4A1/2). Similarly, lambda-cyhalothrin (4 and 8mg/kg bw, for 6days), in a dose-dependent manner, increased significantly hepatic CYP1A1, 1A2, 2A1, 2B1, 2B2, 2E1, 3A1, 3A2 and 4A1 mRNA levels and IL-1ß, NFκB, Nrf2, p53, caspase-3 and Bax gene expressions. PCR array analysis showed from 84 genes examined (P<0.05; fold change>1.5), changes in mRNA levels in 18 genes: 13 up-regulated and 5 down-regulated. A greater fold change reversion than 3-fold was observed on the up-regulated ALDH1A1, CYP2B2, CYP2C80 and CYP2D4 genes. Ingenuity Pathway Analysis (IPA) groups the expressed genes into biological mechanisms that are mainly related to drug metabolism. In the top canonical pathways, Oxidative ethanol degradation III together with Fatty Acid α-oxidation may be significant pathways for lambda-cyhalothrin. Our results may provide further understanding of molecular aspects involved in lambda-cyhalothrin-induced liver injury.

Unaltered cocaine self-administration in the prenatal LPS rat model of schizophrenia.

Although cocaine abuse is up to three times more frequent among schizophrenic patients, it remains unclear why this should be the case and whether sex influences this relationship. Using a maternal immune activation model of schizophrenia, we tested whether animals at higher risk of developing a schizophrenia-like state are more prone to acquire cocaine self-administration behavior, and whether they show enhanced sensitivity to the reinforcing actions of cocaine or if they are resistant to extinction. Pregnant rats were injected with lipopolysaccharide on gestational day 15 and 16, and the offspring (both male and female) were tested in working memory (T-maze), social interaction and sensorimotor gating (prepulse inhibition of the acoustic startle response) paradigms. After performing these tests, the rats were subjected to cocaine self-administration regimes (0.5mg/kg), assessing their dose-response and extinction. Male rats born to dams administered lipopolysaccharide showed impaired working memory but no alterations to their social interactions, and both male and female rats showed prepulse inhibition deficits. Moreover, similar patterns of cocaine self-administration acquisition, responsiveness to dose shifts and extinction curves were observed in both control and experimental rats. These results suggest that the higher prevalence of cocaine abuse among schizophrenic individuals is not due to a biological vulnerability directly associated to the disease and that other factors (social, educational, economic, familial, etc.) should be considered given the multifactorial nature of this illness.

Strain differences in the expression of endocannabinoid genes and in cannabinoid receptor binding in the brain of Lewis and Fischer 344 rats.

The Lewis (LEW) and Fischer 344 (F344) rat strains have been proposed as a model to study certain genetic influences on drug use. These strains differ in terms of the self-administration of several drugs, and in their expression of various components of the dopaminergic, glutamatergic, GABAergic and endogenous opioid neurotransmitter systems. As the endocannabinoid system is linked to these systems, we investigated whether these two strains exhibit differences in cannabinoid receptor binding and in the expression of cannabinoid-related genes. Quantitative autoradiography of [(3)H]-CP 55,940 binding levels and real-time PCR assays were used. F344 rats displayed higher levels of cannabinoid receptor binding in the lateral globus pallidus and weaker CNR1 gene expression in the prefrontal cortex (PFc) than LEW rats. Moreover, the N-acyl phosphatidylethanolamine-specific phospholipase D/fatty acid amide hydrolase ratio was greater in the PFc and NAcc of F344 rats. Our results suggest that the endocannabinoid system may be a mediator of the individual differences that exist in the susceptibility to the rewarding effects of drugs of abuse.