Communal nesting shapes the sex-dependent glutamatergic response to early life stress in the rat prefrontal cortex.

Introduction: Early social environment, either positive or negative, shapes the adult brain. Communal nesting (CN), a naturalistic setting in which 2-3 females keep their pups in a single nest sharing care-giving behavior, provides high level of peer interaction for pups. Early social isolation (ESI) from dam and siblings represents, instead, an adverse condition providing no peer interaction. Methods: We investigated whether CN (enrichment setting) might influence the response to ESI (impoverishment setting) in terms of social behavior and glutamate system in the medial prefrontal cortex (mPFC) of adult and adolescent male and female rats. Results: Pinning (a rewarding component of social play behavior) was significantly more pronounced in males than in females exposed to the combination of CN and ESI. CN sensitized the glutamate synapse in the mPFC of ESI-exposed male, but not female, rats. Accordingly, we observed (i) a potentiation of the glutamatergic neurotransmission in the mPFC of both adolescent and adult males, as shown by the recruitment of NMDA receptor subunits together with increased expression/activation of PSD95, SynCAM 1, Synapsin I and αCaMKII; (ii) a de-recruiting of NMDA receptors from active synaptic zones of same-age females, together with reduced expression/activation of the above-mentioned proteins, which might reduce the glutamate transmission. Whether similar sex-dependent glutamate homeostasis modulation occurs in other brain areas remains to be elucidated. Discussion: CN and ESI interact to shape social behavior and mPFC glutamate synapse homeostasis in an age- and sex-dependent fashion, suggesting that early-life social environment may play a crucial role in regulating the risk to develop psychopathology.

Behavioral characterization of co-exposure to cannabinoids and hormonal contraceptives in female rats.

Hormonal contraceptives are among the most widely used drugs by young healthy women to block ovulation and avoid pregnancy. They reduce the ovarian secretion of estradiol and progesterone, hormones that also modulate neuronal plasticity, cognitive functions, emotions and mood. Cannabis is the most commonly used illicit drug worldwide and its use is increasing among young women, many of which regularly take the "pill". Despite evidence of a bidirectional interaction between the endocannabinoid system and gonadal hormones, only very few studies have examined the consequences of cannabis consumption in young females under hormonal contraceptives treatment. To fill this gap, this study evaluated the behavioral effects of co-exposure to chronic 1) hormonal contraceptives, i.e., ethinyl estradiol (EE) plus levonorgestrel (LNG), one of the synthetic estrogen-progestin combinations of hormonal contraceptives, and 2) cannabinoid receptor agonist, i.e., WIN 55,212-2 (WIN), on motor activity, emotional state and cognitive functions in young adult female rats (8-11/experimental group). Hormonal and cannabinoid treatment started at post-natal day (PND) 52 and 56, respectively, while behavioral testing occurred between PND 84-95. The results show that chronic EE-LNG treatment, at doses (0.020 and 0.060 mg/rat, respectively) known to drastically reduce plasma progesterone levels, and the contextual exposure to WIN, at a dose (12.5 µg/kg/infusion) known to be rewarding in the rat, alters the hormonal milieu but does not cause further changes in locomotor activity compared to EE-LNG or WIN alone, and does not modify anxiety-like state (as measured by the elevated plus maze and the marble burying tests) and cognitive abilities (as measured by the novel object recognition and the prepulse inhibition tests) in young adult female rats. Although exposure to EE-LNG and WIN tends to increase the duration of immobility and to reduce the time spent swimming in the forced swimming test, there was not a significant additive effect suggestive of a depressive-like state. These findings allow deepening the current knowledge on the interaction between cannabinoid agonists and hormonal contraceptives and suggest that low, rewarding doses of cannabinoids do not significantly alter the motor and cognitive skills and do not induce anxiety or depressive-like states in females that use hormonal contraceptives.

Sex-specific behavioural, metabolic, and immunohistochemical changes after repeated administration of the synthetic cannabinoid AKB48 in mice.

BACKGROUND AND PURPOSE: AKB48 is a synthetic cannabinoid illegally sold for its psychoactive cannabis-like effects that have been associated with acute intoxication and whose effects are poorly known. EXPERIMENTAL APPROACH: Using a behavioural, neurochemical, and immunohistochemical approach, we investigated the pharmaco-toxicological effects, pharmacokinetics, and neuroplasticity at cannabinoid CB1 receptors in the cerebellum and cortex induced by repeated AKB48 administration in male and female mice. KEY RESULTS: The effects of AKB48 varied significantly depending on sex and treatment duration. The first injection impaired sensorimotor responses and reduced body temperature, analgesia, and breath rate to a greater extent in females than in males; the second injection induced stronger effects in males while the third injection of AKB48 induced weaker responses in both sexes, suggesting emergence of tolerance. The CB1 receptor antagonist NESS-0327 prevented the effects induced by repeated AKB48, confirming a CB1 receptor-mediated action. Blood AKB48 levels were higher in females than in males and repeated administration caused a progressive rise of AKB48 levels in both sexes, suggesting an inhibitory effect on cytochrome activity. Finally, immunohistochemical analysis revealed higher expression of CB1 receptors in the cerebellum and cortex of females, and a rapid CB1 receptor down-regulation in cerebellar and cortical areas following repeated AKB48 injections, with neuroadaptation occurring generally more rapidly in females than in males. CONCLUSION AND IMPLICATIONS: We have shown for the first time that AKB48 effects significantly vary with prolonged use and that sex affects the pharmacodynamic/pharmacokinetic responses to repeated administration, suggesting a sex-tailored approach in managing AKB48-induced intoxication.

Communal nesting differentially attenuates the impact of pre-weaning social isolation on behavior in male and female rats during adolescence and adulthood.

Introduction: Early social isolation (ESI) disrupts neurodevelopmental processes, potentially leading to long-lasting emotional and cognitive changes in adulthood. Communal nesting (CN), i.e., the sharing of parental responsibilities between multiple individuals in a nest, creates a socially enriching environment known to impact social and anxiety-related behaviors. Methods: This study examines the effects of (i) the CN condition and of (ii) ESI during the 3rd week of life (i.e., pre-weaning ESI) on motor, cognitive, and emotional domains during adolescence and adulthood in male and female rats reared in the two different housing conditions, as well as (iii) the potential of CN to mitigate the impact of ESI on offspring. Results: We found that in a spontaneous locomotor activity test, females exhibited higher activity levels compared to males. In female groups, adolescents reared in standard housing (SH) condition spent less time in the center of the arena, suggestive of increased anxiety levels, while the CN condition increased the time spent in the center during adolescence, but not adulthood, independently from ESI. The prepulse inhibition (PPI) test showed a reduced PPI in ESI adolescent animals of both sexes and in adult males (but not in adult females), with CN restoring PPI in males, but not in adolescent females. Further, in the marble burying test SH-ESI adolescent males exhibited higher marble burying behavior than all other groups, suggestive of obsessive-compulsive traits. CN completely reversed this stress-induced effect. Interestingly, ESI and CN did not have a significant impact on burying behavior in adult animals of both sexes. Discussion: Overall, our findings (i) assess the effects of ESI on locomotion, sensorimotor gating, and compulsive-like behaviors, (ii) reveal distinct vulnerabilities of males and females within these domains, and (iii) show how early-life social enrichment may successfully counteract some of the behavioral alterations induced by early-life social stress in a sex-dependent manner. This study strengthens the notion that social experiences during early-life can shape emotional and cognitive outcomes in adulthood, and points to the importance of social enrichment interventions for mitigating the negative effects of early social stress on neurodevelopment.

Olfactory Bulbectomy Model of Depression Lowers Responding for Food in Male and Female Rats: The Modulating Role of Caloric Restriction and Response Requirement.

Depression is a psychiatric disorder characterized by a marked decrease in reward sensitivity. By using the olfactory bulbectomy (OBX) model of depression, it was shown that OBX rats display enhanced drug-taking and seeking behaviors in a self-administration paradigm than sham-operated (SHAM) controls, and sex is an important regulating factor. To reveal potential strain effects, we compared the operant behavior of male and female Sprague-Dawley and Wistar OBX and SHAM rats trained to self-administer palatable food pellets. Results showed that Sprague-Dawley OBX rats of both sexes exhibited lower operant responding rates and food intake than SHAM controls. Food restriction increased responding in both OBX and SHAM groups. Female rats responded more than males, but the OBX lesion abolished this effect. In Wistar rats, bulbectomy lowered food self-administration only during the last training days. Food self-administration was not significantly affected in Wistar rats by sex. In summary, this study showed that bulbectomy significantly reduces operant responding and food intake in male and female Sprague-Dawley rats while inducing a mild reducing effect only in the Wistar strain. Strain-dependent effects were also observed in the modulating role of sex and food restriction on operant responding and palatable food intake.

Outcomes of early social experiences on glucocorticoid and endocannabinoid systems in the prefrontal cortex of male and female adolescent rats.

Social and emotional experiences differently shape individual's neurodevelopment inducing substantial changes in neurobiological substrates and behavior, particularly when they occur early in life. In this scenario, the present study was aimed at (i) investigating the impact of early social environments on emotional reactivity of adolescent male and female rats and (ii) uncovering the underlying molecular features, focusing on the cortical endocannabinoid (eCB) and glucocorticoid systems. To this aim, we applied a protocol of environmental manipulation based on early postnatal socially enriched or impoverished conditions. Social enrichment was realized through communal nesting (CN). Conversely, an early social isolation (ESI) protocol was applied (post-natal days 14-21) to mimic an adverse early social environment. The two forms of social manipulation resulted in specific behavioral and molecular outcomes in both male and female rat offspring. Despite the combination of CN and ESI did not affect emotional reactivity in both sexes, the molecular results reveal that the preventive exposure to CN differently altered mRNA and protein expression of the main components of the glucocorticoid and eCB systems in male and female rats. In particular, adolescent females exposed to the combination of CN and ESI showed increased corticosterone levels, unaltered genomic glucocorticoid receptor, reduced cannabinoid receptor type-1 and fatty acid amide hydrolase protein levels, suggesting that the CN condition evokes different reorganization of these systems in males and females.

Effects of the Phenethylamine 2-Cl-4,5-MDMA and the Synthetic Cathinone 3,4-MDPHP in Adolescent Rats: Focus on Sex Differences.

The illicit drug market of novel psychoactive substances (NPSs) is expanding, becoming an alarming threat due to increasing intoxication cases and insufficient (if any) knowledge of their effects. Phenethylamine 2-chloro-4,5-methylenedioxymethamphetamine (2-Cl-4,5-MDMA) and synthetic cathinone 3,4-methylenedioxy-α-pyrrolidinohexanophenone (3,4-MDPHP) are new, emerging NPSs suggested to be particularly dangerous. This study verified whether these two new drugs (i) possess abuse liability, (ii) alter plasma corticosterone levels, and (iii) interfere with dopaminergic transmission; male and female adolescent rats were included to evaluate potential sex differences in the drug-induced effects. Findings show that the two NPSs are not able to sustain reliable self-administration behavior in rats, with cumulatively earned injections of drugs being not significantly different from cumulatively earned injections of saline in control groups. Yet, at the end of the self-administration training, females (but not males) exhibited higher plasma corticosterone levels after chronic exposure to low levels of 3,4-MDPHP (but not of 2-Cl-4,5-MDMA). Finally, electrophysiological patch-clamp recordings in the rostral ventral tegmental area (rVTA) showed that both drugs are able to increase the firing rate of rVTA dopaminergic neurons in males but not in females, confirming the sex dimorphic effects of these two NPSs. Altogether, this study demonstrates that 3,4-MDPHP and 2-Cl-4,5-MDMA are unlikely to induce dependence in occasional users but can induce other effects at both central and peripheral levels that may significantly differ between males and females.

New insights into methoxetamine mechanisms of action: Focus on serotonergic 5-HT2 receptors in pharmacological and behavioral effects in the rat.

Methoxetamine (MXE) is a dissociative substance of the arylcyclohexylamine class that has been present on the designer drug market as a ketamine-substitute since 2010. We have previously shown that MXE (i) possesses ketamine-like discriminative and positive rewarding effects in rats, (ii) affects brain processing involved in cognition and emotional responses, (iii) causes long-lasting behavioral abnormalities and neurotoxicity in rats and (iv) induces neurological, sensorimotor and cardiorespiratory alterations in mice. To shed light on the mechanisms through which MXE exerts its effects, we conducted a multidisciplinary study to evaluate the various neurotransmitter systems presumably involved in its actions on the brain. In vivo microdialysis study first showed that a single administration of MXE (0.25 and 0.5 mg/kg, i.v.) is able to significantly alter serotonin levels in the rat medial prefrontal cortex (mPFC) and nucleus accumbens. Then, we observed that blockade of the serotonin 5-HT2 receptors through two selective antagonists, ketanserin (0.1 mg/kg, i.p.) and MDL 100907 (0.03 mg/kg, i.p.), at doses not affecting animals behavior per se, attenuated the facilitatory motor effect and the inhibition on visual sensory responses induced by MXE (3 mg/kg, i.p.) and ketamine (3 mg/kg, i.p.), and prevented MXE-induced reduction of the prepulse inhibition in rats, pointing to the 5-HT2 receptors as a key target for the recently described MXE-induced sensorimotor effects. Finally, in-vitro electrophysiological studies revealed that the GABAergic and glutamatergic systems are also likely involved in the mechanisms through which MXE exerts its central effects since MXE inhibits, in a concentration-dependent manner, NMDA-mediated field postsynaptic potentials and GABA-mediated spontaneous currents. Conversely, MXE failed to alter both the AMPA component of field potentials and presynaptic glutamate release, and seems not to interfere with the endocannabinoid-mediated effects on mPFC GABAergic synapses. Altogether, our results support the notion of MXE as a NMDA receptor antagonist and shed further lights into the central mechanisms of action of this ketamine-substitute by pointing to serotonin 5-HT2 receptors as crucial players in the expression of its sensorimotor altering effects and to the NMDA and GABA receptors as potential further important targets of action.

Repeated exposure to JWH-018 induces adaptive changes in the mesolimbic and mesocortical dopaminergic pathways, glial cells alterations, and behavioural correlates.

BACKGROUND AND PURPOSE: Spice/K2 herbal mixtures, containing synthetic cannabinoids such as JWH-018, have been marketed as marijuana surrogates since 2004. JWH-018 has cannabinoid CB1 receptor-dependent reinforcing properties and acutely increases dopaminergic transmission selectively in the NAc shell. Here, we tested the hypothesis that repeated administration of JWH-018 (i) modulates behaviour, (ii) affects dopaminergic transmission and its responsiveness to motivational stimuli, and (iii) is associated with a neuroinflammatory phenotype. EXPERIMENTAL APPROACH: Rats were administered with JWH-018 once a day for 14 consecutive days. We then performed behavioural, electrophysiological, and neurochemical evaluation at multiple time points after drug discontinuation. KEY RESULTS: Repeated JWH-018 exposure (i) induced anxious and aversive behaviours, transitory attentional deficits, and withdrawal signs; (ii) decreased spontaneous activity and number of dopamine neurons in the VTA; and (iii) reduced stimulation of dopaminergic transmission in the NAc shell while potentiating that in the NAc core, in response to acute JWH-018 challenge. Moreover, (iv) we observed a decreased dopamine sensitivity in the NAc shell and core, but not in the mPFC, to a first chocolate exposure; conversely, after a second exposure, dialysate dopamine fully increased in the NAc shell and core but not in the mPFC. Finally, selected dopamine brain areas showed (v) astrogliosis (mPFC, NAc shell and core, VTA), microgliosis (NAc shell and core), and downregulation of CB1 receptors (mPFC, NAc shell and core). CONCLUSION AND IMPLICATIONS: Repeated exposure to JWH-018 may provide a useful model to clarify the detrimental effects of recurring use of Spice/K2 drugs.

Gender-specific approach in psychiatric diseases: Because sex matters.

In both animals and human beings, males and females differ in their genetic background and hormonally driven behaviour and show sex-related differences in brain activity and response to internal and external stimuli. Gender-specific medicine has been a neglected dimension of medicine for long time, and only in the last three decades it is receiving the due scientific and clinical attention. Research has recently begun to identify factors that could provide a neurobiological basis for gender-based differences in health and disease and to point to gonadal hormones as important determinants of male-female differences. Animal studies have been of great help in understanding factors contributing to sex-dependent differences and sex hormones action. Here we review and discuss evidence provided by clinical and animal studies in the last two decades showing gender (in humans) and sex (in animals) differences in selected psychiatric disorders, namely eating disorders (anorexia nervosa, bulimia nervosa, binge eating disorder), schizophrenia, mood disorders (anxiety, depression, obsessive-compulsive disorder) and neurodevelopmental disorders (autism spectrum disorders, attention-deficit/hyperactivity disorder).

Transcranial Magnetic Stimulation: A review about its efficacy in the treatment of alcohol, tobacco and cocaine addiction.

Substance Use Disorder (SUD) is a chronic and relapsing disease characterized by craving, loss of control, tolerance and physical dependence. At present, the combination of pharmacotherapy and psychosocial intervention is the most effective management strategy in preventing relapse to reduce dropout rates and promote abstinence in SUD patients. However, only few effective medications are available. Transcranial Magnetic Stimulation (TMS) is a non-invasive brain stimulation technique that modulates the cellular activity of the cerebral cortex through a magnetic pulse applied on selected brain areas. Recently, the efficacy of TMS has been investigated in various categories of SUD patients. The present review analyzes the application of repetitive TMS in patients with alcohol, tobacco, and cocaine use disorder. Although the number of clinical studies is still limited, repetitive TMS yields encouraging results in these patients, suggesting a possible role of TMS in the treatment of SUD.

The hypodopaminergic state ten years after: transcranial magnetic stimulation as a tool to test the dopamine hypothesis of drug addiction.

An altered dopamine transmission has been described for different types of addiction for a long time. Preclinical and clinical evidence support the hypodopaminergic hypothesis and underpin the need to increase dopamine transmission to obtain therapeutic benefit. Repetitive transcranial magnetic stimulation (rTMS) of prefrontal cortex shows efficacy in treating some forms of addiction. Recent imaging studies confirmed that the therapeutic effect of rTMS is correlated with an enhancement of dopamine transmission. Novel targets for rTMS are under evaluation to increase its effectiveness in treating addiction, and research is ongoing to find the optimal protocol to boost dopaminergic transmission in the addicted brain. TMS can thus be considered a useful tool to test the dopamine hypothesis of drug addiction and instrumental in the search for addiction therapeutics.

Conditioned Place Preference (CPP) in Rats: From Conditioning to Reinstatement Test.

Opioid addiction in humans is a chronically relapsing disorder characterized by discontinuous periods of drug use and abstinence resulting in dependence. With time, the probability of falling into renewed drug consumption becomes particularly high and constitutes a considerable problem in the management of opioid addicts. Opioid addiction represents an important health concern and animal models have been crucial in understanding the neurobiology and pathophysiology of this complex disease. Although animal models of addiction do not fully reproduce the human condition, they do permit investigation of specific elements of the process as well as identification of potential therapeutic targets. In this chapter, we provide a step-by-step description of the morphine-conditioned place preference (CPP) model that represents a useful preclinical animal model extensively used to study the rewarding/aversive effect of drugs.

Analysis of Opioid-Seeking Behavior Through the Intravenous Self-Administration Reinstatement Model in Rats.

The inability to maintain drug abstinence is often referred to as relapse and consists of a process by which an abstaining individual slips back into old behavioral patterns and substance use. Animal models of relapse have been developed over the last decades and significantly contributed to shed light on the neurobiological mechanisms underlying vulnerability to relapse. The most common procedure to study drug-seeking and relapse-like behavior in animals is the "extinction-reinstatement model." Originally elaborated by Pavlov and Skinner, the concepts of reinforced operant responding were applied to addiction research not before 1971 (Stretch et al., Can J Physiol Pharmacol 49:581-589, 1971), and the first report of a reinstatement animal model as it is now used worldwide was published only 10 years later (De Wit and Stewart, Psychopharmacology 75:134-143, 1981). According to the proposed model, opioids are typically self-administered intravenously, as humans do, and although rodents are most often employed in these studies, a variety of species including nonhuman primates, dogs, cats, and pigeons can be used. Several operant responses are available, depending on the species studied. For example, a lever press or a nose poke response typically is used for rodents, whereas a panel press response typically is used for nonhuman primates. In this chapter we describe a simple and easily reproducible protocol of heroin-seeking reinstatement in rats, which proved useful to study the neurobiological mechanisms underlying relapse to heroin and vulnerability factors enhancing the resumption of heroin-seeking behavior.