Pregnenolone-methyl-ether enhances CLIP170 and microtubule functions improving spine maturation and hippocampal deficits related to CDKL5 deficiency.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) cause CDKL5 deficiency disorder (CDD), a neurodevelopmental disease characterized by severe infantile seizures and intellectual disability. The absence of CDKL5 in mice causes defective spine maturation that can at least partially explain the cognitive impairment in CDKL5 patients and CDD mouse models. The molecular basis for such defect may depend on the capacity of CDKL5 to regulate microtubule (MT) dynamics through its association with the MT-plus end tracking protein CLIP170 (cytoplasmic linker protein 170). Indeed, we here demonstrate that the absence of CDKL5 causes CLIP170 to be mainly in a closed inactive conformation that impedes its binding to MTs. Previously, the synthetic pregnenolone analogue, pregnenolone-methyl-ether (PME), was found to have a positive effect on CDKL5-related cellular and neuronal defects in vitro. Here, we show that PME induces the open active conformation of CLIP170 and promotes the entry of MTs into dendritic spines in vitro. Furthermore, the administration of PME to symptomatic Cdkl5-knock-out mice improved hippocampal-dependent behavior and restored spine maturation and the localization of MT-related proteins in the synaptic compartment. The positive effect on cognitive deficits persisted for 1 week after treatment withdrawal. Altogether, our results suggest that CDKL5 regulates spine maturation and cognitive processes through its control of CLIP170 and MT dynamics, which may represent a novel target for the development of disease-modifying therapies.

Maternal immune activation impairs endocannabinoid signaling in the mesolimbic system of adolescent male offspring.

Prenatal infections can increase the risk of developing psychiatric disorders such as schizophrenia in the offspring, especially when combined with other postnatal insults. Here, we tested, in a rat model of prenatal immune challenge by the viral mimic polyriboinosinic-polyribocytidilic acid, whether maternal immune activation (MIA) affects the endocannabinoid system and endocannabinoid-mediated modulation of dopamine functions. Experiments were performed during adolescence to assess i) the behavioral endophenotype (locomotor activity, plus maze, prepulse inhibition of startle reflex); ii) the locomotor activity in response to Δ9-Tetrahydrocannabinol (THC) and iii) the properties of ventral tegmental area (VTA) dopamine neurons in vivo and their response to THC; iv) endocannabinoid-mediated synaptic plasticity in VTA dopamine neurons; v) the expression of cannabinoid receptors and enzymes involved in endocannabinoid synthesis and catabolism in mesolimbic structures and vi) MIA-induced neuroinflammatory scenario evaluated by measurements of levels of cytokine and neuroinflammation markers. We revealed that MIA offspring displayed an altered locomotor activity in response to THC, a higher bursting activity of VTA dopamine neurons and a lack of response to cumulative doses of THC. Consistently, MIA adolescence offspring showed an enhanced 2-arachidonoylglycerol-mediated synaptic plasticity and decreased monoacylglycerol lipase activity in mesolimbic structures. Moreover, they displayed a higher expression of cyclooxygenase 2 (COX-2) and ionized calcium-binding adaptor molecule 1 (IBA-1), associated with latent inflammation and persistent microglia activity. In conclusion, we unveiled neurobiological mechanisms whereby inflammation caused by MIA influences the proper development of endocannabinoid signaling that negatively impacts the dopamine system, eventually leading to psychotic-like symptoms in adulthood.

Use of Marijuana: Effect on Brain Health: A Scientific Statement From the American Heart Association.

Marijuana is perceived as a harmless drug, and its recreational use has gained popularity among young individuals. The concentration of active ingredients in recreational formulations has gradually increased over time, and high-potency illicit cannabinomimetics have become available. Thus, the consumption of cannabis in the general population is rising. Data from preclinical models demonstrate that cannabinoid receptors are expressed in high density in areas involved in cognition and behavior, particularly during periods of active neurodevelopment and maturation. In addition, growing evidence highlights the role of endogenous cannabinoid pathways in the regulation of neurotransmitter release, synaptic plasticity, and neurodevelopment. In animal models, exogenous cannabinoids disrupt these important processes and lead to cognitive and behavioral abnormalities. These data correlate with the higher risk of cognitive impairment reported in some observational studies done in humans. It is unclear whether the effect of cannabis on cognition reverts after abstinence. However, this evidence, along with the increased risk of stroke reported in marijuana users, raises concerns about its potential long-term effects on cognitive function. This scientific statement reviews the safety of cannabis use from the perspective of brain health, describes mechanistically how cannabis may cause cognitive dysfunction, and advocates for a more informed health care worker and consumer about the potential for cannabis to adversely affect the brain.

Dos(e)Age: Role of Dose and Age in the Long-Term Effect of Cannabinoids on Cognition.

Cannabis is still the most widely used illicit drug around the world. While its use has always been prevalent among adolescents, recent evidence suggests that its consumption is also increasing among other population groups, such as pregnant women and aged people. Given the known impact of cannabis on brain development and behavior, it is important to dissect the possible long-term impact of its use across different age groups, especially on measures of cognitive performance. Animal models of cannabinoid exposure have represented a fundamental tool to characterize the long-lasting consequences of cannabinoids on cognitive performance and helped to identify possible factors that could modulate cannabinoids effects in the long term, such as the age of exposure and doses administered. This scoping review was systematically conducted using PubMed and includes papers published from 2015 to December 2021 that examined the effects of cannabinoids, either natural or synthetic, on cognitive performance in animal models where exposure occurred in the prenatal period, during adolescence, or in older animals. Overall, available data clearly point to a crucial role of age in determining the long-term effect of cannabinoid on cognition, highlighting possible detrimental consequences during brain development (prenatal and adolescent exposure) and beneficial outcomes in old age. In contrast, despite the recent advances in the field, it appears difficult to clearly establish a possible role of dosage in the effects of cannabinoids on cognition, especially when the adolescent period is taken into account.

Long-Term Consequences of Adolescent Exposure to THC-Rich/CBD-Poor and CBD-Rich/THC-Poor Combinations: A Comparison with Pure THC Treatment in Female Rats.

Cannabis is the most-used recreational drug worldwide, with a high prevalence of use among adolescents. In animal models, long-term adverse effects were reported following chronic adolescent exposure to the main psychotomimetic component of the plant, delta-9-tetrahydrocannabinol (THC). However, these studies investigated the effects of pure THC, without taking into account other cannabinoids present in the cannabis plant. Interestingly, cannabidiol (CBD) content seems to mitigate some of the side effects of THC, at least in adult animals. Thus, in female rats, we evaluated the long-term consequences of a co-administration of THC and CBD at a 3:1 ratio, chosen based on the analysis of recently confiscated illegal cannabis samples in Europe. CBD content is able to mitigate some of the long-term behavioral alterations induced by adolescent THC exposure as well as long-term changes in CB1 receptor and microglia activation in the prefrontal cortex (PFC). We also investigated, for the first time, possible long-term effects of chronic administration of a THC/CBD combination reminiscent of "light cannabis" (CBD:THC in a 33:1 ratio; total THC 0.3%). Repeated administration of this CBD:THC combination has long-term adverse effects on cognition and leads to anhedonia. Concomitantly, it boosts Glutamic Acid Decarboxylase-67 (GAD67) levels in the PFC, suggesting a possible lasting effect on GABAergic neurotransmission.

Termination of acute stress response by the endocannabinoid system is regulated through lysine-specific demethylase 1-mediated transcriptional repression of 2-AG hydrolases ABHD6 and MAGL.

Acute environmental stress rarely implies long-lasting neurophysiological and behavioral alterations. On the contrary, chronic stress exerts a potent toxic effect at the glutamatergic synapse whose altered physiology has been recognized as a core trait of neuropsychiatric disorders. The endocannabinoid system (ECS) plays an important role in the homeostatic response to acute stress. In particular, stress induces synthesis of endocannabinoid (eCB) 2-arachidonyl glycerol (2-AG). 2-AG stimulates presynaptic cannabinoid 1 (CB1) receptor contributing to stress response termination through inhibition of glutamate release, restraining thereafter anxiety arousal. We employ mouse models of stress response coupled to gene expression analyses, unravelling that in response to acute psychosocial stress in the mouse hippocampus, ECS-mediated synaptic modulation is enhanced via transcriptional repression of two enzymes involved in 2-AG degradation: α/ß-hydrolase domain containing 6 (ABHD6) and monoacylglycerol lipase (MAGL). Such a process is orchestrated by the epigenetic corepressor LSD1 who directly interacts with promoter regulatory regions of Abhd6 and Magl. Remarkably, negative transcriptional control of Abhd6 and Magl is lost in the hippocampus upon chronic psychosocial stress, possibly contributing to trauma-induced drift of synapse physiology toward uncontrolled glutamate transmission. We previously showed that in mice lysine-specific demethylase 1 (LSD1) increases its hippocampal expression in response to psychosocial stress preventing excessive consolidation of anxiety-related plasticity. In this work, we unravel a nodal epigenetic modulation of eCB turn over, shedding new light on the molecular substrate of converging stress-terminating effects displayed by ECS and LSD1.

Endocannabinoid-Epigenetic Cross-Talk: A Bridge toward Stress Coping.

There is no argument with regard to the physical and psychological stress-related nature of neuropsychiatric disorders. Yet, the mechanisms that facilitate disease onset starting from molecular stress responses are elusive. Environmental stress challenges individuals' equilibrium, enhancing homeostatic request in the attempt to steer down arousal-instrumental molecular pathways that underlie hypervigilance and anxiety. A relevant homeostatic pathway is the endocannabinoid system (ECS). In this review, we summarize recent discoveries unambiguously listing ECS as a stress coping mechanism. As stress evokes huge excitatory responses in emotional-relevant limbic areas, the ECS limits glutamate release via 2-arachydonilglycerol (2-AG) stress-induced synthesis and retrograde cannabinoid 1 (CB1)-receptor activation at the synapse. However, ECS shows intrinsic vulnerability as 2-AG overstimulation by chronic stress rapidly leads to CB1-receptor desensitization. In this review, we emphasize the protective role of 2-AG in stress-response termination and stress resiliency. Interestingly, we discuss ECS regulation with a further nuclear homeostatic system whose nature is exquisitely epigenetic, orchestrated by Lysine Specific Demethylase 1. We here emphasize a remarkable example of stress-coping network where transcriptional homeostasis subserves synaptic and behavioral adaptation, aiming at reducing psychiatric effects of traumatic experiences.

Adult Cellular Neuroadaptations Induced by Adolescent THC Exposure in Female Rats Are Rescued by Enhancing Anandamide Signaling.

Background: In rodent models, chronic exposure to cannabis' psychoactive ingredient, Δ9-tetrahydrocannabinol, during adolescence leads to abnormal behavior in adulthood. In female rats, this maladaptive behavior is characterized by endophenotypes for depressive-like and psychotic-like disorders as well as cognitive deficits. We recently reported that most depressive-like behaviors triggered by adolescent Δ9-tetrahydrocannabinol exposure can be rescued by manipulating endocannabinoid signaling in adulthood with the anandamide-inactivating enzyme FAAH inhibitor, URB597. However, the molecular mechanisms underlying URB597's antidepressant-like properties remain to be established. Methods: Here we examined the impact of adult URB597 treatment on the cellular and functional neuroadaptations that occurred in the prefrontal cortex and dentate gyrus of the hippocampus upon Δ9-tetrahydrocannabinol during adolescence through biochemical, morphofunctional, and electrophysiological studies. Results: We found that the positive action of URB597 is associated with the rescue of Δ9-tetrahydrocannabinol-induced deficits in endocannabinoid-mediated signaling and synaptic plasticity in the prefrontal cortex and the recovery of functional neurogenesis in the dentate gyrus of the hippocampus. Moreover, the rescue property of URB597 on depressive-like behavior requires the activity of the CB1 cannabinoid receptor. Conclusions: By providing novel insights into the cellular and molecular mechanisms of URB597 at defined cortical and hippocampal circuits, our results highlight that positive modulation of endocannabinoid-signaling could be a strategy for treating mood alterations secondary to adolescent cannabis use.

Adolescent THC exposure in female rats leads to cognitive deficits through a mechanism involving chromatin modifications in the prefrontal cortex.

BACKGROUND: Increasing cannabis consumption among adolescents, studies that link its early use with mental illnesses, and the political debate on cannabis legalization together call for an urgent need to study molecular underpinnings of adolescent brain vulnerability. The emerging role of epigenetic mechanisms in psychiatric diseases led us to hypothesize that epigenetic alterations could play a role in causes and subsequent development of the depressive/psychotic-like phenotype induced by adolescent, but not adult, Δ9-tetrahydrocannabinol (THC) exposure in female rats. METHODS: We performed a time-course analysis of histone modifications, chromatin remodelling enzymes and gene expression in the prefrontal cortex of female rats after adolescent and adult THC exposure. We also administered a specific epigenetic drug (chaetocin) with THC to investigate its impact on THC-induced behavioural alterations. RESULTS: Adolescent THC exposure induced alterations of selective histone modifications (mainly H3K9me3), impacting the expression of genes closely associated with synaptic plasticity. Changes in both histone modifications and gene expression were more widespread and intense after adolescent treatment, suggesting specific adolescent susceptibility. Adolescent THC exposure significantly increased Suv39H1 levels, which could account for the enhanced H3K9me3. Pharmacological blockade of H3K9me3 during adolescent THC treatment prevented THC-induced cognitive deficits, suggesting the relevant role played by H3K9me3 in THC-induced effects. LIMITATIONS: Only female rats were investigated, and the expression studies were limited to a specific subset of genes. CONCLUSION: Through a mechanism involving SUV39H1, THC modifies histone modifications and, thereby, expression of plasticity genes. This pathway appears to be relevant for the development of cognitive deficits.

Lifelong imbalanced LA/ALA intake impairs emotional and cognitive behavior via changes in brain endocannabinoid system.

Imbalanced dietary n-3 and n-6 PUFA content has been associated with a number of neurological conditions. Endocannabinoids are n-6 PUFA derivatives, whose brain concentrations are sensitive to modifications of fatty acid composition of the diet and play a central role in the regulation of mood and cognition. As such, the endocannabinoid system appears to be an ideal candidate for mediating the effects of dietary fatty acids on mood and cognition. Lifelong administration of isocaloric α-linolenic acid (ALA)-deficient and -enriched diets induced short-term memory deficits, whereas only dietary ALA enrichment altered emotional reactivity in adult male rats compared with animals fed a standard diet that was balanced in ALA/linoleic acid (LA) ratio. In the prefrontal cortex, both diets reduced 2-AG levels and increased MAG lipase expression, whereas only the enriched diet reduced AEA levels, simultaneously increasing FAAH expression. In the hippocampus, an ALA-enriched diet decreased AEA content and NAPE-PLD expression, and reduced 2-AG content while increasing MAG lipase expression. These findings highlight the importance of a diet balanced in fatty acid content for normal brain functions and to support a link between dietary ALA, the brain endocannabinoid system, and behavior, which indicates that dietary ALA intake is a sufficient condition for altering the endocannabinoid system in brain regions modulating mood and cognition.

The anabolic steroid nandrolone alters cannabinoid self-administration and brain CB1 receptor density and function.

Clinical and pre-clinical observations indicate that anabolic-androgenic steroids can induce neurobiological changes that alter the rewarding effects of drugs of abuse. In this study, we investigated the effect of the anabolic steroid nandrolone on the rewarding properties of the cannabinoid CB1 receptor agonist WIN55,212-2 (WIN) in rats. Lister Hooded male rats were treated intramuscularly with nandrolone (15mg/kg) or vehicle for 14 consecutive days, and then allowed to self-administer WIN (12.5µg/kg/infusion) intravenously. After reaching stable drug intake, self-administration behavior was extinguished to examine drug- and cue-induced reinstatement of cannabinoid-seeking behavior. Other behavioral parameters presumed to influence drug-taking and drug-seeking behaviors were examined to gain more insight into the behavioral specificity of nandrolone treatment. Finally, animals were sacrificed for analysis of CB1 receptor density and function in selected brain areas. We found that nandrolone-treated rats self-administered up to 2 times more cannabinoid than vehicle-treated rats, but behaved similarly to control rats when tested for drug- and cue-induced reinstatement of cannabinoid-seeking behavior. Enhanced cannabinoid intake by nandrolone-treated rats was not accompanied by changes in locomotor activity, sensorimotor gating, or memory function. However, our molecular data show that after chronic WIN self-administration nandrolone-treated rats display altered CB1 receptor density and function in selected brain areas. We hypothesize that increased cannabinoid self-administration in nandrolone-treated rats results from a nandrolone-induced decrease in reward function, which rats seem to compensate by voluntarily increasing their cannabinoid intake. Altogether, our findings corroborate the hypothesis that chronic exposure to anabolic-androgenic steroids induces dysfunction of the reward pathway in rats and might represent a potential risk factor for abuse of cannabis and other drugs in humans.

Chronic Δ8-THC Exposure Differently Affects Histone Modifications in the Adolescent and Adult Rat Brain.

Adolescence represents a vulnerable period for the psychiatric consequences of delta9-tetrahydrocannabinol (Δ8-THC) exposure, however, the molecular underpinnings of this vulnerability remain to be established. Histone modifications are emerging as important epigenetic mechanisms involved in the etiopathogenesis of psychiatric diseases, thus, we investigated the impact of chronic Δ8-THC exposure on histone modifications in different brain areas of female rats. We checked histone modifications associated to both transcriptional repression (H3K9 di- and tri-methylation, H3K27 tri-methylation) and activation (H3K9 and H3K14 acetylation) after adolescent and adult chronic Δ8-THC exposure in the hippocampus, nucleus accumbens, and amygdala. Chronic exposure to increasing doses of Δ8-THC for 11 days affected histone modifications in a region- and age-specific manner. The primary effect in the adolescent brain was represented by changes leading to transcriptional repression, whereas the one observed after adult treatment led to transcriptional activation. Moreover, only in the adolescent brain, the primary effect was followed by a homeostatic response to counterbalance the Δ8-THC-induced repressive effect, except in the amygdala. The presence of a more complex response in the adolescent brain may be part of the mechanisms that make the adolescent brain vulnerable to Δ8-THC adverse effects.

Long-term hippocampal glutamate synapse and astrocyte dysfunctions underlying the altered phenotype induced by adolescent THC treatment in male rats.

Cannabis use has been frequently associated with sex-dependent effects on brain and behavior. We previously demonstrated that adult female rats exposed to delta-9-tetrahydrocannabinol (THC) during adolescence develop long-term alterations in cognitive performances and emotional reactivity, whereas preliminary evidence suggests the presence of a different phenotype in male rats. To thoroughly depict the behavioral phenotype induced by adolescent THC exposure in male rats, we treated adolescent animals with increasing doses of THC twice a day (PND 35-45) and, at adulthood, we performed a battery of behavioral tests to measure affective- and psychotic-like symptoms as well as cognition. Poorer memory performance and psychotic-like behaviors were present after adolescent THC treatment in male rats, without alterations in the emotional component. At cellular level, the expression of the NMDA receptor subunit, GluN2B, as well as the levels of the AMPA subunits, GluA1 and GluA2, were significantly increased in hippocampal post-synaptic fractions from THC-exposed rats compared to controls. Furthermore, increases in the levels of the pre-synaptic marker, synaptophysin, and the post-synaptic marker, PSD95, were also present. Interestingly, KCl-induced [(3)H]D-ASP release from hippocampal synaptosomes, but not gliosomes, was significantly enhanced in THC-treated rats compared to controls. Moreover, in the same brain region, adolescent THC treatment also resulted in a persistent neuroinflammatory state, characterized by increased expression of the astrocyte marker, GFAP, increased levels of the pro-inflammatory markers, TNF-α, iNOS and COX-2, as well as a concomitant reduction of the anti-inflammatory cytokine, IL-10. Notably, none of these alterations was observed in the prefrontal cortex (PFC). Together with our previous findings in females, these data suggest that the sex-dependent detrimental effects induced by adolescent THC exposure on adult behavior may rely on its ability to trigger different region-dependent changes in glutamate synapse and glial cells. The phenotype observed in males is mainly associated with marked dysregulations in the hippocampus, whereas the prevalence of alterations in the emotional sphere in females is associated with profound changes in the PFC.

Adolescent exposure to THC in female rats disrupts developmental changes in the prefrontal cortex.

Current concepts suggest that exposure to THC during adolescence may act as a risk factor for the development of psychiatric disorders later in life. However, the molecular underpinnings of this vulnerability are still poorly understood. To analyze this, we investigated whether and how THC exposure in female rats interferes with different maturational events occurring in the prefrontal cortex during adolescence through biochemical, pharmacological and electrophysiological means. We found that the endocannabinoid system undergoes maturational processes during adolescence and that THC exposure disrupts them, leading to impairment of both endocannabinoid signaling and endocannabinoid-mediated LTD in the adult prefrontal cortex. THC also altered the maturational fluctuations of NMDA subunits, leading to larger amounts of gluN2B at adulthood. Adult animals exposed to THC during adolescence also showed increased AMPA gluA1 with no changes in gluA2 subunits. Finally, adolescent THC exposure altered cognition at adulthood. All these effects seem to be triggered by the disruption of the physiological role played by the endocannabinoid system during adolescence. Indeed, blockade of CB1 receptors from early to late adolescence seems to prevent the occurrence of pruning at glutamatergic synapses. These results suggest that vulnerability of adolescent female rats to long-lasting THC adverse effects might partly reside in disruption of the pivotal role played by the endocannabinoid system in the prefrontal cortex maturation.

Endocannabinoids and Mental Disorders.

Preclinical and clinical data fully support the involvement of the endocannabinoid system in the etiopathogenesis of several mental diseases. In this review we will briefly summarize the most common alterations in the endocannabinoid system, in terms of cannabinoid receptors and endocannabinoid levels, present in mood disorders (anxiety, posttraumatic stress disorder, depression, bipolar disorder, and suicidality) as well as psychosis (schizophrenia) and autism. The arising picture for each pathology is not always straightforward; however, both animal and human studies seem to suggest that pharmacological modulation of this system might represent a novel approach for treatment.

Alterations of prefrontal cortex GABAergic transmission in the complex psychotic-like phenotype induced by adolescent delta-9-tetrahydrocannabinol exposure in rats.

Although several findings indicate an association between adolescent cannabis abuse and the risk to develop schizophrenia later in life, the evidence for a causal relationship is still inconclusive. In the present study, we investigated the emergence of psychotic-like behavior in adult female rats chronically exposed to delta-9-tetrahydrocannabinol (THC) during adolescence. To this aim, female Sprague-Dawley rats were treated with THC during adolescence (PND 35-45) and, in adulthood (PND 75), a series of behavioral tests and biochemical assays were performed in order to investigate the long-term effects of adolescent THC exposure. Adolescent THC pretreatment leads to long-term behavioral alterations, characterized by recognition memory deficits, social withdrawal, altered emotional reactivity and sensitization to the locomotor activating effects of acute PCP. Moreover, since cortical disinhibition seems to be a key feature of many different animal models of schizophrenia and GABAergic hypofunction in the prefrontal cortex (PFC) has been observed in postmortem brains from schizophrenic patients, we then investigated the long-lasting consequences of adolescent THC exposure on GABAergic transmission in the adult rat PFC. Biochemical analyses revealed that adolescent THC exposure results in reduced GAD67 and basal GABA levels within the adult PFC. GAD67 expression is reduced both in parvalbumin (PV)- and cholecystokinin (CCK)-containing interneurons; this alteration may be related to the altered emotional reactivity triggered by adolescent THC, as silencing PFC GAD67 expression through a siRNA-mediated approach is sufficient to impact rats' behavior in the forced swim test. Finally, the cellular underpinnings of the observed sensitized response to acute PCP in adult THC-treated rats could be ascribed to the increased cFos immunoreactivity and glutamate levels in the PFC and dorsal striatum. The present findings support the hypothesis that adolescent THC exposure may represent a risk factor for the development of a complex psychotic-like behavior in adulthood.

Sex Differences in Response-Contingent Cannabis Vapor Administration During Adolescence Mediate Enduring Effects on Behavioral Flexibility and Prefrontal Microglia Activation in Rats.

Introduction: Cannabis is the most used illicit drug in the United States. With many states passing legislation to permit its recreational use, there is concern that cannabis use among adolescents could increase dramatically in the coming years. Historically, it has been difficult to model real-world cannabis use to investigate the causal relationship between cannabis use in adolescence and behavioral and neurobiological effects in adulthood. Materials and Methods: We used a response-contingent vapor administration model to investigate long-term effects of cannabis use during adolescence on the medial prefrontal cortex (mPFC) and mPFC-dependent behaviors in male and female rats. Results: Adolescent (35- to 55-day-old) female rats had significantly higher rates of responding for vaporized Δ9-tetrahydrocannabinol (THC)-dominant cannabis extract (CANTHC) compared with adolescent males. In adulthood (70-110 days old), female, but not male, CANTHC rats also took more trials to reach criterion and made more regressive errors in an automated attentional set-shifting task compared with vehicle rats, thereby indicating sex differences in behavioral flexibility impairments. Notably, sex-treatment interactions were not observed when rats of each sex were exposed to a noncontingent CANTHC vapor dosing regimen that approximated CANTHC vapor deliveries earned by females. No differences were observed in effort-based decision making in either sex. In the mPFC, female (but not male) CANTHC rats displayed more reactive microglia with no changes in myelin basic protein expression or dendritic spine density. Conclusion: Altogether, these data reveal important sex differences in rates of responding for CANTHC vapor in adolescence that may confer enduring alterations to mPFC structure and function and suggest that there may be subtle differences in the effects of response-contingent versus noncontingent cannabis exposure that should be systematically examined in future studies.

Extracellular vesicles released by microglia and macrophages carry endocannabinoids which foster oligodendrocyte differentiation.

Introduction: Microglia and macrophages can influence the evolution of myelin lesions through the production of extracellular vesicles (EVs). While microglial EVs promote in vitro differentiation of oligodendrocyte precursor cells (OPCs), whether EVs derived from macrophages aid or limit OPC maturation is unknown. Methods: Immunofluorescence analysis for the myelin protein MBP was employed to evaluate the impact of EVs from primary rat macrophages on cultured OPC differentiation. Raman spectroscopy and liquid chromatography-mass spectrometry was used to define the promyelinating lipid components of myelin EVs obtained in vitro and isolated from human plasma. Results and discussion: Here we show that macrophage-derived EVs do not promote OPC differentiation, and those released from macrophages polarized towards an inflammatory state inhibit OPC maturation. However, their lipid cargo promotes OPC maturation in a similar manner to microglial EVs. We identify the promyelinating endocannabinoids anandamide and 2-arachidonoylglycerol in EVs released by both macrophages and microglia in vitro and circulating in human plasma. Analysis of OPC differentiation in the presence of the endocannabinoid receptor antagonists SR141716A and AM630 reveals a key role of vesicular endocannabinoids in OPC maturation. From this study, EV-associated endocannabinoids emerge as important mediators in microglia/macrophage-oligodendrocyte crosstalk, which may be exploited to enhance myelin repair.

Sex-dependent changes in brain CB1R expression and functionality and immune CB2R expression as a consequence of maternal deprivation and adolescent cocaine exposure.

Early life stress has been associated with several psychiatric disorders, including drug addiction. Actually, maternal deprivation (MD) alters the endocannabinoid system, which participates in motivation and reward for drugs, including cocaine. At youth, the rate of cocaine abuse is alarmingly increasing. Herein, we have investigated the consequences of MD and/or adolescent cocaine exposure in brain CB1Rs and CB2Rs in immune tissues. Control and maternally deprived (24h on postnatal day, pnd, 9) male and female Wistar rats were administered cocaine (8mg/kg/day) or saline during adolescence (pnd 28-42). At adulthood, [(3)H]-CP-55,940 autoradiographic binding was employed for the analysis of CB1R density and CP-55,940-stimulated [(35)S]-GTPgammaS binding for CB1R functionality; CB2R expression was analyzed by Western blotting. Sex differences in CB1R expression and functionality were found, and MD induced important and enduring sex-dependent changes. In addition, the plastic changes induced by adolescent cocaine administration in brain CB1Rs were differentially influenced by early life events. MD increased spleen CB2R expression while adolescent cocaine administration attenuated this effect; cocaine exposure also diminished CB2R expression in bone marrow. Present findings provide evidence for changes in brain CB1R expression and functionality and immune CB2R expression as a consequence of early life stress and adolescent cocaine exposure, and indicate functional interactions between both treatments, which in many regions differ between males and females.

Assay of GTPγS Binding in Autoradiography.

Autoradiography of radiolabeled GTPγS ([35S]GTPγS) binding is a relevant technique to study the function of G protein-coupled receptors (GPCRs) ex vivo. Here, we describe the protocol for such a method, suitable for investigating CB1 receptor functionality in tissue slices from rodent brains.