Ricardo Llorente

Endocannabinoid System and Synaptic Plasticity: Implications for Emotional Responses

The endocannabinoid system has been involved in the regulation of anxiety, and proposed as an inhibitory modulator of neuronal, behavioral and adrenocortical responses to stressful stimuli. Brain regions such as the amygdala, hippocampus and cortex, which are directly involved in the regulation of emotional behavior, contain high densities of cannabinoid CB1 receptors. Mutant mice lacking CB1 receptors show anxiogenic and depressive-like behaviors as well as an altered hypothalamus pituitary adrenal axis activity, whereas enhancement of endocannabinoid signaling produces anxiolytic and antidepressant-like effects. Genetic and pharmacological approaches also support an involvement of endocannabinoids in extinction of aversive memories. Thus, the endocannabinoid system appears to play a pivotal role in the regulation of emotional states. Endocannabinoids have emerged as mediators of short- and long-term synaptic plasticity in diverse brain structures. Despite the fact that most of the studies on this field have been performed using in vitro models, endocannabinoid-mediated plasticity might be considered as a plausible candidate underlying some of the diverse physiological functions of the endogenous cannabinoid system, including developmental, affective and cognitive processes. In this paper, we will focus on the functional relevance of endocannabinoid-mediated plasticity within the framework of emotional responses. Alterations of the endocannabinoid system may constitute an important factor in the aetiology of certain neuropsychiatric disorders, and, in turn, enhancers of endocannabinoid signaling could represent a potential therapeutical tool in the treatment of both anxiety and depressive symptoms.

Early maternal deprivation induces gender‐dependent changes on the expression of hippocampal CB1 and CB2 cannabinoid receptors of neonatal rats

Early maternal deprivation (MD) in rats (24 h, postnatal day 9–10) is a model for neurodevelopmental stress. There are some data proving that MD affects the endocannabinoid system (ECS) in a gender‐dependent manner, and that these changes may account for the proposed schizophrenia‐like phenotype of MD rats. The impact of MD on cannabinoid receptor distribution in the hippocampus is unknown. The aim of this study is to evaluate the expression of CB1 and CB2 receptors in diverse relevant subregions (DG, CA1, and CA3) of the hippocampus in 13‐day‐old rats by immunohistochemistry and densitometry. MD induced a significant decrease in CB1 immunoreactivity (more marked in males than in females), which was mainly associated with fibers in the strata pyramidale and radiatum of CA1 and in the strata oriens, pyramidale, and radiatum of CA3. In contrast, MD males and females showed a significant increase in CB2 immunoreactivity in the three hippocampal areas analyzed that was detected in neuropil and puncta in the stratum oriens of CA1 and CA3, and in the polymorphic cell layer of the dentate gyrus. A marked sex dimorphism was observed in CA3, with females exhibiting higher CB1 immunoreactivity than males, and in dentate gyrus, with females exhibiting lower CB2 immunoreactivity than males. These results point to a clear association between developmental stress and dysregulation of the ECS. The present MD procedure may provide an interesting experimental model to further address the role of the ECS in neurodevelopmental mental illnesses such as schizophrenia.

Behavioural and neuroendocrine effects of cannabinoids in critical developmental periods.

The present article focuses on psychoneuroendocrine effects of cannabinoids in developing animals, with special emphasis on the perinatal, periweanling and periadolescent periods. We describe and discuss published data dealing with acute and long-term effects of exposure to cannabinoid agonists in such critical periods. Human studies have demonstrated that the consumption of marijuana by women during pregnancy affects the neurobehavioural development of their children. Investigations using animal models provide useful information for a better understanding of the long-lasting deleterious consequences of cannabis exposure during pregnancy and lactation. The increasing use of cannabis among adolescents and its associated public health problems have led to a parallel increase in basic research on appropriate animal models. Chronic administration of cannabinoid agonists during the periadolescent period causes persistent behavioural alterations in adult animals. Some of these alterations may be related to a possible increased risk of psychosis and other neuropsychiatric disorders in early onset cannabis users.

Early maternal deprivation and neonatal single administration with a cannabinoid agonist induce long-term sex-dependent psychoimmunoendocrine effects in adolescent rats

Maternal deprivation [24h on postnatal day 9] might represent an animal model of schizophrenia and behavioural and neurochemical alterations observed in adulthood may be mediated by hippocampal impairments induced by abnormally increased glucocorticoids due to neonatal stress. We aimed to provide new data for psychoimmunoendocrine characterization of this animal model by evaluating its effects in adolescent rats of both genders. In previous studies we found that cannabinoid compounds counteracted the enhanced impulsivity of maternally deprived animals and that the cannabinoid receptor agonist WIN 55,212-2 showed neuroprotective properties in neonatal rats. So, we hypothesised that this compound could counteract at least some of the detrimental effects that we expected to find in maternally deprived animals. Accordingly, the drug was administered immediately after the maternal deprivation period. Maternally deprived males showed significantly decreased motor activity in the holeboard and the plus-maze. The cannabinoid agonist induced, exclusively in males, a significant anxiogenic-like effect, which was reversed by maternal deprivation. In the forced swimming test, both treatments independently induced depressive-like responses. Maternal deprivation reduced immunological function whereas the drug exerted tissue-dependent effects on the immune parameters analysed. Maternally deprived females showed reduced corticosterone levels whereas the cannabinoid agonist increased hormone concentration in all groups. In general, the results show detrimental effects of both treatments as well as intriguing interactions, notably in relation to emotional behaviour and certain immunological responses.

Early maternal deprivation in rats induces gender-dependent effects on developing hippocampal and cerebellar cells

Adult animals submitted to a single prolonged episode of maternal deprivation [24 h, postnatal day 9–10] show behavioral alterations that resemble specific symptoms of schizophrenia. According to the neurodevelopmental theory, these behavioral deficits might be mediated by detrimental neurodevelopmental processes that might be associated, at least partially, with stress‐induced corticosterone responses. In order to address this hypothesis, we have focused on the hippocampus and cerebellar cortex, two brain regions that show high density of glucocorticoid receptors, and analyzed possible neuronal and glial alterations by immunohistochemical techniques. To evaluate the presence of degenerated neurons we used Fluoro‐Jade‐C (FJ‐C) staining and for the study of astrocytes we employed glial fibrillary acidic protein (GFAP). Within control animals, females showed significantly more GFAP positive cells than males and a trend towards more FJ‐C positive cells. Maternal deprivation induced neuronal degeneration and astroglial changes in the hippocampus and cerebellar cortex of neonatal rats that, in general, were more marked in males. This differential effect may be attributable to a greater vulnerability of males to this kind of early environmental insult and/or to sex‐dependent differences in the onset and/or progression of the effects. The present experimental procedure may be instrumental in elucidating sex‐dependent mechanisms of neurodevelopmental psychiatric disorders with a basis in early environmental insults.

Sex-dependent alterations in response to maternal deprivation in rats

We review here our latest results regarding short- and long-term effects of a neonatal maternal deprivation (MD) stress [24h at postnatal day (PND) 9] on diverse psychoneuroimmunoendocrine parameters, pointing out the existence of numerous sexual dimorphisms. Behavioral changes observed in MD animals might be at least in part attributable to neurodevelopmental effects of MD-induced elevated corticosterone levels. Our findings of short-term effects of MD on hippocampal and cerebellar neurons and glial cells appear to support this hypothesis. However, it is important to note that these cellular effects were more marked in males than in females. Moreover, in analyzing the effects of this neonatal stress on the endocannabinoid system (hippocampal endocannabinoid levels and CB1 receptors) we have also found that males were more affected by MD. Since all these sexual dimorphisms were found at an early neonatal age (PND 13), they are attributable to organizational effects of gonadal steroids. We discuss the potential implications of the elevated corticosterone and decreased leptin levels shown by MD animals in their diverse functional alterations, including the above mentioned neural effects as well as the intriguing persistent deficit in their immunological system. We also emphasize the necessity of analyzing the important influence of sex as regards the specific consequences of early life stress.

Detrimental psychophysiological effects of early maternal deprivation in adolescent and adult rodents: altered responses to cannabinoid exposure.

Environmental rearing conditions during the neonatal period are critical for the establishment of neurobiological factors controlling behavior and stress responsiveness. Early maternal deprivation (MD), consisting of a single 24-h maternal deprivation episode during early neonatal life, has been proposed as an animal model for certain psychopathologies including anxiety, depression and schizophrenic-related disorders. Despite first onset of mental disorders usually occur during adolescence, characterization of MD has been mostly developed in adult animals. We review here a series of experiments that were conducted on rats and mice, in which we analyzed the psychoimmunoendocrine outcomes of MD at both adolescence and adulthood. As a whole our results indicate that MD might promote a depressive-like trait that may be present from adolescence to maturity. Maternally deprived adolescent animals also displayed altered locomotor responses, a reduced interest for social investigation and seemed prone for impulsive behavior. Therefore, MD in rodents is further confirmed as a suitable animal model for the study of neuropsychiatric disorders that might become evident during adolescence. Given the increasing consumption of cannabis derivatives among the juvenile population and the reported comorbidity of neuropsychiatric symptoms with cannabis abuse, we also discuss our results indicating altered responses of maternally deprived adolescent animals to cannabinoid compounds.

Gender-dependent cellular and biochemical effects of maternal deprivation on the hippocampus of neonatal rats: A possible role for the endocannabinoid system

Adult animals submitted to a single prolonged episode of maternal deprivation (MD) [24 h, postnatal days (PND) 9–10] show behavioral alterations that resemble specific symptoms of schizophrenia. These behavioral impairments may be related to neuronal loss in the hippocampus triggered by elevated glucocorticoids. Furthermore, our previous data suggested functional relationships between MD stress and the endocannabinoid system. In this study, we addressed the effects of MD on hippocampal glial cells and the possible relationship with changes in plasma corticosterone (CORT) levels. In addition, we investigated the putative involvement of the endocannabinoid system by evaluating (a) the effects of MD on hippocampal levels of endocannabinoids (b) The modulation of MD effects by two inhibitors of endocannabinoids inactivation, the fatty acid amide hydrolase inhibitor N‐arachidonoyl‐serotonin (AA‐5‐HT), and the endocannabinoid reuptake inhibitor, OMDM‐2. Drug treatments were administered once daily from PND 7 to PND 12 at a dose of 5 mg/kg, and the animals were sacrificed at PND 13. MD induced increased CORT levels in both genders. MD males also showed an increased number of astrocytes in CA1 and CA3 areas and a significant increase in hippocampal 2‐arachidonoylglycerol. The cannabinoid compounds reversed the endocrine and cellular effects of maternal deprivation. We provide direct evidence for gender‐dependent cellular and biochemical effects of MD on developmental hippocampus, including changes in the endocannabinoid system.

The endocannabinoid system in critical neurodevelopmental periods: sex differences and neuropsychiatric implications

This review focuses on the endocannabinoid system as a crucial player during critical periods of brain development, and how its disturbance either by early life stressful events or cannabis consumption may lead to important neuropsychiatric signs and symptoms. First we discuss the advantages and limitations of animal models within the framework of neuropsychiatric research and the crucial role of genetic and environmental factors for the establishment of vulnerable phenotypes. We are becoming aware of important sex differences that have emerged in relation to the psychobiology of cannabinoids. We will discuss sexual dimorphisms observed within the endogenous cannabinoid system, as well as those observed with exogenously administered cannabinoids. We start with how the expression of cannabinoid CB1 receptors is regulated throughout development. Then, we discuss recent results showing how an experimental model of early maternal deprivation, which induces long-term neuropsychiatric symptoms, interacts in a sex-dependent manner with the brain endocannabinoid system during development. This is followed by a discussion of differential vulnerability to the pathological sequelae stemming from cannabinoid exposure during adolescence. Next we talk about sex differences in the interactions between cannabinoids and other drugs of abuse. Finally, we discuss the potential implications that organizational and activational actions of gonadal steroids may have in establishing and maintaining sex dependence in the neurobiological actions of cannabinoids and their interaction with stress.

Early maternal deprivation induces changes on the expression of 2-AG biosynthesis and degradation enzymes in neonatal rat hippocampus

Early maternal deprivation (MD) in rats (24h, PND 9-10) is a model for neurodevelopmental stress. Our previous data showed that MD altered the hippocampal levels of the endocannabinoid 2-AG and the expression of hippocampal cannabinoid receptors in 13-day-old rats, with males being more markedly affected. The aim of this study was to analyze the impact of MD on the enzymes involved in 2-AG biosynthesis (DAGLalpha and DAGLbeta) and degradation (MAGL) in relevant areas (DG, CA1, CA3) of the hippocampus in 13-day-old neonatal rats. The expression of the enzymes was evaluated by quantitative RT-PCR, immunohistochemistry, and densitometry. MD induced a significant increase in DAGLalpha immunoreactivity in both males and females, which was mainly associated with fibers in the polymorphic cell layer of the dentate gyrus and in the stratum pyramidale of CA3. In contrast, the molecular layer of the dentate gyrus showed a significant decrease in DAGLalpha immunoreactivity in MD males and females. No changes were observed in DAGLbeta immunoreactivity. MD induced a significant decrease in MAGL immunoreactivity in hippocampal CA3 and CA1 areas, more marked in males than in females, and that was mainly associated with fibers in all strata of CA3 and CA1. The results also showed a significant decrease of MAGL mRNA levels in MD males. These data support a clear association between neurodevelopmental stress and dysregulation of the endocannabinoid system. This association may be relevant for schizophrenia and other neurodevelopmental psychiatric disorders.