Microbiota influence on behavior: Integrative analysis of serotonin metabolism and behavioral profile in germ-free mice.

Previous studies on germ-free (GF) animals have described altered anxiety-like and social behaviors together with dysregulations in brain serotonin (5-HT) metabolism. Alterations in circulating 5-HT levels and gut 5-HT metabolism have also been reported in GF mice. In this study, we conducted an integrative analysis of various behaviors as well as markers of 5-HT metabolism in the brain and along the GI tract of GF male mice compared with conventional (CV) ones. We found a strong decrease in locomotor activity, accompanied by some signs of increased anxiety-like behavior in GF mice compared with CV mice. Brain gene expression analysis showed no differences in HTR1A and TPH2 genes. In the gut, we found decreased TPH1 expression in the colon of GF mice, while it was increased in the cecum. HTR1A expression was dramatically decreased in the colon, while HTR4 expression was increased both in the cecum and colon of GF mice compared with CV mice. Finally, SLC6A4 expression was increased in the ileum and colon of GF mice compared with CV mice. Our results add to the evidence that the microbiota is involved in regulation of behavior, although heterogeneity among studies suggests a strong impact of genetic and environmental factors on this microbiota-mediated regulation. While no impact of GF status on brain 5-HT was observed, substantial differences in gut 5-HT metabolism were noted, with tissue-dependent results indicating a varying role of microbiota along the GI tract.

Effects of a Lacticaseibacillus Mix on Behavioural, Biochemical, and Gut Microbial Outcomes of Male Mice following Chronic Restraint Stress.

The effect of supplementation with Lactobacillus strains to prevent the consequences of chronic stress on anxiety in mouse strains sensitive to stress and the consequences on gut microbiota have been relatively unexplored. Thus, we administered a Lacticaseibacillus casei LA205 and Lacticaseibacillus paracasei LA903 mix to male BALB/cByJrj mice two weeks before and during 21-day chronic restraint stress (CRS) (non-stressed/solvent (NS-PBS), non-stressed/probiotics (NS-Probio), CRS/solvent (S-PBS), CRS/probiotics (S-Probio)). CRS resulted in lower body weight and coat state alteration, which were attenuated by the probiotic mix. S-Probio mice showed less stress-associated anxiety-like behaviours than their NS counterpart, while no difference was seen in PBS mice. Serum corticosterone levels were significantly higher in the S-Probio group than in other groups. In the hippocampus, mRNA expression of dopamine and serotonin transporters was lower in S-Probio than in S-PBS mice. Few differences in bacterial genera proportions were detected, with a lower relative abundance of Alistipes in S-Probio vs. S-PBS. CRS was accompanied by a decrease in the proportion of caecal acetate in S-PBS mice vs. NS-PBS, but not in the intervention groups. These data show that the probiotic mix could contribute to better coping with chronic stress, although the precise bacterial mechanism is still under investigation.

Male mice engaging differently in emotional eating present distinct plasmatic and neurological profiles.

Objective: Stressed individuals tend to turn to calorie-rich food, also known as 'comfort food' for the temporary relief it provides. The emotional eating drive is highly variable among subjects. Using a rodent model, we explored the plasmatic and neurobiological differences between 'high and low emotional eaters' (HEE and LEE).Methods: 40 male mice were exposed for 5 weeks to a protocol of unpredictable chronic mild stress. Every 3 or 4 days, they were submitted to a 1-h restraint stress, immediately followed by a 3-h period during which a choice between chow and chocolate sweet cereals was proposed. The dietary intake was measured by weighing. Plasmatic and neurobiological characteristics were compared in mice displaying high vs low intakes.Results: Out of 40 mice, 8 were considered as HEE because of their high post-stress eating score, and 8 as LEE because of their consistent low intake. LEE displayed higher plasma corticosterone and lower levels of NPY than HEE, but acylated and total ghrelin were similar in both groups. In the brain, the abundance of NPY neurons in the arcuate nucleus of the hypothalamus was similar in both groups, but was higher in the ventral hippocampus and the basal lateral amygdala of LEE. The lateral hypothalamus LEE had also more orexin (OX) positive neurons. Both NPY and OX are orexigenic peptides and mood regulators.Discussion: Emotional eating difference was reflected in plasma and brain structures implicated in emotion and eating regulation. These results concur with the psychological side of food consumption.

The Impact of Gut Microbiota-Derived Metabolites in Autism Spectrum Disorders.

Autism Spectrum Disorder (ASD) is a set of neurodevelopmental disorders characterised by behavioural impairment and deficiencies in social interaction and communication. A recent study estimated that 1 in 89 children have developed some form of ASD in European countries. Moreover, there is no specific treatment and since ASD is not a single clinical entity, the identification of molecular biomarkers for diagnosis remains challenging. Besides behavioural deficiencies, individuals with ASD often develop comorbid medical conditions including intestinal problems, which may reflect aberrations in the bidirectional communication between the brain and the gut. The impact of faecal microbial composition in brain development and behavioural functions has been repeatedly linked to ASD, as well as changes in the metabolic profile of individuals affected by ASD. Since metabolism is one of the major drivers of microbiome-host interactions, this review aims to report emerging literature showing shifts in gut microbiota metabolic function in ASD. Additionally, we discuss how these changes may be involved in and/or perpetuate ASD pathology. These valuable insights can help us to better comprehend ASD pathogenesis and may provide relevant biomarkers for improving diagnosis and identifying new therapeutic targets.

Relation between Mood and the Host-Microbiome Co-Metabolite 3-Indoxylsulfate: Results from the Observational Prospective NutriNet-Santé Study.

Gut microbiota metabolizes tryptophan into indole, which can influence brain and behavior. Indeed, some oxidized derivatives of indole, formed in the liver, have neuroactive properties, and indole overproduction by the gut microbiota induces an anxio-depressive phenotype in rodents. The aim of this study was to investigate in humans whether there was a relationship between recurrent depressive symptoms and indole production by the gut microbiota. A case-control study was conducted in 45-65-year-old women, who were participants in the observational prospective NutriNet-Santé Study. Cases were defined as having two Center for Epidemiological Studies-Depression Scales (CES-D) scores ≥ 23 at a two-year interval (recurrent depressive symptoms, n = 87). Each case was matched with two controls (two CES-D <23; n = 174). Urinary excretion of 3-indoxylsulfate, the major final metabolite of indole, was used as a biomarker of indole production by the gut microbiota. Conditional logistic regression models for paired data showed a positive association between urinary 3-indoxylsulfate concentrations, grouped in tertiles, and recurrent depressive symptoms (odds ratio = 2.46, p for trend = 0.0264 in the final model adjusted for confounding factors). This association suggested that indole production by the gut microbiota may play a role in the onset of mood disorders in humans.

Depressive symptoms, fruit and vegetables consumption and urinary 3-indoxylsulfate concentration: a nested case-control study in the French Nutrinet-Sante cohort.

PURPOSE: Previous epidemiologic studies have provided some evidence of an inverse association between fruit and vegetables consumption and risk of developing recurrent depressive symptoms. This association could possibly be explained by the role of such dietary factors on the gut microbiota. Especially, indole, a metabolite of tryptophan produced by gut bacteria, may be associated with the development of mood disorders. Thus, the purpose of this study was to investigate relationships between fruit and vegetables intake, recurrent depressive symptoms and indole, using measurement of its main urinary excretion form, i.e., 3-indoxylsulfate, as a biomarker. METHODS: A nested case-control study was conducted in 891 women (aged 45-65) participating to the web-based NutriNet-Santé cohort with available dietary data and biological samples. Cases (individuals with recurrent depressive symptoms, n = 297) were defined as having two Center for Epidemiologic Studies-Depression Scale (CES-D) scores ≥ 16 during the follow-up and were matched with 2 controls having two CES-D scores < 16. Urinary 3-indoxylsulfate concentration was measured as a biomarker of indole production by the gut microbiota. Multivariable conditional logistic regression models were used to test the association of both fruit and vegetables consumption and urine 3-indoxylsulfate measurements with recurrent depressive symptoms. We also tested the association between fruit and vegetables consumption and urinary 3-indoxylsulfate levels using multivariate analysis of variance models. RESULTS: We found a significant inverse association between fruit and vegetables consumption and the risk of having recurrent depressive symptoms over a 2-year period. Fruit and vegetables consumption was inversely associated to urinary 3-indoxylsulfate concentration. However, no significant association was observed between urinary 3-indoxylsulfate levels and recurrent depressive symptoms within this sample. CONCLUSIONS: Our results confirm that low fruit and vegetables consumption could be associated with recurrent depressive symptoms. We also found an inverse association between fruit and vegetable intake and urinary levels of 3-indoxylsulfate. However, it is not possible to conclude to a possible mediation role of the indole produced by the gut microbiota from tryptophan, since there was no relationship between 3-indoxylsulfate and recurrent depressive symptoms.

A Probiotic Mixture Induces Anxiolytic- and Antidepressive-Like Effects in Fischer and Maternally Deprived Long Evans Rats.

A role of the gut microbiota in psychiatric disorders is supported by a growing body of literature. The effects of a probiotic mixture of four bacterial strains were studied in two models of anxiety and depression, naturally stress-sensitive Fischer rats and Long Evans rats subjected to maternal deprivation. Rats chronically received either the probiotic mixture (1.109 CFU/day) or the vehicle. Anxiety- and depressive-like behaviors were evaluated in several tests. Brain monoamine levels and gut RNA expression of tight junction proteins (Tjp) and inflammatory markers were quantified. The gut microbiota was analyzed in feces by 16S rRNA gene sequencing. Untargeted metabolite analysis reflecting primary metabolism was performed in the cecal content and in serum. Fischer rats treated with the probiotic mixture manifested a decrease in anxiety-like behaviors, in the immobility time in the forced swimming test, as well as in levels of dopamine and its major metabolites, and those of serotonin metabolites in the hippocampus and striatum. In maternally deprived Long Evans rats treated with the probiotic mixture, the number of entries into the central area in the open-field test was increased, reflecting an anxiolytic effect. The probiotic mixture increased Tjp1 and decreased Ifnγ mRNA levels in the ileum of maternally deprived rats. In both models, probiotic supplementation changed the proportions of several Operational Taxonomic Units (OTU) in the gut microbiota, and the levels of certain cecal and serum metabolites were correlated with behavioral changes. Chronic administration of the tested probiotic mixture can therefore beneficially affect anxiety- and depressive-like behaviors in rats, possibly owing to changes in the levels of certain metabolites, such as 21-deoxycortisol, and changes in brain monoamines.

Genome, Environment, Microbiome and Metabolome in Autism (GEMMA) Study Design: Biomarkers Identification for Precision Treatment and Primary Prevention of Autism Spectrum Disorders by an Integrated Multi-Omics Systems Biology Approach.

Autism Spectrum Disorder (ASD) affects approximately 1 child in 54, with a 35-fold increase since 1960. Selected studies suggest that part of the recent increase in prevalence is likely attributable to an improved awareness and recognition, and changes in clinical practice or service availability. However, this is not sufficient to explain this epidemiological phenomenon. Research points to a possible link between ASD and intestinal microbiota because many children with ASD display gastro-intestinal problems. Current large-scale datasets of ASD are limited in their ability to provide mechanistic insight into ASD because they are predominantly cross-sectional studies that do not allow evaluation of perspective associations between early life microbiota composition/function and later ASD diagnoses. Here we describe GEMMA (Genome, Environment, Microbiome and Metabolome in Autism), a prospective study supported by the European Commission, that follows at-risk infants from birth to identify potential biomarker predictors of ASD development followed by validation on large multi-omics datasets. The project includes clinical (observational and interventional trials) and pre-clinical studies in humanized murine models (fecal transfer from ASD probands) and in vitro colon models. This will support the progress of a microbiome-wide association study (of human participants) to identify prognostic microbiome signatures and metabolic pathways underlying mechanisms for ASD progression and severity and potential treatment response.

Role of the Gut Microbiota in the Pathophysiology of Autism Spectrum Disorder: Clinical and Preclinical Evidence.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder affecting 1 in 160 people in the world. Although there is a strong genetic heritability to ASD, it is now accepted that environmental factors can play a role in its onset. As the prevalence of gastrointestinal (GI) symptoms is four-times higher in ASD patients, the potential implication of the gut microbiota in this disorder is being increasingly studied. A disturbed microbiota composition has been demonstrated in ASD patients, accompanied by altered production of bacterial metabolites. Clinical studies as well as preclinical studies conducted in rodents have started to investigate the physiological functions that gut microbiota might disturb and thus underlie the pathophysiology of ASD. The first data support an involvement of the immune system and tryptophan metabolism, both in the gut and central nervous system. In addition, a few clinical studies and a larger number of preclinical studies found that modulation of the microbiota through antibiotic and probiotic treatments, or fecal microbiota transplantation, could improve behavior. Although the understanding of the role of the gut microbiota in the physiopathology of ASD is only in its early stages, the data gathered in this review highlight that this role should be taken in consideration.

The gut microbiota metabolite indole increases emotional responses and adrenal medulla activity in chronically stressed male mice.

BACKGROUND AND AIMS: The gut microbiota produces metabolites that are an integral part of the metabolome and, as such, of the host physiology. Changes in gut microbiota metabolism could therefore contribute to pathophysiological processes. We showed previously that a chronic and moderate overproduction of indole from tryptophan in male individuals of the highly stress-sensitive F344 rat strain induced anxiety-like and helplessness behaviors. The aim of the present study was to extend the scope of these findings by investigating whether emotional behaviors of male mice that are moderately stress-sensitive but chronically exposed to environmental stressors would also be affected by indole. METHODS: We colonized germ-free male C3H/HeN mice with a wild-type indole-producing Escherichia coli strain, or with the non-indole producing mutant. Gnotobiotic mice were subjected to an unpredictable chronic mild stress procedure, then to a set of tests aimed at assessing anxiety-like (novelty and elevated plus maze tests) and depression-like behaviors (coat state, splash, nesting, tail suspension and sucrose tests). Results of the individual tests were aggregated into a common z-score to estimate the overall emotional response to chronic mild stress and chronic indole production. We also carried out biochemical and molecular analyses in gut mucosa, plasma, brain hippocampus and striatum, and adrenal glands, to examine biological correlates that are usually associated with stress, anxiety and depression. RESULTS: Chronic mild stress caused coat state degradation and anhedonia in both indole-producing and non-indole producing mice, but it did not influence behaviors in the other tests. Chronic indole production did not influence mice behavior when tests were considered individually, but it increased the overall emotionality z-score, specifically in mice under chronic mild stress. Interestingly, in the same mice, indole induced a dramatic increase of the expression of the adrenomedullary Pnmt gene, which is involved in catecholamine biosynthesis. By contrast, systemic tryptophan bioavailability, brain serotonin and dopamine levels and turnover, as well as expression of gut and brain genes involved in cytokine production and tryptophan metabolism along the serotonin and kynurenine pathways, remained similar in all mice. CONCLUSIONS: Chronic indole production by the gut microbiota increased the vulnerability of male mice to the adverse effects of chronic mild stress on emotional behaviors. It also targeted catecholamine biosynthetic pathway of the adrenal medulla, which plays a pivotal role in body's physiological adaptation to stressful events. Future studies will aim to investigate the action mechanisms responsible for these effects.

BAHD1 haploinsufficiency results in anxiety-like phenotypes in male mice.

BAHD1 is a heterochomatinization factor recently described as a component of a multiprotein complex associated with histone deacetylases HDAC1/2. The physiological and patho-physiological functions of BAHD1 are not yet well characterized. Here, we examined the consequences of BAHD1 deficiency in the brains of male mice. While Bahd1 knockout mice had no detectable defects in brain anatomy, RNA sequencing profiling revealed about 2500 deregulated genes in Bahd1-/- brains compared to Bahd1+/+ brains. A majority of these genes were involved in nervous system development and function, behavior, metabolism and immunity. Exploration of the Allen Brain Atlas and Dropviz databases, assessing gene expression in the brain, revealed that expression of the Bahd1 gene was limited to a few territories and cell subtypes, particularly in the hippocampal formation, the isocortex and the olfactory regions. The effect of partial BAHD1 deficiency on behavior was then evaluated on Bahd1 heterozygous male mice, which have no lethal or metabolic phenotypes. Bahd1+/- mice showed anxiety-like behavior and reduced prepulse inhibition (PPI) of the startle response. Altogether, these results suggest that BAHD1 plays a role in chromatin-dependent gene regulation in a subset of brain cells and support recent evidence linking genetic alteration of BAHD1 to psychiatric disorders in a human patient.

First step of odorant detection in the olfactory epithelium and olfactory preferences differ according to the microbiota profile in mice.

We have previously provided the first evidence that the microbiota modulates the physiology of the olfactory epithelium using germfree mice. The extent to which changes to the olfactory system depend on the microbiota is still unknown. In the present work, we explored if different microbiota would differentially impact olfaction. We therefore studied the olfactory function of three groups of mice of the same genetic background, whose parents had been conventionalized before mating with microbiota from three different mouse strains. Caecal short chain fatty acids profiles and 16S rRNA gene sequencing ascertained that gut microbiota differed between the three groups. We then used a behavioural test to measure the attractiveness of various odorants and observed that the three groups of mice differed in their attraction towards odorants. Their olfactory epithelium properties, including electrophysiological responses recorded by electro-olfactograms and expression of genes related to the olfactory transduction pathway, also showed several differences. Overall, our data demonstrate that differences in gut microbiota profiles are associated with differences in olfactory preferences and in olfactory epithelium functioning.

Indole, a Signaling Molecule Produced by the Gut Microbiota, Negatively Impacts Emotional Behaviors in Rats.

Gut microbiota produces a wide and diverse array of metabolites that are an integral part of the host metabolome. The emergence of the gut microbiome-brain axis concept has prompted investigations on the role of gut microbiota dysbioses in the pathophysiology of brain diseases. Specifically, the search for microbe-related metabolomic signatures in human patients and animal models of psychiatric disorders has pointed out the importance of the microbial metabolism of aromatic amino acids. Here, we investigated the effect of indole on brain and behavior in rats. Indole is produced by gut microbiota from tryptophan, through the tryptophanase enzyme encoded by the tnaA gene. First, we mimicked an acute and high overproduction of indole by injecting this compound in the cecum of conventional rats. This treatment led to a dramatic decrease of motor activity. The neurodepressant oxidized derivatives of indole, oxindole and isatin, accumulated in the brain. In addition, increase in eye blinking frequency and in c-Fos protein expression in the dorsal vagal complex denoted a vagus nerve activation. Second, we mimicked a chronic and moderate overproduction of indole by colonizing germ-free rats with the indole-producing bacterial species Escherichia coli. We compared emotional behaviors of these rats with those of germ-free rats colonized with a genetically-engineered counterpart strain unable to produce indole. Rats overproducing indole displayed higher helplessness in the tail suspension test, and enhanced anxiety-like behavior in the novelty, elevated plus maze and open-field tests. Vagus nerve activation was suggested by an increase in eye blinking frequency. However, unlike the conventional rats dosed with a high amount of indole, the motor activity was not altered and neither oxindole nor isatin could be detected in the brain. Further studies are required for a comprehensive understanding of the mechanisms supporting indole effects on emotional behaviors. As our findings suggest that people whose gut microbiota is highly prone to produce indole could be more likely to develop anxiety and mood disorders, we addressed the issue of the inter-individual variability of indole producing potential in humans. An in silico investigation of metagenomic data focused on the tnaA gene products definitively proved this inter-individual variability.

Olfactory epithelium changes in germfree mice.

Intestinal epithelium development is dramatically impaired in germfree rodents, but the consequences of the absence of microbiota have been overlooked in other epithelia. In the present study, we present the first description of the bacterial communities associated with the olfactory epithelium and explored differences in olfactory epithelium characteristics between germfree and conventional, specific pathogen-free, mice. While the anatomy of the olfactory epithelium was not significantly different, we observed a thinner olfactory cilia layer along with a decreased cellular turn-over in germfree mice. Using electro-olfactogram, we recorded the responses of olfactory sensitive neuronal populations to various odorant stimulations. We observed a global increase in the amplitude of responses to odorants in germfree mice as well as altered responses kinetics. These changes were associated with a decreased transcription of most olfactory transduction actors and of olfactory xenobiotic metabolising enzymes. Overall, we present here the first evidence that the microbiota modulates the physiology of olfactory epithelium. As olfaction is a major sensory modality for most animal species, the microbiota may have an important impact on animal physiology and behaviour through olfaction alteration.

Vulnerability to opiate intake in maternally deprived rats: implication of MeCP2 and of histone acetylation.

We previously showed that maternal deprivation predisposes male rats to anxiety, accompanied with an increase in their opiate consumption. In the present report, we searched for brain epigenetic mechanisms that possibly underlie this increase. For that, we examined the expression of the methyl-CpG-binding protein MeCP2 and of the histone deacetylases HDAC2 and HDAC3, as well as the acetylation status of histone H3 and H4 in mesolimbic structures of adult maternally deprived rats, using immunohistochemistry and Western blot analysis. A long-lasting increase in MeCP2 expression was found throughout the striatum of deprived rats. Enhanced HDAC2 expression and increased nuclear HDAC activity in the nucleus accumbens of deprived rats were associated with lower acetylation levels of histone H3 and H4. Treatment for 3 weeks with the HDAC inhibitor sodium valproate abolished HDAC activation together with the decrease in the acetylation levels of histone H4, and was accompanied with normalized oral morphine consumption. The data indicate that epigenetic mechanisms induced by early adverse environment memorize life experience to trigger greater opiate vulnerability during adult life. They suggest that sodium valproate may lessen vulnerability to opiate intake, particularly in subgroups of individuals subjected to adverse postnatal environments.

Absence of the gut microbiota enhances anxiety-like behavior and neuroendocrine response to acute stress in rats.

BACKGROUND AND AIMS: Establishment of the gut microbiota is one of the most important events in early life and emerging evidence indicates that the gut microbiota influences several aspects of brain functioning, including reactivity to stress. To better understand how the gut microbiota contributes to a vulnerability to the stress-related psychiatric disorders, we investigated the relationship between the gut microbiota, anxiety-like behavior and HPA axis activity in stress-sensitive rodents. We also analyzed the monoamine neurotransmitters in the brain upper structures involved in the regulation of stress and anxiety. METHODS: Germfree (GF) and specific pathogen free (SPF) F344 male rats were first subjected to neurological tests to rule out sensorimotor impairments as confounding factors. Then, we examined the behavior responses of rats to social interaction and open-field tests. Serum corticosterone concentrations, CRF mRNA expression levels in the hypothalamus, glucocorticoid receptor (GR) mRNA expression levels in the hippocampus, and monoamine concentrations in the frontal cortex, hippocampus and striatum were compared in rats that were either exposed to the open-field stress or not. RESULTS: GF rats spent less time sniffing an unknown partner than SPF rats in the social interaction test, and displayed a lower number of visits to the aversive central area, and an increase in latency time, time spent in the corners and number of defecations in the open-field test. In response to the open-field stress, serum corticosterone concentrations were 2.8-fold higher in GF than in SPF rats. Compared to that of SPF rats, GF rats showed elevated CRF mRNA expression in the hypothalamus and reduced GR mRNA expression in the hippocampus. GF rats also had a lower dopaminergic turnover rate in the frontal cortex, hippocampus and striatum than SPF rats. CONCLUSIONS: In stress-sensitive F344 rats, absence of the gut microbiota exacerbates the neuroendocrine and behavioral responses to acute stress and the results coexist with alterations of the dopaminergic turnover rate in brain upper structures that are known to regulate reactivity to stress and anxiety-like behavior.

The dopamine D1 receptor agonist SKF 38393 improves temporal order memory performance in maternally deprived rats.

Previously, we showed that maternal deprivation (MD) (3h/day, postnatal-day 1-14) impaired the performance at adulthood in the object temporal order memory task (TMT) that principally implicates the medial prefrontal cortex (mPFC). Dopamine (DA) transmission in the PFC may play a critical role in the achievement of the TMT. Here, to investigate whether MD could results in dysfunction of the DA system in the mPFC, we assessed in this region the tissue contents and extracellular levels of DA and its metabolites, as the density of D1 receptor. Besides we examined whether an agonist of the DA receptor D1, the SKF38393, could have a beneficial effect on the performance of deprived (D) rats in the TMT. We observed that MD induced a significant reduction of the extracellular level of DOPAC in the mPFC and in the density of the D1 receptor in the anterior cingulate cortex, a sub-region of mPFC. On the other hand, we observed that an acute systemic injection of a D1 receptor agonist, SKF38393, was effective to correct the memory deficiency of D rats in the TMT, when administered before the retrieval phase. We showed that a stress suffered by rats during the perinatal period led to dysfunction of the adult DA system, possibly triggering greater vulnerability to cognitive and mood disorders. Interestingly, an acute administration of a D1 receptor agonist in adulthood was sufficient to improve the deficit in the temporal memory. A better understanding of this phenomenon would permit the development of treatments adapted to patients with a history of early traumatic experiences.

Possible involvement of endocannabinoids in the increase of morphine consumption in maternally deprived rat.

Whether adolescent exposure to chronic delta-9-tetrahydrocannabinol (THC) facilitates progression to opioid consumption is still controversial. In a maternal deprivation model (3 h daily from postnatal day 1-14), we previously reported that adolescent exposure to chronic THC blocks morphine dependence in maternally deprived (D) rats. Owing to the existence of a functional cross-interaction between the opioid and cannabinoid systems in reward, we evaluated if the vulnerability to opiate reward in D rats, may involve an alteration of the endocannabinoid system. Anandamide and 2-arachidonoylglycerol (2-AG), were quantified in the striatum and mesencephalon of adolescent and adult D and non-deprived (animal facility rearing, AFR) rats by isotope dilution liquid chromatography-mass spectrometry. Oral morphine self-administration behavior was analyzed for 14 weeks, 24 days after chronic injection of the cannabinoid CB1 receptor antagonist/inverse agonist, SR141716A (3 mg/kg) for 2 weeks during adolescence (PND 35-48). Adolescent D rats exhibited higher basal levels of anandamide than adolescent AFR rats in the nucleus accumbens (38%), the caudate-putamen nucleus (62%) and the mesencephalon (320%), whereas adult D rats showed an increase of anandamide and 2-AG levels in the nucleus accumbens (50% and 24%, respectively) and of 2-AG in the caudate-putamen nucleus (48%), compared to adult AFR rats. Chronic administration of SR141716A to adolescent D rats blocked the escalation behavior in the morphine consumption test. Our data suggest that altered brain endocannabinoid levels may contribute to the escalation behavior in the morphine consumption test in a maternal deprivation model.

Maternal deprivation induces deficits in temporal memory and cognitive flexibility and exaggerates synaptic plasticity in the rat medial prefrontal cortex.

Early life adverse events can lead to structural and functional impairments in the prefrontal cortex (PFC). Here, we investigated whether maternal deprivation (MD) alters PFC-dependent executive functions, neurons and astrocytes number and synaptic plasticity in adult male Long-Evans rats. The deprivation protocol consisted of a daily separation of newborn Long-Evans pups from their mothers and littermates 3h/day postnatal day 1-14. Cognitive performances were assessed in adulthood using the temporal order memory task (TMT) and the attentional set-shifting task (ASST) that principally implicates the PFC and the Morris water maze task (WMT) that does not essentially rely on the PFC. The neurons and astrocytes of the prelimbic (PrL) area of the medial PFC (mPFC) were immunolabelled respectively with anti-NeuN and anti-GFAP antibodies and quantified by stereology. The field potentials evoked by electrical stimulation of ventral hippocampus (ventral HPC) were recorded in vivo in the PrL area. In adulthood, MD produced cognitive deficits in two PFC-dependent tasks, the TMT and ASST, but not in the WMT. In parallel, MD induced in the prelimbic area of the medial PFC an upregulation of long-term potentiation (LTP), without any change in the number of neurons and astrocytes. We provide evidence that MD leads in adults to an alteration of the cognitive abilities dependent on the PFC, and to an exaggerated synaptic plasticity in this region. We suggest that this latter phenomenon may contribute to the impairments in the cognitive tasks.

Early stress leads to effects on estrous cycle and differential responses to stress.

Women are more susceptible than men to stress-related mental disorders. However, few animal studies have been conducted on females. Given the interactions between gonadic hormones and the hypothalamo-pituitary-adrenal (HPA) axis, we hypothesized that the effects of early stress may be different between males and females depending on the state of their estrous cycle. Using adult Long-Evans rats of both genders, the effects of maternal deprivation were investigated on the estrous cycle length, corticosterone levels after food deprivation or restraint stress procedures, and the negative feedback efficiency of dexamethasone on the HPA axis. The individual length of the estrous cycle was evaluated using vaginal smears. Non-deprived (AFR) females mainly exhibited regular 5-day cycles (40% of the population) and 4-5-day cycles (26%), with fewer 4-day cycles (18%) and irregular cycles (16%). Comparatively, deprived (D) females displayed a significant decrease of 5-day cycles (24%) and a significant increase of irregular cycles (28%). After the restraint stress procedure, D females exhibited higher corticosterone level than AFR females during proestrous. After the food deprivation procedure, D and AFR females maintained dose-response sensitivity to the negative feedback induced by dexamethasone but only during proestrous. No differences were observed between D and AFR males under these experimental conditions. These data highlight the importance of early environmental factors in regulating the spontaneous pattern of the estrous cycle as well as gender- and stressor-dependent sensitivity of the HPA axis according to steroid levels.