The Impact of Mild Chronic Stress and Maternal Experience in the Fmr1 Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is a pervasive developmental disorder and the most common monogenic cause of autism spectrum disorder (ASD). Female heterozygous (HET) carriers play a major role in the transmission of the pathology and present several FXS- and ASD-like behavioral alterations. Despite their clear genetic origins, FXS symptoms are known to be modulated by environmental factors, e.g., exposure to chronic stress, especially during critical life periods, such as pregnancy. Pregnancy, together with pups' care, constitutes maternal experience, i.e., another powerful environmental factor affecting several neurobehavioral functions in females. Here we investigated the impact of maternal experience on the long-term effects of stress in Fmr1-HET female mice. Our findings demonstrated that the behavioral abnormalities of HET females, i.e., hyperactivity and memory deficits, were unaffected by stress or maternal experience. In contrast, stress, independently of maternal experience, induced the appearance of cognitive deficits in WT mice. Maternal experience increased anxiety levels in all mice and enhanced their corticosterone levels, concomitantly promoting the effects of stress on social communication and adrenal glands. In translational terms, these results advance our understanding of the environmental modulation of the behavioral alterations observed in FXS female carriers and highlight the long-term impact of maternal experience and its interactions with chronic stress.

The temporal origin of dentate granule neurons dictates their role in spatial memory.

The dentate gyrus is one of the only brain regions that continues its development after birth in rodents. Adolescence is a very sensitive period during which cognitive competences are programmed. We investigated the role of dentate granule neurons (DGNs) born during adolescence in spatial memory and compared them with those generated earlier in life (in embryos or neonates) or during adulthood by combining functional imaging, retroviral and optogenetic tools to tag and silence DGNs. By imaging DGNs expressing Zif268, a proxy for neuronal activity, we found that neurons generated in adolescent rats (and not embryos or neonates) are transiently involved in spatial memory processing. In contrast, adult-generated DGNs are recruited at a later time point when animals are older. A causal relationship between the temporal origin of DGNs and spatial memory was confirmed by silencing DGNs in behaving animals. Our results demonstrate that the emergence of spatial memory depends on neurons born during adolescence, a function later assumed by neurons generated during adulthood.

Therapeutic efficacy of the BKCa channel opener chlorzoxazone in a mouse model of Fragile X syndrome.

Fragile X syndrome (FXS) is an X-linked neurodevelopmental disorder characterized by several behavioral abnormalities, including hyperactivity, anxiety, sensory hyper-responsiveness, and autistic-like symptoms such as social deficits. Despite considerable efforts, effective pharmacological treatments are still lacking, prompting the need for exploring the therapeutic value of existing drugs beyond their original approved use. One such repurposed drug is chlorzoxazone which is classified as a large-conductance calcium-dependent potassium (BKCa) channel opener. Reduced BKCa channel functionality has been reported in FXS patients, suggesting that molecules activating these channels could serve as promising treatments for this syndrome. Here, we sought to characterize the therapeutic potential of chlorzoxazone using the Fmr1-KO mouse model of FXS which recapitulates the main phenotypes of FXS, including BKCa channel alterations. Chlorzoxazone, administered either acutely or chronically, rescued hyperactivity and acoustic hyper-responsiveness as well as impaired social interactions exhibited by Fmr1-KO mice. Chlorzoxazone was more efficacious in alleviating these phenotypes than gaboxadol and metformin, two repurposed treatments for FXS that do not target BKCa channels. Systemic administration of chlorzoxazone modulated the neuronal activity-dependent gene c-fos in selected brain areas of Fmr1-KO mice, corrected aberrant hippocampal dendritic spines, and was able to rescue impaired BKCa currents recorded from hippocampal and cortical neurons of these mutants. Collectively, these findings provide further preclinical support for BKCa channels as a valuable therapeutic target for treating FXS and encourage the repurposing of chlorzoxazone for clinical applications in FXS and other related neurodevelopmental diseases.

Long-lasting plasticity of hippocampal adult-born neurons.

Adult neurogenesis occurs in the dentate gyrus of the hippocampus, which is a key structure in learning and memory. It is believed that adult-born neurons exert their unique role in information processing due to their high plasticity during immature stage that renders them malleable in response to environmental demands. Here, we demonstrate that, in rats, there is no critical time window for experience-induced dendritic plasticity of adult-born neurons as spatial learning in the water maze sculpts the dendritic arbor of adult-born neurons even when they are several months of age. By ablating neurogenesis within a specific period of time, we found that learning was disrupted when the delay between ablation and learning was extended to several months. Together, these results show that mature adult-born neurons are still plastic when they are functionally integrated into dentate network. Our results suggest a new perspective with regard to the role of neo-neurons by highlighting that even mature ones can provide an additional source of plasticity to the brain to process memory information.

Early Administration of the Phytocannabinoid Cannabidivarin Prevents the Neurobehavioral Abnormalities Associated with the Fmr1-KO Mouse Model of Fragile X Syndrome.

Phytocannabinoids, including the non-addictive cannabis component cannabidivarin (CBDV), have been reported to hold therapeutic potential in several neurodevelopmental disorders (NDDs). Nonetheless, the therapeutic value of phytocannabinoids for treating Fragile X syndrome (FXS), a major NDD, remains unexplored. Here, we characterized the neurobehavioral effects of CBDV at doses of 20 or 100 mg/kg in the Fmr1-knockout (Fmr1-KO) mouse model of FXS using two temporally different intraperitoneal regimens: subchronic 10-day delivery during adulthood (Study 1: rescue treatment) or chronic 5-week delivery at adolescence (Study 2: preventive treatment). Behavioral tests assessing FXS-like abnormalities included anxiety, locomotor, cognitive, social and sensory alterations. Expression of inflammatory and plasticity markers was investigated in the hippocampus and prefrontal cortex. When administered during adulthood (Study 1), the effects of CBDV were marginal, rescuing at the lower dose only the acoustic hyper-responsiveness of Fmr1-KO mice and at both doses their altered hippocampal expression of neurotrophins. When administered during adolescence (Study 2), CBDV at both doses prevented the cognitive, social and acoustic alterations of adult Fmr1-KO mice and modified the expression of several inflammatory brain markers in both wild-type littermates and mutants. These findings warrant the therapeutic potential of CBDV for preventing neurobehavioral alterations associated with FXS, highlighting the relevance of its early administration.

Long-term behavioral effects of prenatal stress in the Fmr1-knock-out mouse model for fragile X syndrome.

Fragile X syndrome (FXS) is a major neurodevelopmental disorder and the most common monogenic cause of autism spectrum disorder (ASD). FXS is caused by a mutation in the X-linked FMR1 gene leading to the absence of the FMRP protein, inducing several behavioral deficits, including motor, emotional, cognitive, and social abnormalities. Beside its clear genetic origins, FXS can be modulated by environmental factors, e.g., stress exposure: indeed the behavioral phenotype of FXS, as well as of ASD patients can be exacerbated by the repeated experience of stressful events, especially early in life. Here we investigated the long-term effects of prenatal exposure to unpredictable chronic stress on the behavioral phenotype of the Fmr1-knock-out (KO) mouse model for FXS and ASD. Mice were tested for FXS- and ASD-relevant behaviors first at adulthood (3 months) and then at aging (18 months), in order to assess the persistence and the potential time-related progression of the stress effects. Stress induced the selective emergence of behavioral deficits in Fmr1-KO mice that were evident in spatial memory only at aging. Stress also exerted several age-specific behavioral effects in mice of both genotypes: at adulthood it enhanced anxiety levels and reduced social interaction, while at aging it enhanced locomotor activity and reduced the complexity of ultrasonic calls. Our findings underline the relevance of gene-environment interactions in mouse models of neurodevelopmental syndromes and highlight the long-term behavioral impact of prenatal stress in laboratory mice.

Autistic-like behavioral effects of prenatal stress in juvenile Fmr1 mice: the relevance of sex differences and gene-environment interactions.

Fragile X Syndrome (FXS) is the most common heritable form of mental retardation and monogenic cause of autism spectrum disorder (ASD). FXS is due to a mutation in the X-linked FMR1 gene and is characterized by motor, cognitive and social alterations, mostly overlapping with ASD behavioral phenotypes. The severity of these symptoms and their timing may be exacerbated and/or advanced by environmental adversity interacting with the genetic mutation. We therefore tested the effects of the prenatal exposure to unpredictable chronic stress on the behavioral phenotype of juveniles of both sexes in the Fmr1 knock-out (KO) mouse model of FXS. Mice underwent behavioral tests at 7-8 weeks of age, that is, when most of the relevant behavioral alterations are absent or mild in Fmr1-KOs. Stress induced the early appearance of deficits in spontaneous alternation in KO male mice, without exacerbating the behavioral phenotype of mutant females. In males stress also altered social interaction and communication, but mostly in WT mice, while in females it induced effects on locomotion and communication in mice of both genotypes. Our data therefore highlight the sex-dependent relevance of early environmental stressors to interact with genetic factors to influence the appearance of selected FXS- and ASD-like phenotypes.

Spatial learning depends on both the addition and removal of new hippocampal neurons.

The role of adult hippocampal neurogenesis in spatial learning remains a matter of debate. Here, we show that spatial learning modifies neurogenesis by inducing a cascade of events that resembles the selective stabilization process characterizing development. Learning promotes survival of relatively mature neurons, apoptosis of more immature cells, and finally, proliferation of neural precursors. These are three interrelated events mediating learning. Thus, blocking apoptosis impairs memory and inhibits learning-induced cell survival and cell proliferation. In conclusion, during learning, similar to the selective stabilization process, neuronal networks are sculpted by a tightly regulated selection and suppression of different populations of newly born neurons.

Age-dependent effect of prenatal stress on hippocampal cell proliferation in female rats.

Stressors occurring during pregnancy can alter the developmental trajectory of offspring and lead to, among other deleterious effects, cognitive deficits and hyperactivity of the hypothalamo-pituitary-adrenal axis. A recent feature of the prenatal stress (PS) model is its reported influence on structural plasticity in hippocampal formation, which sustains both cognitive functions and stress responsiveness. Indeed, we and others have previously reported that males exposed to stress in utero are characterized by a decrease in hippocampal cell proliferation, and consequently neurogenesis, from adolescence to senescence. Recent studies in females submitted to PS have reported conflicting results, ranging from no effect to a decrease in cell proliferation. We hypothesized that changes in cell proliferation in PS female rats are age dependent. To address this issue, we examined the impact of PS on hippocampal cell proliferation in juvenile, young, middle-aged and old females. As hypothesized, we found an age-dependent effect of PS in female rats as cell proliferation was significantly decreased only when animals reached senescence, a time when adrenal gland weight also increased. These data suggest that the deleterious effects of PS on hippocampal cell proliferation in females are either specific to senescence or masked during adulthood by protective factors.

Postnatal stimulation of the pups counteracts prenatal stress-induced deficits in hippocampal neurogenesis.

BACKGROUND: Prenatal stress constitutes a developmental risk factor for later psychopathology. The behavioral disorders are sustained by neurobiological alterations including long-term reduction of hippocampal neurogenesis; its deregulation has been involved in cognitive impairments, mood disorders and addiction. A major goal is to define periods in development and strategies for intervening to prevent the effects of early stressful events. We investigated the ability of a postnatal infantile stimulation to prevent prenatal stress-induced alteration in hippocampal neurogenesis. METHODS: The influence of postnatal handling on prenatal stress-induced changes in hippocampal neurogenesis was examined in 4 and 26 month-old male rats. Three distinct phases of the neurogenesis were studied: proliferation, survival and neuronal differentiation. RESULTS: Prenatal stress reduced hippocampal cell proliferation all throughout life. Furthermore, the survival rate of newborn cells, the number of immature neurons and the number of differentiated new neurons were reduced in young and old prenatally-stressed rats. All those deleterious effects were counteracted by neonatal handling. CONCLUSIONS: These data show that finer aspects of brain shaping can be rewired by environmental influences occurring at sensitive phase of development. They also suggest that infantile stimulation may reverse the appearance of behavioral disorders induced by early life stress.

Does prenatal stress affect latent inhibition? It depends on the gender.

The present experiment was designed to examine the effects of prenatal stress and gender in latent inhibition. Prenatal stress has been proposed as a risk factor both for depression and for schizophrenia, and both of these syndromes are associated with alterations in the functional state of the dopamine system. There is also some evidence that prenatal stress can produce changes in dopamine activity, although the details of the stress-induced changes are a matter of debate. Latent inhibition (LI), which is strongly dependent on dopaminergic activity, consists in delayed Pavlovian conditioning about a stimulus that previously signalled no consequence. We induced prenatal stress by exposing gestating dams to a daily constraint stress during the last week of pregnancy. We tested the rats for LI of conditioned taste aversion by exposing them to sucrose for 3 days prior to conditioning. Irrespective of stress or gender, latent inhibition was observed but the degree of LI varied as a function of both prenatal stress and gender. Unstressed males showed less LI than unstressed females, but prenatal stress increased the amount of LI only in the males. These results are inconsistent with the use of prenatal stress as an animal model for schizophrenia. A model is proposed that accounts for the relationships between gender, dopamine function, cognitive changes and psychiatric pathology.

Influence of ontogenetic age on the role of dentate granule neurons.

New neurons are continuously produced in the adult dentate gyrus of the hippocampus, a key structure in learning and memory. It has been shown that adult neurogenesis is crucial for normal memory processing. However, it is not known whether neurons born during the developmental period and during adulthood support the same functions. Here, we demonstrate that neurons born in neonates (first postnatal week) are activated in different memory processes when they are mature compared to neurons born in adults. By imaging the activation of these two different neuron generations in the same rat and using the IEG Zif268 and Fos, we show that these neurons are involved in discriminating dissimilar contexts and spatial problem solving, respectively. These findings demonstrate that the ontogenetic stage during which neurons are generated is crucial for their function within the memory network.

Individual vulnerability to substance abuse and affective disorders: role of early environmental influences.

One of the most important questions raised by modern psychiatry and experimental psychopathology is the origin of mental diseases. More concisely, clinical and experimental neurosciences are increasingly concerned with the factors that render one individual more vulnerable than another to a given pathological outcome. Animal models are now available to understand the sources of individual differences for specific phenotypes prone to behavioral disadaptations. Over the last 10 years we have explored the consequences of environmental perinatal manipulations in the rat. We have shown that prenatal stress is at the origin of a wide range of physiological and behavioral aberrances such as alterations in the activity of the hormonal stress axis, increased vulnerability to drug of abuse, emotional liability, cognitive impairments and predisposition to pathological aging. Taken together, these abnormalities define a bio-behavioral syndrome. Furthermore, the cognitive disabilities observed in prenatally-stressed rats were recently related to an alteration of neurogenesis in the dentate gyrus, thus confirming the impact of early life events on brain morphology. A second model (handling model) has also been developed in which pups are briefly separated from their mothers during early postnatal life. In contrast with prenatally-stressed animals, handled rats exhibited a reduced emotion response when confronted with novel situations and were protected against age-induced impairments of both the hormonal stress axis and cognitive functions. Taken together, the results of these investigations show that the bio-behavioral phenotype that characterizes each individual is strongly linked to the nature and timing of perinatal experience. Furthermore, data collected in prenatally-stressed animals indicate that this model could be used profitably to understand the etiology and pathophysiology of affective disorders.