The herbicides glyphosate and glufosinate and the cyanotoxin ß-N-methylamino-l-alanine induce long-term motor disorders following postnatal exposure: the importance of prior asymptomatic maternal inflammatory sensitization.

Background: Prenatal maternal immune activation (MIA) and/or perinatal exposure to various xenobiotics have been identified as risk factors for neurological disorders, including neurodegenerative diseases. Epidemiological data suggest an association between early multi-exposures to various insults and neuropathologies. The "multiple-hit hypothesis" assumes that prenatal inflammation makes the brain more susceptible to subsequent exposure to several kinds of neurotoxins. To explore this hypothesis and its pathological consequences, a behavioral longitudinal procedure was performed after prenatal sensitization and postnatal exposure to low doses of pollutants. Methods: Maternal exposure to an acute immune challenge (first hit) was induced by an asymptomatic lipopolysaccharide (LPS) dose (0.008 mg/kg) in mice. This sensitization was followed by exposing the offspring to environmental chemicals (second hit) postnatally, by the oral route. The chemicals used were low doses of the cyanotoxin ß-N-methylamino-l-alanine (BMAA; 50 mg/kg), the herbicide glufosinate ammonium (GLA; 0.2 mg/kg) or the pesticide glyphosate (GLY; 5 mg/kg). After assessing maternal parameters, a longitudinal behavioral assessment was carried out on the offspring in order to evaluate motor and emotional abilities in adolescence and adulthood. Results: We showed that the low LPS immune challenge was an asymptomatic MIA. Even though a significant increase in systemic pro-inflammatory cytokines was detected in the dams, no maternal behavioral defects were observed. In addition, as shown by rotarod assays and open field tests, this prenatal LPS administration alone did not show any behavioral disruption in offspring. Interestingly, our data showed that offspring subjected to both MIA and post-natal BMAA or GLA exposure displayed motor and anxiety behavioral impairments during adolescence and adulthood. However, this synergistic effect was not observed in the GLY-exposed offspring. Conclusion: These data demonstrated that prenatal and asymptomatic immune sensitization represents a priming effect to subsequent exposure to low doses of pollutants. These double hits act in synergy to induce motor neuron disease-related phenotypes in offspring. Thus, our data strongly emphasize that multiple exposures for developmental neurotoxicity regulatory assessment must be considered. This work paves the way for future studies aiming at deciphering cellular pathways involved in these sensitization processes.

Disruption of Astrocyte-Dependent Dopamine Control in the Developing Medial Prefrontal Cortex Leads to Excessive Grooming in Mice.

BACKGROUND: Astrocytes control synaptic activity by modulating perisynaptic concentrations of ions and neurotransmitters including dopamine (DA) and, as such, could be involved in the modulating aspects of mammalian behavior. METHODS: We produced a conditional deletion of the vesicular monoamine transporter 2 (VMAT2) specifically in astrocytes (aVMTA2cKO mice) and studied the effects of the lack of VMAT2 in prefrontal cortex (PFC) astrocytes on the regulation of DA levels, PFC circuit functions, and behavioral processes. RESULTS: We found a significant reduction of medial PFC (mPFC) DA levels and excessive grooming and compulsive repetitive behaviors in aVMAT2cKO mice. The mice also developed a synaptic pathology, expressed through increased relative AMPA versus NMDA receptor currents in synapses of the dorsal striatum receiving inputs from the mPFC. Importantly, behavioral and synaptic phenotypes were rescued by re-expression of mPFC VMAT2 and L-DOPA treatment, showing that the deficits were driven by mPFC astrocytes that are critically involved in developmental DA homeostasis. By analyzing human tissue samples, we found that VMAT2 is expressed in human PFC astrocytes, corroborating the potential translational relevance of our observations in mice. CONCLUSIONS: Our study shows that impairment of the astrocytic control of DA in the mPFC leads to symptoms resembling obsessive-compulsive spectrum disorders such as trichotillomania and has a profound impact on circuit function and behaviors.

Synaptic Plasticity Dysfunctions in the Pathophysiology of 22q11 Deletion Syndrome: Is There a Role for Astrocytes?

The 22q11 deletion syndrome (DS) is the most common microdeletion syndrome in humans and gives a high probability of developing psychiatric disorders. Synaptic and neuronal malfunctions appear to be at the core of the symptoms presented by patients. In fact, it has long been suggested that the behavioural and cognitive impairments observed in 22q11DS are probably due to alterations in the mechanisms regulating synaptic function and plasticity. Often, synaptic changes are related to structural and functional changes observed in patients with cognitive dysfunctions, therefore suggesting that synaptic plasticity has a crucial role in the pathophysiology of the syndrome. Most interestingly, among the genes deleted in 22q11DS, six encode for mitochondrial proteins that, in mouse models, are highly expressed just after birth, when active synaptogenesis occurs, therefore indicating that mitochondrial processes are strictly related to synapse formation and maintenance of a correct synaptic signalling. Because correct synaptic functioning, not only requires correct neuronal function and metabolism, but also needs the active contribution of astrocytes, we summarize in this review recent studies showing the involvement of synaptic plasticity in the pathophysiology of 22q11DS and we discuss the relevance of mitochondria in these processes and the possible involvement of astrocytes.

3Rs-based optimization of mice behavioral testing: The habituation/dishabituation olfactory test.

BACKGROUND: There is clear evidence that most of the paradigms that are used in the field of behavioral neuroscience suffer from a lack of reliability mainly because of oversimplification of both testing procedures and interpretations. In the present study we show how an already existing behavioral test, the olfactory habituation / dishabituation task, can be optimized in such a way that animal number and animal distress could be minimized, number/confidence of behavioral outcomes and number of explored behavioral dimensions could be increased. NEW METHOD: We used ethologically relevant technical and procedural changes associated with videotracking-based automated quantification of sniffing behavior to validate our new setup. Mainly internal and construct validity were challenged through the implementation of a series of simple experiments. RESULTS: We show that the new version of the test: 1) has very good within and inter laboratory replicability, 2) is sensitive to some environmental / experimental factors while insensitive to others, 3) allows investigating hedonism, both state and trait anxiety, efficacy of anxiolytic molecules, acute stress, mental retardation-related social impairments and learning and memory. 4) We also show that interest for both nonsocial and social odors is stable over time which makes repetitive testing possible. CONCLUSIONS: This work paves the way for future studies showing how behavioral tests / procedures may be improved by using ethologically relevant changes, in order to question laboratory animals more adequately. Refining behavioral tests may considerably increase predictivity of preclinical tests and, ultimately, help reinforcing translational research.

Multiple effects of the herbicide glufosinate-ammonium and its main metabolite on neural stem cells from the subventricular zone of newborn mice.

The globally used herbicide glufosinate-ammonium (GLA) is structurally analogous to the excitatory neurotransmitter glutamate, and is known to interfere with cellular mechanisms involved in the glutamatergic system. In this report, we used an in vitro model of murine primary neural stem cell culture to investigate the neurotoxicity of GLA and its main metabolite, 4-methylphosphinico-2-oxobutanoic acid (PPO). We demonstrated that GLA and PPO disturb ependymal wall integrity in the ventricular-subventricular zone (V-SVZ) and alter the neuro-glial differentiation of neural stem cells. GLA and PPO impaired the formation of cilia, with reduced Celsr2 expression after PPO exposure. GLA promoted the differentiation of neuronal and oligodendroglial cells while PPO increased B1 cell population and impaired neuronal fate of neural stem cells. These results confirm our previous in vivo report that developmental exposure to GLA alters neurogenesis in the SVZ, and neuroblast migration along the rostral migratory stream. They also highlight the importance of investigating the toxicity of pesticide degradation products. Indeed, not only GLA, but also its metabolite PPO disrupts V-SVZ homeostasis and provides a novel cellular mechanism underlying GLA-induced neurodevelopmental toxicity. Furthermore, we were able to demonstrate a neurotoxic activity of a metabolite of GLA different from that of GLA active substance for the very first time.

Perinatal Exposure to the Cyanotoxin ß-N-Méthylamino-L-Alanine (BMAA) Results in Long-Lasting Behavioral Changes in Offspring-Potential Involvement of DNA Damage and Oxidative Stress.

We recently demonstrated that perinatal exposure to the glutamate-related herbicide, glufosinate ammonium, has deleterious effects on neural stem cell (NSC) homeostasis within the sub-ventricular zone (SVZ), probably leading to ASD-like symptoms in offspring later in life. In the present study, we aimed to investigate whether perinatal exposure to another glutamate-related toxicant, the cyanobacterial amino acid ß-N-methylamino-L-alanine (BMAA), might also trigger neurodevelopmental disturbances. With this aim, female mice were intranasally exposed to low doses of BMAA, 50 mg kg-1 three times a week from embryonic days 7-10 to postnatal day 21. Behavioral analyses were performed during the offspring's early life and during adulthood. Developmental analyses revealed that perinatal exposure to BMAA hastened the appearance of some reflexes and communicative skills. BMAA-exposed offspring displayed sex-dependent changes in emotional cognition shortly after exposure. Later in life, the female offspring continued to express emotional defects and to display abnormal sociability, while males were less affected. To assess whether early exposure to BMAA had deleterious effects on NSC homeostasis, we exposed mice NSCs to 1 and 3 mM BMAA during 24 h. We found that BMAA-exposed NSCs produced high levels of ROS, highlighting the ability of BMAA to induce oxidative stress. We also showed that BMAA exposure increased the number of γH2AX/53BP1 foci per nucleus, suggesting that BMAA-induced DNA damage in NSCs. Collectively, this data strongly suggests that perinatal exposure to the cyanobacteria BMAA, even at low doses, results in neurobehavioral disturbances during both the postnatal period and adulthood. This is considered to be underpinned at the cellular level through dysregulation of NSC homeostasis in the developing brain.

In utero and lactational exposure to low-doses of the pyrethroid insecticide cypermethrin leads to neurodevelopmental defects in male mice-An ethological and transcriptomic study.

Accumulating evidence suggests that developmental exposure to environmental chemicals may modify the course of brain development, ultimately leading to neuropsychiatric / neurodegenerative disorders later in life. In the present study, we assessed the impact of one of the most frequently used pesticides in both residential and agricultural applications - the synthetic pyrethroid cypermethrin (CYP) - on developmental neurotoxicity (DNT). Female mice were perinatally exposed to low doses of CYP (5 and 20 mg/kg body weight) from gestation to postnatal day 15. Behavioral analyses were performed during the offspring's early life and during adulthood. Postnatal analyses revealed that perinatal exposure to CYP disturbed motor development without modifying sensory and communicative skills. We found that later in life, CYP-exposed offspring expressed maladaptive behaviors in response to highly challenging tasks and abnormal sociability. Transcriptomic analyses performed in the offspring's brain at the end of the exposure, highlighted mitochondrial dysfunction as a relevant pathomechanism underlying CYP-induced DNT. Interestingly, several genes involved in proteostasis maintenance were also shown to be dysregulated suggesting that alterations in biogenesis, folding, trafficking and degradation of proteins may significantly contribute to CYP-related DNT. From a regulatory perspective, this study highlights that behavioral and transcriptomic analyses are complementary tools providing useful direction for better DNT characterization, and as such, should be used together more systematically.

IL-33 receptor ST2 regulates the cognitive impairments associated with experimental cerebral malaria.

Cerebral malaria (CM) is associated with a high mortality rate and long-term neurocognitive impairment in survivors. The murine model of experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA (PbA)-infection reproduces several of these features. We reported recently increased levels of IL-33 protein in brain undergoing ECM and the involvement of IL-33/ST2 pathway in ECM development. Here we show that PbA-infection induced early short term and spatial memory defects, prior to blood brain barrier (BBB) disruption, in wild-type mice, while ST2-deficient mice did not develop cognitive defects. PbA-induced neuroinflammation was reduced in ST2-deficient mice with low Ifng, Tnfa, Il1b, Il6, CXCL9, CXCL10 and Cd8a expression, associated with an absence of neurogenesis defects in hippocampus. PbA-infection triggered a dramatic increase of IL-33 expression by oligodendrocytes, through ST2 pathway. In vitro, IL-33/ST2 pathway induced microglia expression of IL-1ß which in turn stimulated IL-33 expression by oligodendrocytes. These results highlight the IL-33/ST2 pathway ability to orchestrate microglia and oligodendrocytes responses at an early stage of PbA-infection, with an amplification loop between IL-1ß and IL-33, responsible for an exacerbated neuroinflammation context and associated neurological and cognitive defects.

Chronic Treatment with the IDO1 Inhibitor 1-Methyl-D-Tryptophan Minimizes the Behavioural and Biochemical Abnormalities Induced by Unpredictable Chronic Mild Stress in Mice - Comparison with Fluoxetine.

We demonstrated that confronting mice to the Unpredictable Chronic Mild Stress (UCMS) procedure-a validated model of stress-induced depression-results in behavioural alterations and biochemical changes in the kynurenine pathway (KP), suspected to modify the glutamatergic neurotransmission through the imbalance between downstream metabolites such as 3-hydroxykynurenine, quinolinic and kynurenic acids. We showed that daily treatment with the IDO1 inhibitor 1-methyl-D-tryptophan partially rescues UCMS-induced KP alterations as does the antidepressant fluoxetine. More importantly we demonstrated that 1-methyl-D-tryptophan was able to alleviate most of the behavioural changes resulting from UCMS exposure. We also showed that both fluoxetine and 1-methyl-D-tryptophan robustly reduced peripheral levels of proinflammatory cytokines in UCMS mice suggesting that their therapeutic effects might occur through anti-inflammatory processes. KP inhibition might be involved in the positive effects of fluoxetine on mice behaviour and could be a relevant strategy to counteract depressive-like symptoms.

Perinatal Exposure to Glufosinate Ammonium Herbicide Impairs Neurogenesis and Neuroblast Migration through Cytoskeleton Destabilization.

Neurogenesis, a process of generating functional neurons from neural precursors, occurs throughout life in restricted brain regions such as the subventricular zone (SVZ). During this process, newly generated neurons migrate along the rostral migratory stream to the olfactory bulb to replace granule cells and periglomerular neurons. This neuronal migration is pivotal not only for neuronal plasticity but also for adapted olfactory based behaviors. Perturbation of this highly controlled system by exogenous chemicals has been associated with neurodevelopmental disorders. We reported recently that perinatal exposure to low dose herbicide glufosinate ammonium (GLA), leads to long lasting behavioral defects reminiscent of Autism Spectrum Disorder-like phenotype in the offspring (Laugeray et al., 2014). Herein, we demonstrate that perinatal exposure to low dose GLA induces alterations in neuroblast proliferation within the SVZ and abnormal migration from the SVZ to the olfactory bulbs. These disturbances are not only concomitant to changes in cell morphology, proliferation and apoptosis, but are also associated with transcriptomic changes. Therefore, we demonstrate for the first time that perinatal exposure to low dose GLA alters SVZ neurogenesis. Jointly with our previous work, the present results provide new evidence on the link between molecular and cellular consequences of early life exposure to the herbicide GLA and the onset of ASD-like phenotype later in life.

Pre- and postnatal exposure to low dose glufosinate ammonium induces autism-like phenotypes in mice.

Glufosinate ammonium (GLA) is one of the most widely used herbicides in agriculture. As is the case for most pesticides, potential adverse effects of GLA have not been studied from the perspective of developmental neurotoxicity. Early pesticides exposure may weaken the basic structure of the developing brain and cause permanent changes leading to a wide range of lifelong effects on health and/or behavior. Here, we addressed the developmental impact of GLA by exposing female mice to low dose GLA during both pre- and postnatal periods and analyzed potential developmental and behavioral changes of the offspring during infancy and adulthood. A neurobehavioral test battery revealed significant effects of GLA maternal exposure on early reflex development, pup communication, affiliative behaviors, and preference for social olfactory cues, but emotional reactivity and emotional memory remained unaltered. These behavioral alterations showed a striking resemblance to changes seen in animal models of Autistic Spectrum Disorders. At the brain level, GLA maternal exposure caused some increase in relative brain weight of the offspring. In addition, reduced expression of Pten and Peg3 - two genes implicated in autism-like deficits - was observed in the brain of GLA-exposed pups at postnatal day 15. Our work thus provides new data on the link between pre- and postnatal exposure to the herbicide GLA and the onset of autism-like symptoms later in life. It also raises fundamental concerns about the ability of current safety testing to assess risks of pesticide exposure during critical developmental periods.

Rescue of fragile X syndrome phenotypes in Fmr1 KO mice by a BKCa channel opener molecule.

BACKGROUND: Fragile X Syndrome (FXS) is the most common form of inherited intellectual disability and is also associated with autism spectrum disorders. Previous studies implicated BKCa channels in the neuropathogenesis of FXS, but the main question was whether pharmacological BKCa stimulation would be able to rescue FXS neurobehavioral phenotypes. METHODS AND RESULTS: We used a selective BKCa channel opener molecule (BMS-204352) to address this issue in Fmr1 KO mice, modeling the FXS pathophysiology. In vitro, acute BMS-204352 treatment (10 µM) restored the abnormal dendritic spine phenotype. In vivo, a single injection of BMS-204352 (2 mg/kg) rescued the hippocampal glutamate homeostasis and the behavioral phenotype. Indeed, disturbances in social recognition and interaction, non-social anxiety, and spatial memory were corrected by BMS-204352 in Fmr1 KO mice. CONCLUSION: These results demonstrate that the BKCa channel is a new therapeutic target for FXS. We show that BMS-204352 rescues a broad spectrum of behavioral impairments (social, emotional and cognitive) in an animal model of FXS. This pharmacological molecule might open new ways for FXS therapy.

Evidence for a key role of the peripheral kynurenine pathway in the modulation of anxiety- and depression-like behaviours in mice: focus on individual differences.

We previously reported that confronting mice to the Unpredictable Chronic Mild Stress procedure (UCMS) resulted in peripheral and cerebral alterations of the kynurenine pathway (KP). The present study tested whether KP disturbances are associated with differences in anxiety- and depressive-like behaviors in both naïve and UCMS mice. Non-stressed and UCMS mice were subjected to the elevated plus maze test and to the forced swim test. Mice were then sacrificed for quantification of tryptophan (TRP)-serotonin (5-HT) and TRP-kynurenine (KYN) metabolites in two corticolimbic structures involved in the regulation of mood (cingulate cortex=CC; amygdala=AMY). We showed that an elevated peripheral (lung) KYN/TRP ratio is correlated to the magnitude of anxiodepressive-like phenotypes only in UCMS mice. We also observed, in UCMS mice, that a high peripheral (lung) KYN/TRP ratio is associated with an increased metabolism of 5-HT in CC and a reduced level of kynurenic acid (KYNA) in AMY. Our results suggest that elevated peripheral KP might underlie cerebral biochemical changes and might consequently be involved in the modulation of behavior but only in UCMS mice. These findings make more complete the comprehension of the KP involvement in behavioral changes induced by chronic stress and suggest that it could play a crucial role in the modulation of emotional states.

Peripheral and cerebral metabolic abnormalities of the tryptophan-kynurenine pathway in a murine model of major depression.

Occurring both peripherally and centrally, the kynurenine pathway (KP) - an alternative pathway to 5-HT synthesis from tryptophan (TRP) - could be of particular value to better understand the link between peripheral changes of circulating levels of glucocorticoids (GC)/proinflammatory cytokines and altered neurotransmission observed in depressed patients. Indeed, it is activated by these mediators of stress and can produce several neuroactive compounds like quinolinic acid (QUIN) and kynurenic acid (KYNA) that can respectively increase and decrease glutamate concentration in brain. In order to characterize the role of both the peripheral and cerebral KP in the pathophysiology of depressive disorders, we used the Unpredictable Chronic Mild Stress (UCMS) to induce a depressive-like syndrome and we then measured the level of relevant TRP-KYN pathway metabolites: KYN, 3-hydroxykynurenine (3HK; precursor of QUIN) and KYNA. We also measured TRP-5HT pathway metabolites: TRP, 5-HT, 5-HIAA. We showed that UCMS increased TRP catabolism along the KP in the periphery. 5-HT and KYN were found to be strongly negatively correlated in all brain structures of control mice and of UCMS mice except in the hippocampus. More importantly we found that KYN was preferentially metabolized along the QUIN pathway at the subcortical level (amygdala/striatum) whereas, at the cortical level (cingulate cortex), the QUIN pathway was reduced. Considering the role of these metabolites on the glutamatergic neurotransmission, we propose that such KP alterations could participate to the cortical/subcortical glutamatergic alterations reported in depressed patients.

Effects of neuronal and inducible NOS inhibitor 1-[2-(trifluoromethyl) phenyl] imidazole (TRIM) in unpredictable chronic mild stress procedure in mice.

Nitric oxide is an intracellular messenger which is involved in several functions and pathologies such as depression, anxiety, learning and memory. In many studies nitric oxide synthase inhibitors (NOSI) were shown to possess antidepressant-like effects in animal models of depression. The aim of this study is to investigate the effects of a selective neuronal and inducible nitric oxide synthase inhibitor TRIM (30 mg/kg/day, 35 days) in mice subjected to unpredictable chronic mild stress and then compare it's effect with a conventional selective serotonin reuptake inhibitor fluoxetine (15 mg/kg/day, 35 days). Stressed vehicle animals showed a significant disturbed coat state when compared with nonstressed animals and this effect was reversed by TRIM or fluoxetine. Both TRIM and fluoxetine prevented the stress-induced deficit in the grooming behaviour in the splash test. TRIM and fluoxetine also significantly decreased the attack frequency when compared to the stressed control group in the resident-intruder test. These results support the assumption that NOS inhibitors can be a new class of antidepressant drugs possibly acting on neuronal NOS.