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.

XLF/Cernunnos loss impairs mouse brain development by altering symmetric proliferative divisions of neural progenitors.

XLF/Cernunnos is a component of the ligation complex used in classical non-homologous end-joining (cNHEJ), a major DNA double-strand break (DSB) repair pathway. We report neurodevelopmental delays and significant behavioral alterations associated with microcephaly in Xlf-/- mice. This phenotype, reminiscent of clinical and neuropathologic features in humans deficient in cNHEJ, is associated with a low level of apoptosis of neural cells and premature neurogenesis, which consists of an early shift of neural progenitors from proliferative to neurogenic divisions during brain development. We show that premature neurogenesis is related to an increase in chromatid breaks affecting mitotic spindle orientation, highlighting a direct link between asymmetric chromosome segregation and asymmetric neurogenic divisions. This study reveals thus that XLF is required for maintaining symmetric proliferative divisions of neural progenitors during brain development and shows that premature neurogenesis may play a major role in neurodevelopmental pathologies caused by NHEJ deficiency and/or genotoxic stress.

Haslea ostrearia Pigment Marennine Affects Key Actors of Neuroinflammation and Decreases Cell Migration in Murine Neuroglial Cell Model.

Haslea ostrearia, a cosmopolitan marine pennate diatom, produces a characteristic blue pigment called marennine that causes the greening of filter-feeding organisms, such as oysters. Previous studies evidenced various biological activities of purified marennine extract, such as antibacterial, antioxidant and antiproliferative effects. These effects could be beneficial to human health. However, the specific biological activity of marennine remains to be characterized, especially regarding primary cultures of mammals. In the present study, we aimed to determine in vitro the effects of a purified extract of marennine on neuroinflammatory and cell migratory processes. These effects were assessed at non-cytotoxic concentrations of 10 and 50µg/mL on primary cultures of neuroglial cells. Marennine strongly interacts with neuroinflammatory processes in the immunocompetent cells of the central nervous system, represented by astrocytes and microglial cells. An anti-migratory activity based on a neurospheres migration assay has also been observed. These results encourage further study of Haslea blue pigment effects, particularly the identification of molecular and cellular targets affected by marennine, and strengthen previous studies suggesting that marennine has bioactivities which could be beneficial for human health applications.

ß-N-Methyl-Amino-L-Alanine cyanotoxin promotes modification of undifferentiated cells population and disrupts the inflammatory status in primary cultures of neural stem cells.

ß-N-Methyl-Amino-L-Alanine (BMAA) produced by 95% of cyanobacteria is in constant augmentation with cyanobacteria worldwide proliferation due to global warming and eutrophication. Previously, it has been shown that this contaminant induced neurological disorders, notably by acting as a developmental toxin. However, very few studies focus on the impact of BMAA on neuroglial cells, like astrocytes and microglial cells, in a developmental context. In the present study, we investigated whether BMAA disturbs neurogenesis from mice subventricular zone (SVZ) cells and whether this neurotoxin induces neuroinflammation. We show that BMAA at 100 µM disturbs the population of undifferentiated cells (B1 and C cells) and promotes their proliferation. Further, BMAA affects the organization of neuroblasts, indicating that SVZ function could be impaired. BMAA affects neuroinflammatory processes by increasing the release of proinflammatory cytokines IL-1ß, IL-6 and TNFα. Our study adds to evidence that BMAA may disturb the central nervous system homeostasis by targeting glial cells. We highlighted that BMAA may impair SVZ niches and drives astrocytes and microglial cells into a proinflammatory status, with an ameboid shape for microglia.

When pharmaceutical drugs become environmental pollutants: Potential neural effects and underlying mechanisms.

Pharmaceutical drugs have become consumer products, with a daily use for some of them. The volume of production and consumption of drugs is such that they have become environmental pollutants. Their transfer to wastewater through urine, feces or rinsing in case of skin use, associated with partial elimination by wastewater treatment plants generalize pollution in the hydrosphere, including drinking water, sediments, soils, the food chain and plants. Here, we review the potential effects of environmental exposure to three classes of pharmaceutical drugs, i.e. antibiotics, antidepressants and non-steroidal anti-inflammatory drugs, on neurodevelopment. Experimental studies analyzing their underlying modes of action including those related to endocrine disruption, and molecular mechanisms including epigenetic modifications are presented. In addition, the contribution of brain imaging to the assessment of adverse effects of these three classes of pharmaceuticals is approached.

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.

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.

Glufosinate aerogenic exposure induces glutamate and IL-1 receptor dependent lung inflammation.

Glufosinate-ammonium (GLA), the active component of an herbicide, is known to cause neurotoxicity. GLA shares structural analogy with glutamate. It is a powerful inhibitor of glutamine synthetase (GS) and may bind to glutamate receptors. Since these potentials targets of GLA are present in lung and immune cells, we asked whether airway exposure to GLA may cause lung inflammation in mice. A single GLA exposure (1 mg/kg) induced seizures and inflammatory cell recruitment in the broncho-alveolar space, and increased myeloperoxidase (MPO), inducible NO synthase (iNOS), interstitial inflammation and disruption of alveolar septae within 6-24 h. Interleukin 1ß (IL-1ß) was increased and lung inflammation depended on IL-1 receptor 1 (IL-1R1). We demonstrate that glutamate receptor pathway is central, since the N-methyl-D-aspartate (NMDA) receptor inhibitor MK-801 prevented GLA-induced lung inflammation. Chronic exposure (0.2 mg/kg 3× per week for 4 weeks) caused moderate lung inflammation and enhanced airway hyperreactivity with significant increased airway resistance. In conclusion, GLA aerosol exposure causes glutamate signalling and IL-1R-dependent pulmonary inflammation with airway hyperreactivity in mice.

Characterization of seizures induced by acute exposure to an organophosphate herbicide, glufosinate-ammonium.

Glufosinate-ammonium (GLA), the active component of a widely used herbicide, induces convulsions in rodents and humans. In mouse, intraperitoneal treatment with 75 mg/kg GLA generates repetitive tonic-clonic seizures associated with 100% mortality within 72 h after treatment. In this context, we characterized GLA-induced seizures, their histological consequences and the effectiveness of diazepam treatment. Epileptic discharges on electroencephalographic recordings appeared simultaneously in the hippocampus and the cerebral cortex. Diazepam treatment at 6 h immediately stopped the seizures and prevented animal death. However, intermittent seizures were recorded on electroencephalogram from 6 h after diazepam treatment until 24 h, but had disappeared after 15 days. In our model, neuronal activation (c-Fos immunohistochemistry) was observed 6 h after GLA exposure in the dentate gyrus, CA1, CA3, amygdala, piriform and entorhinal cortices, indicating the activation of the limbic system. In these structures, Fluoro-Jade C and Cresyl violet staining did not show neuronal suffering. However, astroglial activation was clearly observed at 24 h and 15 days after GLA treatment in the amygdala, piriform and entorhinal cortices by PCR quantitative, western blot and immunohistochemistry. Concomitantly, glutamine synthetase mRNA expression (PCR quantitative), protein expression (western blot) and enzymatic activity were upregulated. In conclusion, our study suggests that GLA-induced seizures: (a) involved limbic structures and (b) induced astrocytosis without neuronal degeneration as an evidence of a reactive astrocyte beneficial effect for neuronal protection.

Spinal cord oligodendrocyte-derived alarmin IL-33 mediates neuropathic pain.

Neuropathic pain from injury to the peripheral and CNS represents a major health care issue. We have investigated the role of IL-33/IL-33 receptor (ST2) signaling in experimental models of neuropathic pain in mice. Chronic constriction injury (CCI) of the sciatic nerve induced IL-33 production in the spinal cord. IL-33/citrine reporter mice revealed that oligodendrocytes are the main cells expressing IL-33 within the spinal cord together with a minor expression by neurons, microglia. and astrocytes. CCI-induced mechanical hyperalgesia was reduced in IL-33R (ST2)(-/ -) mice compared with wild-type (WT) mice. Intrathecal treatment of WT mice with soluble IL-33 receptor (IL-33 decoy receptor) markedly reduced CCI-induced hyperalgesia. Consistent with these observations, intrathecal injection of IL-33 enhanced CCI hyperalgesia and induced hyperalgesia in naive mice. IL-33-mediated hyperalgesia during CCI was dependent on a reciprocal relationship with TNF-α and IL-1ß. IL-33-induced hyperalgesia was markedly attenuated by inhibitors of PI3K, mammalian target of rapamycin, MAPKs (p38, ERK, and JNK), NF-κB, and also by the inhibitors of glial cells (microglia and astrocytes). Furthermore, targeting these signaling pathways and cells inhibited IL-33-induced TNF-α and IL-1ß production in the spinal cord. Our study, therefore, reveals an important role of oligodendrocyte-derived IL-33 in neuropathic pain.

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.

Visual sensorial impairments in neurodevelopmental disorders: evidence for a retinal phenotype in Fragile X Syndrome.

Visual sensory impairments are common in Mental Deficiency (MD) and Autism Spectrum Disorder (ASD). These defects are linked to cerebral dysfunction in the visual cortical area characterized by the deregulation of axon growth/guidance and dendrite spine immaturity of neurons. However, visual perception had not been addressed, although the retina is part of the central nervous system with a common embryonic origin. Therefore, we investigated retinal perception, the first event of vision, in a murine model of MD with autistic features. We document that retinal function is altered in Fmr1 KO mice, a model of human Fragile X Syndrome. Indeed, In Fmr1 KO mice had a lower retinal function characterized by a decreased photoreceptors neuron response, due to a 40% decrease in Rhodopsin content and to Rod Outer Segment destabilization. In addition, we observed an alteration of the visual signal transmission between photoreceptors and the inner retina which could be attributed to deregulations of pre- and post- synaptic proteins resulting in retinal neurons synaptic destabilization and to retinal neurons immaturity. Thus, for the first time, we demonstrated that retinal perception is altered in a murine model of MD with autistic features and that there are strong similarities between cerebral and retinal cellular and molecular defects. Our results suggest that both visual perception and integration must be taken into account in assessing visual sensory impairments in MD and ASD.

Coupling between hydrodynamic forces and planar cell polarity orients mammalian motile cilia.

In mammals, motile cilia cover many organs, such as fallopian tubes, respiratory tracts and brain ventricles. The development and function of these organs critically depend on efficient directional fluid flow ensured by the alignment of ciliary beating. To identify the mechanisms involved in this process, we analysed motile cilia of mouse brain ventricles, using biophysical and molecular approaches. Our results highlight an original orientation mechanism for ependymal cilia whereby basal bodies first dock apically with random orientations, and then reorient in a common direction through a coupling between hydrodynamic forces and the planar cell polarity (PCP) protein Vangl2, within a limited time-frame. This identifies a direct link between external hydrodynamic cues and intracellular PCP signalling. Our findings extend known PCP mechanisms by integrating hydrodynamic forces as long-range polarity signals, argue for a possible sensory role of ependymal cilia, and will be of interest for the study of fluid flow-mediated morphogenesis.

Brain pathways mediating the pro-aggressive effect of the steroid sulfatase (Sts) gene.

STS is the single enzyme that converts all steroid sulfates into their free steroid forms. Initiation of attack behavior against conspecific male mice appeared to be linked to Sts. Here we have confirmed the role of Sts through an association study with attack behavior. Previous studies indicated a positive correlation between the initiation of attack behavior and liver STS concentration levels in male mice, but this finding was not compatible with established knowledge of STS mechanisms. High STS concentrations induce low concentrations of sulfated steroids. Sulfated and un-sulfated steroids are GABA(A) receptor agonists and NMDA receptor positive allosteric modulators. This synaptic pattern of functioning can generate attack behavior and we have confirmed here that an injection of the sulfated steroid dehydroepiandrosterone sulfate (DHEA-S) increases attack behavior. To solve the paradox, we measured the transcription activity of the genes underlying the pathways involved in the hydrolysis of sulfated steroids and leading to the formation of un-conjugated steroids in the mouse brain. We observed that the genes monitoring the steroid biosynthesis pathways exhibited a transcription pattern resulting in an increased sulfotransferase activity in the attacking males that could counterbalance the de-sulfating activity of Sts in the attacking mice.

Attack behaviors in mice: from factorial structure to quantitative trait loci mapping.

The emergence or non-emergence of attack behavior results from interaction between the genotype and the conditions under which the mice are tested. Inbred mice of the same strain reared or housed under conditions do not react the same way; reactions also vary according to the place selected for testing and the different opponents. A factor analysis showed that the attack behavior in non-isolated males, tested in neutral area covaried with high testosterone and steroid sulfatase and low brain 5-hydroxytriptamine (5-HT), beta-endorphin and Adrenocorticotropic Hormone (ACTH) concentration, whereas, for isolated males tested in their own housing cage, it covaried with high testosterone activity and low brain 5-HT concentration. A wide genome scan was performed with two independent populations derived from C57BL/6J and NZB/BlNJ, each being reared, housed and tested under highly contrasting conditions, as described above, and confronted with A/J standard males. Common Quantitative Trait Loci emerged for two rearing/testing conditions. For rattling latency we detected Quantitative Trait Loci on Mus musculus chromosome 8 (MMU8) (at 44, LOD score=3.51 and 47 cM, LOD score=6.22, for the first and the second conditions) and on MMU12 (at 39 cM, LOD score=3.69 and at 41 cM, LOD score=2.99, respectively). For the number of attacks, Quantitative Trait Loci were common: on MMU11 at 39 cM LOD score=4.51 and 45 cM, LOD score=3.05, respectively, and on MMU12 (17 cM, LOD score=2.71 and 24 cM, LOD score=3.10). The steroid sulfatase gene (Sts), located on the X-Y pairing region, was linked, but only in non-isolated males, tested in neutral area for rattling latency, first attack latency, and number of attacks (LOD scores=4.9, 4.79 and 3.57, respectively). We found also that the Quantitative Trait Locus encompassing Sts region interacted with other Quantitative Trait Loci. These results indicate that attack behavior measured in different rearing and testing conditions have different biological and genetic correlates. This suggests that further explorations should be done with standardized tests and, in addition, with a wide range of tests, so as to gain an understanding of the true impact of genes or pharmacological treatments on specific categories of aggressive behavior.