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International audience; The reparative ability of the central nervous system varies widely in the animal kingdom. In the mammalian brain, the regenerative mechanisms are very limited and newly formed neurons do not survive longer, probably due to a non-suitable local environment. On the opposite, fish can repair the brain after injury, with fast and complete recovery of damaged area. The brain of zebrafish, a teleost fish widely used as vertebrate model, also possesses high regenerative properties after injury. Taking advantage of this relevant model, the aim of the present study was to investigate the role of brain-derived neurotrophic factor (BDNF) in the regenerative ability of adult brain, after stab wound telencephalic injury. BDNF is involved in many brain functions and plays key roles in the repair process after traumatic brain lesions. It has been reported that BDNF strengthens the proliferative activity of neuronal precursor cells, facilitates the neuronal migration toward injured areas, and shows survival properties due to its anti-apoptotic effects. BDNF mRNA levels, assessed by quantitative PCR and in situ hybridization at 1, 4, 7, and 15 days after the lesion, were increased in the damaged telencephalon, mostly suddenly after the lesion. Double staining using in situ hybridization and immunocytochemistry revealed that BDNF mRNA was restricted to cells identified as mature neurons. BDNF mRNA expressing neurons mostly increased in the area around the lesion, showing a peak 1 day after the lesion. Taken together, these results highlight the role of BDNF in brain repair processes and reinforce the value of zebrafish for the study of regenerative neurogenesis.

International audience; Natural and synthetic estrogens and progestins are widely used in human and veterinary medicine and are detected in waste and surface waters. Our previous studies have clearly shown that a number of these substances targets the brain to induce the estrogen-regulated brain aromatase expression but the consequences on brain development remain virtually unexplored. The aim of the present study was therefore to investigate the effect of estradiol (E2), progesterone (P4) and norethindrone (NOR), a 19-nortestosterone progestin, on zebrafish larval neurogenesis. We first demonstrated using real-time quantitative PCR that nuclear estrogen and progesterone receptor brain expression is impacted by E2, P4 and NOR. We brought evidence that brain proliferative and apoptotic activities were differentially affected depending on the steroidal hormone studied, the concentration of steroids and the region investigated. Our findings demonstrate for the first time that steroid compounds released in aquatic environment have the capacity to disrupt key cellular events involved in brain development in zebrafish embryos further questioning the short- and long-term consequences of this disruption on the physiology and behavior of organisms.

International audience; Glyphosate is found in a large array of non-selective herbicides such as Roundup® (Monsanto, Creve Coeur, MO, USA) and is by far the most widely used herbicide. Recent work in rodent models suggests that glyphosate-based herbicides during development can affect neuronal communication and result in altered behaviours, albeit through undefined mechanisms of action. To our knowledge, no study has investigated the effects glyphosate or its formulation in herbicide on maternal behaviour and physiology. In the present study, relatively low doses of glyphosate (5 mg kg-1 d-1 ), Roundup® (5 mg kg-1 d-1 glyphosate equivalent), or vehicle were administered by ingestion to Sprague-Dawley rats from gestational day (GD) 10 to postpartum day (PD)22. The treatments significantly altered licking behaviour toward pups between PD2 and PD6. We also show in the dams at PD22 that Roundup exposure affected the maturation of doublecortin-immunoreactive new neurones in the dorsal dentate gyrus of the hippocampus of the mother. In addition, the expression of synaptophysin was up-regulated by glyphosate in the dorsal and ventral dentate gyrus and CA3 regions of the hippocampus, and down-regulated in the cingulate gyrus. Although a direct effect of glyphosate alone or its formulation on the central nervous system is currently not clear, we show that gut microbiota is significantly altered by the exposure to the pesticides, with significant alteration of the phyla Bacteroidetes and Firmicutes. This is the first study to provide evidence that glyphosate alone or in formulation (Roundup) differentially affects maternal behaviour and modulates neuroplasticity and gut microbiota in the mother.

The epigenetic mark 5-hydroxymethylcytosine (5hmC) is a cytosine modification that is abundant in the central nervous system of mammals and which results from 5-methylcytosine oxidation by TET enzymes. Such a mark is suggested to play key roles in the regulation of chromatin structure and gene expression. However, its precise functions still remain poorly understood and information about its distribution in non-mammalian species is still lacking. Here, the distribution of 5hmC was investigated in the brain of adult zebrafish, African claw frog, and mouse in a comparative manner. We show that zebrafish neurons are endowed with high levels of 5hmC, whereas quiescent or proliferative neural progenitors show low to undetectable levels of the modified cytosine. In the brain of larval and juvenile Xenopus, 5hmC is also detected in neurons, while ventricular proliferative cells do not display this epigenetic mark. Similarly, 5hmC is enriched in neurons compared to neural progenitors of the ventricular zone in the mouse developing cortex. Interestingly, 5hmC colocalized with the methylated DNA binding protein MeCP2 and with the active chromatin histone modification H3K4me2 in mouse neurons. Taken together, our results show an evolutionarily conserved cerebral distribution of 5hmC between fish and tetrapods and reinforce the idea that 5hmC fulfills major functions in the control of chromatin activity in vertebrate neurons. J. Comp. Neurol. 525:478-497, 2017. © 2016 Wiley Periodicals, Inc.

The present study was conducted to assess the effects of Cd exposure on estrogen signaling in the zebrafish brain, as well as the potential protective role of Zn against Cd-induced toxicity. For this purpose, the effects on transcriptional activation of the estrogen receptors (ERs), aromatase B (Aro-B) protein expression and molecular expression of related genes were examined in vivo using wild-type and transgenic zebrafish embryos. For in vitro studies, an ER-negative glial cell line (U251MG) transfected with different zebrafish ER subtypes (ERα, ERβ1 and ERβ2) was also used. Embryos were exposed either to estradiol (E2 ), Cd, E2 +Cd or E2 +Cd+Zn for 72 h and cells were exposed to the same treatments for 30 h. Our results show that E2 treatment promoted the transcriptional activation of ERs and increased Aro-B expression, at both the protein and mRNA levels. Although exposure to Cd, does not affect the studied parameters when administered alone, it significantly abolished the E2 -stimulated transcriptional response of the reporter gene for the three ER subtypes in U251-MG cells, and clearly inhibited the E2 induction of Aro-B in radial glial cells of zebrafish embryos. These inhibitory effects were accompanied by a significant downregulation of the expression of esr1, esr2a, esr2b and cyp19a1b genes compared to the E2 -treated group used as a positive control. Zn administration during simultaneous exposure to E2 and Cd strongly stimulated zebrafish ERs transactivation and increased Aro-B protein expression, whereas mRNA levels of the three ERs as well as the cyp19a1b remained unchanged in comparison with Cd-treated embryos. In conclusion, our results clearly demonstrate that Cd acts as a potent anti-estrogen in vivo and in vitro, and that Cd-induced E2 antagonism can be reversed, at the protein level, by Zn supplement. Copyright © 2016 John Wiley & Sons, Ltd.

Kisspeptins are now considered key players in the neuroendocrine control of puberty and reproduction, at least in mammals. Most teleosts have two kiss genes, kiss1 and kiss2, but their sites of expression are still poorly documented. As a first step in investigating the role of kisspeptins in the European sea bass, a perciform fish, we studied the distribution of kiss1 and kiss2-expressing cells in the brain of males and females undergoing their first sexual maturation. Animals were examined at early and late in the reproductive season. We also examined the putative expression of estrogen receptors in kiss-expressing cells and, finally, we investigated whether kisspeptins are expressed in the pituitary gland. We show that kiss1-expressing cells were consistently detected in the habenula and, in mature males and females, in the rostral mediobasal hypothalamus. In both sexes, kiss2-expressing cells were consistently detected at the level of the preoptic area, but the main kiss2 mRNA-positive population was observed in the dorsal hypothalamus, above and under the lateral recess. No obvious sexual differences in kiss1 and kiss2 mRNA expression were detected. Additional studies based on confocal imaging clearly showed that most kiss1 mRNA-containing cells of the mediobasal hypothalamus strongly express ERα and slightly express ERβ2. At the pituitary level, both sexes exhibited kiss1 mRNA expression in most FSHβ-positive cells and never in LHβ-positive cells. © 2012 Wiley Periodicals, Inc., Grant sponsor: EU Project LIFECYCLE; Grant number: FP7-222719-1 (to O.K., S.Z.); Grant sponsor: JAE-Predoc (CSIC, Spain; to S.E.).

The brain of teleosts is known for its strong aromatase expression, exhibiting unique features compared with other vertebrates. Among these features is the high sensitivity of aromatase B (the product of cyp19a1b) to estrogens. This effect involves the binding of estrogen receptors on an estrogen-responsive element (ERE) of the cyp19a1b promoter. Given the presence of potential androgen-responsive elements (AREs) on this promoter, in vivo and in vitro effects of androgens were studied. Using immunohistochemistry and quantitative PCR on zebrafish embryos, we found that cyp19a1b is upregulated by testosterone, an aromatizable androgen, and by 5alpha-dihydrotestosterone (DHT), a nonaromatizable androgen, suggesting a potential androgenic regulation of cyp19a1b through androgen receptors (ARs). To assess a putative direct regulation of the cyp19a1b gene by ARs, we transfected U251MG cells with zebrafish AR together with a luciferase reporter gene driven by 3000 bp of the proximal cyp19a1b promoter containing the ERE and potential AREs. Interestingly, although zebrafish AR activated luciferase reporter genes controlled by AREs, they failed to induce the cyp19a1b-luciferase construct. These data suggest that the androgenic regulation of cyp19a1b does not involve AR. We further showed that regulation of the cyp19a1b gene by testosterone is, in fact, due to aromatization, whereas the effect of DHT involves conversion into 5alpha-androstane-3beta,17beta-diol (betadiol), a metabolite of DHT with known estrogenic activity. The blockage of the androgen regulation of cyp19a1b expression using antiestrogens further confirmed the involvement of estrogen receptors in mediating these effects.

Kisspeptins are well known as mediators of the coordinated communication between the brain-pituitary axis and the gonads in many vertebrates. To test the hypothesis that gonadal steroids regulate kiss1 and kiss2 mRNA expression in European sea bass (a teleost fish), we examined the brains of gonad-intact (control) and castrated animals, as well as castrated males (GDX) and ovariectomized females (OVX) that received testosterone (T) and estradiol (E2) replacement, respectively, during recrudescence. In GDX males, low expression of kiss1 mRNA is observed by in situ hybridization in the caudal hypothalamus (CH) and the mediobasal hypothalamus (MBH), although hypothalamic changes in kiss1 mRNA levels were not statistically different among the groups, as revealed by real-Time PCR. However, T strongly decreased kiss2 expression levels in the hypothalamus, which was documented in the MBH and the nucleus of the lateral recess (NRLd) in GDX T-Treated sea bass males. Conversely, it appears that E2 evokes lowkiss1mRNAin the CH, while there were cells expressing kiss2 in the MBH and NRLd in these OVX females. These results demonstrate that kisspeptin neurons are presumably sensitive to the feedback actions of sex steroids in the sea bass, suggesting that the MBH represents a major site for sex steroid actions on kisspeptins in this species. Also, recent data provide evidence that both positive and negative actions occur in key factors involved in sea bass reproductive function, including changes in the expression of gnrh-1/gonadotropin, cyp19b, er and ar genes and sex steroid and gonadotropin plasma levels in this teleost fish., This work was supported by the Spanish Ministry of Science and Innovation (Ministerio de Ciencia e Innovación; MICINN) and the Spanish Ministry of Economy and Competitiveness (Ministerio de Economía y Competitividad; MINECO) [AGL2009-11086 KISSCONTROL to A.F. and M.C., CSD2007-00002 AQUAGENOMICS]; the Regional Government of Valencia (Generalitat Valenciana) [REPROBASS; PROMETEOII/2014/051]; and the Seventh Framework Programme [FP7-222719 LIFECYCLE]. M.V.A. was supported by MICINN and MINECO (Spain) [AGL2010-036443 FPI fellowship].

Estrogens are known as steroid hormones affecting the brain in many different ways and a wealth of data now document effects on neurogenesis. Estrogens are provided by the periphery but can also be locally produced within the brain itself due to local aromatization of circulating androgens. Adult neurogenesis is described in all vertebrate species examined so far, but comparative investigations have brought to light differences between vertebrate groups. In teleost fishes, the neurogenic activity is spectacular and adult stem cells maintain their mitogenic activity in many proliferative areas within the brain. Fish are also quite unique because brain aromatase expression is limited to radial glia cells, the progenitor cells of adult fish brain. The zebrafish has emerged as an interesting vertebrate model to elucidate the cellular and molecular mechanisms of adult neurogenesis, and notably its modulation by steroids. The main objective of this review is to summarize data related to the functional link between estrogens production in the brain and neurogenesis in fish. First, we will demonstrate that the brain of zebrafish is an endogenous source of steroids and is directly targeted by local and/or peripheral steroids. Then, we will present data demonstrating the progenitor nature of radial glial cells in the brain of adult fish. Next, we will emphasize the role of estrogens in constitutive neurogenesis and its potential contribution to the regenerative neurogenesis. Finally, the negative impacts on neurogenesis of synthetic hormones used in contraceptive pills production and released in the aquatic environment will be discussed.

International audience; Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has emerged as an active mediator in many essential functions in the central nervous system of mammals. BDNF plays significant roles in neurogenesis, neuronal maturation and/or synaptic plasticity and is involved in cognitive functions such as learning and memory. Despite the vast literature present in mammals, studies devoted to BDNF in the brain of other animal models are scarse. Zebrafish is a teleost fish widely known for developmental genetic studies and is emerging as model for translational neuroscience research. In addition, its brain shows many sites of adult neurogenesis allowing higher regenerative properties after traumatic injuries. To add further knowledge on neurotrophic factors in vertebrate brain models, we decided to determine the distribution of bdnf mRNAs in the larval and adult zebrafish brain and to characterize the phenotype of cells expressing bdnf mRNAs by means of double staining studies. Our results showed that bdnf mRNAs were widely expressed in the brain of 7 days old larvae and throughout the whole brain of mature female and male zebrafish. In adults, bdnf mRNAs were mainly observed in the dorsal telencephalon, preoptic area, dorsal thalamus, posterior tuberculum, hypothalamus, synencephalon, optic tectum and medulla oblongata. By combining immunohistochemistry with in situ hybridization, we showed that bdnf mRNAs were never expressed by radial glial cells or proliferating cells. By contrast, bdnf transcripts were expressed in cells with neuronal phenotype in all brain regions investigated. Our results provide the first demonstration that the brain of zebrafish expresses bdnf mRNAs in neurons and open new fields of research on the role of the BDNF factor in brain mechanisms in normal and brain repairs situations.

This study, conducted in the brain of a perciform fish, the European sea bass, aimed at raising antibodies against the precursor of the kisspeptins in order to map the kiss systems and to correlate the expression of kisspeptins, kiss1 and kiss2, with that of kisspeptin receptors (kiss-R1 and kiss-R2). Specific antibodies could be raised against the preprokiss2, but not the preoprokiss1. The data indicate that kiss2 neurons are mainly located in the hypothalamus and project widely to the subpallium and pallium, the preoptic region, the thalamus, the pretectal area, the optic tectum, the torus semicircularis, the mediobasal medial and caudal hypothalamus, and the neurohypophysis. These results were compared to the expression of kiss-R1 and kiss-R2 messengers, indicating a very good correlation between the wide distribution of Kiss2-positive fibers and that of kiss-R2 expressing cells. The expression of kiss-R1 messengers was more limited to the habenula, the ventral telencephalon and the proximal pars distalis of the pituitary. Attempts to characterize the phenotype of the numerous cells expressing kiss-R2 showed that neurons expressing tyrosine hydroxylase, neuropeptide Y and neuronal nitric oxide synthase are targets for kisspeptins, while GnRH1 neurons did not appear to express kiss-R1 or kiss-R2 messengers. In addition, a striking result was that all somatostatin-positive neurons expressed-kissR2. These data show that kisspeptins are likely to regulate a wide range of neuronal systems in the brain of teleosts. © 2013 Escobar et al.

International audience; The cyp19a1 gene that encodes aromatase, the only enzyme permitting conversion of C19 aromatizable androgens into estrogens, is present as a single copy in the genome of most vertebrate species, except in teleosts in which it has been duplicated. This study aimed at investigating the brain expression of a cyp19a1 gene expressed in both gonad and brain of Japanese eel, a basal teleost. By means of immunohistochemistry and in situ hybridization, we show that cyp19a1 is expressed only in radial glial cells of the brain and in pituitary cells. Treatments with salmon pituitary homogenates (female) or human chorionic gonadotrophin (male), known to turn on steroid production in immature eels, strongly stimulated cyp19a1 messenger and protein expression in radial glial cells and pituitary cells. Using double staining studies, we also showed that aromatase-expressing radial glial cells exhibit proliferative activity in both the brain and the pituitary. Altogether, these data indicate that brain and pituitary expression of Japanese eel cyp19a1 exhibits characteristics similar to those reported for the brain specific cyp19a1b gene in teleosts having duplicated cyp19a1 genes. This supports the hypothesis that, despite the fact that eels also underwent the teleost specific genome duplication, they have a single cyp19a1 expressed in both brain and gonad. Such data also suggest that the intriguing features of brain aromatase expression in teleost fishes were not gained after the whole genome duplication and may reflect properties of the cyp19a1 gene of ancestral Actinopterygians.

International audience; In rodents, there is increasing evidence that nuclear progesterone receptors are transiently expressed in many regions of the developing brain, notably outside the hypothalamus. This suggests that progesterone and/or its metabolites could be involved in functions not related to reproduction, particularly in neurodevelopment. In this context, the adult fish brain is of particular interest, as it exhibits constant growth and high neurogenic activity that is supported by radial glia progenitors. However, although synthesis of neuroprogestagens has been documented recently in the brain of zebrafish, information on the presence of progesterone receptors is very limited. In zebrafish, a single nuclear progesterone receptor (pgr) has been cloned and characterized. Here, we demonstrate that this pgr is widely distributed in all regions of the zebrafish brain. Interestingly, we show that Pgr is strongly expressed in radial glial cells and more weakly in neurons. Finally, we present evidence, based on quantitative PCR and immunohistochemistry, that nuclear progesterone receptor mRNA and proteins are upregulated by estrogens in the brain of adult zebrafish. These data document for the first time the finding that radial glial cells are preferential targets for peripheral progestagens and/or neuroprogestagens. Given the crucial roles of radial glial cells in adult neurogenesis, the potential effects of progestagens on their activity and the fate of daughter cells require thorough investigation.