Biofilm exopolysaccharides alter sensory-neuron-mediated sickness during lung infection.

Infections of the lung cause observable sickness thought to be secondary to inflammation. Signs of sickness are crucial to alert others via behavioral-immune responses to limit contact with contagious individuals. Gram-negative bacteria produce exopolysaccharide (EPS) that provides microbial protection; however, the impact of EPS on sickness remains uncertain. Using genome-engineered Pseudomonas aeruginosa (P. aeruginosa) strains, we compared EPS-producers versus non-producers and a virulent Escherichia coli (E. coli) lung infection model in male and female mice. EPS-negative P. aeruginosa and virulent E. coli infection caused severe sickness, behavioral alterations, inflammation, and hypothermia mediated by TLR4 detection of the exposed lipopolysaccharide (LPS) in lung TRPV1+ sensory neurons. However, inflammation did not account for sickness. Stimulation of lung nociceptors induced acute stress responses in the paraventricular hypothalamic nuclei by activating corticotropin-releasing hormone neurons responsible for sickness behavior and hypothermia. Thus, EPS-producing biofilm pathogens evade initiating a lung-brain sensory neuronal response that results in sickness.

Hypothalamic vasopressin sex differentiation is observed by embryonic day 15 in mice and is disrupted by the xenoestrogen bisphenol A.

Arginine vasopressin (AVP) neurons of the hypothalamic paraventricular region (AVPPVN) mediate sex-biased social behaviors across most species, including mammals. In mice, neural sex differences are thought to be established during a critical window around birth ( embryonic (E) day 18 to postnatal (P) day 2) whereby circulating testosterone from the fetal testis is converted to estrogen in sex-dimorphic brain regions. Here, we found that AVPPVN neurons are sexually dimorphic by E15.5, prior to this critical window, and that gestational bisphenol A (BPA) exposure permanently masculinized female AVPPVN neuronal numbers, projections, and electrophysiological properties, causing them to display male-like phenotypes into adulthood. Moreover, we showed that nearly twice as many neurons that became AVP+ by P0 were born at E11 in males and BPA-exposed females compared to control females, suggesting that AVPPVN neuronal masculinization occurs between E11 and P0. We further narrowed this sensitive period to around the timing of neurogenesis by demonstrating that exogenous estrogen exposure from E14.5 to E15.5 masculinized female AVPPVN neuronal numbers, whereas a pan-estrogen receptor antagonist exposed from E13.5 to E15.5 blocked masculinization of males. Finally, we showed that restricting BPA exposure to E7.5-E15.5 caused adult females to display increased social dominance over control females, consistent with an acquisition of male-like behaviors. Our study reveals an E11.5 to E15.5 window of estrogen sensitivity impacting AVPPVN sex differentiation, which is impacted by prenatal BPA exposure.

Gestational Exposure to Common Endocrine Disrupting Chemicals and Their Impact on Neurodevelopment and Behavior.

Endocrine disrupting chemicals are common in our environment and act on hormone systems and signaling pathways to alter physiological homeostasis. Gestational exposure can disrupt developmental programs, permanently altering tissues with impacts lasting into adulthood. The brain is a critical target for developmental endocrine disruption, resulting in altered neuroendocrine control of hormonal signaling, altered neurotransmitter control of nervous system function, and fundamental changes in behaviors such as learning, memory, and social interactions. Human cohort studies reveal correlations between maternal/fetal exposure to endocrine disruptors and incidence of neurodevelopmental disorders. Here, we summarize the major literature findings of endocrine disruption of neurodevelopment and concomitant changes in behavior by four major endocrine disruptor classes:bisphenol A, polychlorinated biphenyls, organophosphates, and polybrominated diphenyl ethers. We specifically review studies of gestational and/or lactational exposure to understand the effects of early life exposure to these compounds and summarize animal studies that help explain human correlative data.

Ascl1 is required to specify a subset of ventromedial hypothalamic neurons.

Despite clear physiological roles, the ventromedial hypothalamus (VMH) developmental programs are poorly understood. Here, we asked whether the proneural gene achaete-scute homolog 1 (Ascl1) contributes to VMH development. Ascl1 transcripts were detected in embryonic day (E) 10.5 to postnatal day 0 VMH neural progenitors. The elimination of Ascl1 reduced the number of VMH neurons at E12.5 and E15.5, particularly within the VMH-central (VMHC) and -dorsomedial (VMHDM) subdomains, and resulted in a VMH cell fate change from glutamatergic to GABAergic. We observed a loss of Neurog3 expression in Ascl1-/- hypothalamic progenitors and an upregulation of Neurog3 when Ascl1 was overexpressed. We also demonstrated a glutamatergic to GABAergic fate switch in Neurog3-null mutant mice, suggesting that Ascl1 might act via Neurog3 to drive VMH cell fate decisions. We also showed a concomitant increase in expression of the central GABAergic fate determinant Dlx1/2 in the Ascl1-null hypothalamus. However, Ascl1 was not sufficient to induce an ectopic VMH fate when overexpressed outside the normal window of competency. Combined, Ascl1 is required but not sufficient to specify the neurotransmitter identity of VMH neurons, acting in a transcriptional cascade with Neurog3.

kcna1a mutant zebrafish model episodic ataxia type 1 (EA1) with epilepsy and show response to first-line therapy carbamazepine.

OBJECTIVE: KCNA1 mutations are associated with a rare neurological movement disorder known as episodic ataxia type 1 (EA1), and epilepsy is a common comorbidity. Current medications provide only partial relief for ataxia and/or seizures, making new drugs needed. Here, we characterized zebrafish kcna1a-/- as a model of EA1 with epilepsy and compared the efficacy of the first-line therapy carbamazepine in kcna1a-/- zebrafish to Kcna1-/- rodents. METHODS: CRISPR/Cas9 mutagenesis was used to introduce a mutation in the sixth transmembrane segment of the zebrafish Kcna1 protein. Behavioral and electrophysiological assays were performed on kcna1a-/- larvae to assess ataxia- and epilepsy-related phenotypes. Real-time quantitative polymerase chain reaction (qPCR) was conducted to measure mRNA levels of brain hyperexcitability markers in kcna1a-/- larvae, followed by bioenergetics profiling to evaluate metabolic function. Drug efficacies were tested using behavioral and electrophysiological assessments, as well as seizure frequency in kcna1a-/- zebrafish and Kcna1-/- mice, respectively. RESULTS: Zebrafish kcna1a-/- larvae showed uncoordinated movements and locomotor deficits, along with scoliosis and increased mortality. The mutants also exhibited impaired startle responses when exposed to light-dark flashes and acoustic stimulation as well as hyperexcitability as measured by extracellular field recordings and upregulated fosab transcripts. Neural vglut2a and gad1b transcript levels were disrupted in kcna1a-/- larvae, indicative of a neuronal excitatory/inhibitory imbalance, as well as a significant reduction in cellular respiration in kcna1a-/- , consistent with dysregulation of neurometabolism. Notably, carbamazepine suppressed the impaired startle response and brain hyperexcitability in kcna1a-/- zebrafish but had no effect on the seizure frequency in Kcna1-/- mice, suggesting that this EA1 zebrafish model might better translate to humans than rodents. SIGNIFICANCE: We conclude that zebrafish kcna1a-/- show ataxia and epilepsy-related phenotypes and are responsive to carbamazepine treatment, consistent with EA1 patients. These findings suggest that kcna1-/- zebrafish are a useful model for drug screening as well as studying the underlying disease biology.

Pharmacological and Genetic Disruption of C-Type Natriuretic Peptide (nppcl) Expression in Zebrafish (Danio rerio) Causes Stunted Growth during Development.

Human patients with mutations within NPPC or NPR2 genes (encoding C-type natriuretic peptide (CNP) and guanylyl cyclase-B (GC-B), respectively) display clinical signs associated with skeletal abnormalities, such as overgrowth or short stature. Mice with induced models of Nppc or Npr2 deletion display profound achondroplasia, dwarfism and early death. Recent pharmacological therapies to treat short stature are utilizing long-acting CNP analogues, but the effects of manipulating CNP expression during development remain unknown. Here, we use Danio rerio (zebrafish) as a model for vertebrate development, employing both pharmacological and reverse genetics approaches to alter expression of genes encoding CNP in zebrafish. Four orthologues of CNP were identified in zebrafish, and spatiotemporal expression profiling confirmed their presence during development. Bioinformatic analyses suggested that nppcl is the most likely the orthologue of mammalian CNP. Exogenous CNP treatment of developing zebrafish embryos resulted in impaired growth characteristics, such as body length, head width and eye diameter. This reduced growth was potentially caused by increased apoptosis following CNP treatment. Expression of endogenous nppcl was downregulated in these CNP-treated embryos, suggesting that negative feedback of the CNP system might influence growth during development. CRISPR knock-down of endogenous nppcl in developing zebrafish embryos also resulted in impaired growth characteristics. Collectively, these data suggest that CNP in zebrafish is crucial for normal embryonic development, specifically with regard to growth.

The effects of prenatal bisphenol A exposure on brain volume of children and young mice.

Bisphenol A (BPA) is a synthetic chemical used for the manufacturing of plastics, epoxy resin, and many personal care products. This ubiquitous endocrine disruptor is detectable in the urine of over 80% of North Americans. Although adverse neurodevelopmental outcomes have been observed in children with high gestational exposure to BPA, the effects of prenatal BPA on brain structure remain unclear. Here, using magnetic resonance imaging (MRI), we studied the associations of maternal BPA exposure with children's brain structure, as well as the impact of comparable BPA levels in a mouse model. Our human data showed that most maternal BPA exposure effects on brain volumes were small, with the largest effects observed in the opercular region of the inferior frontal gyrus (ρ = -0.2754), superior occipital gyrus (ρ = -0.2556), and postcentral gyrus (ρ = 0.2384). In mice, gestational exposure to an equivalent level of BPA (2.25 µg BPA/kg bw/day) induced structural alterations in brain regions including the superior olivary complex (SOC) and bed nucleus of stria terminalis (BNST) with larger effect sizes (1.07≤ Cohens d ≤ 1.53). Human (n = 87) and rodent (n = 8 each group) sample sizes, while small, are considered adequate to perform the primary endpoint analysis. Combined, these human and mouse data suggest that gestational exposure to low levels of BPA may have some impacts on the developing brain at the resolution of MRI.

Neurog2 Acts as a Classical Proneural Gene in the Ventromedial Hypothalamus and Is Required for the Early Phase of Neurogenesis.

The tuberal hypothalamus is comprised of the dorsomedial, ventromedial, and arcuate nuclei, as well as parts of the lateral hypothalamic area, and it governs a wide range of physiologies. During neurogenesis, tuberal hypothalamic neurons are thought to be born in a dorsal-to-ventral and outside-in pattern, although the accuracy of this description has been questioned over the years. Moreover, the intrinsic factors that control the timing of neurogenesis in this region are poorly characterized. Proneural genes, including Achate-scute-like 1 (Ascl1) and Neurogenin 3 (Neurog3) are widely expressed in hypothalamic progenitors and contribute to lineage commitment and subtype-specific neuronal identifies, but the potential role of Neurogenin 2 (Neurog2) remains unexplored. Birthdating in male and female mice showed that tuberal hypothalamic neurogenesis begins as early as E9.5 in the lateral hypothalamic and arcuate and rapidly expands to dorsomedial and ventromedial neurons by E10.5, peaking throughout the region by E11.5. We confirmed an outside-in trend, except for neurons born at E9.5, and uncovered a rostrocaudal progression but did not confirm a dorsal-ventral patterning to tuberal hypothalamic neuronal birth. In the absence of Neurog2, neurogenesis stalls, with a significant reduction in early-born BrdU+ cells but no change at later time points. Further, the loss of Ascl1 yielded a similar delay in neuronal birth, suggesting that Ascl1 cannot rescue the loss of Neurog2 and that these proneural genes act independently in the tuberal hypothalamus. Together, our findings show that Neurog2 functions as a classical proneural gene to regulate the temporal progression of tuberal hypothalamic neurogenesis.SIGNIFICANCE STATEMENT Here, we investigated the general timing and pattern of neurogenesis within the tuberal hypothalamus. Our results confirmed an outside-in trend of neurogenesis and uncovered a rostrocaudal progression. We also showed that Neurog2 acts as a classical proneural gene and is responsible for regulating the birth of early-born neurons within the ventromedial hypothalamus, acting independently of Ascl1 In addition, we revealed a role for Neurog2 in cell fate specification and differentiation of ventromedial -specific neurons. Last, Neurog2 does not have cross-inhibitory effects on Neurog1, Neurog3, and Ascl1 These findings are the first to reveal a role for Neurog2 in hypothalamic development.

A non-canonical role for the proneural gene Neurog1 as a negative regulator of neocortical neurogenesis.

Neural progenitors undergo temporal identity transitions to sequentially generate the neuronal and glial cells that make up the mature brain. Proneural genes have well-characterised roles in promoting neural cell differentiation and subtype specification, but they also regulate the timing of identity transitions through poorly understood mechanisms. Here, we investigated how the highly related proneural genes Neurog1 and Neurog2 interact to control the timing of neocortical neurogenesis. We found that Neurog1 acts in an atypical fashion as it is required to suppress rather than promote neuronal differentiation in early corticogenesis. In Neurog1-/- neocortices, early born neurons differentiate in excess, whereas, in vitro, Neurog1-/- progenitors have a decreased propensity to proliferate and form neurospheres. Instead, Neurog1-/- progenitors preferentially generate neurons, a phenotype restricted to the Neurog2+ progenitor pool. Mechanistically, Neurog1 and Neurog2 heterodimerise, and while Neurog1 and Neurog2 individually promote neurogenesis, misexpression together blocks this effect. Finally, Neurog1 is also required to induce the expression of neurogenic factors (Dll1 and Hes5) and to repress the expression of neuronal differentiation genes (Fezf2 and Neurod6). Neurog1 thus employs different mechanisms to temper the pace of early neocortical neurogenesis.

Emerging roles for hypothalamic microglia as regulators of physiological homeostasis.

The hypothalamus is a crucial brain region that responds to external stressors and functions to maintain physiological homeostatic processes, such as core body temperature and energy balance. The hypothalamus regulates homeostasis by producing hormones that thereby influence the production of other hormones that then control the internal milieu of the body. Microglia are resident macrophages and phagocytic immune cells of the central nervous system (CNS), classically known for surveying the brain's environment, responding to neural insults, and disposing of cellular debris. Recent evidence has shown that microglia are also responsive to external stressors and can influence both the development and function of the hypothalamus in a sex-dependent manner. This emerging microglia-hypothalamic interaction raises the intriguing notion that microglia might play an unappreciated role in hypothalamic control of physiological homeostasis. In this review, we briefly outline how the hypothalamus regulates physiological homeostasis and then describe how this literature overlaps with our understanding of microglia's role in the CNS. We also outline the current literature demonstrating how microglia loss or activation affects the hypothalamus, and ultimately homeostasis. We conclude by proposing how microglia could be key regulators of homeostatic processes by sensing cues external to the CNS and transmitting them through the hypothalamus.

Embryonic microglia influence developing hypothalamic glial populations.

BACKGROUND: Although historically microglia were thought to be immature in the fetal brain, evidence of purposeful interactions between these immune cells and nearby neural progenitors is becoming established. Here, we examined the influence of embryonic microglia on gliogenesis within the developing tuberal hypothalamus, a region later important for energy balance, reproduction, and thermoregulation. METHODS: We used immunohistochemistry to quantify the location and numbers of glial cells in the embryonic brain (E13.5-E17.5), as well as a pharmacological approach (i.e., PLX5622) to knock down fetal microglia. We also conducted cytokine and chemokine analyses on embryonic brains in the presence or absence of microglia, and a neurosphere assay to test the effects of the altered cytokines on hypothalamic progenitor behaviors. RESULTS: We identified a subpopulation of activated microglia that congregated adjacent to the third ventricle alongside embryonic Olig2+ neural progenitor cells (NPCs) that are destined to give rise to oligodendrocyte and astrocyte populations. In the absence of microglia, we observed an increase in Olig2+ glial progenitor cells that remained at the ventricle by E17.5 and a concomitant decrease of these Olig2+ cells in the mantle zone, indicative of a delay in migration of these precursor cells. A further examination of maturing oligodendrocytes in the hypothalamic grey and white matter area in the absence of microglia revealed migrating oligodendrocyte progenitor cells (OPCs) within the grey matter at E17.5, a time point when OPCs begin to slow their migration. Finally, quantification of cytokine and chemokine signaling in ex vivo E15.5 hypothalamic cultures +/- microglia revealed decreases in the protein levels of several cytokines in the absence of microglia. We assayed the influence of two downregulated cytokines (CCL2 and CXCL10) on neurosphere-forming capacity and lineage commitment of hypothalamic NPCs in culture and showed an increase in NPC proliferation as well as neuronal and oligodendrocyte differentiation. CONCLUSION: These data demonstrate that microglia influence gliogenesis in the developing tuberal hypothalamus.

Granulocyte-colony-stimulating factor (G-CSF) signaling in spinal microglia drives visceral sensitization following colitis.

Pain is a main symptom of inflammatory diseases and often persists beyond clinical remission. Although we have a good understanding of the mechanisms of sensitization at the periphery during inflammation, little is known about the mediators that drive central sensitization. Recent reports have identified hematopoietic colony-stimulating factors as important regulators of tumor- and nerve injury-associated pain. Using a mouse model of colitis, we identify the proinflammatory cytokine granulocyte-colony-stimulating factor (G-CSF or Csf-3) as a key mediator of visceral sensitization. We report that G-CSF is specifically up-regulated in the thoracolumbar spinal cord of colitis-affected mice. Our results show that resident spinal microglia express the G-CSF receptor and that G-CSF signaling mediates microglial activation following colitis. Furthermore, healthy mice subjected to intrathecal injection of G-CSF exhibit pronounced visceral hypersensitivity, an effect that is abolished by microglial depletion. Mechanistically, we demonstrate that G-CSF injection increases Cathepsin S activity in spinal cord tissues. When cocultured with microglia BV-2 cells exposed to G-CSF, dorsal root ganglion (DRG) nociceptors become hyperexcitable. Blocking CX3CR1 or nitric oxide production during G-CSF treatment reduces excitability and G-CSF-induced visceral pain in vivo. Finally, administration of G-CSF-neutralizing antibody can prevent the establishment of persistent visceral pain postcolitis. Overall, our work uncovers a DRG neuron-microglia interaction that responds to G-CSF by engaging Cathepsin S-CX3CR1-inducible NOS signaling. This interaction represents a central step in visceral sensitization following colonic inflammation, thereby identifying spinal G-CSF as a target for treating chronic abdominal pain.

Neurog2 and Ascl1 together regulate a postmitotic derepression circuit to govern laminar fate specification in the murine neocortex.

A derepression mode of cell-fate specification involving the transcriptional repressors Tbr1, Fezf2, Satb2, and Ctip2 operates in neocortical projection neurons to specify six layer identities in sequence. Less well understood is how laminar fate transitions are regulated in cortical progenitors. The proneural genes Neurog2 and Ascl1 cooperate in progenitors to control the temporal switch from neurogenesis to gliogenesis. Here we asked whether these proneural genes also regulate laminar fate transitions. Several defects were observed in the derepression circuit in Neurog2-/-;Ascl1-/- mutants: an inability to repress expression of Tbr1 (a deep layer VI marker) during upper-layer neurogenesis, a loss of Fezf2+/Ctip2+ layer V neurons, and precocious differentiation of normally late-born, Satb2+ layer II-IV neurons. Conversely, in stable gain-of-function transgenics, Neurog2 promoted differentiative divisions and extended the period of Tbr1+/Ctip2+ deep-layer neurogenesis while reducing Satb2+ upper-layer neurogenesis. Similarly, acute misexpression of Neurog2 in early cortical progenitors promoted Tbr1 expression, whereas both Neurog2 and Ascl1 induced Ctip2. However, Neurog2 was unable to influence the derepression circuit when misexpressed in late cortical progenitors, and Ascl1 repressed only Satb2. Nevertheless, neurons derived from late misexpression of Neurog2 and, to a lesser extent, Ascl1, extended aberrant subcortical axon projections characteristic of early-born neurons. Finally, Neurog2 and Ascl1 altered the expression of Ikaros and Foxg1, known temporal regulators. Proneural genes thus act in a context-dependent fashion as early determinants, promoting deep-layer neurogenesis in early cortical progenitors via input into the derepression circuit while also influencing other temporal regulators.

A novel metabolism-based phenotypic drug discovery platform in zebrafish uncovers HDACs 1 and 3 as a potential combined anti-seizure drug target.

Despite the development of newer anti-seizure medications over the past 50 years, 30-40% of patients with epilepsy remain refractory to treatment. One explanation for this lack of progress is that the current screening process is largely biased towards transmembrane channels and receptors, and ignores intracellular proteins and enzymes that might serve as efficacious molecular targets. Here, we report the development of a novel drug screening platform that harnesses the power of zebrafish genetics and combines it with in vivo bioenergetics screening assays to uncover therapeutic agents that improve mitochondrial health in diseased animals. By screening commercially available chemical libraries of approved drugs, for which the molecular targets and pathways are well characterized, we were able to reverse-identify the proteins targeted by efficacious compounds and confirm the physiological roles that they play by utilizing other pharmacological ligands. Indeed, using an 870-compound screen in kcna1-morpholino epileptic zebrafish larvae, we uncovered vorinostat (Zolinza™; suberanilohydroxamic acid, SAHA) as a potent anti-seizure agent. We further demonstrated that vorinostat decreased average daily seizures by ∼60% in epileptic Kcna1-null mice using video-EEG recordings. Given that vorinostat is a broad histone deacetylase (HDAC) inhibitor, we then delineated a specific subset of HDACs, namely HDACs 1 and 3, as potential drug targets for future screening. In summary, we have developed a novel phenotypic, metabolism-based experimental therapeutics platform that can be used to identify new molecular targets for future drug discovery in epilepsy.

Bisphenol A and microglia: could microglia be responsive to this environmental contaminant during neural development?

There is a growing interest in the functional role of microglia in the developing brain. In our laboratory, we have become particularly intrigued as to whether fetal microglia in the embryonic brain are susceptible to maternal challenges in utero (e.g., maternal infection, stress) and, if so, whether their precocious activation could then adversely influence brain development. One such challenge that is newly arising in this field is whether microglia might be downstream targets to endocrine-disrupting chemicals, such as the plasticizer bisphenol A (BPA), which functions in part by mimicking estrogen structure and function. A growing body of evidence demonstrates that gestational exposure to BPA has adverse effects on brain development, although the exact mechanisms are still emerging. Given that microglia express estrogen receptors and steroid-producing enzymes, microglia might be an unappreciated target of BPA. Mechanistically, we propose that BPA binding to estrogen receptors within microglia initiates transcription of downstream target genes, which then leads to activation of microglia that can then perhaps adversely influence brain development. Here, we first briefly outline the current understanding of how microglia may influence brain development and then describe how this literature overlaps with our understanding of BPA's effects during similar time points. We also outline the current literature demonstrating that BPA exposure affects microglia. We conclude by discussing our thoughts on the mechanisms through which exposure to BPA could disrupt normal microglia functions, ultimately affecting brain development that could potentially lead to lasting behavioral effects and perhaps even neuroendocrine diseases such as obesity.

In utero electroporation induces cell death and alters embryonic microglia morphology and expression signatures in the developing hypothalamus.

BACKGROUND: Since its inception in 2001, in utero electroporation (IUE) has been widely used by the neuroscience community. IUE is a technique developed to introduce plasmid DNA into embryonic mouse brains without permanently removing the embryos from the uterus. Given that IUE labels cells that line the ventricles, including radial fibers and migrating neuroblasts, this technique is an excellent tool for studying factors that govern neural cell fate determination and migration in the developing mouse brain. Whether IUE has an effect on microglia, the immune cells of the central nervous system (CNS), has yet to be investigated. METHODS: We used IUE and the pCIG2, pCIC-Ascl1, or pRFP-C-RS expression vectors to label radial glia lining the ventricles of the embryonic cortex and/or hypothalamus. Specifically, we conducted IUE at E14.5 and harvested the brains at E15.5 or E17.5. Immunohistochemistry, along with cytokine and chemokine analyses, were performed on embryonic brains with or without IUE exposure. RESULTS: IUE using the pCIG2, pCIC-Ascl1, or pRFP-C-RS vectors alone altered microglia morphology, where the majority of microglia near the ventricles were amoeboid and displayed altered expression signatures, including the upregulation of Cd45 and downregulation of P2ry12. Moreover, IUE led to increases in P2ry12- cells that were Iba1+/IgG+ double-positive in the brain parenchyma and resembled macrophages infiltrating the brain proper from the periphery. Furthermore, IUE resulted in a significant increase in cell death in the developing hypothalamus, with concomitant increases in cytokines and chemokines known to be released during pro-inflammatory states (IL-1ß, IL-6, MIP-2, RANTES, MCP-1). Interestingly, the cortex was protected from elevated cell death following IUE, implying that microglia that reside in the hypothalamus might be particularly sensitive during embryonic development. CONCLUSIONS: Our results suggest that IUE might have unintended consequences of activating microglia in the embryonic brain, which could have long-term effects, particularly within the hypothalamus.

Depletion of embryonic microglia using the CSF1R inhibitor PLX5622 has adverse sex-specific effects on mice, including accelerated weight gain, hyperactivity and anxiolytic-like behaviour.

Microglia are the resident immune cells in the central nervous system (CNS). Originally thought to be primarily responsible for disposing of cellular debris and responding to neural insults, emerging research now shows that microglia are highly dynamic cells involved in a variety of neurodevelopmental processes. The hypothalamus is a brain region critical for maintaining homeostatic processes such as energy balance, thirst, food intake, reproduction, and circadian rhythms. Given that microglia colonize the embryonic brain alongside key steps of hypothalamic development, here we tested whether microglia are required for the proper establishment of this brain region. The Colony-stimulating factor-1 receptor (Csf1r) is expressed by microglia, macrophages and osteoclasts, and is required for their proliferation, differentiation, and survival. Therefore, to eliminate microglia from the fetal brain, we treated pregnant dams with the CSF1R inhibitor PLX5622. We showed that approximately 99% of microglia were eliminated by embryonic day 15.5 (E15.5) after pregnant dams were placed on a PLX5622 diet starting at E3.5. Following microglia depletion, we observed elevated numbers of apoptotic cells accumulating throughout the developing hypothalamus. Once the PLX5622 diet was removed, microglia repopulated the postnatal brain within 7 days and did not appear to repopulate from Nestin+ precursors. Embryonic microglia depletion also resulted in a decreased litter size, as well as an increase in the number of pups that died within the first two postnatal days of life. In pups that survived, the elimination of microglia in the fetal brain resulted in a decrease in the number of Pro-opiomelanocortin (POMC) neurons and a concomitant accelerated weight gain starting at postnatal day 5 (P5), suggesting that microglia could be important for the development of cell types key to hypothalamic satiety centers. Moreover, surviving PLX5622 exposed animals displayed craniofacial and dental abnormalities, perhaps due to non-CNS effects of PLX5622 on macrophages and/or osteoclasts. Finally, depletion of microglia during embryogenesis had long-term sex-specific effects on behaviour, including the development of hyperactivity and anxiolytic-like behaviour in juvenile and adult female mice, respectively. Together, these data demonstrate an important role for microglia during the development of the embryonic hypothalamus, and perhaps the CNS more broadly.

Opening the black box of endocrine disruption of brain development: Lessons from the characterization of Bisphenol A.

Bisphenol A (BPA) is among the best-studied endocrine disrupting chemicals, known to act via multiple steroid hormone receptors to mediate a myriad of cellular effects. Pre-, peri-, and postnatal BPA exposure have been linked to a variety of altered behaviors in multiple model organisms, ranging from zebrafish to frogs to mammalian models. Given that BPA can cross the human placental barrier and has been found in the serum of human fetuses during gestation, BPA has been postulated to adversely affect ongoing neurodevelopment, ultimately leading to behavioral disorders later in life. Indeed, the brain has been identified as a key developmental target for BPA disruption. Despite these known associations between gestational BPA exposure and adverse developmental outcomes, as well as an extensive body of evidence existing in the literature, the mechanisms by which BPA induces its cellular- and tissue-specific effects on neurodevelopmental processes still remains poorly understood at a mechanistic level. In this review we will briefly summarize the effects of gestational BPA exposure on neural developmental mechanisms and resulting behaviors, and then present suggestions for how we might address gaps in our knowledge to develop a fuller understanding of endocrine neurodevelopmental disruption to better inform governmental policy against the use of BPA or other endocrine disruptors.

Low-dose exposure to bisphenol A and replacement bisphenol S induces precocious hypothalamic neurogenesis in embryonic zebrafish.

Bisphenol A (BPA), a ubiquitous endocrine disruptor that is present in many household products, has been linked to obesity, cancer, and, most relevant here, childhood neurological disorders such as anxiety and hyperactivity. However, how BPA exposure translates into these neurodevelopmental disorders remains poorly understood. Here, we used zebrafish to link BPA mechanistically to disease etiology. Strikingly, treatment of embryonic zebrafish with very low-dose BPA (0.0068 µM, 1,000-fold lower than the accepted human daily exposure) and bisphenol S (BPS), a common analog used in BPA-free products, resulted in 180% and 240% increases, respectively, in neuronal birth (neurogenesis) within the hypothalamus, a highly conserved brain region involved in hyperactivity. Furthermore, restricted BPA/BPS exposure specifically during the neurogenic window caused later hyperactive behaviors in zebrafish larvae. Unexpectedly, we show that BPA-mediated precocious neurogenesis and the concomitant behavioral phenotype were not dependent on predicted estrogen receptors but relied on androgen receptor-mediated up-regulation of aromatase. Although human epidemiological results are still emerging, an association between high maternal urinary BPA during gestation and hyperactivity and other behavioral disturbances in the child has been suggested. Our studies here provide mechanistic support that the neurogenic period indeed may be a window of vulnerability and uncovers previously unexplored avenues of research into how endocrine disruptors might perturb early brain development. Furthermore, our results show that BPA-free products are not necessarily safer and support the removal of all bisphenols from consumer merchandise.

Mice lacking the transcription factor SHOX2 display impaired cerebellar development and deficits in motor coordination.

Purkinje cells of the developing cerebellum secrete the morphogen sonic hedgehog (SHH), which is required to maintain the proliferative state of granule cell precursors (GCPs) prior to their differentiation and migration to form the internal granule layer (IGL). Despite a wealth of knowledge regarding the function of SHH during cerebellar development, the upstream regulators of Shh expression during this process remain largely unknown. Here we report that the murine short stature homeobox 2 (Shox2) gene is required for normal Shh expression in dorsal-residing Purkinje cells. Using two different Cre drivers, we show that elimination of Shox2 in the brain results in developmental defects in the inferior colliculus and cerebellum. Specifically, loss of Shox2 in the cerebellum results in precocious differentiation and migration of GCPs from the external granule layer (EGL) to the IGL. This correlates with premature bone morphogenetic protein 4 (Bmp4) expression in granule cells of the dorsal cerebellum. The size of the neonatal cerebellum is reduced in Shox2-mutant animals, which is consistent with a reduction in the number of GCPs present in the EGL, and could account for the smaller vermis and thinner IGL present in adult Shox2mutants. Shox2-mutant mice also display reduced exploratory activity, altered gait and impaired motor coordination. Our findings are the first to show a role for Shox2 in brain development. We provide evidence that Shox2 plays an important role during cerebellar development, perhaps to maintain the proper balance of Shh and Bmp expression levels in the dorsal vermis, and demonstrate that in the absence of Shox2, mice display both cerebellar impairments and deficits in motor coordination, ultimately highlighting the importance of Shox2 in the cerebellum.