Automated high content image analysis of dendritic arborization in primary mouse hippocampal and rat cortical neurons in culture.

BACKGROUND: Primary neuronal cell cultures are useful for studying mechanisms that influence dendritic morphology during normal development and in response to various stressors. However, analyzing dendritic morphology is challenging, particularly in cultures with high cell density, and manual methods of selecting neurons and tracing dendritic arbors can introduce significant bias, and are labor-intensive. To overcome these challenges, semi-automated and automated methods are being developed, with most software solutions requiring computer-assisted dendrite tracing with subsequent quantification of various parameters of dendritic morphology, such as Sholl analysis. However fully automated approaches for classic Sholl analysis of dendritic complexity are not currently available. NEW METHOD: The previously described Omnisphero software, was extended by adding new functions to automatically assess dendritic mass, total length of the dendritic arbor and the number of primary dendrites, branch points, and terminal tips, and to perform Sholl analysis. RESULTS: The new functions for assessing dendritic morphology were validated using primary mouse hippocampal and rat cortical neurons transfected with a fluorescently tagged MAP2 cDNA construct. These functions allow users to select specific populations of neurons as a training set for subsequent automated selection of labeled neurons in high-density cultures. COMPARISON WITH EXISTING SEMI-AUTOMATED METHODS: Compared to manual or semi-automated analyses of dendritic arborization, the new functions increase throughput while significantly decreasing researcher bias associated with neuron selection, tracing, and thresholding. CONCLUSION: These results demonstrate the importance of using unbiased automated methods to mitigate experimenter-dependent bias in analyzing dendritic morphology.

Void sorcerer: an open source, open access framework for mouse uroflowmetry.

Observational and experimental studies of rodent voiding behaviors have greatly contributed to our understanding of lower urinary tract function including the complex social, environmental, and internal stimuli that affect voiding in health and models of disease. Void spot assays (VSA), cystometry (awake or anesthetized), and uroflowmetry are techniques commonly used in rodent models to assess voiding. Uroflowmetry is non-invasive and can be performed multiple times in the same freely moving animals and can be used to generate synchronized video corresponding to each void to characterize micturition patterns (e.g., droplets versus solid stream). However, approaches to evaluate uroflowmetry in rodent models vary widely across laboratories. Most importantly, an open access software to run these tests is not freely available (although complete systems are commercially available), limiting use of this important assay. We developed the Void Sorcerer, an uroflowmetry system for mice for reliable determination of frequency, voided volume, voiding duration, interval times between micturitions, and flow rate. This report provides a detailed description of how to build this system and includes open access software for developing uroflowmetry capability in their laboratories and improve upon it in a cost-effective manner. Our goals are to improve access, increase reproducibility among laboratories, and facilitate standardizing testing procedures.

Developmental exposure to polychlorinated biphenyls (PCBs) in the maternal diet causes host-microbe defects in weanling offspring mice.

The gut microbiota is important for maintaining homeostasis of the host. Gut microbes represent the initial site for toxicant processing following dietary exposures to environmental contaminants. The diet is the primary route of exposure to polychlorinated biphenyls (PCBs), which are absorbed via the gut, and subsequently interfere with neurodevelopment and behavior. Developmental exposures to PCBs have been linked to increased risk of neurodevelopmental disorders (NDD), including autism spectrum disorder (ASD), which are also associated with a high prevalence of gastrointestinal (GI) distress and intestinal dysbiosis. We hypothesized that developmental PCB exposure impacts colonization of the gut microbiota, resulting in GI pathophysiology, in a genetically susceptible host. Mouse dams expressing two heritable human mutations (double mutants [DM]) that result in abnormal Ca2+ dynamics and produce behavioral deficits (gain of function mutation in the ryanodine receptor 1 [T4826I-RYR1] and a human CGG repeat expansion [170-200 CGG repeats] in the fragile X mental retardation gene 1 [FMR1 premutation]). DM and congenic wild type (WT) controls were exposed to PCBs (0-6 mg/kg/d) in the diet starting 2 weeks before gestation and continuing through postnatal day 21 (P21). Intestinal physiology (Ussing chambers), inflammation (qPCR) and gut microbiome (16S sequencing) studies were performed in offspring mice (P28-P30). Developmental exposure to PCBs in the maternal diet caused significant mucosal barrier defects in ileum and colon (increased secretory state and tight junction permeability) of juvenile DM mice. Furthermore, PCB exposure increased the intestinal inflammatory profile (Il6, Il1ß, and Il22), and resulted in dysbiosis of the gut microbiota, including altered ß-diversity, in juvenile DM mice developmentally exposed to 1 mg/kg/d PCBs when compared to WT controls. Collectively, these findings demonstrate a novel interaction between PCB exposure and the gut microbiota in a genetically susceptible host that provide novel insight into environmental risk factors for neurodevelopmental disorders.

Edar is a downstream target of beta-catenin and drives collagen accumulation in the mouse prostate.

Beta-catenin (CTNNB1) directs ectodermal appendage spacing by activating ectodysplasin A receptor (EDAR) transcription, but whether CTNNB1 acts by a similar mechanism in the prostate, an endoderm-derived tissue, is unclear. Here we examined the expression, function, and CTNNB1 dependence of the EDAR pathway during prostate development. In situ hybridization studies reveal EDAR pathway components including Wnt10b in the developing prostate and localize these factors to prostatic bud epithelium where CTNNB1 target genes are co-expressed. We used a genetic approach to ectopically activate CTNNB1 in developing mouse prostate and observed focal increases in Edar and Wnt10b mRNAs. We also used a genetic approach to test the prostatic consequences of activating or inhibiting Edar expression. Edar overexpression does not visibly alter prostatic bud formation or branching morphogenesis, and Edar expression is not necessary for either of these events. However, Edar overexpression is associated with an abnormally thick and collagen-rich stroma in adult mouse prostates. These results support CTNNB1 as a transcriptional activator of Edar and Wnt10b in the developing prostate and demonstrate Edar is not only important for ectodermal appendage patterning but also influences collagen organization in adult prostates.This article has an associated First Person interview with the first author of the paper.

Polychlorinated biphenyls influence on autism spectrum disorder risk in the MARBLES cohort.

BACKGROUND: Autism spectrum disorder (ASD) is suspected to have environmental and genetic contributions. Polychlorinated biphenyls (PCBs) are environmental risk factors of interest due to their potential as neurodevelopmental toxicants and environmental persistence despite a US production ban in the 1970s. METHODS: Participants were mother-child pairs from MARBLES, a high-risk pregnancy cohort that enrolls families who have one child diagnosed with ASD and are planning to have another child. PCB concentrations were measured in maternal blood at each trimester of pregnancy using gas chromatography coupled with triple quadruple mass spectrometry. Concentrations were summed into total PCB and two categories based on function/mechanisms of action: dioxin-like (DL), and ryanodine receptor (RyR)-activating PCBs. Multinomial logistic regression assessed risk of clinical outcome classification of ASD and non-typical development (Non-TD) compared to typically developing (TD) in the children at 3 years old. RESULTS: A total of 104 mother-child pairs were included. There were no significant associations for total PCB; however, there were borderline significant associations between DL-PCBs and decreased risk for Non-TD outcome classification (adjusted OR: 0.41 (95% CI 0.15-1.14)) and between RyR-activating PCBs and increased risk for ASD outcome classification (adjusted OR: 2.63 (95% CI 0.87-7.97)). CONCLUSION: This study does not provide strong supporting evidence that PCBs are risk factors for ASD or Non-TD. However, these analyses suggest the need to explore more deeply into subsets of PCBs as risk factors based on their function and structure in larger cohort studies where non-monotonic dose-response patterns can be better evaluated.

Genetic mutations in Ca2+ signaling alter dendrite morphology and social approach in juvenile mice.

Dendritic morphology is a critical determinant of neuronal connectivity, and calcium signaling plays a predominant role in shaping dendrites. Altered dendritic morphology and genetic mutations in calcium signaling are both associated with neurodevelopmental disorders (NDDs). In this study we tested the hypothesis that dendritic arborization and NDD-relevant behavioral phenotypes are altered by human mutations that modulate calcium-dependent signaling pathways implicated in NDDs. The dendritic morphology of pyramidal neurons in CA1 hippocampus and somatosensory cortex was quantified in Golgi-stained brain sections from juvenile mice of both sexes expressing either a human gain-of-function mutation in ryanodine receptor 1 (T4826I-RYR1), a human CGG repeat expansion (170-200 CGG repeats) in the fragile X mental retardation gene 1 (FMR1 premutation), both mutations (double mutation; DM), or wildtype mice. In hippocampal neurons, increased dendritic arborization was observed in male T4826I-RYR1 and, to a lesser extent, male FMR1 premutation neurons. Dendritic morphology of cortical neurons was altered in both sexes of FMR1 premutation and DM animals with the most pronounced differences seen in DM females. Genotype also impaired behavior, as assessed using the three-chambered social approach test. The most striking lack of sociability was observed in DM male and female mice. In conclusion, mutations that alter the fidelity of calcium signaling enhance dendritic arborization in a brain region- and sex-specific manner and impair social behavior in juvenile mice. The phenotypic outcomes of these mutations likely provide a susceptible biological substrate for additional environmental stressors that converge on calcium signaling to determine individual NDD risk.

Sex-Dependent Effects of 2,2',3,5',6-Pentachlorobiphenyl on Dendritic Arborization of Primary Mouse Neurons.

Early life exposures to environmental contaminants are implicated in the pathogenesis of many neurodevelopmental disorders (NDDs). These disorders often display sex biases, but whether environmental neurotoxicants act in a sex-dependent manner to modify neurodevelopment is largely unknown. Since altered dendritic morphology is associated with many NDDs, we tested the hypothesis that male and female primary mouse neurons are differentially susceptible to the dendrite-promoting activity of 2,2',3,5',6-pentachlorobiphenyl (PCB 95). Hippocampal and cortical neuron-glia co-cultures were exposed to vehicle (0.1% dimethylsulfoxide) or PCB 95 (100 fM-1 µM) from day in vitro 7-9. As determined by Sholl analysis, PCB 95-enhanced dendritic growth in female but not male hippocampal and cortical neurons. In contrast, both male and female neurons responded to bicuculline with increased dendritic complexity. Detailed morphometric analyses confirmed that PCB 95 effects on the number and length of primary and nonprimary dendrites varied depending on sex, brain region and PCB concentration, and that female neurons responded more consistently with increased dendritic growth and at lower concentrations of PCB 95 than their male counterparts. Exposure to PCB 95 did not alter cell viability or the ratio of neurons to glia in cultures of either sex. These results demonstrate that cultured female mouse hippocampal and cortical neurons are more sensitive than male neurons to the dendrite-promoting activity of PCB 95, and suggest that mechanisms underlying PCB 95-induced dendritic growth are sex-dependent. These data highlight the importance of sex in neuronal responses to environmental neurotoxicants.

3,3'-Dichlorobiphenyl (PCB 11) promotes dendritic arborization in primary rat cortical neurons via a CREB-dependent mechanism.

PCB 11 (3,3'-dichlorobiphenyl), a contemporary congener produced as a byproduct of current pigment production processes, has recently emerged as a prevalent worldwide pollutant. We recently demonstrated that exposure to PCB 11 increases dendritic arborization in vitro, but the mechanism(s) mediating this effect remain unknown. To address this data gap, primary cortical neuron-glia co-cultures derived from neonatal Sprague-Dawley rats were exposed for 48 h to either vehicle (0.1% DMSO) or PCB 11 at concentrations ranging from 1 fM to 1 nM in the absence or presence of pharmacologic antagonists of established molecular targets of higher chlorinated PCBs. Reporter cell lines were used to test activity of PCB 11 at the aryl hydrocarbon receptor (AhR) and thyroid hormone receptor (THR). PCB 11 lacked activity at the AhR and THR, and antagonism of these receptors had no effect on the dendrite-promoting activity of PCB 11. Pharmacologic antagonism of various calcium channels or treatment with antioxidants also did not alter PCB 11-induced dendritic arborization. In contrast, pharmacologic blockade or shRNA knockdown of cAMP response element-binding protein (CREB) significantly decreased dendritic growth in PCB 11-exposed cultures, suggesting PCB 11 promotes dendritic growth via CREB-mediated mechanisms. Since CREB signaling is crucial for normal neurodevelopment, and perturbations of CREB signaling have been associated with neurodevelopmental disorders, our findings suggest that this contemporary pollutant poses a threat to the developing brain, particularly in individuals with heritable mutations that promote CREB signaling.

PCB 95 promotes dendritic growth in primary rat hippocampal neurons via mTOR-dependent mechanisms.

Polychlorinated biphenyls (PCBs), and in particular non-dioxin-like (NDL) congeners, continue to pose a significant risk to the developing nervous system. PCB 95, a prevalent NDL congener in the human chemosphere, promotes dendritic growth in rodent primary neurons by activating calcium-dependent transcriptional mechanisms that normally function to link activity to dendritic growth. Activity-dependent dendritic growth is also mediated by calcium-dependent translational mechanisms involving mechanistic target of rapamycin (mTOR), suggesting that the dendrite-promoting activity of PCB 95 may also involve mTOR signaling. Here, we test this hypothesis using primary neuron-glia co-cultures derived from the hippocampi of postnatal day 0 Sprague Dawley rats. PCB 95 (1 nM) activated mTOR in hippocampal cultures as evidenced by increased phosphorylation of mTOR at ser2448. Pharmacologic inhibition of mTOR signaling using rapamycin (20 nM), FK506 (5 nM), or 4EGI-1 (1 µM), and siRNA knockdown of mTOR, or the mTOR complex binding proteins, raptor or rictor, blocked PCB 95-induced dendritic growth. These data identify mTOR activation as a novel molecular mechanism contributing to the effects of PCB 95 on dendritic arborization. In light of clinical data linking gain-of-function mutations in mTOR signaling to neurodevelopmental disorders, our findings suggest that mTOR signaling may represent a convergence point for gene by environment interactions that confer risk for adverse neurodevelopmental outcomes.

In vivo and in vitro sex differences in the dendritic morphology of developing murine hippocampal and cortical neurons.

Altered dendritic morphology is common in neurodevelopmental disorders (NDDs), many of which show sex biases in prevalence, onset and/or severity. However, whether dendritic morphology varies as a function of sex in juvenile mice or primary neuronal cell cultures is largely unknown even though both are widely used models for studying NDDs. To address this gap, we quantified dendritic morphology in CA1 pyramidal hippocampal and adjacent somatosensory pyramidal cortical neurons from male and female postnatal day (P)28 C57BL/6J mice. As determined by Sholl analysis of Golgi-stained brain sections, dendritic arbors of male hippocampal neurons are more complex than females. Conversely, dendritic morphology of female cortical neurons is more complex than males. In primary neuron-glia co-cultures from P0 mouse hippocampi, male neurons have more complex dendritic arbors than female neurons. Sex differences are less pronounced in cortical cultures. In vitro sex differences in dendritic morphology are driven in part by estrogen-dependent mechanisms, as evidenced by decreased dendritic complexity in male hippocampal neurons cultured in phenol red-free media or in the presence of an estrogen receptor antagonist. Evidence that sex influences dendritic morphogenesis in two models of neurodevelopment in a region-specific manner has significant mechanistic implications regarding sex biases in NDDs.

Detection of 3,3'-Dichlorobiphenyl in Human Maternal Plasma and Its Effects on Axonal and Dendritic Growth in Primary Rat Neurons.

3,3'-Dichlorobiphenyl (PCB 11), a byproduct of pigment production, is increasingly detected in environmental samples. While more highly chlorinated PCB congeners are known developmental neurotoxicants, nothing is known about the potential developmental neurotoxicity of PCB 11. To address this critical data gap, we measured PCB 11 levels in human maternal plasma and quantified the effects of PCB 11 and its major metabolites on morphometric parameters of neuronal connectivity in cultured primary neurons. Mass spectrometry analyses of plasma from 241 pregnant women enrolled in the MARBLES study (University of California, Davis) detected PCB 11 in all samples at concentrations ranging from 0.005 to 1.717 ng/ml. Morphometric analyses of primary neuron-glia co-cultures dissociated from the neocortices or hippocampi of neonatal Sprague Dawley rats exposed to vehicle or concentrations ranging from 1 attamolar (aM) to 1 micromolar (µM) of PCB 11, OH-PCB 11, or PCB 11 sulfate indicated that PCB 11 and both metabolites significantly increased axonal and dendritic growth in cortical and hippocampal pyramidal neurons. PCB 11 significantly altered neuronal morphogenesis at concentrations as low as 1 femtomolar (fM), which is ∼0.22 ng/ml. These data suggest the potential for the developing human brain to be exposed to PCB 11, and demonstrate that environmentally relevant levels of PCB 11 alter axonal and dendritic growth in neuronal cell types critically involved in cognitive and higher-order behaviors. These findings identify PCB 11 as a potential environmental risk factor for adverse neurodevelopmental outcomes in humans.

Valid statistical approaches for analyzing sholl data: Mixed effects versus simple linear models.

BACKGROUND: The Sholl technique is widely used to quantify dendritic morphology. Data from such studies, which typically sample multiple neurons per animal, are often analyzed using simple linear models. However, simple linear models fail to account for intra-class correlation that occurs with clustered data, which can lead to faulty inferences. NEW METHOD: Mixed effects models account for intra-class correlation that occurs with clustered data; thus, these models more accurately estimate the standard deviation of the parameter estimate, which produces more accurate p-values. While mixed models are not new, their use in neuroscience has lagged behind their use in other disciplines. RESULTS: A review of the published literature illustrates common mistakes in analyses of Sholl data. Analysis of Sholl data collected from Golgi-stained pyramidal neurons in the hippocampus of male and female mice using both simple linear and mixed effects models demonstrates that the p-values and standard deviations obtained using the simple linear models are biased downwards and lead to erroneous rejection of the null hypothesis in some analyses. COMPARISON WITH EXISTING METHODS: The mixed effects approach more accurately models the true variability in the data set, which leads to correct inference. CONCLUSIONS: Mixed effects models avoid faulty inference in Sholl analysis of data sampled from multiple neurons per animal by accounting for intra-class correlation. Given the widespread practice in neuroscience of obtaining multiple measurements per subject, there is a critical need to apply mixed effects models more widely.

Developing and applying the adverse outcome pathway concept for understanding and predicting neurotoxicity.

The Adverse Outcome Pathway (AOP) concept has recently been proposed to support a paradigm shift in regulatory toxicology testing and risk assessment. This concept is similar to the Mode of Action (MOA), in that it describes a sequence of measurable key events triggered by a molecular initiating event in which a stressor interacts with a biological target. The resulting cascade of key events includes molecular, cellular, structural and functional changes in biological systems, resulting in a measurable adverse outcome. Thereby, an AOP ideally provides information relevant to chemical structure-activity relationships as a basis for predicting effects of structurally similar compounds. AOPs could potentially also form the basis for qualitative and quantitative predictive modeling of the human adverse outcome resulting from molecular initiating or other key events for which higher-throughput testing methods are available or can be developed. A variety of cellular and molecular processes are known to be critical for normal function of the central (CNS) and peripheral nervous systems (PNS). Because of the biological and functional complexity of the CNS and PNS, it has been challenging to establish causative links and quantitative relationships between key events that comprise the pathways leading from chemical exposure to an adverse outcome in the nervous system. Following introduction of the principles of MOA and AOPs, examples of potential or putative adverse outcome pathways specific for developmental or adult neurotoxicity are summarized and aspects of their assessment considered. Their possible application in developing mechanistically informed Integrated Approaches to Testing and Assessment (IATA) is also discussed.

Beta-catenin and estrogen signaling collaborate to drive cyclin D1 expression in developing mouse prostate.

Androgen, beta-catenin (CTNNB1), and estrogen pathways stimulate proliferative growth of developing mouse prostate but how these pathways interact is not fully understood. We previously found that androgens induce CTNNB1 signaling in mouse urogenital sinus (UGS) epithelium from which prostatic ductal epithelium derives. Others have shown that low estradiol concentrations induce UGS epithelial proliferative growth. Here, we found that CTNNB1 signaling overlaps cyclin D1 (CCND1) expression in prostatic buds and we used a genetic approach to test whether CTNNB1 signaling induces CCND1 expression. We observed an unexpected sexually dimorphic response to hyperactive CCNTB1 signaling: in male mouse UGS it increased Ccnd1 mRNA abundance without increasing its protein abundance but in female UGS it increased Ccnd1 mRNA and protein abundance, suggesting a potential role for estrogens in stabilizing CCND1 protein. Treating wild type male UGS explants with androgen and either 17ß-estradiol or a proteasome inhibitor increased CCND1 protein and KI67 labeling in prostatic bud epithelium. Together, our results are consistent with an epithelial proliferative growth mechanism linking CTNNB1-driven Ccnd1 transcription and estrogen-mediated CCND1 protein stabilization.

Species and Sex Differences in the Morphogenic Response of Primary Rodent Neurons to 3,3'-Dichlorobiphenyl (PCB 11).

PCB 11 is an emerging global pollutant that we recently showed promotes axonal and dendritic growth in primary rat neuronal cell cultures. Here, we address the influence of sex and species on neuronal responses to PCB 11. Neuronal morphology was quantified in sex-specific primary hippocampal and cortical neuron-glia co-cultures derived from neonatal C57BL/6J mice and Sprague Dawley rats exposed for 48 h to vehicle (0.1% DMSO) or PCB 11 at concentrations ranging from 1 fM to 1 nM. Total axonal length was quantified in tau-1 immunoreactive neurons at day in vitro (DIV) 2; dendritic arborization was assessed by Sholl analysis at DIV 9 in neurons transfected with MAP2B-FusRed. In mouse cultures, PCB 11 enhanced dendritic arborization in female, but not male, hippocampal neurons and male, but not female, cortical neurons. In rat cultures, PCB 11 promoted dendritic arborization in male and female hippocampal and cortical neurons. PCB 11 also increased axonal growth in mouse and rat neurons of both sexes and neuronal cell types. These data demonstrate that PCB 11 exerts sex-specific effects on neuronal morphogenesis that vary depending on species, neurite type, and neuronal cell type. These findings have significant implications for risk assessment of this emerging developmental neurotoxicant.

DNA methylation: a mechanism linking environmental chemical exposures to risk of autism spectrum disorders?

There is now compelling evidence that gene by environment interactions are important in the etiology of autism spectrum disorders (ASDs). However, the mechanisms by which environmental factors interact with genetic susceptibilities to confer individual risk for ASD remain a significant knowledge gap in the field. The epigenome, and in particular DNA methylation, is a critical gene expression regulatory mechanism in normal and pathogenic brain development. DNA methylation can be influenced by environmental factors such as diet, hormones, stress, drugs, or exposure to environmental chemicals, suggesting that environmental factors may contribute to adverse neurodevelopmental outcomes of relevance to ASD via effects on DNA methylation in the developing brain. In this review, we describe epidemiological and experimental evidence implicating altered DNA methylation as a potential mechanism by which environmental chemicals confer risk for ASD, using polychlorinated biphenyls (PCBs), lead, and bisphenol A (BPA) as examples. Understanding how environmental chemical exposures influence DNA methylation and how these epigenetic changes modulate the risk and/or severity of ASD will not only provide mechanistic insight regarding gene-environment interactions of relevance to ASD but may also suggest potential intervention strategies for these and potentially other neurodevelopmental disorders.

In Utero and Lactational TCDD Exposure Increases Susceptibility to Lower Urinary Tract Dysfunction in Adulthood.

Benign prostatic hyperplasia, prostate cancer, and changes in the ratio of circulating testosterone and estradiol often occur concurrently in aging men and can lead to lower urinary tract (LUT) dysfunction. To explore the possibility of a fetal basis for the development of LUT dysfunction in adulthood, Tg(CMV-cre);Nkx3-1(+/-);Pten(fl/+) mice, which are genetically predisposed to prostate neoplasia, were exposedin uteroand during lactation to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 1 µg/kg po) or corn oil vehicle (5 ml/kg) after a single maternal dose on 13 days post coitus, and subsequently were aged without further manipulation, or at 8 weeks of age were exposed to exogenous 17 ß-estradiol (2.5 mg) and testosterone (25 mg) (T+E2) via slow release subcutaneous implants.In uteroand lactational (IUL) TCDD exposure in the absence of exogenous hormone treatment reduced voiding pressure in adult mice, but otherwise had little effect on mouse LUT anatomy or function. By comparison, IUL TCDD exposure followed by exogenous hormone treatment increased relative kidney, bladder, dorsolateral prostate, and seminal vesicle weights, hydronephrosis incidence, and prostate epithelial cell proliferation, thickened prostate periductal smooth muscle, and altered prostate and bladder collagen fiber distribution. We propose a 2-hit model whereby IUL TCDD exposure sensitizes mice to exogenous-hormone-induced urinary tract dysfunction later in life.

Influence of animal husbandry practices on void spot assay outcomes in C57BL/6J male mice.

AIMS: Mice are increasingly being used as models to investigate aspects of urinary dysfunction that humans with lower urinary tract symptoms (LUTS) experience. One method used to examine voiding function is the spontaneous void spot assay. The purpose of this study was to characterize and identify animal husbandry conditions that might confound results of the spontaneous void spot assay in male C57Bl/6J mice. METHODS: Mice were placed in cages lined with filter paper for 4 hr and urine was visualized with UV transillumination. Voiding parameters including urine spot number, spot size, total urine area, primary void area, corner and center voiding were quantified. RESULTS: Adult male mice void more frequently with advancing age and a subpopulation (5-10%) display a frequent spotting pattern at 6-9 weeks of age. Voiding was not significantly different in male mice weaned to group housing (4-6 per cage) versus single housing, and was not altered when they were used as breeders. Voiding was changed upon transferring group housed adult males to single density cages, which decreased total urine area. Repeated assays of male voiding behavior over three consecutive days increased primary void area by the third day of monitoring and revealed that voiding behavior is impacted by routine cage changes and time of day. CONCLUSIONS: Together these results identify housing and husbandry practices that influence male voiding behaviors in the spontaneous void spot assay and will inform voiding behavior analyses conducted with male C57Bl/6J mice.

Histone acetylation regulates prostate ductal morphogenesis through a bone morphogenetic protein-dependent mechanism.

BACKGROUND: Epigenetic factors influence stem cell function and other developmental events but their role in prostate morphogenesis is not completely known. We tested the hypothesis that histone deacetylase (HDAC) activity is required for prostate morphogenesis. RESULTS: We identified the presence of class I nuclear HDACs in the mouse urogenital sinus (UGS) during prostate development and found that Hdac 2 mRNA abundance diminishes as development proceeds which is especially evident in prostatic epithelium. Blockade of HDACs with the inhibitor trichostatin A (TSA) decreased the number of prostatic buds formed in UGS explant cultures but not the number of buds undergoing branching morphogenesis. In the latter, TSA promoted an extensive branching phenotype that was reversed by exogenous NOGGIN protein, which functions as a bone morphogenetic protein (BMP) inhibitor. TSA also increased Bmp2 promoter H3K27ac abundance, Bmp2 and Bmp4 mRNA abundance, and the percentage of epithelial cells marked by BMP-responsive phosphorylated SMAD1/5/8 protein. TSA exposed UGS explants grafted under the kidney capsule of untreated host mice for continued development achieved a smaller size without an obvious difference in glandular histology compared with control treated grafts. CONCLUSIONS: These results are consistent with an active role for HDACs in shaping prostate morphogenesis by regulating Bmp abundance.

DNA methylation as a dynamic regulator of development and disease processes: spotlight on the prostate.

Prostate development, benign hyperplasia and cancer involve androgen and growth factor signaling as well as stromal-epithelial interactions. We review how DNA methylation influences these and related processes in other organ systems such as how proliferation is restricted to specific cell populations during defined temporal windows, how androgens elicit their actions and how cells establish, maintain and remodel DNA methylation in a time and cell specific fashion. We also discuss mechanisms by which hormones and endocrine disrupting chemicals reprogram DNA methylation in the prostate and elsewhere and examine evidence for a reawakening of developmental epigenetic pathways as drivers of prostate cancer and benign prostate hyperplasia.