Maternal immune activation with toll-like receptor 7 agonist during mid-gestation alters juvenile and adult developmental milestones and behavior.

Infections during pregnancy are associated with increased risk for adult neuropsychiatric disease, such as major depressive disorder, schizophrenia, and autism spectrum disorder. In mouse models of maternal immune activation (MIA), different toll-like receptors (TLRs) are stimulated to initiate inflammatory responses in mother and fetus. The goal of this study was to determine sex-dependent aspects of MIA using a TLR7/8 agonist, Resiquimod (RQ), on neurodevelopment. RQ was administered to timed-pregnant mice on embryonic day (E) 12.5. At E15, maternal/fetal plasma cytokines were measured by enzyme-linked immunosorbent assay (ELISA). Maternal cytokines interleukin (IL)-6 and IL-10 were higher while tumor necrosis factor (TNF)-α and IL-17 were lower in pregnant dams exposed to RQ. Fetal cytokines (E15) were altered at the same timepoint with fetal plasma IL-6 and IL-17 greater after RQ compared to vehicle, while IL-10 and TNF-α were higher in male fetuses but not female. Other timed-pregnant dams were allowed to give birth. MIA with RQ did not alter the female to male ratio of offspring born per litter. Body weights were reduced significantly in both sexes at birth, and over the next 5 weeks. Offspring from RQ-injected mothers opened their eyes 5 days later than controls. Similarly, female offspring from RQ-injected mothers exhibited pubertal delay based on vaginal opening 2-3 days later than control females. On the behavioral side, juvenile and adult male and female MIA offspring exhibited less social-like behavior in a social interaction test. Anhedonia-like behavior was greater in MIA adult female mice. This study provides support for sex-dependent influences of fetal antecedents for altered brain development and behavioral outputs that could be indicative of increased susceptibility for adult disorders through immune mechanisms. Future studies are needed to determine neural cellular and molecular mechanisms for such programming effects.

A Microphysiological System for Studying Barrier Health of Live Tissues in Real Time.

Epithelial cells create barriers that protect many different components in the body from their external environment. The gut in particular carries bacteria and other infectious agents. A healthy gut epithelial barrier prevents unwanted substances from accessing the underlying lamina propria while maintaining the ability to digest and absorb nutrients. Increased gut barrier permeability, better known as leaky gut, has been linked to several chronic inflammatory diseases. Yet understanding the cause of leaky gut and developing effective interventions are still elusive due to the lack of tools to maintain tissue's physiological environment while elucidating cellular functions under various stimuli ex vivo. This paper presents a microphysiological system capable of recording real-time barrier permeability of mouse gut tissues in a realistic physiological environment over extended durations. Key components of the microphysiological system include a microfluidic chamber designed to hold the live tissue explant and create a sufficient microphysiological environment to maintain tissue viability; proper media composition that preserves a microbiome and creates necessary oxygen gradients across the barrier; integrated sensor electrodes and supporting electronics for acquiring and calculating transepithelial electrical resistance (TEER); and a scalable system architecture to allow multiple chambers running in parallel for increased throughput. The experimental results demonstrate that the system can maintain tissue viability for up to 72 hours. The results also show that the custom-built and integrated TEER sensors are sufficiently sensitive to distinguish differing levels of barrier permeability when treated with collagenase and low pH media compared to control. Permeability variations in tissue explants from different positions in the intestinal tract were also investigated using TEER revealing their disparities in permeability. Finally, the results also quantitatively determine the effect of the muscle layer on total epithelial resistance.

Evaluating different models of maternal stress on stress-responsive systems in prepubertal mice.

Introduction: Maternal adversity during pregnancy influences neurodevelopment in human and model animal offspring. Adversity can result from stressors coming from many different directions ranging from environmental to nutritional and physiological to immune (e.g., infection). Most stressors result in fetal overexposure to glucocorticoids that have been directly linked to long- and short-term negative impacts on neurological health of offspring. Neuropsychiatric diseases postulated to have fetal origins are diverse and include such things cardiovascular disease, obesity, affective disorders, and metabolic and immune disorders. Methods: The experiments in the current study compare 3 stressors: prenatal exposure to dexamethasone (DEX), maternal high fat diet (HFD), and maternal caloric restriction (CR). Offspring of mothers with these treatments were examined prepubertally to evaluate stress responsiveness and stress-related behaviors in in male and female mice. Results: Prenatal exposure to synthetic glucocorticoid, DEX, resulted in decreased neonatal body weights, reduced social interaction behavior, and hypoactive stress response offspring exposed to maternal DEX. Maternal CR resulted in decreased body weights and social interaction behavior in males and females and increased anxiety-like behavior and acute stress response only in males. HFD resulted in altered body weight gain in both sex offspring with decreased anxiety-like behavior in a female-biased manner. Discussion: The idea that glucocorticoid responses to different stressors might serve as a common stimulus across stress paradigms is insufficient, given that different modes of prenatal stress produced differential effects. Opposite nutritional stressors produced similar outcomes for anxiety-like behavior in both sexes, social-like behavior in females, and a hyperactive adrenal stress response in males. One common theme among the three models of maternal stress (DEX, CR, and HFD) was consistent data showing their role in activating the maternal and fetal immune response. By tuning in on the more immediate immunological aspect on the developing fetus (e.g., hormones, cytokines), additional studies may tease out more direct outcomes of maternal stress in rodents and increase their translational value to human studies.

Microfluidic organotypic device to test intestinal mucosal barrier permeability ex vivo.

To protect the body from external pathogens, the intestines have sophisticated epithelial and mucosal barriers. Disruptions to barrier integrity are associated with a variety of disorders such as irritable bowel disease, Crohn's disease, and celiac disease. One critical component of all barriers are collagens in the extracellular matrix. While the importance of the intestinal barrier is established, current models lack the ability to represent the complex biology that occurs at these barriers. For the current study a microfluidic device model was modified to determine the effectiveness of collagen breakdown to cause barrier disruption. Bacterial collagenase was added for 48 h to the luminal channel of a dual flow microfluidic device to examine changes in intestinal barrier integrity. Tissues exhibited dose-dependent alterations in immunoreactive collagen-1 and claudin-1, and coincident disruption of the epithelial monolayer barrier as indicated by goblet cell morphologies. This ex vivo model system offers promise for further studies exploring factors that affect gut barrier integrity and potential downstream consequences that cannot be studied in current models.

1H-NMR Profiling of Short-Chain Fatty Acid Content from a Physiologically Accurate Gut-on-a-Chip Device.

It has been shown that short-chain fatty acids (SCFAs) produced by the gut microbiome are of importance to host tissue health; however, measuring such compounds in biological samples is often limited to using hours to days old fecal and blood plasma samples. Organ-on-a-chip models have been created to simplify the complexity but struggle to reproduce the full biology of the gut specifically. We recently reported a tissue-in-a-chip gut model that incorporates gut explanted tissue into a microfluidic device. The system maintains a biologically relevant oxygen gradient and tissue ex vivo for days at a time, but minimal characterization of biological activity was reported. Herein, we use 1H-NMR to analyze the SCFA content of tissue media effluents from gut explants cultured in the recently developed microfluidic organotypic device (MOD). 1H-NMR can identify key SCFAs in the complex samples with minimal sample preparation. Our findings show that maintaining physiologically relevant oxygen conditions, something often missing from many other culture systems, significantly impacts the SCFA profile. Additionally, we noted the changes in SCFAs with culture time and potential variability between SCFA levels in male and female mouse tissue explants cultured in the MOD system based on 1H-NMR spectral profiles.

Sex-Dependent Shared and Nonshared Genetic Architecture Across Mood and Psychotic Disorders.

BACKGROUND: Sex differences in incidence and/or presentation of schizophrenia (SCZ), major depressive disorder (MDD), and bipolar disorder (BIP) are pervasive. Previous evidence for shared genetic risk and sex differences in brain abnormalities across disorders suggest possible shared sex-dependent genetic risk. METHODS: We conducted the largest to date genome-wide genotype-by-sex (G×S) interaction of risk for these disorders using 85,735 cases (33,403 SCZ, 19,924 BIP, and 32,408 MDD) and 109,946 controls from the PGC (Psychiatric Genomics Consortium) and iPSYCH. RESULTS: Across disorders, genome-wide significant single nucleotide polymorphism-by-sex interaction was detected for a locus encompassing NKAIN2 (rs117780815, p = 3.2 × 10-8), which interacts with sodium/potassium-transporting ATPase (adenosine triphosphatase) enzymes, implicating neuronal excitability. Three additional loci showed evidence (p < 1 × 10-6) for cross-disorder G×S interaction (rs7302529, p = 1.6 × 10-7; rs73033497, p = 8.8 × 10-7; rs7914279, p = 6.4 × 10-7), implicating various functions. Gene-based analyses identified G×S interaction across disorders (p = 8.97 × 10-7) with transcriptional inhibitor SLTM. Most significant in SCZ was a MOCOS gene locus (rs11665282, p = 1.5 × 10-7), implicating vascular endothelial cells. Secondary analysis of the PGC-SCZ dataset detected an interaction (rs13265509, p = 1.1 × 10-7) in a locus containing IDO2, a kynurenine pathway enzyme with immunoregulatory functions implicated in SCZ, BIP, and MDD. Pathway enrichment analysis detected significant G×S interaction of genes regulating vascular endothelial growth factor receptor signaling in MDD (false discovery rate-corrected p < .05). CONCLUSIONS: In the largest genome-wide G×S analysis of mood and psychotic disorders to date, there was substantial genetic overlap between the sexes. However, significant sex-dependent effects were enriched for genes related to neuronal development and immune and vascular functions across and within SCZ, BIP, and MDD at the variant, gene, and pathway levels.

Sex differences in anatomic plasticity of gut neuronal-mast cell interactions.

The gut wall houses mast cells that are anatomically situated near enteric neuronal fibers. Roles of specific neuropeptides in modulating function of immune components like mast cells in response to challenge with bacterial components are relatively unknown. Investigating such interactions requires models that include diverse cellular elements in native anatomic arrangements. Using an organotypic slice model that maintains gut wall cellular diversity ex vivo, the present study compared responses between tissues derived from male and female mice to examine neural-immune signaling in the gut wall after selected treatments. Ileum slices were treated with pharmacological reagents that block neuronal function (e.g., tetrodotoxin) or vasoactive intestinal peptide (VIP) receptors prior to challenge with lipopolysaccharide (LPS) to assess their influence on anatomic plasticity of VIP fibers and activation of mast cells. Sex differences were observed in the number of mucosal mast cells (c-kit/ACK2 immunoreactive) at baseline, regardless of treatment, with female ileum tissue having 46% more ACK2-IR mast cells than males. After challenge with LPS, male mast cell counts rose to female levels. Furthermore, sex differences were observed in the percentage of ACK2-IR cells within 1 µm of a VIP+ neuronal fiber, and mast cell size, a metric previously tied to activation, with females having larger cells at baseline. Male mast cell sizes reached female levels after LPS challenge. This study suggests sex differences in neural-immune plasticity and in mast cell activation both basally and in response to challenge with LPS. These sex differences could potentially impact functional neuroimmune response to pathogens.

Vasoactive intestinal peptide regulates ileal goblet cell production in mice.

Innervation of the intestinal mucosa has gained more attention with demonstrations of tuft and enteroendocrine cell innervation. However, the role(s) these fibers play in maintaining the epithelial and mucus barriers are still poorly understood. This study therefore examines the proximity of mouse ileal goblet cells to neuronal fibers, and the regulation of goblet cell production by vasoactive intestinal peptide (VIP). An organotypic intestinal slice model that maintains the cellular diversity of the intestinal wall ex vivo was used. An ex vivo copper-free click-reaction to label glycosaminoglycans was used to identify goblet cells. Pharmacological treatment of slices was used to assess the influence of VIP receptor antagonism on goblet cell production and neuronal fiber proximity. Goblet cells were counted and shown to have at least one peripherin immunoreactive fiber within 3 µm of the cell, 51% of the time. Treatment with a VIP receptor type I and II antagonist (VPACa) resulted in an increase in the percentage of goblet cells with peripherin fibers. Pharmacological treatments altered goblet cell counts in intestinal crypts and villi, with tetrodotoxin and VPACa substantially decreasing goblet cell counts. When cultured with 5-Ethynyl-2'-deoxyuridine (EdU) as an indicator of cell proliferation, colocalization of labeled goblet cells and EdU in ileal crypts was decreased by 77% when treated with VPACa. This study demonstrates a close relationship of intestinal goblet cells to neuronal fibers. By using organotypic slices from mouse ileum, vasoactive intestinal peptide receptor regulation of gut wall goblet cell production was revealed.

Human colon function ex vivo: Dependence on oxygen and sensitivity to antibiotic.

BACKGROUND: Human intestines contain a heterogeneous collection of cells that include immune, neural and epithelial elements interacting in a highly complex physiology that is challenging to maintain ex vivo. There is an extreme oxygen gradient across the intestinal wall due in part to microbiota in the lumen and close to the gut wall, which complicates the design of tissue culture systems. The current study established the use of an organotypic slice model of human intestinal tissue derived from colonoscopy biopsies to study host-microbial interactions after antibiotic treatment, and the influence of oxygen concentration on gut wall function. METHODS: Organotypic slices from human colon biopsies collected during routine colonoscopy provided three-dimensional environments that maintained cellular morphology ex vivo. Biopsy slices were used to study impacts of oxygen concentrations and antibiotic treatments on epithelial proliferation rates, and metabolites from tissue culture supernatants. RESULTS: Immune function was validated via demonstration of a T lymphocyte response to Salmonella enterica serovar Typhimurium. Following 24 h of Salmonella exposure there was a significant increase in CD3+ T-lymphocytes in biopsy slices. Metabolite profiling of tissue culture supernatants validated the influence of antibiotic treatment under varied oxygen culture conditions on both host and microbiome-mediated metabolism. Epithelial health was influenced by oxygen and antibiotic. Increased epithelial proliferation was measured in lowered oxygen conditions (1% = 5.9 mmHg) compared to atmospheric conditions standard at 5000 feet above sea level in Colorado (~17% = 100 mmHg). Antibiotic treatment reduced epithelial proliferation only in 5.9 mmHg oxygen cultured slices. CONCLUSIONS: A human colon organotypic slice model was established for applications ranging from gut epithelial proliferation to enteric pathogen influence on mucosal immune functions ex vivo. The results further support the need to account for oxygen concentration in primary tissue cultures, and that antibiotic use impacts gut-microbe-immune interactions.

Estradiol induces BDNF/TrkB signaling in triple-negative breast cancer to promote brain metastases.

Breast cancer brain metastases (BM) affect younger women disproportionally, including those lacking estrogen receptor (ER), progesterone receptor, and HER2 (known as triple-negative breast cancer; TNBC). Previous studies in preclinical models showed that pre-menopausal levels of estradiol (E2) promote TNBC-BM through incompletely understood mechanisms involving reactive astrocytes. Herein, a novel mechanism involving E2-dependent upregulation of brain-derived neurotrophic factor (BDNF) in astrocytes, and subsequent activation of tumor cell tropomyosin kinase receptor B (TrkB), is identified. E2 increased experimental BM of TNBC 4T1BR5 and E0771 cells by 21 and 3.6 fold, respectively, compared to E2-depleted mice. ERα+ reactive astrocytes were found at early and late stages of BM, and E2 upregulated BDNF in ER+ reactive astrocytes in vitro and in vivo. TrkB was expressed in TNBC brain-trophic cell lines, BM-patient-derived xenografts, and breast cancer BM. Conditioned media from E2-treated astrocytes (CM-E2) activated TrkB and downstream AKT, ERK, and PLC-γ signaling in TNBC cells, increasing their invasiveness and tumor-initiating capability in vitro. The promotion of BM by E2-activated astrocytes was found to be more complex, involving feedback loops and other receptor tyrosine kinases. In 4T1BR5 cells, there was a positive feedback loop whereby astrocytic BDNF induced cancer cell BDNF translation. Upregulation of cancer cell BDNF was required to promote full invasiveness of 4T1BR5 in response to CM-E2, and was observed in brain metastatic cells in E2-treated mice in vivo. Moreover, the non-competitive BDNF/TrkB inhibitor ANA-12 reduced E2-induced 4T1BR5 BM to levels similar to OVX mice. BDNF also activated EGFR in TrkB+EGFR+ TNBC cells, suggesting that E2 action through astrocytes activates redundant pathways promoting BM. These findings have important therapeutic implications, as they provide a rationale to use E2-depletion therapies or TrkB inhibitors to prevent or delay development of BM in younger women.

From organotypic culture to body-on-a-chip: A neuroendocrine perspective.

The methods used to study neuroendocrinology have been as diverse as the discoveries to come out of the field. Maintaining live neurones outside of a body in vitro was important from the beginning, building on methods that dated back to at least the first decade of the 20th Century. Neurosecretion defines an essential foundation of neuroendocrinology based on work that began in the 1920s and 1930s. Throughout the first half of the 20th Century, many paradigms arose for studying everything from single neurones to whole organs in vitro. Two of these survived as preeminent systems for use throughout the second half of the century: cell cultures and explant systems. Slice cultures and explants that emerged as organotypic technologies included such neuroendocrine organs such as the brain, pituitary, adrenals and intestine. The vast majority of these studies were carried out in static cultures for which media were changed over a time scale of days. Tissues were used for experimental techniques such as electrical recording of neuronal physiology in single cells and observation by live microscopy. When maintained in vitro, many of these systems only partially capture the in vivo physiology of the organ system of interest, often because of a lack of cellular diversity (eg, neuronal cultures lacking glia). Modern microfluidic methodologies show promise for organ systems, ranging from the reproductive to the gastrointestinal to the brain. Moving forward and striving to understand the mechanisms that drive neuroendocrine signalling centrally and peripherally, there will always be a need to consider the heterogeneous cellular compositions of organs in vivo.

Sex differences in major depression and comorbidity of cardiometabolic disorders: impact of prenatal stress and immune exposures.

Major depressive disorder topped ischemic heart disease as the number one cause of disability worldwide in 2012, and women have twice the risk of men. Further, the comorbidity of depression and cardiometabolic disorders will be one of the primary causes of disability worldwide by 2020, with women at twice the risk. Thus, understanding the sex-dependent comorbidities has public health consequences worldwide. We propose here that sex differences in MDD-cardiometabolic comorbidity originate, in part, from pathogenic processes initiated in fetal development that involve sex differences in shared pathophysiology between the brain, the vascular system, the CNS control of the heart and associated hormonal, immune, and metabolic physiology. Pathways implicate neurotrophic and angiogenic growth factors, gonadal hormone receptors, and neurotransmitters such as gamma amino butyric acid (GABA) on neuronal and vascular development of HPA axis regions, such as the paraventricular nucleus (PVN), in addition to blood pressure, in part through the renin-angiotensin system, and insulin and glucose metabolism. We show that the same prenatal exposures have consequences for sex differences across multiple organ systems that, in part, share common pathophysiology. Thus, we believe that applying a sex differences lens to understanding shared biologic substrates underlying these comorbidities will provide novel insights into the development of sex-dependent therapeutics. Further, taking a lifespan perspective beginning in fetal development provides the opportunity to target abnormalities early in the natural history of these disorders in a sex-dependent way.

Electrochemical biosensor system using a CMOS microelectrode array provides high spatially and temporally resolved images.

The ability to view biological events in real time has contributed significantly to research in the life sciences. While video capture of real time changes in anatomical relationships is important, it is equally important to visualize real time changes in the chemical communications that drive cell behaviors. This paper describes an electrochemical imaging system capable of capturing changes in chemical gradients in live tissue slices. The system consists of a CMOS microchip with 8192 configurable Pt surface electrodes, on-chip potentiostat, on-chip control logic, and a microfluidic device designed to interface with the CMOS chip to support ex vivo tissue experimentation. All data processing and visualization methods, sensor calibrations, microfluidics fabrication, and tissue preparation and handling procedures are described. Using norepinephrine as a target analyte for proof of concept, the system is capable of differentiating concentrations of norepinephrine as low as 8 µM and up to 1024 µM with a linear response and a spatial resolution of 25.5 µm × 30.4 µm. Electrochemical imaging was tested using murine adrenal tissue as a biological model and successfully showed caffeine-stimulated release of catecholamines from live slices of adrenal tissue with temporal sensitivity. This system successfully demonstrates the use of a high-density microelectrode array for electrochemical analysis with high spatiotemporal resolution to gather chemical gradient information in parallel with optical microscopy recordings.

Powering ex vivo tissue models in microfluidic systems.

This Frontiers review analyzes the rapidly growing microfluidic strategies that have been employed in attempts to create physio relevant 'organ-on-chip' models using primary tissue removed from a body (human or animal). Tissue harvested immediately from an organism, and cultured under artificial conditions is referred to as ex vivo tissue. The use of primary (organotypic) tissue offers unique benefits over traditional cell culture experiments, and microfluidic technology can be used to further exploit these advantages. Defining the utility of particular models, determining necessary constituents for acceptable modeling of in vivo physiology, and describing the role of microfluidic systems in tissue modeling processes is paramount to the future of organotypic models ex vivo. Virtually all tissues within the body are characterized by a large diversity of cellular composition, morphology, and blood supply (e.g., nutrient needs including oxygen). Microfluidic technology can provide a means to help maintain tissue in more physiologically relevant environments, for tissue relevant time-frames (e.g., matching the natural rates of cell turnover), and at in vivo oxygen tensions that can be controlled within modern microfluidic culture systems. Models for ex vivo tissues continue to emerge and grow in efficacy as mimics of in vivo physiology. This review addresses developments in microfluidic devices for the study of tissues ex vivo that can serve as an important bridge to translational value.

Embryonic Exposure to Dexamethasone Affects Nonneuronal Cells in the Adult Paraventricular Nucleus of the Hypothalamus.

Neurons in the paraventricular nucleus of the hypothalamus (PVN) integrate peripheral signals and coordinate responses that maintain numerous homeostatic functions. An excess of glucocorticoids during fetal development results in long-lasting consequences tied to disrupted PVN development. The PVN contains a distinct neuronal population and a threefold greater vascular density than the surrounding brain regions that prepubertally is reduced in offspring exposed to excess glucocorticoids in utero. This study expands the examination of sex-specific nonneuronal PVN composition by examining astrocytes, astrocytic endfeet, and pericytes. Blood-brain barrier (BBB) competency and composition were examined along with depressive-like behavior and hypothalamic-pituitary-adrenal function in male and female mice. For PVN vasculature, female offspring of vehicle (veh)-treated mothers had significantly more astrocytes and pericytes than male offspring from the same litters. Female offspring from dexamethasone (dex)-treated mothers had significantly lower levels of astrocytes than female offspring from veh-treated mothers, whereas male offspring from dex-treated mothers had greater levels of pericytes compared with veh-treated male offspring. Using the tail-suspension test, male and female offspring from dex-treated mothers had significantly shorter latencies to immobility, indicating an increase in depression-like behavior, and showed greater plasma corticosterone after restraint stress, which was significantly greater in female offspring from dex-treated mothers even after recovery. Therefore, in addition to long-term sex differences in cellular components of the BBB in the PVN that were differentially regulated by fetal glucocorticoid exposure, there were behavioral differences observed into early adulthood in a sex-specific manner.

An organotypic slice model for ex vivo study of neural, immune, and microbial interactions of mouse intestine.

Organotypic tissue slices provide seminatural, three-dimensional microenvironments for use in ex vivo study of specific organs and have advanced investigative capabilities compared with isolated cell cultures. Several characteristics of the gastrointestinal tract have made in vitro models for studying the intestine challenging, such as maintaining the intricate structure of microvilli, the intrinsic enteric nervous system, Peyer's patches, the microbiome, and the active contraction of gut muscles. In the present study, an organotypic intestinal slice model was developed that allows for functional investigation across regions of the intestine. Intestinal tissue slices were maintained ex vivo for several days in a physiologically relevant environment that preserved normal enterocyte structure, intact and proliferating crypt cells, submucosal organization, and muscle wall composure. Cell death was measured by a membrane-impermeable DNA binding indicator, ethidium homodimer, and less than 5% of cells were labeled in all regions of the villi and crypt epithelia at 24 h ex vivo. This tissue slice model demonstrated intact myenteric and submucosal neuronal plexuses and functional interstitial cells of Cajal to the extent that nonstimulated, segmental contractions occurred for up to 48 h ex vivo. To detect changes in physiological responses, slices were also assessed for segmental contractions in the presence and absence of antibiotic treatment, which resulted in slices with lesser or greater amounts of commensal bacteria, respectively. Segmental contractions were significantly greater in slices without antibiotics and increased native microbiota. This model renders mechanisms of neuroimmune-microbiome interactions in a complex gut environment available to direct observation and controlled perturbation.

The Influence of Gonadal Steroid Hormones on Immunoreactive Kisspeptin in the Preoptic Area and Arcuate Nucleus of Developing Agonadal Mice with a Genetic Disruption of Steroidogenic Factor 1.

Kisspeptin, a regulator of reproductive function and puberty in mammals, is expressed in the rostral (anteroventral) periventricular nucleus (AVPV) and arcuate nucleus (Arc), and its expression is at least partially regulated by estradiol in rodents. The aim of the present study was to determine contributions of genetic factors and gonadal steroid hormones to the sexual differentiation of kisspeptin-immunoreactive (kisspeptin-ir) cell populations in the AVPV and Arc during postnatal development using agonadal steroidogenic factor 1 (SF-1) knockout (KO) mice. To examine the effects of gonadal hormones on pubertal development of kisspeptin neurons, SF-1 KO mice were treated with estradiol benzoate (EB) from postnatal day (P)25 to P36, and their brains were examined at P36. No sex differences were observed in the SF-1 KO mice during postnatal development and after treatment with EB - which failed to increase the number of kisspeptin-ir cells at P36 to the levels found in wild-type (WT) control females. This suggests that specific time periods of estradiol actions or other factors are needed for sexual differentiation of the pattern of immunoreactive kisspeptin in the AVPV. Kisspeptin immunoreactivity in the Arc was significantly higher in gonadally intact WT and SF-1 KO females than in male mice at P36 during puberty. Further, in WT and SF-1 KO females, but not in males, adult levels were reached at P36. This suggests that maturation of the kisspeptin system in the Arc differs between sexes and is regulated by gonad-independent mechanisms.

Spatiotemporal norepinephrine mapping using a high-density CMOS microelectrode array.

A high-density amperometric electrode array containing 8192 individually addressable platinum working electrodes with an integrated potentiostat fabricated using Complementary Metal Oxide Semiconductor (CMOS) processes is reported. The array was designed to enable electrochemical imaging of chemical gradients with high spatiotemporal resolution. Electrodes are arranged over a 2 mm × 2 mm surface area into 64 subarrays consisting of 128 individual Pt working electrodes as well as Pt pseudo-reference and auxiliary electrodes. Amperometric measurements of norepinephrine in tissue culture media were used to demonstrate the ability of the array to measure concentration gradients in complex media. Poly(dimethylsiloxane) microfluidics were incorporated to control the chemical concentrations in time and space, and the electrochemical response at each electrode was monitored to generate electrochemical heat maps, demonstrating the array's imaging capabilities. A temporal resolution of 10 ms can be achieved by simultaneously monitoring a single subarray of 128 electrodes. The entire 2 mm × 2 mm area can be electrochemically imaged in 64 seconds by cycling through all subarrays at a rate of 1 Hz per subarray. Monitoring diffusional transport of norepinephrine is used to demonstrate the spatiotemporal resolution capabilities of the system.

GnRH Neuron-Specific Ablation of Gαq/11 Results in Only Partial Inactivation of the Neuroendocrine-Reproductive Axis in Both Male and Female Mice: In Vivo Evidence for Kiss1r-Coupled Gαq/11-Independent GnRH Secretion.

The gonadotropin-releasing hormone (GnRH) is the master regulator of fertility and kisspeptin (KP) is a potent trigger of GnRH secretion from GnRH neurons. KP signals via KISS1R, a Gαq/11-coupled receptor, and mice bearing a global deletion of Kiss1r (Kiss1r(-/-)) or a GnRH neuron-specific deletion of Kiss1r (Kiss1r(d/d)) display hypogonadotropic hypogonadism and infertility. KISS1R also signals via ß-arrestin, and in mice lacking ß-arrestin-1 or -2, KP-triggered GnRH secretion is significantly diminished. Based on these findings, we hypothesized that ablation of Gαq/11 in GnRH neurons would diminish but not completely block KP-triggered GnRH secretion and that Gαq/11-independent GnRH secretion would be sufficient to maintain fertility. To test this, Gnaq (encodes Gαq) was selectively inactivated in the GnRH neurons of global Gna11 (encodes Gα11)-null mice by crossing Gnrh-Cre and Gnaq(fl/fl);Gna11(-/-) mice. Experimental Gnaq(fl/fl);Gna11(-/-);Gnrh-Cre (Gnaq(d/d)) and control Gnaq(fl/fl);Gna11(-/-) (Gnaq(fl/fl)) littermate mice were generated and subjected to reproductive profiling. This process revealed that testicular development and spermatogenesis, preputial separation, and anogenital distance in males and day of vaginal opening and of first estrus in females were significantly less affected in Gnaq(d/d) mice than in previously characterized Kiss1r(-/-) or Kiss1r(d/d) mice. Additionally, Gnaq(d/d) males were subfertile, and although Gnaq(d/d) females did not ovulate spontaneously, they responded efficiently to a single dose of gonadotropins. Finally, KP stimulation triggered a significant increase in gonadotropins and testosterone levels in Gnaq(d/d) mice. We therefore conclude that the milder reproductive phenotypes and maintained responsiveness to KP and gonadotropins reflect Gαq/11-independent GnRH secretion and activation of the neuroendocrine-reproductive axis in Gnaq(d/d) mice. SIGNIFICANCE STATEMENT: The gonadotropin-releasing hormone (GnRH) is the master regulator of fertility. Over the last decade, several studies have established that the KISS1 receptor, KISS1R, is a potent trigger of GnRH secretion and inactivation of KISS1R on the GnRH neuron results in infertility. While KISS1R is best understood as a Gαq/11-coupled receptor, we previously demonstrated that it could couple to and signal via non-Gαq/11-coupled pathways. The present study confirms these findings and, more importantly, while it establishes Gαq/11-coupled signaling as a major conduit of GnRH secretion, it also uncovers a significant role for non-Gαq/11-coupled signaling in potentiating reproductive development and function. This study further suggests that by augmenting signaling via these pathways, GnRH secretion can be enhanced to treat some forms of infertility.

Novel Interaction of Class IIb Histone Deacetylase 6 (HDAC6) with Class IIa HDAC9 Controls Gonadotropin Releasing Hormone (GnRH) Neuronal Cell Survival and Movement.

The impact of histone deacetylases (HDACs) in the control of gonadotropin releasing hormone (GnRH) neuronal development is unknown. We identified an increase in many HDACs in GT1-7 (differentiated) compared with NLT (undifferentiated) GnRH neuronal cell lines. Increased HDAC9 mRNA and protein and specific deacetylase activity in GT1-7 cells suggested a functional role. Introduction of HDAC9 in NLT cells protected from serum withdrawal induced apoptosis and impaired basal neuronal cell movement. Conversely, silencing of endogenous HDAC9 in GT1-7 cells increased apoptosis and cell movement. Comparison of WT and mutant HDAC9 constructs demonstrated that the HDAC9 pro-survival effects required combined cytoplasmic and nuclear localization, whereas the effects on cell movement required a cytoplasmic site of action. Co-immunoprecipitation demonstrated a novel interaction of HDAC9 selectively with the Class IIb HDAC6. HDAC6 was also up-regulated at the mRNA and protein levels, and HDAC6 catalytic activity was significantly increased in GT1-7 compared with NLT cells. HDAC9 interacted with HDAC6 through its second catalytic domain. Silencing of HDAC6, HDAC9, or both, in GT1-7 cells augmented apoptosis compared with controls. HDAC6 and -9 had additive effects to promote cell survival via modulating the BAX/BCL2 pathway. Silencing of HDAC6 resulted in an activation of movement of GT1-7 cells with induction in acetylation of α-tubulin. Inhibition of HDAC6 and HDAC9 together resulted in an additive effect to increase cell movement but did not alter the acetylation of αtubulin. Together, these studies identify a novel interaction of Class IIa HDAC9 with Class IIb HDAC6 to modulate cell movement and survival in GnRH neurons.