Study Objective: We aim to determine the antigen presentation capacity of enteric glial cells in response to challenge with Epstein-Barr virus (EBV). Hypothesis: Enteric glial cells are innate immune competent and express antigens via HLA/MHC class I and II moieties in addition to their roles in supporting neuronal function. We recently showed that enteric glial cells are reactive in the progressive multiple sclerosis (MS) colon and found stool levels of the enteric glial proteins glial fibrillary acidic protein (GFAP) and S100b to be starkly elevated in progressive MS vs. relapsing-remitting MS (RRMS) and healthy controls (Neurol Neuroimm Neurinflamm, 12(6):e200466, 2025. Downstream impacts of enteric glial antigen presentation in the human gut wall remain unknown. Enteric glial cells express myelin proteins including proteolipid protein 1 (PLP-1) yet are non-myelinating glia. The infective capacity of enteric glial cells, particularly with viruses relevant to MS etiology like EBV is not known. We hypothesize that infection of enteric glial cells by EBV induces antigen presentation of peptides with myelin protein sequence homology, triggering the induction of autoreactive T cells. Methods: Duodenum and colon tissue from healthy controls and MS subjects was analyzed via immunohistochemistry for PLP-1, GFAP, S100b, and HLA class I and class II alleles. An immortalized human enteric glial cell line was used to investigate responses to EBV challenge via immunohistochemistry. We investigated peptide expression by HLAs in EBV, heat-killed EBV, and vehicle treated glial cell cultures before processing for global immunopeptidomics for HLA-(A/B/C) and HLA-DR/DQ. Results: Intestinal tissue from MS subjects showed elevated levels of enteric glial cells as marked by immunoreactivity to PLP-1, S100b, and GFAP. We found that enteric glia from MS subjects expressed substantially more immunoreactive EBV receptor (anti-CD21) and EBV nuclear antigen (EBNA1) when compared to healthy control tissue. When we investigated the response of human enteric glial cell cultures to EBV challenge, we found an elevation in immunoreactive HLAs from class I and II. Immunopeptidomics showed an elevation in peptides presented by both HLA class I and class II alleles after challenge with EBV vs. heat killed EBV or vehicle controls. Conclusions: The results indicate an intestinal neural phenotype in MS, with enteric glial cell reactivity and antigen presentation in the MS gut and in response to EBV challenge in vitro. Peptide presentation profiles will guide future investigations that allow for targeted immunotherapies at the enteric glial – T cell interface. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Maternal immune activation (MIA) can occur during pregnancy due to infectious diseases or other inflammatory conditions. Resiquimod (RQ), a toll-like receptor (TLR) 7 agonist, induces inflammatory responses and mimics responses to viral infections. We previously showed that prenatal stressors increase anxiety-like behavior and dysregulation of neuroendocrine and autonomic function in adult offspring. The current project further investigates the degree to which late-gestation MIA with RQ impacts offspring development, anxiety-like behavior, and neuroendocrine function. Pregnant rat dams were injected with vehicle (phosphate-buffered saline) or RQ (1 mg/kg, s.c.) on gestation day 18. Tests were conducted in offspring before (3-4wks old) and after (8-10wks old) puberty. Open field tests assessed anxiety-like behavior, and a 20-minute restraint tube stress test assessed products of hypothalamic-pituitary-adrenal (HPA) axis activation. In-utero MIA exposure did not affect birth weights or early-life developmental milestones (i.e., the timing of eye opening, righting reflex). The female offspring of MIA dams experienced a significant delay (~1 day) in vaginal opening, suggesting delayed pubertal onset. Before puberty, MIA did not affect offspring open field assessments, nor were there differences in levels of the glucocorticoid corticosterone (CORT) in plasma following restraint. After puberty, male and female offspring of MIA dams showed increased anxiety-like behavior (F(1,42)=4.701, p=0.0359). Plasma CORT concentrations following restraint also trended higher in the adult female offspring of dams exposed to RQ (F(1,22)=3.337, p=0.0813), but with a higher suggested average in all the adult offspring of MIA dams compared to controls. We also measured proteins involved in the synthesis of norepinephrine/epinephrine (i.e., tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH)), the degradation of norepinephrine/epinephrine (i.e., catechol-o-methyltransferase (COMT)), and the degradation of acetylcholine (i.e., acetylcholinesterase (AChE)) within the left ventricle of the heart. Adult female offspring express higher levels of TH (F(1,13) = 4.365, p=0.0569), DBH (F(1,13)=7.628, P=0.0162), COMT (F(1,13)=9.009, p=0.0102), and AChE (F(1,13)=4.105, p=0.0638) compared to males, regardless of prenatal MIA exposure. The results support MIA-induced dysregulation of the HPA axis and anxiety-like behavior only in adult offspring, suggesting that gonadal hormones and aging play a critical role in programming changes associated with in-utero MIA exposure. This project is funded by ORWH-NIMH 3U54MH118919 and ORWH-NIMH 3U54MH118919-04S3. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

The autonomic nervous system (ANS) coordinates the body's response to stress. Both physical and psychological stressors trigger neuroendocrine reactions from the hypothalamus, influencing peripheral systems such as cardiovascular, pulmonary, and gastrointestinal systems. Chronic stress can activate immune responses, leading to elevated levels of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNFα), which has been associated with psychiatric disorders, including major depressive disorder. In this study, we investigated the impact of pro-inflammatory cytokines on microglia in the dorsal vagal complex (DVC) and the ventral lateral medulla (VLM), key brainstem regions involved in autonomic signal integration. The DVC includes the area postrema (AP), a sensory circumventricular organ lacking an intact blood-brain barrier. This structural feature makes the AP particularly vulnerable to circulating factors, allowing it to relay peripheral changes to brainstem neurons. We administered an acute peripheral injection of TNFα (63 µg/kg) and, two hours later, perfused the animals with paraformaldehyde. The number of cFOS immunoreactive cells, indicative of neuronal activation, was significantly elevated in the DVC (44.33 ± 7.513; p < 0.05) and the VLM (9.00 ± 1.37; p < 0.05). Microglia, being resident immune cells in the CNS, respond to pro-inflammatory cytokines, such as TNFα. To examine the role of microglia in mediating neuronal responses to peripheral TNFα, we administered minocycline (160 µg/kg) into the fourth ventricle to inhibit microglia responses caused by pro-inflammatory signaling. This treatment reduced IBA1 immunoreactivity by 71% in the AP (223.6 µm 2 ± 30.79; p < 0.002) and by 57% in the NTS (123.9 µm 2 ± 39.63; p < 0.01). Subsequently, a TNFα injection was given 24 hours after minocycline administration. With microglial polarization inhibited, the number of cFOS-positive cells was significantly reduced in both the DVC (p < 0.05) and the VLM (p < 0.002). These findings highlight the impact of acute pro-inflammatory signaling on brainstem autonomic circuits and suggest that brainstem neurons can relay peripheral signals independently of microglial activation. Future research will aim to identify the specific neuronal phenotypes affected by TNFα following microglial inhibition and their role in ANS regulation. ORWH U54-MH118919 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

BACKGROUND:Parkinson's Disease (PD) is a neurodegenerative disorder with prodromal gastrointestinal (GI) issues often emerging decades before motor symptoms. Pathologically, PD can be driven by the accumulation of misfolded alpha synuclein (aSyn) protein in the brain and periphery, including the GI tract. Disease epidemiology differs by sex, with men twice as likely to develop PD. Women, however, experience faster disease progression, higher mortality, and more severe GI symptoms. Gut calcitonin gene-related peptide (CGRP) is a key regulator of intestinal contractions and visceral pain. The current study tests the hypothesis that sex differences in GI symptomatology in PD are the result of aSyn aggregation altering enteric CGRP signaling pathways. METHODS:To facilitate peripheral aSyn aggregation, the pesticide rotenone was administered intraperitoneally once daily for 2 weeks to male and female mice. Mice were sacrificed 2 weeks after the last rotenone injection, and immunohistochemistry was performed on sections of proximal colon. KEY RESULTS:Levels of aSyn were heightened in PGP9.5 immunoreactive myenteric plexus neurons, a subset of which were immunoreactive to CGRP and showed a similar increase in aSyn immunoreactivity in rotenone-treated mice. Female mice exhibited 153% more myenteric aSyn, 26% more apical CGRP immunoreactivity in the mucosa, and 66.7% more aSyn in apical CGRP+ fibers after rotenone when compared to males. Goblet cell numbers were diminished, but the individual cells were larger in the apical regions of crypts in the colons of rotenone-treated mice with no difference between males and females. CONCLUSIONS:This study used a mouse model of PD to uncover sex-specific alterations in enteric neuronal and epithelial populations, underscoring the importance of considering sex as a biological variable while investigating prodromal GI symptoms.

Parkinson’s Disease (PD) is a neurodegenerative disorder with prodromal symptoms often emerging decades before motor issues, including gastrointestinal (GI) symptoms such as constipation and visceral pain. The pathological hallmark of PD is accumulation of misfolded α-synuclein (aSyn) protein in the brain and periphery, including the GI tract. Men have two times the risk of developing PD compared to women. However, disease progression and mortality are higher in women, who also report worse GI symptoms. Gut calcitonin gene related peptide (CGRP) is involved in modulating intestinal contractions and pain, and CGRP receptor antagonists lead to constipation. We hypothesize that sex differences in GI symptoms of PD are the result of sex specific aSyn aggregation and differential enteric CGRP signaling. Intraperitoneal injections of the pesticide rotenone were given to C57BL/6J mice to facilitate aggregation of aSyn. Immunohistochemistry was performed on colon sections using antibodies to aSyn, protein gene product (PGP) 9.5, and CGRP. Quantities of aSyn were analyzed in myenteric PGP9.5+ neuronal fibers. Vehicle treated females had 48% (mean +/- SEM = 0.66 μm2 +/- 0.04) less immunoreactive (ir) enteric aSyn than males (1.3 μm2 +/- 0.13; [F(1,22) = 4.8, p < 0.05). Rotenone treatment increased aSyn-ir by 56% (1.5 μm2 +/- 0.13) in females and 28% (1.08 μm2 +/- 0.14) in males. When interrogated by region, rotenone treatment increased the average precent area of CGRP-ir in the apical crypt (male 9% +/- 0.6; female 10% +/- 0.6) compared to vehicle (male 7% +/- 0.8; female 7% +/- 0.7). This increase was 14% higher in females compared to males (F(1,12) = 5.6, p < 0.05). In males and females, the average percent area of CGRP-ir in the mid-basal crypt region decreased with rotenone treatment (male 4% +/- 0.4; female 2.6% +/- 0.1) compared to vehicle (male 3%+/-0.2, female 1.7%+/-0.06; [F(1,12) = 19.62, p < 0.001). Microbiota have been shown to modulate visceral pain via the release of CGRP, and PD patients have altered microbiotas. The larger increase in CGRP-ir at the apical region of crypts after rotenone treatment in females may contribute to sensitivity to noxious stimuli from the lumen, including microbiota and their metabolites. In addition, decreased mid-basal CGRP-ir and aSyn increases in females after rotenone treatment provide valuable data towards explaining increased GI symptoms seen in PD females. Females are significantly understudied within the context of PD, however, investigating the underlying pathophysiology of GI symptoms while addressing sex as a variable could provide clues to the sex differences seen in clinical outcomes. Since these symptoms often arise years before motor dysfunction and diagnosis, it is critical to determine sex specific mechanisms of aSyn aggregation and influence on enteric neural involvement in prodromal GI symptoms. SCORE MGH ORWH U54-MH118919. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract Disclosure: S. Ariyanfar: None. C.K. Thompson: None. S. Tobet: None. D.J. Good: None. Prader–Willi syndrome (PWS) is a multisystemic neurodevelopmental disorder, characterized by biphasic symptoms, including diminished weight gain during development, along with neonatal hypotonia and failure to thrive, with later onset hyperphagia, severe weight gain, and morbid obesity. PWS arises from the deletion or inactivation of paternally inherited genes, with the smallest region encompassing a group of 30 small nucleolar RNAs ‘snoRNAs’ known as the SNORD116@ locus. SNORD116@ snoRNAs are cleaved from a larger long non-coding gene, SNHG14. Whole-body deletion of Nhlh2 in mice results in PWS-like phenotypes, such as later onset weight gain, and delayed puberty. We previously have shown that overexpression of Snord116-3 in a mouse hypothalamic cell line improves the stability of Nhlh2 mRNA, likely through a motif in the 3’ untranslated region of the Nhlh2 mRNA. This motif can be disrupted by the presence of a single nucleotide polymorphism, confirming the mechanism of action. Nevertheless, to date, no published studies are showing the in vivo co-localization between Nhlh2 and Snord116 (or its host gene, Snhg14), and the question of where and when Snord116 snoRNAs interact with target mRNA Nhlh2 remains unanswered. To fill this gap, we mapped simultaneous expression of Snhg14 and Nhlh2 throughout the adult mouse brain, using quantitative RNA multi-plex in situ hybridization “RNAScope”. In our initial analysis, we focused on Pomc neurons of the hypothalamus as targeted deletion of Nhlh2 in these neurons results in obesity levels similar to that of the whole body deletion of Nhlh2, a present phenotype in PWS. Our data for the first time showed the co-expression of Nhlh2 and Snhg14 in Pomc neurons of Arcuate nucleus. In the analysis of 54 Pomc neurons, 72% and 39% were positive for Nhlh2 and Snhg14 expression respectively, with 39% of detected signals co-expressed in the same cell (correlation coefficient 0.78, P < 0.0001). Interestingly, in neurons that only expressed Nhlh2 (33) there were nearly evenly distributed signals in the cytoplasm (9 cells) and nucleus (10 cells). When both Nhlh2 and Snhg14 messages were coexpressed, 17 of the 21 cells expressed Nhlh2 in the nucleus only (P < 0.0003, P< 0.0007 for the Likelihood ratio test and Chi-square test, respectively). Thus, based on the high magnification cell analysis, acquired 3D images, and probe location, we can conclude that the Snhg14 host transcript and Nhlh2 interact spatially. This new in vivo data supports the previously shown regulatory role for Snord 116 snoRNAs towards Nhlh2 mRNA stability and suggests that regulation may involve nuclear sequestration of Nhlh2. We are currently extending our studies to examine expression in the brains from patients with PWS and determine if energy balance signals affect co-localization patterns within the hypothalamus. Presentation: 6/3/2024

Precision cut lung slices (PCLS) bridge a gap between in vivo and in vitro studies by maintaining anatomical organization with structural integrity and intercellular signaling pathways. Applications of PCLS have included the modeling of inflammatory lung diseases, metabolism studies, and drug development. In the lungs, immune responses are carried out by a network of T- and B- cells, the latter of which are resident. The limited resident T-cell population of the lung diminishes accurate representations of pathogen response capacity in PCLS. Addressing this, we set out to increase pulmonary T-cell populations ex vivo. We hypothesized that thymus and bone marrow-derived T-cells would work synergistically to populate the lung in co-culture experiments. A murine organotypic lung co-culture model was developed and characterized for tissue health and T-cell recruitment over 3 days ex vivo using adult neurobasal media with 4 mM glucose + 2% B27 supplement. Lung slices were cultured independently, with bone marrow, thymus, or both. Immune colonization of the lung was assessed using immunohistochemistry for CD3+ T-cells and ACK2+ cells. Cells were counted in alveolar and airway spaces after 3 days of culture. Co-culture of lung slices with bone marrow did not increase CD3+ immunoreactive T-cells while thymus co-culture increased CD3+ T-cells by 76% in the alveolar space and by 39% in the airway, relative to lung alone. When lung slices were cultured with bone marrow plus thymus, CD3+ T-cells increased by 206% in the alveolar space and by 251% in the airway, relative to the lung alone. Co-culture with thymus increased ACK2+ cells by 48% in the airway, while lung culture with both thymus and bone marrow, ACK2+ cells increased by 35% in the alveolar space and 85% in the airway, relative to the lung alone. These results suggest that the increased T-cell population corresponding with thymus and bone marrow co-culture could be a result of cell-cell interaction or the secretion of growth factors. Cell secretions or growth factor release could stimulate thymic secretion of T-cells or could stimulate T-cell proliferation in the lung, suggesting that co-culture with thymus and bone marrow can elicit a T-cell response ex vivo. Future studies will center around differentiating what drives T-cell population to be increased in lung co-cultures and what T-cell subsets are being recruited in PCLS co-cultures while analyzing their capacity for response to pathogens ex vivo. Anschutz Foundation Pandemic Preparedness Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract Disclosure: J.A. Sheng: None. S.A. Tobet: None. Background and Hypothesis: Maternal immune activation (MIA) has been associated with increased risk for neuropsychiatric diseases in the offspring. Recently, our lab demonstrated toll-like receptor 7 (TLR7) activation during impaired stress-related behaviors (anxiety-, anhedonia-, social-like) (Sheng et al., 2023, Front. Neurosci.). Stress-related behaviors are regulated by neurons in the paraventricular nucleus of the hypothalamus (PVN), an anatomic region involved in the hypothalamic-pituitary-adrenal (HPA) stress response. We hypothesize immune system activation by TLR7 during mid-gestation impairs PVN neuroendocrine stress response and brain vasculature. Methods: Timed-pregnant female mice were administered the TLR7 agonist Resiquimod (RQ) or vehicle saline on embryonic day 12.5. Adult offspring underwent 20-minute acute restraint stress with 80-minutes of recovery to examine plasma corticosterone (ELISA assay) as an indicator of HPA function. Mice were transcardially perfused with fluorescein isothiocyanate (FITC; 100mg/mL) to visualize blood vessel integrity (FITC leakage; Frahm & Tobet, 2015, Brain Struct. Funct.). Sections through the PVN of offspring were also immunolabeled for Glial Fibrillary Acidic Protein (GFAP; astrocytic end feet) and IBA-1 (microglia). Only data from brains of offspring that did not undergo restraint stress are presented for baseline changes in blood-brain barrier components by MIA. Results: Plasma corticosterone was elevated in RQ-offspring (males p<0.0001, females p<0.01 vs. VEH) following stressor and recovery, suggesting delayed negative feedback to the HPA axis. Blood-brain barrier integrity diminished in RQ-adult offspring who showed greater FITC leakage in the PVN as indicated by an increase in the ratio of extravascular to intravascular FITC (males p<0.001, females p<0.01). GFAP+ coverage of FITC-labeled vessels was greater in the PVN only in RQ males compared to VEH (p<0.001). Microglia were examined in relation to vasculature, and data show more IBA-1+ cells in the PVN with close proximity (within 2µm) to blood vessels after maternal injection of RQ in females compared to VEH (p<0.05). In RQ-adult males, the average size of IBA-1+ cells in the PVN were greater than VEH counterparts (p<0.05), but number and proximity of IBA-1+ cells to blood vessels were unchanged. No changes in these measures were seen in control brain regions. Conclusions: This study provides support for sex-dependent influences of fetal immune antecedents on brain development and adult neuroendocrine function that could indicate a locus for increased susceptibility to adult neuropsychiatric disorders. While the current experiments found significant post-pubertal changes selectively in adult PVN, ongoing studies are examining whether these effects are evident prior to puberty. Supported by ORWH-NIMH U54 SCORE-MH118919. Presentation: 6/1/2024

Maternal immune activation (MIA), a maternal stressor, increases risk for neuropsychiatric diseases, such as Major Depressive Disorder in offspring. MIA of toll-like receptor 7 (TLR7) initiates an immune response in mother and fetuses in a sex-selective manner. The paraventricular nucleus of the hypothalamus (PVN), a brain region that is sexually dimorphic and regulates hypothalamic-pituitary-adrenal (HPA) stress responses, have been tied to stress-related behaviors (i.e., depression, anxiety, social impairments). The current study characterized the sex-selective impact of mid-gestational TLR7 activation on PVN vasculature of adult offspring based on a prior study of excess prenatal glucocorticoid stress. The PVN of offspring were evaluated to determine if fetal MIA impacted vascular leakage in the brains of adult mice with or without restraint stress. Timed-pregnant female mice were administered the TLR7 agonist Resiquimod (RQ) or saline vehicle on embryonic day (E) 12.5. Basal and restraint stress-induced corticosterone was measured to examine changes in stress response. Mice were perfused transcardially with fluorescein isothiocyanate (FITC) to assess blood vessel integrity. Sections with FITC-labeled blood vessels through the PVN of offspring were immunolabeled for Glial Fibrillary Acidic Protein (GFAP; astrocytic end feet) and IBA-1 (microglia). MIA with RQ led to elevated levels of plasma corticosterone 60-minutes after restraint in offspring, suggesting prenatal RQ impairs glucocorticoid negative feedback. Blood-brain barrier integrity was assessed. Adult offspring of RQ injected dams showed greater leakage in the PVN (greater in males than females). GFAP+ colocalization with FITC-labeled vessels was lower in the PVN of offspring from RQ treated dams, potentially contributing to the observed increased FITC leakage. Microglia were examined in relation to the vasculature as an indicator of a neuroimmune response. Data show IBA-1+ cells greater in size and number in the PVN with closer proximity to blood vessels after maternal injection of RQ in a male-selective manner. Microglia were unchanged in females from RQ-treated dams but were smaller in size after restraint. This study provides support for sex-selective influences of fetal immune antecedents for altered brain vascular and blood brain barrier development and adult neuroendocrine function that could indicate a PVN locus for increased susceptibility for adult disorders.

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.

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.

Abstract Disclosure: E.A. Castellanos: None. S.A. Tobet: None. The autonomic nervous system (ANS) is a major integrator of stress responses throughout the body. Physical or psychological stressors lead to neuroendocrine outputs from the hypothalamus and neurotransmitter mediated alterations in circuitry that control peripheral body systems including cardiovascular, pulmonary, and gastrointestinal, among others. Chronic stress can activate immune components, including microglia, leading to increased levels of proinflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-a) in the blood. Increased levels of these cytokines have been associated with several psychiatric disorders including major depressive disorder. In the current study, the impact of pro-inflammatory cytokines was examined in the dorsal vagal complex (DVC), a key brainstem region responsible for integrating autonomic inputs and outputs. The DVC includes the area postrema (AP), a sensory circumventricular organ, the nucleus of the solitary tract (NTS), and the vagal dorsal motor complex. The AP is in the middle of the DVC region, adjacent to cerebrospinal fluid and lacking a blood brain barrier (i.e., fenestrated capillaries). These traits make the AP uniquely located to integrate peripheral and central interactions between circulating cytokines and select immune cells in the DVC. This study concentrated on the effects of a single peripheral injection on microglia quantities in the DVC. TNF-a (50µg/Kg) and IL-6 (50µg/Kg) were administered by intraperitoneal injection and 2h later animals were perfused with 4% paraformaldehyde. Immunoreactive ionized calcium-binding adapter molecule 1 (IBA1) was used to identify microglia in the brainstem. In the area postrema there was a trend towards more immunoreactive IBA1 total area (µm^2; p = 0.08, ns) in mice that were injected with TNF-a and IL-6 compared to controls. By contrast, in the adjacent NTS there was 1.5x’s more immunoreactive IBA1 in mice that were injected with IL-6 and TNF-a (µm^2; p < 0.001) compared to control vehicle injections. Comparing brain regions in the DVC, there is 1.7x more microglia cells in the area postrema per section (50µm) compared to the NTS (p < 0.001), but in the NTS cells are 1.3x larger in size in comparison to the microglia cells in the AP overall (µm^2/ number of cells; p < 0.05). Finally, in the NTS there was a 1.5x larger total area of the IBA1 immunoreactivity following TNF-a injections compared to IL-6 injected mice (µm^2; p < 0.05). These data reveal a rapid effect of peripherally injected pro-inflammatory cytokines on microglial quantities and size in the brainstem. Future investigations will elucidate the role of neural-immune pathways in influencing the ANS during a chronic immune stress response. Supported by ORWH U54-MH118919. Presentation: 6/3/2024

Background and Hypothesis: Maternal immune activation (MIA) has been associated with increased risk for neuropsychiatric disease. Fetal exposure to maternal infection activates different toll-like receptors (TLRs) to initiate innate inflammatory responses in mother and fetus. The goal of the current study is to determine sex-dependent aspects of MIA during mid-gestation on neurodevelopment. Methods: Timed-pregnant female mice were administered RQ (TLR7 agonist) or vehicle saline on embryonic day (E) 12.5. Maternal and fetal cytokines were measured to ensure infection of pregnant dam (E15). Offspring were assessed postnatally for developmental milestones and behavior. Adult offspring were perfused with fluorescein isothiocyanate (FITC; 100mg/mL) to visualize blood vessel integrity (FITC leakage; Frahm & Tobet, 2015). To determine neural molecular mechanisms for behavioral changes, sections through the paraventricular nucleus of the hypothalamus (PVN) of offspring were immunolabeled for Glial Fibrillary Acidic Protein (GFAP; astrocytic end feet), Desmin (pericytes) and IBA-1 (microglia). Results: Maternal cytokines IL-6 (p<0.05) and IL-10 (p<0.01) were higher while TNFα (p<0.01) and IL-17 were lower 3 days after RQ-exposure. In fetuses (E15), IL-6 (p<0.05) and IL-17 (p<0.0001) were greater, while IL-10 was higher only in males (p<0.001) exposed to maternal infection. RQ-exposed males also had reduced TNFα (p<0.05). Additionally, RQ-exposed offspring had lower body weights at birth and delayed eye openings. Females exposed to maternal RQ exhibited slower onset of puberty with delayed vaginal openings. On the behavioral side, juvenile and adult offspring of RQ injected moms exhibited less social-like behavior (p<0.001 for all groups) with lower hedonic-like behavior selectively in females (p<0.001). RQ-offspring further showed greater leakage in the PVN indicated by more FITC in the extravascular space (males p<0.001, females p<0.01). GFAP + (astrocytic end feet) coverage of FITC-labeled vessels was higher in the PVN selectively in RQ males (p<0.001). Desmin+ (pericyte) coverage was greater in the PVN of RQ males (p<0.01) and females (p<0.001). Finally, there was greater number of IBA-1+ cells in the adult offspring PVN of both sexes (p<0.01) after maternal injection of RQ. Conclusions: 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 will examine how timing of infection during gestation (mid vs. late) changes neurodevelopmental outputs in offspring. Supported by ORWH-NIMH U54 SCORE-MH118919. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background:Current multimodal neuroimaging plays a critical role in studying clinical conditions such as cardiovascular disease, major depression, and other disorders related to chronic stress. These conditions involve the brainstem-hypothalamic network, specifically the locus coeruleus (LC), dorsal vagal complex (DVC), and paraventricular nucleus (PVN) of the hypothalamus, collectively referred to as the "DVC-LC-PVN circuitry." This circuitry is strongly associated with the norepinephrine (NE) and epinephrine (E) neurotransmitter systems, which are implicated in the regulation of key autonomic functions, such as cardiovascular and respiratory control, stress response, and cognitive and emotional behaviors. Objectives:To develop a methodology for delineating the DVC-LC-PVN circuitry in the human brain using multimodal neuroimaging. Methods:We combined structural T1-weighted morphometric magnetic resonance imaging (MRI) and diffusion MRI-based tractography to map the DVC-LC-PVN circuitry in the human brain. This methodology was applied to a pilot sample of brain datasets from five healthy adult subjects obtained from the publicly available Human Connectome Project repository and to one post-mortem human dataset. Results:The DVC-LC-PVN circuitry was delineated in vivo in five human subjects and one ultra-high resolution post-mortem dataset, allowing for refined anatomical observations. Conclusion:NE and E neurotransmitter systems engender substantial interest in both basic and clinical neuroscience due to their roles in the regulation of key autonomic functions, such as cardiovascular and respiratory control, stress responses, and cognitive and emotional behaviors. As demonstrated in this study, multimodal neuroimaging techniques provide a valuable approach for mapping small brainstem and hypothalamic structures and complex circuitries such as the DVC-LC-PVN circuitry.

With an increasing aging population and Alzheimer's disease tsunami, it is critical to identify early antecedents of brain aging to target for intervention and prevention. Women and men develop and age differently, thus using a sex differences lens can contribute to identification of early risk biomarkers and resilience. There is growing evidence for fetal antecedents to adult memory impairments, potentially through disruption of maternal prenatal immune pathways. Here, we hypothesized that in utero exposure to maternal pro-inflammatory cytokines will have sex-dependent effects on specific brain circuitry regulating offspring's memory and immune function that will be retained across the lifespan. Using a unique prenatal cohort, we tested this in 204 adult offspring, equally divided by sex, who were exposed/unexposed to an adverse in utero maternal immune environment and followed into early midlife (~age 50). Functional magnetic resonance imaging results showed exposure to pro-inflammatory cytokines in utero (i.e., higher maternal IL-6 and TNF-α levels) was significantly associated with sex differences in brain activity and connectivity underlying memory circuitry and performance and with a hyperimmune state, 50 years later. In contrast, the anti-inflammatory cytokine, IL-10 alone, was not significantly associated with memory circuitry in midlife. Predictive validity of prenatal exposure was underscored by significant associations with age 7 academic achievement, also associated with age 50 memory performance. Results uniquely demonstrated that adverse levels of maternal in utero pro-inflammatory cytokines during a critical period of the sexual differentiation of the brain produced long-lasting effects on immune function and memory circuitry/function from childhood to midlife that were sex-dependent, brain region-specific, and, within women, reproductive stage-dependent.

Accumulating evidence suggests that Parkinson’s disease (PD) pathology may arise in the gut. This likely occurs through the enteric nervous system (ENS), which facilitates bidirectional communication between the brain and intestines. A hallmark of PD is neuronal accumulation of misfolded α-synuclein (aSyn) proteins which have been shown to travel from the ENS to the brain via the vagus nerve. The goal of this study was to connect related neural and immune phenotypes in ex vivo and in vivo mouse models of PD.Recent data suggests that enteric neuronal fibers containing calcitonin gene related peptide (CGRP) act as chemo-sensors that can be stimulated to release CGRP in response to bacterial and chemical signals. CGRP can activate receptors that influence goblet cells (GCs) and immune cells. GCs are responsible for secreting mucus to maintain mucosal barrier integrity. Deterioration of gut barrier integrity is hallmark of PD patients. We hypothesize that alterations in luminal contents modifies and disturbs GCs disrupting the gut barrier in PD. This may result in increased release of CGRP leading to alterations in mucus production, immune responses, and aSyn aggregation in enteric neurons. In this study, the pesticide rotenone was given to C57BL/6 mice to facilitate aggregation of misfolded aSyn. Lectin and immuno-histochemistry were performed on sections of ileum and colon. GC mucopolysaccharides were fluorescently labeled with the lectin Ulex Europaeus Agglutinin I (UEA) conjugated to rhodamine. UEA-1+ cells in crypts were 25% more prevalent (68.5 +/- 0.5 vs. 52.5 +/-2.5) with rotenone treatment but 53% less prevalent in the luminal portion of crypts (22.5 +/- 2.5 vs. 47.5 +/-2.5). Fewer GCs in luminal positions is consistent with decreased mucus seen in PD patients. T cells were identified by immunoreactive (ir) CD3. Approximately 50% fewer CD3+ cells were in rotenone treated lamina propria (17 +/- 1.4) compared to control (36.2 +/- 3.5) consistent with previous reports. The area of CGRP-ir fibers in the lamina propria was 114% higher in control (6.4%+/-1.3) compared to rotenone treated tissue (5.6%+/-0.6). Rotenone treatment resulted in a 217% increase in the area average of CGRP-ir fibers in the region just below the epithelial monolayer (12.8%+/-1.3) compared to control (5.8%+/-0.7). While we did not observe CGRP fibers beyond the epithelial cell layer, GC cell disruption that could allow luminal contents to access CGRP fibers was suggested by the potential failure of GCs to migrate toward luminal surfaces from crypts. Future studies are needed to test the hypothesis that rotenone disturbance of mitochondrial function in GCs causes intestinal wall failure that leads to altered neuroimmune function. Aggregation of aSyn in enteric neurons may thereby be mediated by altered immune to neural actions as exacerbated by alterations in CGRP containing fibers and T cell populations in the gut. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract Disclosure: J.A. Sheng: None. R.J. Handa: None. S.A. Tobet: None. Background: The blood-brain barrier (BBB) protects the brain from the influx of harmful compounds in the blood. It is comprised of multiple cells, including endothelial cells sharing tight junctions, pericytes, and astrocyte endfeet. The paraventricular nucleus of the hypothalamus (PVN) is 3-5 times more vascularized than surrounding regions in the brain. The current study focuses on the BBB in the PVN as a function of stress. Recent data further suggests a high fat diet (HFD) disrupts the integrity of the BBB and leads to impairment of brain function (Li et al, 2021), suggesting a potential mechanism that may influence stress-related diseases. Methods: Adult male and female mice were placed on standard mouse chow (2918; Tekland) or a HFD (TD.06414, Tekland) for 6 weeks and further divided into control and stressed groups. Control mice were euthanized directly out of their home cage and stressed mice were euthanized 60-min after a 20-min acute restraint stress. Mice were perfused with fluorescein isothiocyanate in phosphate buffered saline followed by fixation with 4% paraformaldehyde to visualize blood vessel integrity (FITC leakage; Frahm & Tobet, 2015). Immunolabeled Glial Fibrillary Acidic Protein (GFAP; astrocytic end feet) and IBA-1 (microglia) were used to further assess BBB integrity and neuroinflammation. Results: Results showed ∼40% more FITC leakage in the PVN vasculature in adult HFD females (p<0.0001) but not males. Similar leakage was not noted in the region lateral to the PVN. IBA-1 immunoreactivity (microglia) showed a HFD-related 50% increase in raw cell counts (p<0.001) and a HFD x restraint decrease in fluorescence intensity (p<0.02) in both sexes. GFAP immunoreactivity (astrocyte end feet) additionally showed 2-fold increase in cell counts in both sexes by HFD (p<0.0001) and a female-specific HFD-induced increase in area (∼40%; p<0.001). Conclusions: Data suggest a chronic high fat diet impairs BBB integrity in PVN vasculature that can be exacerbated further by a brief exposure to stress in adult mice. This effect is being further studied at the cellular level in astrocytes and microglia. Such changes in these BBB components could indicate critical roles for the uniquely dense PVN vasculature, increasing risk of damage to neural functions that could include obesity, cardiovascular autonomic regulation, or depression-like behaviors. Supported by ORWH-NIMH U54 MH118919 SCORE. References: Li C, Shi L, Wang Y, Peng C, Wu L, Zhang Y, Du Z. (2021). High-fat diet exacerbates lead-induced blood-brain barrier disruption by disrupting tight junction integrity. Environ Toxicol. 36(7):1412-1421. doi: 10.1002/tox.23137. Frahm KA, Tobet SA. (2015). Development of the blood-brain barrier within the paraventricular nucleus of the hypothalamus: influence of fetal glucocorticoid excess. Brain Struct Funct. 220(4): 2225-2234. doi: 10.1007/s00429-014-0787-8. Presentation: Thursday, June 15, 2023

Abstract Organotypic lung slices, sometimes known as precision‐cut lung slices (PCLS), provide an environment in which numerous cell types and interactions can be maintained outside the body (ex vivo). PCLS were maintained ex vivo for up to a week and demonstrated health via the presence of neurons, maintenance of tissue morphology, synthesis of mucopolysaccharides, and minimal cell death. Multiple phenotypes of neuronal fibers were present in lung slices with varied size, caliber, and neurotransmitter immunoreactivity. Of the neuropeptides present in fibers, calcitonin gene‐related peptide (CGRP) was the most prevalent. Exposing PCLS to recombinant CGRP resulted in the proliferation and dispersion of CD19+ B cells in slices taken selectively from females. The number of granules containing immunoreactive (ir) surfactant protein C (SPC), which are representative of alveolar type 2 cells, increased in slices from females within 24 h of exposure to CGRP. Additionally, ir‐SPC granule size increased in slices from males and females across 48 h of exposure to CGRP. Exposure of PCLS to exogenous CGRP did not alter the number of solitary pulmonary neuroendocrine cells (PNEC) but did result in neuroendocrine bodies that had significantly more cells. Neuronal fiber numbers were unchanged based on ir‐peripherin; however, ir‐CGRP became non‐detectable in fibers while unchanged in PNECs. The effects of exogenous CGRP provide insight into innate immune and neuroendocrine responses in the lungs that may be partially regulated by neural fibers. The sex‐dependent nature of these changes may point to the basis for sex‐selective outcomes among respiratory diseases.