American Journal of Physiology-Regulatory, Integrative and Comparative Physiology

Regulation of water permeability in the collecting duct is important for osmoregulatory acclimation in teleost fish. In hyperosmotic environments such as seawater (SW), the teleost kidney functions as a site of divalent ion excretion. The collecting ducts reabsorb Na+, Cl-, and water, thereby reducing urine volume and producing small amounts of isotonic urine with high concentrations of divalent ions. In hypoosmotic environments such as freshwater (FW) or low-salinity brackish water (BW), the kidney produces large volumes of hypotonic urine and serves as a site of water excretion; under these conditions, the collecting ducts reabsorb Na+ and Cl- but not water. To identify aquaporins (Aqps) involved in regulating water permeability in the collecting ducts of teleosts, we analyzed renal Aqp expression in a euryhaline marine fish, the Japanese pufferfish (Takifugu rubripes), which possesses 16 Aqp genes in its genome, seven of which (Aqp1aa, 1ab, 3a, 4a, 7, 8bb, and 11a) are expressed in the kidney. Quantitative RT-PCR analysis showed that Aqp1aa and Aqp4a were highly expressed in collecting duct tissues, and that Aqp1aa expression was markedly reduced in fish acclimated to BW. Immunohistochemistry revealed apical localization of Aqp1aa and basolateral localization of Aqp4 in collecting duct cells, with apical Aqp1aa downregulated in BW. These results suggest that Aqp1aa and Aqp4 mediate water reabsorption in SW and that downregulation of Aqp1aa contributes to hypotonic urine production in BW. NEW & NOTEWORTHYRegulation of water permeability in the collecting duct is important for osmoregulation in teleost fish. Expression analyses of aquaporins (Aqps) in the marine pufferfish Takifugu rubripes showed that Aqp1aa and Aqp4a are highly expressed in the collecting duct and localized to the apical and basolateral membranes, respectively. Renal Aqp1aa expression was markedly reduced in fish acclimated to hypoosmotic brackish water. These results indicate that collecting duct water permeability is regulated by Aqp1aa expression.

Hypoxia (low oxygen availability) is a common environmental stressor in estuarine ecosystems that negatively affects fish survival as well as physiological and behavioral responses. However, the effects of hypoxia on the brain remains poorly understood, particularly in non-model species. Here, we investigated how prolonged hypoxia influences neural, vascular, and molecular responses in the brain of the speckled sanddab (Citharichthys stigmaeus), an ecologically relevant estuarine flatfish. Fish were exposed to normoxic or hypoxic conditions for seven days, and responses were assessed using histological analyses of neural proliferation and vascular structure, alongside transcriptomic and proteomic profiling. Hypoxia increased neural cell proliferation and progenitor activation in the hypothalamic nucleus recessus lateralis (NRL) and optic tectum, while reducing survival of newly generated cells. At the tissue level, hypoxia induced region-specific vascular remodeling, characterized by increased vessel area and vessel number without evidence of widespread endothelial proliferation. At the molecular level, transcriptomic and proteomic analyses revealed consistent enrichment of biological processes related to stress responses, development, metabolism, and cellular homeostasis, despite limited overlap between individual genes and proteins. Gene- and protein-level analyses further indicated activation of hypoxia-responsive pathways, including HIF signaling and oxidative stress protection, alongside selective metabolic reprogramming. Together, these findings demonstrate that hypoxia induces multi-level changes in the brain, linking neural plasticity, vascular remodeling, and molecular responses. This integrated response likely supports brain function under reduced oxygen availability in dynamic estuarine environments and highlights the role of the brain in regulating responses to environmental stress.

Cold environments pose substantial metabolic challenges to ectothermic organisms. In amphibians, such as the African clawed frog (Xenopus laevis), exposure to cold temperatures induces pronounced hyperglycemia; however, the molecular mechanisms underlying this response remain unclear. This study investigated the metabolic responses of the liver to cold exposure using transcriptome analysis. Adult frogs were subjected to a temperature of 5{degrees}C for five days, and their liver transcriptome was subsequently analyzed using RNA sequencing. Cold exposure significantly elevated blood glucose levels. Transcriptome analysis revealed extensive alterations in gene expression, including the upregulation of key gluconeogenesis-related genes. Notably, genes involved in FOXO1 signaling exhibited coordinated changes, with increased expression of foxo1 and its regulator prmt1 (arginine methyltransferase) and decreased expression of mdm2 (E3 ubiquitin ligase), suggesting that the phosphorylation of FOXO1 may be suppressed. Consistent with these findings, the expression of gluconeogenic genes (g6pc1 and pck1) was elevated, whereas the glycolytic gene gck was downregulated, indicating a shift towards glucose production. In addition to carbohydrate metabolism, genes involved in lipid and cholesterol metabolism, particularly fatty acid desaturases (scd and fads2), were also upregulated, suggesting that the remodeling of membrane lipid composition may occur under cold conditions. Furthermore, genes related to antioxidant and redox pathways, including those involved in the detoxification of reactive oxygen species and iron sequestration, were induced, indicating enhanced redox regulation. Collectively, these results demonstrate that cold exposure induces coordinated metabolic remodeling in the liver of X. laevis, characterized by enhanced gluconeogenesis, lipid remodeling, and robust redox regulation. SUMMARY STATEMENTCold exposure drives coordinated hepatic metabolic reprogramming in Xenopus laevis, elevating gluconeogenesis, modifying lipid composition, and strengthening antioxidant defenses through integrated transcriptional responses that support survival under a low-temperature environment.

The corticotropin-releasing factor (CRF) system is a major neural regulator of stress responses in vertebrates. However, stress-related roles for the CRF system in other tissues--and whether these roles vary between stressor types--remain unclear. To address this gap, we first characterized the CRF system in the gills and intestine of rainbow trout (Oncorhynchus mykiss) and then evaluated how it is transcriptionally regulated following either an immune (vaccination) or osmotic (seawater transfer) stressor. Additionally, since the CRF system is involved in food intake regulation, we also evaluated whether feeding state affects the intestinal CRF system. Vaccination against Vibrio anguillarum reduced CRF system activity in the intestine--as indicated by elevations in CRF binding protein transcripts paired with reductions in ligand (crfa2) and receptor (crfr1b) transcripts--but did not affect the gill CRF system. In contrast, seawater transfer caused the abundance of most CRF system transcripts to increase in the middle (but not posterior) portion of the intestine, while transcript levels of CRF binding proteins and receptors in the gills declined. Finally, levels of CRF system transcripts in the intestine varied with feeding state in a region-specific manner. In the middle intestine, transcript levels of most components declined with fasting and increased when feeding was resumed, whereas the opposite pattern occurred in the posterior intestine. Overall, our results implicate the peripheral CRF system as a stressor- and epithelial tissue-specific modulator of immune and osmoregulatory functions in teleosts.

Spinal cord injury (SCI) disrupts supraspinal autonomic pathways that regulate cardiovascular function, producing marked blood pressure instability and contributing to secondary injury in peripheral organs. The kidney is particularly vulnerable to these disturbances because renal blood flow (RBF) depends on tightly regulated interactions between neural, myogenic, and vascular control mechanisms. However, how SCI level and chronicity alter dynamic renal autoregulation remains poorly defined. Here, we investigated the effects of high- and low-thoracic SCI on renal hemodynamic control using in vivo blood pressure and RBF recordings in female mice. Hemodynamics were assessed at baseline and during acute sympathetic stimulation induced by norepinephrine (NE; 10 g/kg, i.v.) at 24 h and 4 wk following spinal cord transection at thoracic level 3 (T3) or thoracic level 10 (T10). Time-domain analyses quantified systolic blood pressure recovery, while frequency-domain analyses were used to resolve myogenic and sympathetic contributions to RBF regulation. High-thoracic SCI caused marked disruption of renal vascular responses to acute hypertension, producing paradoxical increases in RBF during NE-induced pressure elevations and sustained reductions in baseline and evoked RBF activity within frequency ranges associated with myogenic and sympathetic vasomotion. These impairments were most pronounced during the chronic phase of injury, consistent with loss of dynamic autoregulatory control and vascular remodeling. In contrast, low-thoracic SCI preserved baseline renal vasomotor activity and demonstrated recovery of dynamic autoregulatory responses over time. These findings identify SCI level and chronicity as critical determinants of renal microvascular regulation and demonstrate that high-thoracic SCI produces persistent autonomic-vascular uncoupling. This disruption of dynamic renal autoregulation represents a previously underappreciated mechanism of secondary organ vulnerability following neurotrauma.

Gastroschisis is a congenital abdominal wall defect in which fetal intestines herniate into the amniotic cavity. Despite 97% surgical repair success rate, 40% of affected infants require hospital readmission due to gastrointestinal complications, where underlying mechanisms remain poorly characterized. We hypopthesized that the cord blood metabolome of neonates with gastroschisis differs systematically from controls and may reveal pathway-level alterations relevant to neonatal physiology. Cord blood plasma collected at delivery (23 samples each group) was analyzed using ultra-performance liquid chromatography coupled with tandem mass spectrometry. Unsupervised principal component analysis and hierarchical clustering demonstrated significant separation between groups (PERMANOVA pseudo-F = 4.632, R{superscript 2} = 0.095, p = 0.001). 53 metabolites met criteria for differential abundance, 75% were lipids. Key alterations included reduced free fatty acids, increased fatty acid amides and ceramides, disrupted steroid and bile acid metabolism, and decreased biliverdin and bilirubin isomers. Our findings provide insight into gastroschisis pathophysiology and identify potential biomarkers for future investigation.

Elastin haploinsufficiency is a primary determinant of arteriopathy and hypertension that hallmark Williams syndrome (WS), a rare genetic disorder resulting from microdeletion of genes on human chromosome 7, including the elastin gene (ELN). Accumulating evidence suggests renal dysfunction, including enhanced sodium and water retention as an underlying cause of blood pressure elevation resulting from heterozygous deletion of Eln (Eln+/-) in mice that recapitulates the cardiovascular phenotype of WS. However, the underlying pathophysiological mechanisms are poorly understood. Here, we determined whether the activity of neuraminidase-1 (NEU1) of the elastin receptor complex (ERC) contributes to abnormal handling of water and electrolytes by the kidney in Eln haploinsufficiency. Adult male and female Eln+/+ and Eln+/- mice were subjected to acute extracellular fluid volume expansion with normal saline, combined with pharmacological intervention targeting vasopressin V2 receptor (V2R), NEU1, ENaC, and NKCC2. In male Eln+/+ mice, V2R blockade induced a dose-dependent increase in urine flow rate without affecting sodium excretion. Conversely, V2R stimulation with desmopressin markedly increased urinary sodium excretion in male Eln+/+ but not Eln+/- mice, while both sexes of Eln+/- mice exhibited marked suppression of urine flow rate. Abrogation of ERC signaling through NEU1 inhibition produced a modest increase in urinary sodium excretion in male mice of both genotypes but augmented urine flow rate only in male Eln+/+mice. NEU1 blockade strikingly enhanced the natriuretic effect of furosemide and amiloride in male Eln+/+ and modestly in Eln+/-mice. Taken together, we conclude that Eln haploinsufficiency disrupts vasopressin-dependent modulation of sodium and water reabsorption by sex-dependently altering ERC-mediated modulation of NKCC2 and ENaC. These findings reveal a novel mechanism by which abnormal ERC activity due to Eln haploinsufficiency potentially contributes to renal dysfunction and hypertension. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/731713v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1870e8org.highwire.dtl.DTLVardef@9cbd8dorg.highwire.dtl.DTLVardef@60b2deorg.highwire.dtl.DTLVardef@7f31f1_HPS_FORMAT_FIGEXP M_FIG C_FIG AC, adenylyl cyclase; AQP2, aquaporin 2; CD, collecting duct; CNT, connecting tubule; DCT, distal convoluted tubule; EBP, elastin binding protein; Eln, elastin allele; ENaC, epithelial sodium channel; ERC, elastin receptor complex; Gs, stimulatory G subunit; NEU1, neuroaminidase1; NKCC2, sodium-potassium-chloride cotransporter; PPCA, protective protein/ cathepsin A; TAL, loop of Henle thick ascending limb; V2R, vasopressin receptor type 2

Background The relative contributions of molecular size, electrostatic charge, and filtration rate to glomerular transport remain controversial. We hypothesized that glomerular sieving data contain a limited number of underlying transport modes that can be identified directly from experimental measurements. Methods Glomerular sieving coefficients were measured in anesthetized rats using neutral and anionic polysucrose during baseline conditions and glucagon-induced hyperfiltration. Data were analyzed using aligned-rank two-factor ANOVA, nonlinear mixed-effects regression of an electrostatic distributed two-pore model, pairwise correlation analysis, and principal component analysis. Results Hyperfiltration reduced the sieving of small and intermediate polysucrose molecules, whereas anionic polysucrose exhibited lower sieving coefficients than neutral polysucrose over a broad range of molecular sizes. An electrostatic distributed two-pore model accurately reproduced the observed effects of filtration rate and molecular charge and yielded an effective pore-wall charge density of 5.4 mC/m2 (95% confidence interval, 4.5 to 6.6). Pairwise correlation analysis revealed strong coupling between neighboring molecular sizes throughout the entire measured size range. Principal component analysis of the 2.5-8.0 nm size-selective region showed that the first principal component explained 96.3% of the variance and the first two principal components explained 99.9% of the variance. Separate analyses of the 2.5-5.0 nm and 5.0-8.0 nm transport regions showed that the first principal component explained 99.4% and 89.5% of the variance, respectively. Conclusions Glomerular sieving curves exhibited a highly constrained low-dimensional structure despite differences in molecular charge, filtration rate, and individual animals. The observed transport structure was consistent with distinct small-pore and large-pore transport domains and enabled highly effective principal component-based denoising of experimental sieving data.

The corticotropin-releasing factor (CRF) system bidirectionally interacts with cytokines and other immune-related components in mammals. However, the nature of these interactions remains poorly characterized in other vertebrates, including teleost fishes. To gain insight into the relationship between immune responses and the CRF system in teleosts, we explored how CRF system components were transcriptionally regulated in immune organs of rainbow trout (Oncorhynchus mykiss). We first characterized the CRF system in the spleen and head kidney--two primary immune organs in teleosts--and found that many CRF system components were present in both tissues, but splenic expression was consistently greater. Changes in the abundance of splenic CRF system components following vaccination (which transiently stimulated inflammatory responses and cytokine production) indicated contrasting and time-dependent regulation of CRF receptor 1 (CRFR1; suppression) and CRFR2 (stimulation) activities in response to an inflammatory challenge. Using spleen explant cultures, we then evaluated whether these effects were mediated by either of nuclear factor kappa B (NF-{kappa}B; a pro-inflammatory transcription factor) or cortisol (an anti-inflammatory hormone). At baseline, cultured spleens increased cytokine production and exhibited transcriptional changes in CRF system components comparable to those observed following vaccination. Cortisol treatment and NF-{kappa}B inhibition both attenuated the rise in cytokine transcription; however, cortisol treatment generally affected transcripts influencing CRFR1 activity, while NF-{kappa}B inhibition reduced CRFR2 activity. Overall, our data provide novel insight into CRF system regulation in the spleen and suggest that cortisol and inflammatory cytokines differentially regulate CRFR1 and CRFR2 activity within this organ.

The carotid body (CB) is the key peripheral oxygen sensor. CB mitochondria are hypothesised to be uniquely adapted with unusually low intrinsic oxygen affinity which, in association with nitric oxide (NO) and reactive oxygen species signalling, enables acute responsiveness to hypoxia. However, CB mitochondrial physiology or intrinsic oxygen affinity have never been measured directly. We sought to address this key gap by isolating sheep CB mitochondria and comprehensively characterising their phenotype and contrasting them to a non-oxygen sensing tissue, left ventricular myocardium (LV). High resolution respirometry, liquid chromatography mass spectrometry, enzymatic assays and in silico modelling were used to characterise mitochondrial content, aerobic capacity, oxygen affinity, complex subunit abundance and activity, H2O2 production and NO sensitivity in ovine CB and LV. Mitochondrial oxygen affinity (P50 = 0.089 mmHg) was lower in the CB than the LV (P50 = 0.058 mmHg; p = 0.005). Whilst mitochondrial content was lower in the CB, CB mitochondria had higher respiratory rates and enzymatic activity than LV. H2O2 production and NO sensitivity were similar in the two tissues. While intrinsic mitochondrial oxygen affinity is slightly lower in the oxygen sensing CB than in the non-oxygen sensing LV, this difference is small. Hence, any role of mitochondria in CB oxygen sensing is not due to an intrinsic difference in the O2 affinity of cytochrome oxidase due to differential expression of its subunits. Instead, this work suggests that differences in O2 affinity in vivo are secondary to other factors, perhaps including NO, that alter mitochondrial O2 affinity.

Cyclooxygenase-1 (COX-1) is classically regarded as a constitutive enzyme that produces eicosanoids with housekeeping functions, but recent evidence indicates that it may also be involved in the acute phase of severe systemic inflammation. There is evidence indicating that COX-1 is selectively activated in the spleen via post-translational mechanisms early the course of LPS-induced systemic inflammation. However, the mechanistic link between COX-1 and the spleen has not yet been demonstrated in direct experiments. The present study was conducted to fill this gap. The effects of the COX-1 inhibitor SC-560 on the LPS-induced severity triad (hypotension, hypothermia and acidosis) were evaluated in rats subjected to splenectomy or in sham-operated controls. In the sham-operated group, SC-560 significantly attenuated the severity triad independently of changes in plasma cytokines (TNF and IL-1{beta}). In the splenectomized rats, SC-560 completely lost its ability to attenuate the hypotension and the acidosis induced by LPS. The effect of SC-560 on LPS-induced hypothermia was also impaired by splenectomy, though not completely. We then conducted a lipidomic screening to identify which COX-1-derived eicosanoids might be responsible for mediating the severity triad. Based on spleen-blood correlations, the screening identified PGE2 and PGD2 as putative candidates. In conclusion, the present study provides direct evidence for a mechanistic link between the spleen and COX-1 in the mediation of severity in systemic inflammation, and identifies PGE2 and PGD2 as putative candidates involved.

Endometriosis is a steroid-dependent gynecologic disease characterized by progesterone (P4) resistance, subfertility/infertility, and pelvic pain; however, the molecular mechanisms underlying impaired P4 responsiveness in endometriosis tissue are not fully understood. RE-1 silencing transcription factor (REST), a transcriptional regulator implicated in steroid hormone signaling, has emerged as a potential mediator of P4 responsiveness. Here, we investigated the role of REST in endometriosis using human tissues and a uterine-specific Rest conditional knockout mouse model. Immunohistochemical analysis of eutopic endometrium and ectopic lesions from patients with endometriosis revealed significantly reduced nuclear REST expression compared with control endometrium, suggesting loss of functional REST in disease. To assess the physiological consequences of REST deficiency, uterine-specific Rest knockout (Rest d/d) mice were generated. Rest d/d females exhibited progressive subfertility and hyper-estrogenic uterine tissue characteristics that displayed a blunted responsiveness to P4 treatment. Loss of Rest selectively altered expression of P4-responsive genes associated with endometriosis pathology, despite preserved P4 receptor expression. Following induction of experimental endometriosis, female mice that developed endometriotic-like lesions using Rest-deficient donor tissue developed significantly larger lesions that were less responsive to P4 treatment compared to lesions induced using control tissue. Mechanical sensitivity was modestly increased in mice receiving Rest-deficient tissue, whereas vaginal hyperalgesia was unaffected. These findings identify loss of nuclear REST as a feature of endometriosis and support a role of REST in subfertility, lesion progression, and blunted response to P4. REST may represent a novel molecular contributor to altered P4 responsiveness and a potential therapeutic target in endometriosis. Significance StatementEndometriosis is a common disease in women characterized by altered steroid hormone signaling, infertility, and pelvic pain. RE-1 silencing transcription factor (REST) is a candidate regulator of steroid hormone signaling in gynecologic disease but a role in endometriosis pathophysiology remains unexplored. To fill this knowledge gap, our study utilizes human endometrial and endometriotic tissues coupled with a conditional knockout mouse model for uterine Rest deficiency. We show that REST is significantly reduced in eutopic and ectopic endometrial tissue from women with endometriosis and that deletion from mouse uterine tissue recapitulates clinical characteristics in women with endometriosis including progesterone resistance, sub-fertility and pelvic pain. These findings will further guide future research to understand impaired steroid signaling in the pathophysiology of endometriosis.

The dural venous sinuses are the main conduit for cerebral blood flow to drain from the head back to the heart. Pathology of these sinuses has significant impacts on cerebral blood volume and intracranial pressure, indicating that these veins have an important role in cerebral hemodynamics. Active and modifiable mechanisms of sinus diameter control may present an opportunity for physiological or pathological regulation of venous blood flow. Here, we characterized the molecular and anatomical properties of dural vascular smooth muscle cells (SMCs) using RNA-sequencing and immunohistochemistry, demonstrating that artery-like SMCs are exclusive to sinus vessels. An ex vivo pressurized sinus preparation from mice was used to functionally characterize the vasodynamics of the sinus and its corresponding bridging veins. We found that the sinus contains dynamically contractile SMCs and constricts to both pressure and contractile agonists, while the bridging veins do not. We further demonstrated that these sinus SMCs exhibit dynamic calcium signaling that is responsive to contractile stimuli. These results reveal a contractile SMC phenotype localized to dural venous sinuses and demonstrate that sinus vessels possess active, artery-like mechanisms for regulating cerebral venous outflow.

Heart rate (HR) and its variability (HRV), mediated by the autonomic nervous system are key indicators of diving physiology and behavioral state, in vertebrates. However, these indicators remain understudied in cetaceans due to the technical challenges of recording electrocardiograms (ECGs) underwater. To overcome these challenges, we developed a waterproof device based on an all-in-one suction-cup that integrate an ECG-accelerometer logger with bipolar electrodes. Using this device, we obtained high-resolution ECG signals in bottlenose dolphins (Tursiops truncatus, n=8), belugas (Delphinapterus leucas, n=2), and orcas (Orcinus orca, n=1) during breathing and apnea. This approach allowed us to highlight species-specific features of the ECG waveform, consistent with a biphasic T wave in the three species of cetaceans and a bifid P wave unique to belugas, which were independent from the respiration state. Resting surface HRs were 70 {+/-} 4 bpm in dolphins, 51 {+/-} 1 bpm in belugas, and 50 {+/-} 2 bpm in the orca and exhibited pronounced oscillation related to the mechanism of respiratory sinus arrhythmia. As expected, short apneas ([~] 1 min) induced bradycardia in all three species (53 {+/-} 5, 33 {+/-} 3, and 37 {+/-} 2 bpm, respectively). In dolphins this bradycardia was coupled with a significant decrease of the coefficient of variability of RR intervals, one of the indices of HRV. Moreover, we were surprised to observe HR oscillations throughout apnea, suggesting a persistent fluctuation of autonomic modulation. Thus, to better understand autonomic modulation in cetaceans we employed food (fishes, squids, gelatin, etc.) as a strong rewarding stimulus. For that we compared HR and HRV during 2-min of food deprivation versus continuous feeding periods. In dolphins, food deprivation produced no significant change in HR or HRV from resting surface values, whereas continuous feeding decreased HR of about 20 % and increased HRV metrics (StDRR, CVRR, RMSSD). Belugas showed similar responses, with a HR decline of about 40 % and an increase HRV indices. These findings established baseline HR and HRV parameters during breathing or apnea for three cetacean species and demonstrate that autonomic responses to appetitive stimuli can be non-invasively quantified, validating a novel tool to investigate cetacean cardiovascular physiology and environmental perception.

Perinatal opioid exposure is a prevalent clinical concern linked to respiratory instability and adverse infant outcomes. The opioid buprenorphine is prescribed as a medication for opioid use disorder during pregnancy and used to treat neonatal opioid withdrawal syndrome, yet its direct effects on neonatal control of breathing have not been examined. Here, we asked how acute buprenorphine exposure affects breathing at rest, and during chemoreceptor stimulation. Using dual-chamber head-out plethysmography, we measured pulmonary ventilation rate ([V]I) and metabolic rate in awake male and female Sprague-Dawley neonatal rats on postnatal days 4-5 (P4-5) during eupnea and a hypoxic-hypercapnic (HH) challenge. The effects of buprenorphine and two opioid receptor antagonists, naloxone hydrochloride, or peripherally restricted naloxone methiodide, were assessed using a repeated measures design. [V]I during eupnea and HH were markedly depressed following buprenorphine administration. Buprenorphine reduced [V]O2 and [V]CO2 and produced ventilatory equivalents for O2 and CO2 consistent with frank hypoventilation, driven by reduced breathing frequency and tidal volume (VT). When administered after buprenorphine, neither naloxone hydrochloride nor naloxone methiodide could rescue the buprenorphine-mediated hypoventilation in eupnea or during HH. In contrast, pre-treatment with either naloxone hydrochloride or naloxone methiodide attenuated buprenorphine-induced hypoventilation by preserving VT. These findings demonstrate that neonatal protective chemoreceptor reflexes are depressed by buprenorphine and suggest that pre-treatment with a peripheral opioid receptor antagonist could mitigate buprenorphine-induced hypoventilation without inducing opioid withdrawal. Key PointsO_LIAcute buprenorphine exposure significantly depressed pulmonary ventilation rate ([V]I) during eupnea and hypoxic hypercapnia (HH) in awake neonatal rats. C_LIO_LIBuprenorphine-induced hypoventilation was driven by reduced tidal volume (VT) and breathing frequency. C_LIO_LIBuprenorphine also reduced oxygen consumption ([V]O2) and carbon dioxide production ([V]CO2). C_LIO_LINaloxone given after buprenorphine failed to reverse hypoventilation. C_LIO_LIIn contrast, pre-treatment with either naloxone hydrochloride or peripherally restricted naloxone methiodide mitigated buprenorphine-induced hypoventilation by preserving VT. C_LI

Long-term winter dormancy in ectotherms (brumation) defines the annual cycle of many temperate-zone reptiles, yet the transcriptional regulation that supports survival across months of cold and aphagy remains poorly understood. We generated time-resolved transcriptomic profiles of liver and testis from male red-sided garter snakes (Thamnophis sirtalis parietalis) at five timepoints spanning the eight-month brumation cycle: pre-brumation, early, mid-, and late brumation, and post-arousal under continued aphagy. Time-course negative-binomial regression (maSigPro) followed by gene-set enrichment analysis identified 3,715 transcripts in liver and 5,828 in testis with significant temporal expression structure organized into five overarching temporal patterns: sustained downregulation, downregulation with post-arousal recovery, sustained upregulation, brumation-specific upregulation and cyclic modulation. Liver showed coordinated upregulation of fatty acid mobilization enzymes (ATGL, FOXO1, PPAR, CPT1A) and gluconeogenic regulators (CREBBP, PCK1) coincident with sustained low temperatures. Additionally, low temperature transcriptional activity was suggestive of a shift toward hepatic lipid mobilization and alanine-supported gluconeogenesis. Testis showed sustained suppression of meiosis, reproduction, and DNA-metabolism gene sets that did not fully recover at arousal consistent with this species dissociated reproductive pattern. Both tissues showed coordinated upregulation of stress-response pathways involving heat-shock proteins, HIF1 and a glutathione-based antioxidant defense. Interestingly, three vitellogenin transcripts and 17{beta}-hydroxysteroid dehydrogenases associated with estradiol-favoring steroid metabolism were upregulated in male liver during late brumation, which is not expected during natural physiology in adult males. Together these data support a framework in which temperature- and starvation-associated transcriptional programs contribute to survival of one of the longest, coldest brumations documented in a squamate. Summary statementA time-resolved transcriptomic analysis of liver and testis spanning eight months of winter brumation in Thamnophis sirtalis parietalis reveals gene expression patterns consistent with a temperature-associated shift toward hepatic lipid mobilization, sustained reproductive suppression, and vitellogenin response in males.

Normal urinary bladder function relies on afferent fibers that detect and integrate mechanical and chemical cues related to bladder distension. Though, the molecular identity and function of the various sensory neuron types involved in bladder function have yet to be fully elucidated. Here, we introduce a novel framework for the functional classification of mechanosensitive bladder afferents based on their differential responses to physiological (15 l/min) and noxious filling (30 s at intravesical pressures of 10, 20, 30, 40, 50, and 60 cmH2O). Our data reveal the presence of three distinct types of mechanosensitive bladder afferents, two that respond to physiological distension (type I and II) and one that is activated by noxious stimulation (type III). Of the two populations that respond to physiological filling, one displays a linear increase in firing with bladder filling (type I), while the firing of the other plateaus as intravesical pressure increases (type II). Fast filling (130 l/min) increases the discharge of all three afferent types, with the effect being most pronounced in those responding to noxious stimulation (type III). Corroborating the existence of three functionally distinct bladder afferent populations, Yoda1, a selective PIEZO1 channel activator, significantly increased the firing rate of types I and III during slow filling and of type III during noxious stimulation. In summary, we present a reliable and reproducible method for studying and classifying bladder afferents, while providing compelling evidence for the existence of functionally distinct populations of mechanosensitive afferents, each activated and regulated by distinct mechanisms. New & NoteworthyUsing a novel approach, we identify three types of mechanosensitive afferents innervating the urinary bladder, two that respond to slow filling and one that is activated only by noxious distension. The three afferent types display distinct firing patterns during rapid filling and in response to the PIEZO1 channel agonist Yoda1.

Adrenal chromaffin cells are key effectors of the sympathoadrenal response and play a central role in the organisms adaptation to environmental and physiological challenges. While cholinergic and pituitary adenylate cyclase-activating polypeptide (PACAP)-dependent mechanisms have long been recognized as major regulators of catecholamine secretion, increasing evidence indicates that connexin-mediated gap junctional communication provides an additional and highly dynamic level of control. Whether early-life experience modifies the adult capacity of chromaffin-cell networks to undergo stress-induced connexin remodeling remains unclear. Here, we examined adrenal medullary connexin expression in adult rats exposed to neonatal maternal separation (MS; 3 h daily, postnatal days 2-15) and later challenged with an 8-day unpredictable mild stress (UMS) protocol. Under basal adult conditions, MS did not produce an overt change in adrenal medullary Cx36 or Cx43 immunoreactivity relative to animal-facility-reared controls. In contrast, UMS increased connexin immunoreactivity in the adrenal medulla, and this response was amplified in animals with a history of MS. MS+UMS animals also displayed enhanced corticosterone responses to acute restraint stress. These findings suggest that neonatal MS does not impose a constitutively altered adult chromaffin-cell phenotype, but instead primes the future stress responsiveness of adrenal medullary connexin remodeling. We propose that chromaffin-cell gap junctions represent a substrate sensitive to stress history, through which developmental experience may influence sympathoadrenal and endocrine adaptation in adulthood.

Stress-related psychiatric disorders and inflammatory bowel diseases share high co-morbidity and contribute to the symptom severity of one another. In mice, ten days of Chronic Social Defeat Stress (CSDS) is sufficient to reduce gut microbiome diversity and the relative abundance of Firmicutes, which are hallmarks of inflammatory bowel diseases. However, mechanisms by which stress causes gut microbiome dysbiosis are largely unknown. Here, we demonstrate that pharmacologically inhibiting {beta}-adrenergic receptors (ARs), which are activated by (nor)adrenaline during stress, mitigates gut dysbiosis otherwise caused by CSDS. Compared to vehicle-treated mice following CSDS, propranolol-treated mice displayed a modest increase in sociability, increased alpha diversity, and increased abundance of anaerobic commensal Clostridia. Abundance of short-chain fatty acid-producing anaerobic Firmicutes abundance correlated with sociability following CSDS across all treatments. Pharmacologically blocking -ARs during stress increased subsequent sociability, but had little effect on gut microbiome composition. Together, our findings support the hypothesis that {beta}-AR activation contributes to stress-induced changes of the gut microbiome. One Sentence SummaryPharmacologically inhibiting beta-adrenergic receptors during chronic stress mitigates reductions in anaerobic, short-chain fatty acid-producing bacteria in the gut.

BackgroundNeonatal sepsis is a major cause of infant morbidity and mortality worldwide, particularly in preterm and very low birthweight babies. Fundamental differences between neonates and adults warrant clinically relevant models of neonatal sepsis. Here, we describe a preclinical fecal-slurry (FS)-induced peritonitis model of polymicrobial sepsis in neonatal rats, along with a novel neonatal rat sepsis score (nRSS) to monitor illness severity. MethodsPeritonitis was induced in 3-day-old Sprague Dawley rats by intraperitoneal injection of various doses (0.3-1.5mg/g body weight) of fecal slurry (FS); control pups received equivalent doses of vehicle. All pups received analgesics (buprenorphine), antibiotics (ampicillin and gentamicin), and fluids (saline) to model clinical standards of sepsis treatment. Time-dependent changes in circulating cytokines (IL-6, IL-1{beta}) and biomarkers of sepsis pathology (hemoglobin, glucose, alanine transaminase [ALT] levels) were assessed and correlated with nRSS scores. ResultsFS administration caused a dose-dependent increase in severity of sepsis over time, as indicated by increases in mortality rates (based on predefined criteria for euthanasia), nRSS scores, as well as time-dependent changes in circulating glucose, hemoglobin, IL-6, IL-1{beta}, and ALT activity levels. nRSS scores correlated with all quantitative measures of sepsis pathology. Notably, females showed higher mortality and higher early NRSS scores than males at moderate to high FS doses, yet biochemical markers and time of death did not differ between sexes, suggesting that the apparent female vulnerability may reflect more conspicuous behavioral manifestations of illness rather than greater underlying physiological severity. ConclusionInduction of peritonitis in rats at postnatal day 3 produced a consistent and reproducible model of polymicrobial neonatal sepsis. Illness severity was monitored using a newly developed nRSS. By minimizing distress and incorporating standards of care, this model and scoring system may serve as a platform for future investigations into the underlying mechanisms and potential therapeutic interventions for neonatal sepsis. ImpactO_LIA clinically relevant rat model of neonatal polymicrobial sepsis was developed, incorporating standards of care (analgesics, antibiotics, and fluid resuscitation) to better reflect the clinical context in which preclinical findings must ultimately translate. C_LIO_LIA novel neonatal rat sepsis scoring system (nRSS) was developed and validated, providing a sensitive, non-invasive measure of disease severity that correlates with biochemical markers and predicts mortality. C_LIO_LIFemale pups showed higher mortality and earlier behavioral signs of illness than males despite equivalent biochemistry, highlighting that clinical scores may capture sex-dependent vulnerability not apparent in standard biochemical measures. C_LIO_LITogether, this model and scoring system offer a refined platform for mechanistic and therapeutic studies of neonatal sepsis while advancing the welfare-conscious 3Rs principles essential to rigorous preclinical research C_LI