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Introduction: In women, the natural loss of ovarian function during menopause is associated with an increase in the number of cases of sleep apnea and its comorbidities, such as hypertension and obesity. However, only a subpopulation of women is affected by these disorders, a trajectory that is misunderstood. We know that stress and childhood adversity are linked with a sex specific increase in disease susceptibility in adults. In mammals, exposure to neonatal maternal separation (NMS) is an established pre-clinical model to address the consequences of early life stress. Therefore, we hypothesized that NMS increases respiratory control disorder and favors obesity in 1-year old female rats. Methodology: Rat pups were either undisturbed (CTRL) or subjected to NMS during which pups were placed in a temperature-controlled incubator 3h/day from postnatal day 3 to 12. At 60 weeks of age, we measured apnea frequency during sleep and chemoreflex response to hypoxia (10% O2; 40s) or hypercapnia (5% CO2; 10 minutes) using a whole-body plethysmography system. We also measured body weight and body length (to calculate the body mass index (BMI – g/cm2)), % of fat (using a magnetic resonance equipment – BRUKER) and leptin levels in the plasma (by Luminex® technology, a bead-based immunoassay). Results: Apnea frequency of NMS females was 20% higher than CTRL; the ventilatory responses to hypoxia and hypercapnia were also significantly greater (30% and 94% respectively). The BMI of NMS was 18% higher than CTRL and greater than the threshold for obesity. Additionally, those animals presented 5% more fat in their body composition, 5% less lean tissue and the same % of fluids. As for leptin, the mean values were similar but the variation in blood levels was 34% higher in NMS than in CTRL. Conclusions: Age-related loss in ovarian function reveals the latent effects of early life stress on respiratory control. By predisposing to obesity and interfering with leptin signaling, NMS also favors metabolic disturbance commonly observed in ageing women. These data suggest that stress is an important factor in the development of sleep apnea and related co-morbidities in a subpopulation of menopausal women. DAM is supported by the FRQ-S (268346) and the ‘Programme de complément de bourse’ from RSRQ (2021-22). This research is supported by the Canadian Institutes of Health Research (RK). 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.

Introduction: The onset of menopause increases the development of cardiovascular diseases and hypertension. However, only a subpopulation of menopausal women develops these disorders. The origins of such distinct aging trajectories are unknown. Since stress has persistent and sex-specific effects on health, we hypothesized that early-life stress exacerbates the cardiovascular consequences of the loss of ovarian function in aging rats. Methodology: Neonatal maternal separation (NMS) is a clinically relevant model of early-life stress. Following birth, rats were either raised under standard conditions (CTRL) or subjected to NMS (3h/day from postnatal days 3 to 12). When the females reached 60 weeks (old age), we measured arterial blood pressure by the “tail-cuff” method. We then measured the activity of the angiotensin-converting enzyme (ACE) and its homolog ACE 2 in serum, two key enzymes involved in the homeostasis of the renin-angiotensin system (RAS). Finally, we compared the expression of FosB, a transcription factor indicating neuronal activity, in the paraventricular nucleus of the hypothalamus (PVN); this structure is involved in the development and maintenance of hypertension and regulation of stress responses. The PVN contains two major anatomical structures: magnocellular (vasopressin secretion) and parvocellular (neuroendocrine-related functional and central autonomic control). Results: The mean arterial blood pressure (MAP) of NMS females was 12% higher than CTRL but the heart rate was 9% lower. Interestingly, in our previous studies, this stress effect was not observed in young adults (8 weeks old). The catalyzed activity of ACE was similar between groups, but ACE2 activity was 60% lower in NMS. The number of neurons expressing FosB was 55% higher in the magnocellular region of the PVN in NMS females. Finally, we observed a strong negative correlation between ACE2 activity and MAP (r2= -0.71, p=0.002). Conclusions: Unlike CTRL, aged NMS females showed an increased risk of hypertension. The lower activity of ACE2 may lead to an accumulation of Angiotensin II, which stimulates the release of vasopressin. Vasopressin, in turn, inhibits the secretion of renin. Increased renin predisposes individuals to develop hypertension. The increased magnocellular activation of PVN indicated that the vasopressin and the RAS may be interacting and contributing increased MAP. Therefore, we conclude that loss of ovarian function reveals the effects of early-life stress on cardiovascular homeostasis in females. DAM is supported by the FRQ-S (268346) and the “Programme de complément de bourse” from RSRQ (2021-22). This research is supported by the Canadian Institutes of Health Research (RK). 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.

Social isolation (SI) is a form of chronic stress that increases the risk for disease (hypertension, obesity, depression) and the COVID-19 pandemic has brought the entire world to appreciate its impact on mental and physical health. However, there is limited information pertaining to the neurobiological mechanisms underlying the deleterious impacts of SI. Within the hypothalamus, orexin-producing neurons (ORX) contribute to the regulation of arousal states, stress response, cardiorespiratory and metabolic homeostasis. Because these functions are all affected by SI, we hypothesised that SI disrupts ORX neurotransmission in rats.The experiments were carried out on two groups of adult female and male Sprague Dawley rats: controls (2/cage, n = 7), and socially isolated (SI; 1/cage, 3 weeks, n = 10). At the end of the isolation protocol, cardiovascular measurements were obtained by the tail cuff method. Rats were deeply anesthetised, and terminal blood samples were taken by cardiac puncture for multiplex assay. The brain was extracted after transcardiac perfusion 4% PFA/borax between 11.00 h and 13.00 h. Fos-B is a transcription factor indicative of neuronal activity; its expression was quantified in the paraventricular nucleus of the hypothalamus (PVN) and in the nucleus of the solitary tract (NTS), which are essential regions in the regulation of the stress response and cardiorespiratory function, respectively. Double immunostaining was performed with Fos-B on ORX neurons.In SI males, the elevated plasma corticosterone levels and increased number of Fos-B expressing neurons in the PVN compared to controls confirm that SI activates the stress pathways. Preliminary results in females indicate no stress activation. Mean arterial pressure of SI rats was higher than controls (+14mmHg); this stress effect was less important in females (+4mmHg). In males, SI reduced the number of ORX-labeled cells by 20% but increased the proportion of cells co-expressing Fos-B by 60%. SI also increased Fos-B expression in the NTS of males compared to controls.We conclude that SI activates the stress pathways in a sex-specific manner. This effect, along with enhanced activation of the NTS and ORX neurons likely play an important role in the etiology of hypertension and metabolic dysfunction. Completion of ongoing analyses in females will determine if the effects of SI on ORX neurotransmission is sex-specific. This research is supported by operating grants from the Canadian Institutes of Health Research (CIRH, RK and VJ). N.J.M. was supported by a Sentinel North Partnered Research Chair in Sleep Pharmacometabolism and a Fonds de Recherche du Quebec Sante (FRQS) Research Scholar J1 award. MG is the recipient of a doctoral scholarship from the FRQS. 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.

Sleep apnea (SA) is a multifactorial and sex-specific respiratory disorder characterized by repeated respiratory arrests during sleep. However, the origins of this disease are not clearly identified. Chronic stress increases risk for obesity, hypertension, and depression, which are common comorbidities of SA. With the increasing prevalence of obesity and about one-third of the world population reported feeling stressed, the study of the impact of stress on respiratory homeostasis has never been so relevant. Since chronic stress induces the main comorbidities of SA, we hypothesized that social isolation (SI) stress induces sex-specific cardiorespiratory and metabolic disorders in rats similar to patients with SA.80 Sprague Dawley male and female rats (8 weeks old) were kept in: 1) standard housing (2/cage; controls) or in 2) social isolation (1/cage). At the end of the three-week protocol, apneas were quantified by whole-body plethysmography during sleep; respiratory variability was assessed by Poincaré plot analysis. Cardiovascular measurements were obtained by tail cuff, body composition by preclinical magnetic resonance, and plasma leptin and corticosterone levels by multiplex immunoassay. The amount of food was weighed before and after the experiments to calculate the intake.By comparison with controls, SI elevated corticosterone levels in male but not females. SI increased the apnea index in intact rat by 21% and augmented respiratory instability. SI also increased blood pressure (+11mmHg) and food intake (+11g/day); however, females had lower increases than males. In males, SI increases weight gains, fat mass, and decreases plasma leptin levels; these effects were not observed in females.SI promotes the emergence of apnea and respiratory instability during sleep. Since the cardiorespiratory and metabolic disturbances are similar to clinical features of SA patient, we propose that stress plays an important role in the etiology of SA. SI offers a novel and unique opportunity to study the origins and pathophysiology of SA. This research is supported by operating grants from the Canadian Institutes of Health Research (CIRH, RK and VJ). N.J.M. was supported by a Sentinel North Partnered Research Chair in Sleep Pharmacometabolism and a Fonds de Recherche du Quebec Sante (FRQS) Research Scholar J1 award. MG is the recipient of a doctoral scholarship from the FRQS. 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.

The respiratory system of women and men develops and functions in distinct neuroendocrine milieus. Despite differences in anatomy and neural control, homeostasis of arterial blood gases is ensured in healthy individuals regardless of sex. This convergence in function differs from the sex-based differences observed in many respiratory diseases. Sleep-disordered breathing (SDB) results mainly from episodes of upper airway closure. This complex and multifactorial respiratory disorder shows significant sexual dimorphism in its clinical manifestations and comorbidities. Guided by recent progress from basic research, this review discusses the hypothesis that stress is necessary to reveal the sexual dimorphism of SDB.

The ectothermic vertebrates are a diverse group that includes the Fishes (Agnatha, Chondrichthyes, and Osteichthyes), and the stem Tetrapods (Amphibians and Reptiles). From an evolutionary perspective, it is within this group that we see the origin of air-breathing and the transition from the use of water to air as a respiratory medium. This is accompanied by a switch from gills to lungs as the major respiratory organ and from oxygen to carbon dioxide as the primary respiratory stimulant. This transition first required the evolution of bimodal breathing (gas exchange with both water and air), the differential regulation of O2 and CO2 at multiple sites, periodic or intermittent ventilation, and unsteady states with wide oscillations in arterial blood gases. It also required changes in respiratory pump muscles (from buccopharyngeal muscles innervated by cranial nerves to axial muscles innervated by spinal nerves). The question of the extent to which common mechanisms of respiratory control accompany this progression is an intriguing one. While the ventilatory control systems seen in all extant vertebrates have been derived from common ancestors, the trends seen in respiratory control in the living members of each vertebrate class reflect both shared-derived features (ancestral traits) as well as unique specializations. In this overview article, we provide a comprehensive survey of the diversity that is seen in the afferent inputs (chemo and mechanoreceptor), the central respiratory rhythm generators, and the efferent outputs (drive to the respiratory pumps and valves) in this group.

The respiratory system of women and men develops and functions in distinct neuroendocrine milieus. Despite differences in anatomy and neural control, homeostasis of arterial blood gases is ensured in healthy individuals regardless of sex. This convergence in function differs from the sex-based differences observed in many respiratory diseases. Sleep-disordered breathing (SDB) results mainly from episodes of upper airway closure. This complex and multifactorial respiratory disorder shows significant sexual dimorphism in its clinical manifestations and comorbidities. Guided by recent progress from basic research, this review discusses the hypothesis that stress is necessary to reveal the sexual dimorphism of SDB.

As our understanding of respiratory control evolves, we appreciate how the basic neurobiological principles of plasticity discovered in other systems shape the development and function of the respiratory control system. While breathing is a robust homeostatic function, there is growing evidence that stress disrupts respiratory control in ways that predispose to disease. Neonatal stress (in the form of maternal separation) affects "classical" respiratory control structures such as the peripheral O2 sensors (carotid bodies) and the medulla (e.g., nucleus of the solitary tract). Furthermore, early life stress disrupts the paraventricular nucleus of the hypothalamus (PVH), a structure that has emerged as a primary determinant of the intensity of the ventilatory response to hypoxia. Although underestimated, the PVH's influence on respiratory function is a logical extension of the hypothalamic control of metabolic demand and supply. In this article, we review the functional and anatomical links between the stress neuroendocrine axis and the medullary network regulating breathing. We then present the persistent and sex-specific effects of neonatal stress on respiratory control in adult rats. The similarities between the respiratory phenotype of stressed rats and clinical manifestations of respiratory control disorders such as sleep-disordered breathing and panic attacks are remarkable. These observations are in line with the scientific consensus that the origins of adult disease are often found among developmental and biological disruptions occurring during early life. These observations bring a different perspective on the structural hierarchy of respiratory homeostasis and point to new directions in our understanding of the etiology of respiratory control disorders. © 2021 American Physiological Society. Compr Physiol 11:1-38, 2021.

Made available in DSpace on 2021-06-25T11:00:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-04-15 In pre-metamorphic tadpoles, the neural network generating lung ventilation is present but actively inhibited; the mechanisms leading to the onset of air breathing are not well understood. Orexin (ORX) is a hypothalamic neuropeptide that regulates several homeostatic functions, including breathing. While ORX has limited effects on breathing at rest, it potentiates reflexive responses to respiratory stimuli mainly via ORX receptor 1 (OX1R). Here, we tested the hypothesis that OX1Rs facilitate the expression of the motor command associated with air breathing in pre-metamorphic bullfrog tadpoles (Lithobates catesbeianus). To do so, we used an isolated diencephalic brainstem preparation to determine the contributions of OX1Rs to respiratory motor output during baseline breathing, hypercapnia and hypoxia. A selective OX1R antagonist (SB-334867; 5-25 µmol l-1) or agonist (ORX-A; 200 nmol l-1 to 1 µmol l-1) was added to the superfusion media. Experiments were performed under basal conditions (media equilibrated with 98.2% O2 and 1.8% CO2), hypercapnia (5% CO2) or hypoxia (5-7% O2). Under resting conditions gill, but not lung, motor output was enhanced by the OX1R antagonist and ORX-A. Hypercapnia alone did not stimulate respiratory motor output, but its combination with SB-334867 increased lung burst frequency and amplitude, lung burst episodes, and the number of bursts per episode. Hypoxia alone increased lung burst frequency and its combination with SB-334867 enhanced this effect. Inactivation of OX1Rs during hypoxia also increased gill burst amplitude, but not frequency. In contrast with our initial hypothesis, we conclude that ORX neurons provide inhibitory modulation of the CO2 and O2 chemoreflexes in pre-metamorphic tadpoles. Department of Animal Morphology and Physiology College of Agricultural and Veterinary Sciences São Paulo State University Unesp. Jaboticabal Department of Pediatrics Institut Universitaire de Cardiologie et de Pneumologie de Québec Université Laval QC Department of Animal Morphology and Physiology College of Agricultural and Veterinary Sciences São Paulo State University Unesp. Jaboticabal

Made available in DSpace on 2021-06-25T10:50:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-04-01 The neural network that regulates breathing shows a significant sexual dimorphism. Ovarian hormones contribute to this distinction as, in rats, ovariectomy reduces the ventilatory response to CO2. Microglia are neuroimmune cells that are sensitive to neuroendocrine changes in their environment. When reacting to challenging conditions, these cells show changes in their morphology that reflect an augmented capacity for producing pro- and anti-inflammatory cytokines. Based on evidence suggesting that microglia contribute to sex-based differences in reflexive responses to hypercapnia, we hypothesized that ovariectomy and hypercapnia promote microglial reactivity in selected brain areas that regulate breathing. We used ionized calcium-binding-adapter molecule-1 (Iba1) immunolabeling to compare the density and morphology of microglia in the locus coeruleus (LC), the caudal medullary raphe, the caudal part of the nucleus of the tractus solitarius (cNTS), and the paraventricular nucleus of the hypothalamus (PVN). Tissue was obtained from SHAM (metaestrus) female rats or following ovariectomy. Rats were exposed to normocapnia or hypercapnia (5% CO2, 20 min). Ovariectomy and hypercapnia did not affect microglial density in any of the structures studied. Ovariectomy promoted a reactive phenotype in the cNTS and LC, as indicated by a larger morphological index. In these structures, hypercapnia had a relatively modest opposing effect; the medullary raphe or the PVN were not affected. We conclude that ovarian hormones attenuate microglial reactivity in CO2/H+ sensing structures. These data suggest that microglia may contribute to neurological diseases in which anomalies of respiratory control are associated with cyclic fluctuations of ovarian hormones or menopause. Département de Pédiatrie Centre de Recherche de l'Institut Universitaire de Cardiologie et Pneumologie de Québec Université Laval Department of Animal Morphology and Physiology Sao Paulo State University UNESP/FCAV at Jaboticabal Département de Psychiatrie et Neurosciences Centre de Recherche du CHU de Québec Université Laval Department of Animal Morphology and Physiology Sao Paulo State University UNESP/FCAV at Jaboticabal

Thyroid hormones (THs) are essential for foetal brain development. Because the gestating mother is the main source of THs to the foetus, maternal hypothyroidism and/or premature birth compromise neurological outcomes in the offspring. Respiratory instability and recurrent apneas due to immaturity of the respiratory control network are major causes of morbidity in infants. Inadequate TH supply may be sufficient to delay perinatal maturation of the respiratory control system; however, this hypothesis remains untested. To address this issue, maternal hypothyroidism was induced by adding methimazole (MMI; 0.02% w/v) to the drinking water of pregnant dams from conception to postpartum day 4 (P4). The effect of TH supplementation on respiratory function was tested by injecting levothyroxine (L-T4) in newborns at P1. Respiratory function was assessed by plethysmography (in vivo) and recording of phrenic output from medullary preparations (in vitro). By comparison with controls, TH deficiency increased the frequency of apneas and decreased basal ventilation in vivo and prevented the age-dependent increase in phrenic burst frequency normally observed in vitro. The effects of TH deficiency on GABAergic modulation of respiratory activity were measured by bath application of muscimol (GABAA agonist) or bicuculline (GABAA antagonist). The phrenic burst frequency responses to GABAergic agents were consistently greater in preparations from TH deficient pups. L-T4 supplementation reversed part of the respiratory anomalies related to MMI treatment in vitro. We conclude that TH deficiency during the perinatal period is sufficient to delay maturation of the respiratory control network development. Excessive GABAergic inhibition may contribute to this effect.

Panic disorder (PD) is ~2 times more frequent in women. An excessive ventilatory response to CO2 inhalation is more likely during the premenstrual phase. While ovarian hormones appear important in the pathophysiology of PD, their role remains poorly understood as female animals are rarely used in pre-clinical studies. Using neonatal maternal separation (NMS) to induce a “PD-like” respiratory phenotype, we tested the hypothesis that NMS disrupts hormonal regulation of the ventilatory response to CO2 in female rats. We then determined whether NMS attenuates the inhibitory actions of 17-β estradiol (E2) on orexin neurons (ORX). Pups were exposed to NMS (3 h/day; postnatal day 3–12). The ventilatory response to CO2-inhalation was tested before puberty, across the estrus cycle, and following ovariectomy. Plasma E2 and hypothalamic ORXA were measured. The effect of an ORX1 antagonist (SB334867; 15 mg/kg) on the CO2 response was tested. Excitatory postsynaptic currents (EPSCs) were recorded from ORX neurons using whole-cell patch-clamp. NMS-related increase in the CO2 response was observed only when ovaries were functional; the largest ventilation was observed during proestrus. SB334867 blocked this effect. NMS augmented levels of ORXA in hypothalamus extracts. EPSC frequency varied according to basal plasma E2 levels across the estrus cycle in controls but not NMS. NMS reproduces developmental and cyclic changes of respiratory manifestations of PD. NMS disrupts the inhibitory actions of E2 on the respiratory network. Impaired E2-related inhibition of ORX neurons during proestrus is a novel mechanism in respiratory manifestations of PD in females.

Made available in DSpace on 2021-06-25T11:08:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Light/dark cycle affects the physiology of vertebrates and hypothalamic orexin neurons (ORX) are involved in this function. The breathing pattern of the green iguana changes from continuous to episodic across the light/dark phases. Since the stimulatory actions of ORX on breathing are most important during arousal, we hypothesized that ORX regulates changes of breathing pattern in iguanas. Thus, we: (1) Localized ORX neurons with immunohistochemistry; (2) Quantified cyclic changes in plasma orexin-A levels by ELISA; (3) Compared breathing pattern at rest and during hypoxia and hypercarbia; (4) Evaluated the participation of the ORX receptors in ventilation with intracerebroventricular microinjections of ORX antagonists during light and dark phases. We show that the ORX neurons of I. iguana are located in the periventricular hypothalamic nucleus. Orexin-A peaks during the light/active phase and breathing parallels these cyclic changes: ventilation is higher during the light phase than during the dark phase. However, inactivation of ORX-receptors does not affect the breathing pattern. Iguanas increase ventilation during hypoxia only during the light phase. Conversely, CO2 promotes post-hypercarbic hyperpnea during both phases. We conclude that ORXs potentiate the post-hypercarbic (but not the hypoxic)-drive to breathe and are not involved in light/dark changes in the breathing pattern. Department of Animal Morphology and Physiology College of Agricultural and Veterinary Sciences São Paulo State University Unesp, Via de Acesso Prof. Paulo Donato Castellane s/n Department of Pediatrics Institut Universitaire de Cardiologie et de Pneumologie de Québec Université Laval Department of Animal Morphology and Physiology College of Agricultural and Veterinary Sciences São Paulo State University Unesp, Via de Acesso Prof. Paulo Donato Castellane s/n FAPESP: 2019/09468-9 CNPq: 407490/2018-3

Impacts of stress on the immune system vary depending on the nature, intensity, and time during which stress is experienced (eg, acute stress is pro-inflammatory vs chronic stress is anti-inflammatory). However, there is limited knowledge on the long-term impact of early life stress on immune responses. Given that immune programming occurs early in life and that immunity and stress both involve sexual dimorphisms, our hypothesis is that neonatal maternal separation (NMS) induces sex-specific immune alterations. Objective Our objective is to evaluate the influence of early life stress on immune response in adult and its dimorphism related to biological sex. Methods We used a well-established rat model of early life stress, NMS, in which we observed sex-specific modulations of the myeloid compartment. Thus, we evaluated the impact of NMS on macrophage activation using LPS stimulation of bone-marrow derived macrophages (BMDM) in vitro. Expression of activation (MHCII, CD40, CD86 and CD80) and maturation markers (SIRPα, CD11b, CD200 and CD200R) were analyzed by flow cytometry. TNF levels were measured by ELISA. Results LPS stimulation of female NMS BMDM had higher expression of activation markers compared to controls. Inflammatory cytokine levels, TNF, increased after LPS stimulation and further increased in female NMS, whereas male NMS level was lower than control male. Futhermore, LPS stimulation lowered CD200 receptor (CD200R) to a greater extent in male NMS than in female NMS. Conclusion These results suggest that NMS accentuates the pro-inflammatory response in adult female (but not in male) and will provide a strong base for improving sex-specific interventions in the context of diseases involving stress and the immune system. Supported by grants from CIHR, QRHN and CRIUCPQ

The neural network that regulates breathing shows a significant sexual dimorphism. Ovarian hormones contribute to this distinction as, in rats, ovariectomy reduces the ventilatory response to CO

International audience; In amphibians, there is some evidence that (1) anatomically separate brainstem respiratory oscillators are involved in rhythm generation, one for the buccal rhythm and another for the lung rhythm and (2) they become functionally coupled during metamorphosis. The present analysis, performed on neurograms recorded using brainstem preparations from Lithobates catesbeianus, aims to investigate the temporal organisation of lung and buccal burst types. Continuous Wavelet Transfom applied to the separated buccal and lung signals of a neurogram revealed that both buccal and lung frequency profiles exhibited the same low frequency peak around 1 Hz. This suggests that a common 'clock' organises both rhythms within an animal. A cross-correlation analysis applied to the buccal and lung burst signals revealed their similar intrinsic oscillation features, occurring at approximately 25 Hz. These observations suggest that a coupling between the lung and buccal oscillators emerges at metamorphosis. This coupling may be related to inter-connectivity between the two oscillators, and to a putative common drive.

Made available in DSpace on 2022-04-28T19:44:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 National Science Foundation Tracing the evolution of the central rhythm generators associated with ventilation in vertebrates is hindered by a lack of information surrounding key transitions. To begin with, central rhythm generation has been studied in detail in only a few species from four vertebrate groups, lamprey, anuran amphibians, turtles, and mammals (primarily rodents). Secondly, there is a lack of information regarding the transition from water breathing fish to air breathing amniotes (reptiles, birds, and mammals). Specifically, the respiratory rhythm generators of fish appear to be single oscillators capable of generating both phases of the respiratory cycle (expansion and compression) and projecting to motoneurons in cranial nerves innervating bucco-pharyngeal muscles. In the amniotes we find oscillators capable of independently generating separate phases of the respiratory cycle (expiration and inspiration) and projecting to pre-motoneurons in the ventrolateral medulla that in turn project to spinal motoneurons innervating thoracic and abdominal muscles (reptiles, birds, and mammals). Studies of the one group of amphibians that lie at this transition (the anurans), raise intriguing possibilities but, for a variety of reasons that we explore, also raise unanswered questions. In this review we summarize what is known about the rhythm generating circuits associated with breathing that arise from the different rhombomeric segments in each of the different vertebrate classes. Assuming oscillating circuits form in every pair of rhombomeres in every vertebrate during development, we trace what appears to be the evolutionary fate of each and highlight the questions that remain to be answered to properly understand the evolutionary transitions in vertebrate central respiratory rhythm generation. Department of Zoology University of British Columbia Département de Pédiatrie Université Laval Department of Biological Sciences California State University Department of Biology University of Ottawa Departamento de Morfologia e Fisiologia Animal UNESP Department of Zoophysiology Aarhus University Departamento de Morfologia e Fisiologia Animal UNESP

Chemokine signaling is important in neuropathic pain, with microglial cells expressing chemokine (C-C motif) receptor CCR2, CCR5 and CCR8, all playing key roles. In the previous report (Padi et al., 2012), oral administration of a short peptide, RAP-103, for 7 days fully prevents mechanical allodynia and inhibits the development of thermal hyperalgesia after partial ligation of the sciatic nerve in rodents. As for the mechanism of the inhibiting effect of RAP-103, it was speculated to be due to dual blockade of CCR2 and CCR5. We report here that RAP-103 exhibits stronger antagonism for CCR8 (half maximal inhibitory concentration [IC50] 7.7 fM) compared to CCR5 (IC50

KEY POINTS Incomplete development of the neural circuits that control breathing contributes to respiratory disorders in pre-term infants. Manifestations include respiratory instability, prolonged apnoeas and poor ventilatory responses to stimuli. Based on evidence suggesting that omega-3 polyunsaturated fatty acids (n-3 PUFA) improves brain development, we determined whether n-3 PUFA supplementation (via the maternal diet) improves respiratory function in 10-11-day-old rat pups. n-3 PUFA treatment prolonged apnoea duration but augmented the relative pulmonary surface area and the ventilatory response to hypoxia. During hypoxia, the drop in body temperature measured in treated pups was 1 °C less than in controls. n-3 PUFA treatment also reduced microglia cell density in the brainstem. Although heterogeneous, the results obtained in rat pups constitute a proof of concept that n-3 PUFA supplementation can have positive effects on neonatal respiration. This includes a more sustained hypoxic ventilatory response and a decreased respiratory inhibition during laryngeal chemoreflex. ABSTRACT Most pre-term infants present respiratory instabilities and apnoeas as a result of incomplete development of the neural circuits that control breathing. Because omega-3 polyunsaturated fatty acids (n-3 PUFA) benefit brain development, we hypothesized that n-3 PUFA supplementation (via the maternal diet) improves respiratory function in rat pups. Pups received n-3 PUFA supplementation from an enriched diet (13 g kg-1 of n-3 PUFA) administered to the mother from birth until the experiments were performed (postnatal days 10-11). Controls received a standard diet (0.3 g kg-1 of n-3 PUFA). Breathing was measured in intact pups at rest and during hypoxia (FiO2 = 0.12; 20 min) using whole body plethysmography. The duration of apnoeas induced by stimulating the laryngeal chemoreflex (LCR) was measured under anaesthesia. Lung morphology was compared between groups. Maternal n-3 PUFA supplementation effectively raised n-3 PUFA levels above control levels both in the blood and brainstem of pups. In intact, resting pups, n-3 PUFA increased the frequency and duration of apnoeas, especially in females. During hypoxia, n-3 PUFA supplemented pups hyperventilated 23% more than controls; their anapyrexic response was 1 °C less than controls. In anaesthetized pups, n-3 PUFA shortened the duration of LCR-induced apnoeas by 32%. The relative pulmonary surface area of n-3 PUFA supplemented pups was 12% higher than controls. Although n-3 PUFA supplementation augments apnoeas, there is no clear evidence of deleterious consequences on these pups. Based on the improved lung architecture and responses to respiratory challenges, this neonatal treatment appears to be beneficial to the offspring. However, further experiments are necessary to establish its overall safety.

Microglia are critical for the refinement of neural networks that takes place during the perinatal period. Their phenotype and actions are guided by the signals produced by neighbouring cells and hormones present in their surrounding milieu. Cell populations and the signals they produce differ between regions. The fact that thyroid hormones (THs) promote the growth and morphological differentiation of microglia within the cortex contributes to the TH’s powerful actions on the developing brain. The brainstem is especially active during early life owing to its role in generation of the rhythmic respiratory motor command. Despite evidences indicating that THs are necessary to proper development of the neural networks regulating this vital homeostatic function, their actions on microglia originating from the brainstem remain unknown. Using primary cultured microglia from newborn mice (C57BL/6J), we first report that regulation of microglial motility by THs is different between cortex and brainstem. Microglial motility (μm traveled over 3 h) was monitored with or without triiodothyronine (T3, 1μM). Exposure to T3 did not stimulate microglial motility from brainstem, but significantly stimulated (316 %) when they were co-cultured with neurons. Motility of cortex microglia was stimulated to the similar extent either with or without neurons. These data suggest that the microglial function in different regions of the brain is determined by the surrounding environment.

Physiological and environmental factors impacting respiratory homeostasis vary throughout the course of an animal’s lifespan from embryo to adult and can shape respiratory development. The developmental emergence of complex neural networks for aerial breathing dates back to ancestral vertebrates, and represents the most important process for respiratory development in extant taxa ranging from fish to mammals. While substantial progress has been made towards elucidating the anatomical and physiological underpinnings of functional respiratory control networks for air-breathing, much less is known about the mechanisms establishing these networks during early neurodevelopment. This is especially true of the complex neurochemical ensembles key to the development of air-breathing. One approach to this issue has been to utilize comparative models such as anuran amphibians, which offer a unique perspective into early neurodevelopment. Here, we review the developmental emergence of respiratory behaviours in anuran amphibians with emphasis on contributions of neurochemicals to this process and highlight opportunities for future research.

Amphibian respiratory development involves a dramatic metamorphic transition from gill to lung breathing and coordination of distinct motor outputs. To determine whether the emergence of adult respiratory motor patterns was associated with similarly dramatic changes in motoneuron (MN) properties, we characterized the intrinsic electrical properties of American bullfrog trigeminal MNs innervating respiratory muscles comprising the buccal pump. In premetamorphic tadpoles (TK stages IX–XVIII) and adult frogs, morphometric analyses and whole cell recordings were performed in trigeminal MNs identified by fluorescent retrograde labeling. Based on the amplitude of the depolarizing sag induced by hyperpolarizing voltage steps, two MN subtypes (I and II) were identified in tadpoles and adults. Compared with type II MNs, type I MNs had larger sag amplitudes (suggesting a larger hyperpolarization-activated inward current), greater input resistance, lower rheobase, hyperpolarized action potential threshold, steeper frequency-current relationships, and fast firing rates and received fewer excitatory postsynaptic currents. Postmetamorphosis, type I MNs exhibited similar sag, enhanced postinhibitory rebound, and increased action potential amplitude with a smaller-magnitude fast afterhyperpolarization. Compared with tadpoles, type II MNs from frogs received higher-frequency, larger-amplitude excitatory postsynaptic currents. Input resistance decreased and rheobase increased postmetamorphosis in all MNs, concurrent with increased soma area and hyperpolarized action potential threshold. We suggest that type I MNs are likely recruited in response to smaller, buccal-related synaptic inputs as well as larger lung-related inputs, whereas type II MNs are likely recruited in response to stronger synaptic inputs associated with larger buccal breaths, lung breaths, or nonrespiratory behaviors involving powerful muscle contractions.

Made available in DSpace on 2020-12-12T02:35:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Canadian Institutes of Health Research Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Introduction: Ovariectomy reduces the hypercapnic ventilatory response and Locus Coeruleus (LC) appears to be involved in this response. Sex hormones as well as CO 2 can act in neurons of LC and also in microglial cells, however what these stimuli promoted in LC monoamine concentrations and microglial morphology is still unknown. Objective: To evaluate monoamine release in the LC, we measured the concentration of norepinephrine (NE), serotonin (5-HT) and dopamine (DA), and the metabolites 5-hydroxyindoleacetic acid (5HIAA; metabolite of 5-HT) and 5-hydroxyphenylacetic acid (DOPAC; metabolite of DA) in the LC, by HPCL method. Material and Methods: Protocols were approved by the local Animal Care and Use Committee (protocol no 8.129/16). For LC microglial analysis, we used ionized calcium-binding adapter molecule-1 (Iba1) immunolabeling to compare the density and morphology of microglia in the LC of OVX and intact female rats during normocapnia or hypercapnia (5%CO2). Protocols were approved by Laval University Animal Care Committee (Permit Number: 2012-023). Results: No significant differences were found in the quantification of the monoamines analyzed as well as their metabolites in OVX female rats compared to intact animals during normocapnia and hypercapnia. Regarding microglia cells in the LC, we observed an increase on morphological index, that indicates a reactive form of the cell, of OVX animals, and also an increase in the nearest neighbor distance (NND) caused by hypercapnia, which may indicate a higher motility of those cells. Conclusions: OVX and hypercapnia can affect microglial cells of LC but not monoamine concentrations. Unesp UFJF Département De Pédiatrie Centre De Recherche De L'institut Universitaire De Cardiologie et Pneumologie de Québec Université Laval Unesp

NEW FINDINGS What is the central question of the study? Progesterone is considered a respiratory stimulant drug, but its effect on medullary respiratory neurons are poorly documented. We investigated whether progesterone alters spontaneous activity of neurons in the nucleus of the solitary tract (NTS). What is the main finding and its importance? In NTS neurons, progesterone decreases the action potential firing frequency in response to current injections and the amplitude of excitatory postsynaptic currents. Based on the established neuroprotective effect of progesterone against excitotoxicity resulting from insults, this inhibitory effect is likely to reflect inhibition of ion fluxes. These results are important because they further our understanding of the mechanisms underlying the diversity of respiratory effects of progesterone. ABSTRACT Progesterone is known to stimulate breathing, but its actions on the respiratory control system have received limited attention. We addressed this issue at the cellular level by testing the hypothesis that progesterone augments excitatory currents at the level of the nucleus tractus solitarii (NTS). Medullary slices from juvenile male rats (14-17 days of age) containing the commissural region of the NTS (NTScom) were incubated with progesterone (1 μm) or vehicle (0.004% DMSO) for 60 min. We performed whole-cell voltage-clamp recordings of spontaneous excitatory postsynaptic currents (EPSCs) in the NTScom and determined membrane properties by applying depolarizing current steps. In comparison to vehicle-treated cells, progesterone exposure attenuates the firing frequency response to current injection and reduces the EPSC amplitude without modifying the EPSC frequency or the basal membrane properties. These data do not support our hypothesis, because they indicate that incubation with progesterone attenuates intrinsic action potential generation and inhibits excitatory synaptic inputs in the NTS. Given that these results are more in line with the protective effect of progesterone against excitotoxicity resulting from various insults, we propose that progesterone acts via inhibition of ionic flux.

During vertebrate development, the neural networks underlying air-breathing undergo changes in connectivity and functionality, allowing lung ventilation to emerge. Yet, the factors regulating development of these critical homeostatic networks remain unresolved. In amphibians, air-breathing occurs sporadically prior to metamorphosis. However, in tadpoles of Lithobates catesbeianus (American bullfrog), hypoxia stimulates gill and lung ventilation during early development. Because accelerated metamorphosis is a useful strategy to escape deterioration of the milieu, we hypothesized that central hypoxia would elicit long-term expression of the lung motor command for air breathing in pre-metamorphic tadpoles (TK stages VI-XIII). To do this, we recorded respiratory activity from cranial nerves V and VII in isolated brainstems before, during, and up to 2 h after exposure to 15 min of mild (PwO

New findings What is the central question of the study? Does neonatal stress, in the form of neonatal maternal separation, influence the maturation of microglial density, morphology and neuronal signalling in medullary regions regulating cardiorespiratory function in rat pups? What is the main finding and its importance? Using Iba-1 immunohistochemistry, we show that neonatal maternal separation augments microglial density and the proportion of cells with an amoeboid morphology in the medulla. Although the current understanding of the effect of early life stress on medullary development is relatively limited, these data show that within this area, microglia are affected by neonatal stress. Microglia could therefore be important effectors in cardiorespiratory disorders resulting from maternal separation. Abstract Neonatal stress has wide-ranging consequences for the developing brain, including the medullary cardiorespiratory network. In rat pups, the reflexive cardiorespiratory inhibition triggered by the presence of liquids near the larynx is augmented by neonatal maternal separation (NMS), especially in males. Sex-specific enhancement of synaptic connectivity by NMS might explain this cardiorespiratory dysfunction. Microglia influence the formation, maturation, activity and elimination of developing synapses, but their role in the wiring of medullary networks is unknown. Owing to their sensitivity to sex hormones and stress hormones, microglial dysfunction could contribute to the abnormal cardiorespiratory phenotype observed in NMS pups. Here, we first used ionized calcium-binding adapter molecule-1 (Iba-1) immunolabelling to compare the density and morphology of microglia in the medulla of male versus female rat pups (14-15 days old) that were either undisturbed or subjected to NMS (3 h day-1 ; postnatal days 3-12). Neonatal maternal separation augmented the density of Iba-1+ cells (caudal region of the NTS), increased the size of the soma and reduced the arborization area (especially in the dorsal motor nucleus of the vagus). Sex-based differences were not observed. Given that the actions of microglia are regulated by neuronal fractalkine (CX3 CL1 ), we then used western blot analysis to compare the expression of CX3 CL1 and its microglial receptor (CX3 CR1 ) in medullary homogenates from control and NMS pups. Although CX3 CR1 expression was 59% greater in males versus females, NMS had no effect on CX3 CL1 /CX3 CR1 signalling. Given that an amoeboid morphology reflects an immature phenotype in developing microglia, NMS could interfere with synaptic pruning via a different mechanism.