Electrophysiological Comparison of Definitive Pro-opiomelanocortin Neurons in the Arcuate Nucleus and the Retrochiasmatic Area of Male and Female Mice.

Pro-opiomelanocortin (POMC)-expressing neurons in the arcuate nucleus of the hypothalamus (ARC) are considered a major site of leptin action. Due to increasing evidence that POMC neurons are highly heterogeneous and indications that the conventional molecular tools to study their functions have important limitations, a reassessment of leptin's effects on definitive POMC neurons is needed. POMC neurons are also expressed in the retrochiasmatic area (RCA), where their function is poorly understood. Furthermore, the response of POMC neurons to leptin in females is largely unknown. Therefore, the present study aimed to determine the differences in leptin responsiveness of POMC neurons in the ARC and the RCA using a mouse model allowing adult-inducible fluorescent labeling. We performed whole-cell patch clamp electrophysiology on 154 POMC neurons from male and female mice. We confirmed and extended the model by which leptin depolarizes POMC neurons, in both the ARC and the RCA. Furthermore, we characterized the electrophysiological properties of an underappreciated subpopulation representing ∼10% of hypothalamic POMC neurons that are inhibited by leptin. We also provide evidence that sex does not appear to be a major determinant of basal properties and leptin responsiveness of POMC neurons, but that females are overall less responsive to leptin compared to males.

Gestational intermittent hypoxia increases FosB-immunoreactive perikaryas in the paraventricular nucleus of the hypothalamus of adult male (but not female) rats.

Sleep-disordered breathing is a respiratory disorder commonly experienced by pregnant women. The recurrent hypoxaemic events associated with sleep-disordered breathing have deleterious consequences for the mother and fetus. Adult male (but not female) rats born to dams subjected to gestational intermittent hypoxia (GIH) have a higher resting blood pressure than control animals and show behavioural/neurodevelopmental disorders. The origin of this persistent, sex-specific effect of GIH in offspring is unknown, but disruption of the neuroendocrine stress pathways is a key mechanism by which gestational stress increases disease risk in progeny. Using FosB immunolabelling as a chronic marker of neuronal activation, we determined whether GIH augments basal expression of FosB in the perikaryas of cells in the paraventricular nucleus of the hypothalamus (PVN), a key structure in the regulation of the stress response and blood pressure. From gestational day 10, female rats were subjected to GIH for 8 h/day (light phase) until the day before delivery (gestational day 21); GIH consisted of 2 min hypoxic bouts (10.5% O2 ) alternating with normoxia. Control rats were exposed to intermittent normoxia over the same period (GNX). At adulthood (10-15 weeks), the brains of male and female rats were harvested for FosB immunohistochemistry. In males, GIH augmented PVN FosB labelling density by 30%. Conversely, PVN FosB density in GIH females was 28% lower than that of GNX females. We conclude that GIH has persistent and sex-specific impacts on the development of stress pathways, thereby offering a plausible mechanism by which GIH can disturb neural development and blood pressure homeostasis in adulthood. NEW FINDINGS: What is the central question of this study? In pregnant women, sleep apnoea increases the risk of disease for the offspring at various life stages. Given that gestational stress disrupts the programming of the stress pathways, we determined whether exposing female rats to gestational intermittent hypoxia (GIH) activates hypothalamic neurons regulating the stress response in adult rats. What is the main finding and its importance? Using FosB immunolabelling as a marker of marker of neuronal activation, we showed that GIH augmented basal activation of the paraventricular nucleus of the hypothalamus in males, but not females. Disruption of the stress pathways is a new hypothesis to explain the persistent and sex-specific impacts of GIH on offspring health.

Intermittent Hypoxia and Weight Loss: Insights into the Etiology of the Sleep Apnea Phenotype.

Sleep apnea (SA) is a major respiratory disorder with increased risk for hypertension and obesity; however, our understanding of the origins of this complex disorder remains limited. Because apneas lead to recurrent drops in O2 during sleep, intermittent hypoxia (IH) is the main animal model to explore the pathophysiology of SA. Here, we assessed the impacts of IH on metabolic function and related signals. Adult male rats were exposed to 1 week of moderate IH (FiO2 = 0.10-30 s, ten cycles/hour, 8 h/day). Using whole-body plethysmography, we measured respiratory variability and apnea index during sleep. Blood pressure and heart rate were measured by the tail-cuff method; blood samples were taken for multiplex assay. At rest, IH augmented arterial blood pressure, respiratory instability, but not apnea index. IH induced weight, fat, and fluid loss. IH also reduced food intake and plasma leptin, adrenocorticotropic hormone (ACTH), and testosterone levels but increased inflammatory cytokines. We conclude that IH does not replicate the metabolic clinical features of SA patient, thus raising our awareness of the limitations of the IH model. The fact that the risk for hypertension occurs before the appearance of apneas provides new insights into the progression of the disease.

Estrogens, age, and, neonatal stress: panic disorders and novel views on the contribution of non-medullary structures to respiratory control and CO2 responses.

CO2 is a fundamental component of living matter. This chemical signal requires close monitoring to ensure proper match between metabolic production and elimination by lung ventilation. Besides ventilatory adjustments, CO2 can also trigger innate behavioral and physiological responses associated with fear and escape but the changes in brain CO2/pH required to induce ventilatory adjustments are generally lower than those evoking fear and escape. However, for patients suffering from panic disorder (PD), the thresholds for CO2-evoked hyperventilation, fear and escape are reduced and the magnitude of those reactions are excessive. To explain these clinical observations, Klein proposed the false suffocation alarm hypothesis which states that many spontaneous panics occur when the brain's suffocation monitor erroneously signals a lack of useful air, thereby maladaptively triggering an evolved suffocation alarm system. After 30 years of basic and clinical research, it is now well established that anomalies in respiratory control (including the CO2 sensing system) are key to PD. Here, we explore how a stress-related affective disorder such as PD can disrupt respiratory control. We discuss rodent models of PD as the concepts emerging from this research has influenced our comprehension of the CO2 chemosensitivity network, especially structure that are not located in the medulla, and how factors such as stress and biological sex modulate its functionality. Thus, elucidating why hormonal fluctuations can lead to excessive responsiveness to CO2 offers a unique opportunity to gain insights into the neuroendocrine mechanisms regulating this key aspect of respiratory control and the pathophysiology of respiratory manifestations of PD.

Persistent augmentation of fictive air breathing by hypoxia: An in vitro study of the role of GABAB signaling in pre-metamorphic tadpoles.

Tadpole development is influenced by environmental cues and hypoxia can favor the emergence of the neural networks driving air breathing. Exposing isolated brainstems from pre-metamorphic tadpoles to acute hypoxia (∼0% O2; 15 min) leads to a progressive increase in fictive air breaths (∼3 fold) in the hours that follow stimulation. Here, we first determined whether this effect persists over longer periods (<18 h); we then evaluated maturity of the motor output by comparing the breathing pattern of hypoxia-exposed brainstems to that of preparations from adult bullfrogs under basal conditions. Because progressive withdrawal of GABAB-mediated inhibition contributes to the developmental increase in fictive lung ventilation, we then hypothesised that hypoxia reduces respiratory sensitivity to baclofen (selective GABAB-agonist). Experiments were performed on isolated brainstem preparations from pre-metamorphic tadpoles (TK stages IV to XIV); respiratory-related neural activity was recorded from cranial nerves V/VII and X before and 18 h after exposure to hypoxia (0% O2 + 2% CO2; 25 min). Time-control experiments (no hypoxia) were performed. Exposing pre-metamorphic tadpoles to hypoxia did not affect gill burst frequency, but augmented the frequency of fictive lung bursts and the incidence of episodic breathing levels intermediate between pre-metamorphic and adult preparations. Addition of baclofen to the aCSF (0,2 µM - 20 min) reduced lung burst frequency, but the response of hypoxia-exposed brainstems was greater than controls. We conclude that acute hypoxia facilitates development and maturation of the motor command driving air breathing. We propose that a greater number of active rhythmogenic neurons expressing GABAb receptors contributes to this effect.

On the origins of sleep disordered breathing, cardiorespiratory and metabolic dysfunction: which came first, the chicken or the egg?

Sleep disordered breathing (SDB) is a complex, sex specific and highly heterogeneous group of respiratory disorders. Nevertheless, sleep fragmentation and repeated fluctuations of arterial blood gases for several hours per night are at the core of the problem; together, they impose significant stress to the organism with deleterious consequences on physical and mental health. SDB increases the risk of obesity, diabetes, depression and anxiety disorders; however, the same health issues are risk factors for SDB. So, which came first, the chicken or the egg? What causes the appearance of the first significant apnoeic events during sleep? These are important questions because although moderate to severe SDB affects ∼500 million adults globally, we still have a poor understanding of the origins of the disease, and the main treatments (and animal models) focus on the symptoms rather than the cause. Because obesity, metabolic dysfunction and stress-related neurological disorders generally appear progressively, we discuss how the development of these diseases can lead to specific anatomical and non-anatomical traits of SDB in males and females while considering the impacts of sex steroids. In light of the growing evidence indicating that the carotid bodies are important sensors of key metabolic and endocrine signals associated with stress and dysmetabolism, we propose that these organs play a key role in the process.

Control of Breathing in Ectothermic Vertebrates.

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. © 2022 American Physiological Society. Compr Physiol 12: 1-120, 2022.

Corrigendum: Testosterone Supplementation Induces Age-Dependent Augmentation of the Hypoxic Ventilatory Response in Male Rats With Contributions From the Carotid Bodies.

[This corrects the article DOI: 10.3389/fphys.2021.781662.].

Amygdala-driven apnea: A breath of fresh air in respiratory neurobiology.

The amygdala is mainly known for its role in the pathogenesis of anxiety and the initiation of fear responses. However, there is growing evidence showing that the amygdala's ability to respond to internal stimuli such as CO2 is limited, thereby challenging its role in the brain-behavior relationship. Based on these results and the strong inhibitory connections between the central nucleus of the amygdala and key brainstem areas regulating the reflexive respiratory responses to CO2, Feinstein et al. propose amygdala-driven apnea as a novel mechanism in the chemoreceptive origin of anxiety.

Stress and Loss of Ovarian Function: Novel Insights into the Origins of Sex-Based Differences in the Manifestations of Respiratory Control Disorders During Sleep.

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.

Thyroid hormones during the perinatal period are necessary to respiratory network development of newborn rats.

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.

Sex-Specific Effects of Stress on Respiratory Control: Plasticity, Adaptation, and Dysfunction.

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.

Orexin-A inhibits fictive air breathing responses to respiratory stimuli in the bullfrog tadpole (Lithobates catesbeianus).

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.

Impact of ovariectomy and CO2 inhalation on microglia morphology in select brainstem and hypothalamic areas regulating breathing in female rats.

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.

Testosterone Supplementation Induces Age-Dependent Augmentation of the Hypoxic Ventilatory Response in Male Rats With Contributions From the Carotid Bodies.

Excessive carotid body responsiveness to O2 and/or CO2/H+ stimuli contributes to respiratory instability and apneas during sleep. In hypogonadal men, testosterone supplementation may increase the risk of sleep-disordered breathing; however, the site of action is unknown. The present study tested the hypothesis that testosterone supplementation potentiates carotid body responsiveness to hypoxia in adult male rats. Because testosterone levels decline with age, we also determined whether these effects were age-dependent. In situ hybridization determined that androgen receptor mRNA was present in the carotid bodies and caudal nucleus of the solitary tract of adult (69 days old) and aging (193-206 days old) male rats. In urethane-anesthetized rats injected with testosterone propionate (2 mg/kg; i.p.), peak breathing frequency measured during hypoxia (FiO2 = 0.12) was 11% greater vs. the vehicle treatment group. Interestingly, response intensity following testosterone treatment was positively correlated with animal age. Exposing ex vivo carotid body preparations from young and aging rats to testosterone (5 nM, free testosterone) 90-120 min prior to testing showed that the carotid sinus nerve firing rate during hypoxia (5% CO2 + 95% N2; 15 min) was augmented in both age groups as compared to vehicle (<0.001% DMSO). Ventilatory measurements performed using whole body plethysmography revealed that testosterone supplementation (2 mg/kg; i.p.) 2 h prior reduced apnea frequency during sleep. We conclude that in healthy rats, age-dependent potentiation of the carotid body's response to hypoxia by acute testosterone supplementation does not favor the occurrence of apneas but rather appears to stabilize breathing during sleep.

Cerebral Erythropoietin Prevents Sex-Dependent Disruption of Respiratory Control Induced by Early Life Stress.

Injuries that occur early in life are often at the root of adult illness. Neonatal maternal separation (NMS) is a form of early life stress that has persistent and sex-specific effects on the development of neural networks, including those that regulate breathing. The release of stress hormones during a critical period of development contributes to the deleterious consequences of NMS, but the role of increased corticosterone (CORT) in NMS-induced respiratory disturbance is unknown. Because erythropoietin (EPO) is a potent neuroprotectant that prevents conditions associated with hyperactivation of the stress neuroaxis in a sex-specific manner, we hypothesized that EPO reduces the sex-specific alteration of respiratory regulation induced by NMS in adult mice. Animals were either raised under standard conditions (controls) or exposed to NMS 3 h/day from postnatal days 3-12. We tested the efficacy of EPO in preventing the effects of NMS by comparing wild-type mice with transgenic mice that overexpress EPO only in the brain (Tg21). In 7-days-old pups, NMS augmented CORT levels ~2.5-fold by comparison with controls but only in males; this response was reduced in Tg21 mice. Respiratory function was assessed using whole-body plethysmography. Apneas were detected during sleep; the responsiveness to stimuli was measured by exposing mice to hypoxia (10% O2; 15 min) and hypercapnia (5% CO2; 10 min). In wild-type, NMS increased the number of apneas and the hypercapnic ventilatory response (HcVR) only in males; with no effect on Tg21. In wild-type males, the incidence of apneas was positively correlated with HcVR and inversely related to the tachypneic response to hypoxia. We conclude that neural EPO reduces early life stress-induced respiratory disturbances observed in males.

Influence of light/dark cycle and orexins on breathing control in green iguanas (Iguana iguana).

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.

Disruption of estradiol regulation of orexin neurons: a novel mechanism in excessive ventilatory response to CO2 inhalation in a female rat model of panic disorder.

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-ß 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.

Facilitation of microglial motility by thyroid hormones requires the presence of neurons in cell culture: A distinctive feature of the brainstem versus the cortex.

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.

Is there a common drive for buccal movements associated with buccal and lung 'breath' in Lithobates catesbeianus?

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.