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.

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.

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.

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.

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.

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.

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.

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.

Development of central respiratory control in anurans: The role of neurochemicals in the emergence of air-breathing and the hypoxic response.

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.

Distinct dampening effects of progesterone on the activity of nucleus tractus solitarii neurons in rat pups.

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.

Respiratory motoneuron properties during the transition from gill to lung breathing in the American bullfrog.

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.

The influence of sex and neonatal stress on medullary microglia in rat pups.

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.

Respiratory consequences of targeted losses of Hoxa5 gene function in mice.

Fetal development of the respiratory tract and diaphragm requires strict coordination between genetically controlled signals and mechanical forces produced by the neural network that generates breathing. HOXA5, which is expressed in the mesenchyme of the trachea, lung and diaphragm, and in phrenic motor neurons, is a key transcription factor regulating lung development and function. Consequently, most Hoxa5-/- mutants die at birth from respiratory failure. However, the extensive effect of the null mutation makes it difficult to identify the origins of respiratory dysfunction in newborns. To address the physiological impact of Hoxa5 tissue-specific roles, we used conditional gene targeting with the Dermo1Cre and Olig2Cre mouse lines to produce specific Hoxa5 deletions in the mesenchyme and motor neurons, respectively. Hoxa5 expression in the mesenchyme is critical for trachea development, whereas its expression in phrenic motor neurons is essential for diaphragm formation. Breathing measurements in adult mice with whole-body plethysmography demonstrated that, at rest, only the motor neuron deletion affects respiration, resulting in higher breathing frequency and decreased tidal volume. But subsequent exposure to a moderate hypoxic challenge (FiO2 =0.12; 10 min) revealed that both mutant mice hyperventilate more than controls. Hoxa5flox/flox;Dermo1+/Cre mice showed augmented tidal volume while Hoxa5flox/flox;Olig2+/Cre mice had the largest increase in breathing frequency. No significant differences were observed between medulla-spinal cord preparations from E18.5 control and Hoxa5flox/flox;Olig2+/Cre mouse embryos that could support a role for Hoxa5 in fetal inspiratory motor command. According to our data, Hoxa5 expression in the mesenchyme and phrenic motor neurons controls distinct aspects of respiratory development.

On the origins of sex-based differences in respiratory disorders: Lessons and hypotheses from stress neuroendocrinology in developing rats.

The environment plays a critical role in shaping development and function of the brain. Stress, especially when experienced early in life, can interfere with these processes. In the context of respiratory control, perinatal stress can therefore alter the ability to achieve the "fine-tuning" necessary for proper detection of chemosensory stimuli and production of an adequate motor (respiratory) command. Depending on the timing, intensity, and duration, the detrimental consequences of perinatal exposure to adverse conditions on the respiratory network become manifest at various life stages and can persist into adulthood. During early life, respiratory diseases commonly associated with dysfunction of neural networks include apnea of prematurity (AOP) and cardio-respiratory failure leading to sudden infant death syndrome (SIDS). Sleep disordered breathing (SDB) can occur at various life stages, including adulthood. Regardless of age, a common element of these disorders is their greater prevalence in males. While this sexual dimorphism points to a potential role of sex hormones, our understanding of the neuroendocrine mechanisms remain poorly understood. In addition to their modulatory influence on breathing, gonadal hormones regulate sexual differentiation of the brain. Stress alters these effects, and over the years our laboratory has used various perinatal stress protocols to gain insight into the origins of sex-based differences in respiratory disorders. This review discusses our recent advances with a focus on the sex-specific impact of early life stress on O2-chemoreflex function both in newborn and adult rats. We conclude by discussing the basic principles emerging from this work, potential mechanisms, and clinical relevance.

Sex-Specific Consequences of Neonatal Stress on Cardio-Respiratory Inhibition Following Laryngeal Stimulation in Rat Pups.

The presence of liquid near the larynx of immature mammals triggers prolonged apneas with significant O2 desaturations and bradycardias. When excessive, this reflex (the laryngeal chemoreflex; LCR) can be fatal. Our understanding of the origins of abnormal LCR are limited; however, perinatal stress and male sex are risk factors for cardio-respiratory failure in infants. Because exposure to stress during early life has deleterious and sex-specific consequences on brain development it is plausible that respiratory reflexes are vulnerable to neuroendocrine dysfunction. To address this issue, we tested the hypothesis that neonatal maternal separation (NMS) is sufficient to exacerbate LCR-induced cardio-respiratory inhibition in anesthetized rat pups. Stressed pups were separated from their mother 3 h/d from postnatal days 3 to 12. At P14-P15, pups were instrumented to monitor breathing, O2 saturation (Spo2), and heart rate. The LCR was activated by water injections near the larynx (10 µl). LCR-induced apneas were longer in stressed pups than controls; O2 desaturations and bradycardias were more profound, especially in males. NMS increased the frequency and amplitude of spontaneous EPSCs (sEPSCs) in the dorsal motor nucleus of the vagus (DMNV) of males but not females. The positive relationship between corticosterone and testosterone observed in stressed pups (males only) suggests that disruption of neuroendocrine function by stress is key to sex-based differences in abnormal LCR. Because testosterone application onto medullary slices augments EPSC amplitude only in males, we propose that testosterone-mediated enhancement of synaptic connectivity within the DMNV contributes to the male bias in cardio-respiratory inhibition following LCR activation in stressed pups.

Sex-specific response to hypoxia in a reduced brainstem preparation from Xenopus laevis.

Respiratory reflexes and tolerance to hypoxia show significant sexual dimorphism. However, the data supporting this notion originates exclusively from mammals. To determine whether this concept is limited to this group of vertebrates, we examined the sex-specific response to acute hypoxia in an adult reduced brainstem preparation from Xenopus laevis. Within the first 5min of exposure to hypoxic aCSF (98% N2/2% CO2), recordings of respiratory-related activity show a stronger increase in fictive breathing frequency in males than females. This initial response was followed by a decrease in respiratory-related activity; this depression occurred 6min sooner in males than females. These results represent new evidences of sexual dimorphism in respiratory control in amphibians and provide potential insight in understanding the homology with other groups of vertebrates, including mammals.

Consequences of gestational stress on GABAergic modulation of respiratory activity in developing newborn pups.

The GABAergic system modulates respiratory activity and undergoes substantial changes during early life. Because this maturation process is sensitive to stress, we tested the hypothesis that gestational stress (GS) alters development of GABAergic modulation of respiratory control in rat pups. The respiratory responses to the selective GABAA receptor agonist muscimol were compared between pups born to dams subjected to GS (bright light and predator odor; 20 min/day from G9 to G19) or maintained under standard (control) conditions. Respiratory activity was measured on 1 and 4 days old pups of both sexes using in vivo (whole body plethysmography) and in vitro (isolated brainstem-spinal cord preparation) approaches. In intact pups, muscimol injection (0.75 mg/kg; i.p.) depressed minute ventilation; this response was less in GS pups, and at P4, muscimol augmented minute ventilation in GS females. Bath application of muscimol (0.01-0.5 µM) onto brainstem preparations decreased inspiratory (C4) burst frequency and amplitude in a dose-dependent manner; the responsiveness decreased with age. However, GS had limited effects on these results. We conclude that the results obtained in vivo are consistent with our hypothesis and show that GS delays maturation of GABAergic modulation of respiratory activity. The differences in the results observed between experimental approaches (in vivo versus in vitro) indicate that the effect of prenatal stress on maturation of GABAergic modulation of respiratory control mainly affects the peripheral/metabolic components of the respiratory control system.

Control of breathing in in vitro brain stem preparation from goldfish (Carassius auratus; Linnaeus).

In vitro brain stem preparations from goldfish (Carassius auratus) were used to first determine whether this species possesses central chemoreceptors able to modulate respiratory activity. Preparations were superfused with an artificial cerebrospinal fluid (aCSF); fictive breathing was recorded extracellularly by placing a suction electrode on cranial nerve VII. Reducing the level of O2 in the gas mixture used to bubble the aCSF from a hyperoxic level (80% or 98.7% O2) to a relative hypoxic level (20% or 40% O2) increased the frequency of the fictive respiratory burst (P = 0.0002). Reducing the pH of the aCSF from 7.9 to 7.4 by increasing CO2 in the superfusate did not affect fictive breathing. Chloride-mediated neurotransmission (GABA/glycine) is a major modulator of respiratory activity; however, its effect on the neural circuits that regulate breathing in teleosts remains unknown. Bath application of GABA (0.5, 5.0 mM) decreased burst frequency but not amplitude; this effect was dose dependent (drug × concentration: P = 0.01). Superfusion of the preparations with aCSF containing 1.25 µM of bicuculline methochloride and 1.50 µM of strychnine hydrochloride (GABAA and glycine receptor antagonists, respectively) increased burst frequency (P = 0.002) and amplitude (P < 0.001). We conclude that respiratory activity produced by the goldfish brain stem is not responsive to the moderate CO2 levels used in this study; it may contain O2 chemoreceptors, but the relatively small response could also reflect nonspecific effects of hypoxia on the central nervous system. Cl(-)-mediated neurotransmission inhibits fictive breathing; this aspect of respiratory regulation is similar to other groups of vertebrates.