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Bullfrog tadpoles ventilate both the buccal cavity and lung. In isolated brainstems, the midbrain/pons influences CO 2 responsiveness and timing of lung ventilatory bursting, depending on larval development. However, little is known about midbrain/pons influences on buccal burst patterns. As such, we investigated how removal of this region affects buccal burst shape and CO 2 responsiveness across development. We measured facial nerve activity in brainstems isolated from tadpoles during early and late developmental stages, under normal and elevated levels of CO 2 . Brainstems were either left intact or transected by removing the midbrain/pons. In late stage preparations, buccal burst pattern differed between intact and reduced preparations, and bursts were responsive to elevated CO 2 in these reduced preparations. These results suggest the midbrain/pons affects tadpole buccal burst pattern and CO 2 responsiveness, perhaps similar to its influences on lung ventilation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Serotonin (5-HT)-synthesizing neurons of the medullary raphe are putative central chemoreceptors, proposed to be one of potentially multiple brain stem chemosensitive cell types and loci interacting to produce the respiratory chemoreflex. Hypocretin-synthesizing neurons of the lateral hypothalamus are important contributors to arousal state, thermoregulation, and feeding behavior and are also reportedly involved in the hypercapnic ventilatory response. Recently, a functional interaction was found between the hypocretin system and 5-HT neurons of the dorsal raphe. The validity and potential significance of hypocretin modulation of medullary raphe 5-HT neurons, however, is unknown. As such, the purpose of this study was to explore functional interactions between the hypocretin system and 5-HT system of the medullary raphe on baseline respiratory output and central chemosensitivity. To explore such interactions, we used the neonatal in vitro medullary slice preparation derived from wild-type (WT) mice (normal 5-HT function) and a knockout strain lacking all central 5-HT neurons (Lmx1b(f/f/p) mice). We examined effects of acidosis, hypocretin-1, a hypocretin receptor antagonist (SB-408124), and the effect of the antagonist on the response to acidosis. We confirmed the critical role of 5-HT neurons in central chemosensitivity given that the increased hypoglossal burst frequency with acidosis, characteristic of WT mice, was absent in preparations derived from Lmx1b(f/f/p) mice. We also found that hypocretin facilitated baseline neural ventilatory output in part through 5-HT neurons. Although the impact of hypocretin on 5-HT neuronal sensitivity to acidosis is still unclear, hypocretins did appear to mediate the burst duration response to acidosis via serotonergic mechanisms.

Serotonergic neurons project widely throughout the CNS and modulate many different brain functions. Particularly important, but controversial, are the contributions of serotonin (5-HT) neurons to respiratory and thermoregulatory control. To better define the roles of 5-HT neurons in breathing and thermoregulation, we took advantage of a unique conditional knock-out mouse in which Lmx1b is genetically deleted in Pet1-expressing cells (Lmx1b(f/f/p)), resulting in near-complete absence of central 5-HT neurons. Here, we show that the hypercapnic ventilatory response in adult Lmx1b(f/f/p) mice was decreased by 50% compared with wild-type mice, whereas baseline ventilation and the hypoxic ventilatory response were normal. In addition, Lmx1b(f/f/p) mice rapidly became hypothermic when exposed to an ambient temperature of 4 degrees C, decreasing core temperature to 30 degrees C within 120 min. This failure of thermoregulation was caused by impaired shivering and nonshivering thermogenesis, whereas thermosensory perception and heat conservation were normal. Finally, intracerebroventricular infusion of 5-HT stimulated baseline ventilation, and rescued the blunted hypercapnic ventilatory response. These data identify a previously unrecognized role of 5-HT neurons in the CO(2) chemoreflex, whereby they enhance the response of the rest of the respiratory network to CO(2). We conclude that the proper function of the 5-HT system is particularly important under conditions of environmental stress and contributes significantly to the hypercapnic ventilatory response and thermoregulatory cold defense.

Inhibitory 5-HTla receptors are located on serotonin (5-HT) neurons (autoreceptors) as well as neurons of the respiratory network (heteroreceptors). Thus, effects on breathing of 5-HT,a agonists, such as (R)-( + )-8-hydroxy-2-(di-N-propylamino) tetralin (8-OH-DPAT), could either be due to decreased firing of 5-HT neurons or direct effects on the respiratory network. Mice in which the transcrip-tion factor LMX1B is genetically deleted selectively in Petl-1 -expressing cells (Lmxf/f/p) essentially have complete absence of central 5-HT neurons, providing a unique opportunity to separate the effect of activation of downstream 5-HTla heteroreceptors from that of autoreceptors. We used rhythmically active medullary slices from wild-type (WT) and Lmxf/f/p neonatal mice to differentiate autore-ceptor versus heteroreceptor effects of 8-OH-DPAT on hypoglossal nerve respiratory output. 8-OH-DPAT transiently increased respira-tory burst frequency in Lmxf/f/p preparations, but not in WT slices. This excitation was abolished when synaptic inhibition was blocked by GABAergic/glycinergic receptor antagonists. Conversely, after 10 min of application, frequency in Lmxf/f/p slices was not different from baseline, whereas it was significantly depressed in WT slices. In WT mice in vivo, subcutaneous injection of 8-OH-DPAT produced similar biphasic respiratory effects as in Lmxf/f/p mice. We conclude that 5-HTla receptor agonists have two competing effects: rapid stimulation of breathing due to excitation of the respiratory network, and delayed inhibition of breathing due to autoreceptor inhibition of 5-HT neurons. The former effect is presumably due to inhibition of inhibitory interneurons embedded in the respiratory network. [ABSTRACT FROM AUTHOR]

Abstract: The role of gap junctions in the brainstem respiratory control system is ambiguous. In the present study, we used juvenile rats to determine whether blocking gap junctions altered eupnea or gasping in the in situ, arterially perfused rat preparation. Blockade of gap junctions with 100μM carbenoxolone or 300μM octanol did not produce any consistent changes in the timing or amplitude of integrated phrenic discharge or in the peak frequency in the power spectrum of phrenic nerve discharge during eupnea or ischemic gasping beyond those changes seen in time-control animals. These findings do not rule out a role for gap junctions in the expression of eupnea or gasping, but they do demonstrate that these intermembrane channels are not obligatory for either rhythm to occur. [Copyright &y& Elsevier]

The perfused in situ juvenile rat preparation produces phrenic discharge patterns comparable to eupnea and gasping in vivo. These ventilatory patterns of eupnea and gasping differ in multiple aspects, including most prominently the rate of rise of inspiratory activity. Because gasping, but not eupnea, appeared similar after vagotomy in spontaneous breathing preparations, it has been assumed that gasping was unresponsive to afferent stimuli from pulmonary stretch receptors. In the present study, efferent activity of the phrenic nerve was recorded during eupnea and gasping in the in situ juvenile rat preparation. Gasping was induced in hypoxichypercapnia or ischemia. An increase in the pressure of tonic lung inflation from 1 to 10 cmH[sub 2]O caused a prolongation of the duration between phrenic bursts in both eupnea or gasping. Bilateral vagotomy eliminated these changes. We conclude that the neural substrate mediating the Hering-Breuer reflex is retained in the in situ preparation and that the brain stem circuitry generating the respiratory patterns responds to tonic activation of pulmonary stretch receptors in a similar manner in eupnea and gasping. These findings support the homology of eupnea-like phrenic discharge patterns in the reduced in situ preparation and eupnea in vivo and disprove the common supposition that gasping is insensitive to vagal afferent feedback from pulmonary stretch receptor mechanisms. [ABSTRACT FROM AUTHOR]

The article reports on a study to test the hypothesis that the nucleus isthmi (NI) in the brain of bullfrogs plays an essential role in the breathing pattern formation. It was found that the NI is not responsible for initiating breathing episodes, but instead offers a tonic excitatory input to respiratory centers in the lower brainstem.

The development of amphibian breathing provides insight into vertebrate respiratory control mechanisms. Neural oscillators in the rostral and caudal medulla drive ventilation in amphibians, and previous reports describe ventilatory oscillators and CO 2 sensitive regions arise during different stages of amphibian metamorphosis. However, inconsistent findings have been enigmatic, and make comparisons to potential mammalian counterparts challenging. In the current study we assessed amphibian central CO 2 responsiveness and respiratory rhythm generation during two different developmental stages. Whole-nerve recordings of respiratory burst activity in cranial and spinal nerves were made from intact or transected brainstems isolated from tadpoles during early or late stages of metamorphosis. Brainstems were transected at the level of the trigeminal nerve, removing rostral structures including the nucleus isthmi, midbrain, and locus coeruleus, or transected at the level of the glossopharyngeal nerve, removing the putative buccal oscillator and caudal medulla. Removal of caudal structures stimulated the frequency of lung ventilatory bursts and revealed a hypercapnic response in normally unresponsive preparations derived from early stage tadpoles. In preparations derived from late stage tadpoles, removal of rostral or caudal structures reduced lung burst frequency, while CO 2 responsiveness was retained. Our results illustrate that structures within the rostral medulla are capable of sensing CO 2 throughout metamorphic development. Similarly, the region controlling lung ventilation appears to be contained in the rostral medulla throughout metamorphosis. This work offers insight into the consistency of rhythmic respiratory and chemosensitive capacities during metamorphosis. [ABSTRACT FROM AUTHOR]

Serotonergic dysfunction compromises ventilatory chemosensitivity and may enhance vulnerability to pathologies such as the Sudden Infant Death Syndrome (SIDS). We have shown raphé contributions to central chemosensitivity involving serotonin (5-HT)-and &#947-aminobutyric acid (GABA)-mediated mechanisms. We tested the hypothesis that mild intermittent hypercapnia (IHc) induces respiratory plasticity, due in part to strengthening of GABA mechanisms. Rat pups were IHc-pretreated (eight consecutive cycles; 5min 5% CO2 - air, 10min air) or constant normocapnia-pretreated as a control, each day for 5 consecutive days beginning at P12. We subsequently assessed CO2 responsiveness using the in situ perfused brainstem preparation. Hypercapnic responses were determined with and without pharmacological manipulation. Results show IHc-pretreatment induces plasticity sufficient for responsiveness despite removal of otherwise critical ketanserin-sensitive mechanisms. Responsiveness following IHc-pretreatment was absent if ketanserin was combined with GABAergic antagonism, indicating that plasticity depends on GABAergic mechanisms. We propose that IHc-induced plasticity could reduce the severity of reflex dysfunctions underlying pathologies such as SIDS. [ABSTRACT FROM AUTHOR]

Abstract: Evidence from in vivo and in vitro experiments conclude that serotonin (5-HT) neurons are involved in and play an important role in central respiratory CO2/H+ chemosensitivity. This study was designed to assess the importance of 5-HT neurons and 5-HT receptor activation in the frequency and amplitude components of the hypercapnic response of the respiratory network in the unanesthetized perfused in situ juvenile rat brainstem preparation that exhibits patterns of phrenic nerve discharge similar to breathing in vivo. Exposure to a hypercapnic perfusate increased phrenic burst frequency and/or amplitude, the neural correlates of breathing frequency and tidal volume in vivo. Hypercapnic responses were also assessed during exposure to ketanserin (5-HT2 receptor antagonist), and 8-OH-DPAT (inhibiting 5-HT neurons via 5-HT1A autoreceptors). Neither of these drugs substantially altered baseline activity, however, both abolished hypercapnic responses of the respiratory network. These data illustrate that 5-HT neurons and 5-HT receptor activation are not required for respiratory rhythm generation per se, but are critical for CO2 responses in situ, supporting the hypothesis that 5-HT neurons play an important role in central ventilatory chemosensitivity in vivo. [Copyright &y& Elsevier]

• Amphibians transition from aquatic to aerial breathing during metamorphosis. • Diverse neurochemicals orchestrate development of respiratory control networks. • Maturation of the central hypoxic ventilatory response depends on GABA development. • Thyroid hormones are master regulators of metamorphosis and neurochemical signaling. • We propose an updated classification of developmental stages based on current data. 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. [ABSTRACT FROM AUTHOR]