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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.)

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 γ-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; 5 min 5% CO2 - air, 10 min 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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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 © 2013 Elsevier B.V. All rights reserved.)

Nitric oxide (NO) is a potent central neuromodulator of respiration, yet its scope and site of action are unclear. We used 7-nitroindazole (7-NI), a selective inhibitor of endogenous neuronal NO synthesis, to investigate the neurogenesis of respiration in larval bullfrog (Rana catesbeiana) isolated brain stems. 7-NI treatment (0.0625-0.75 mM) increased the specific frequency of buccal ventilation (BV) events, indicating influence on BV central rhythm generators (CRGs). The drug reduced occurrence, altered burst shape, and disrupted clustering of lung ventilation (LV) events, without altering their specific frequency. LV burst occurrence and clustering also differed between pH conditions. We conclude that NO has diverse effects on respiratory rhythmogenesis, being necessary for the expression of respiratory rhythms, inhibiting the frequency of BV CRG, and affecting both shape and clustering of LV bursts through conditional modulation of LV CRG. We confirm central chemosensitivity in these preparations and demonstrate chemomodulation of LV burst clustering and occurrence but not specific frequency. Results support distinct oscillators underlying LV and BV CRGs.

Brainstem central chemoreceptors are critical to the hypercapnic ventilatory response, but their location and identity are poorly understood. When studied in vitro, serotonin-synthesizing (5-HT) neurons within the rat medullary raphé are intrinsically stimulated by CO²/ acidosis. The contributions of these neurons to central chemosensitivity in vivo, however, are controversial. Lacking is documentation of CO²-sensitive 5-HT neurons in intact experimental preparations and understanding of their spatial and proportional distribution. Here we test the hypothesis that 5-HT neurons in the rat medullary raphé are sensitive to arterial hypercapnia. We use extracellular recording and hypercapnic challenge of spontaneously active medullary raphé neurons in the unanesthetized in situ perfused decerebrate brainstem preparation to assess chemosensitivity of individual cells. Juxtacellular labeling of a subset of recorded neurons and subsequent immunohistochemistry for the 5-HT-synthesizing enzyme tryptophan hydroxylase (TPH) identify or exclude this neurotransmitter phenotype in electrophysiologically characterized chemosensitive and insensitive cells. We show that the medullary raphé houses a heterogeneous population, including chemosensitive and insensitive 5-HT neurons. Of 124 recorded cells, 16 cells were juxtacellularly filled, visualized, and immunohistochemically identified as 5-HT synthesizing, based on TPH-immunoreactivity. Forty-four percent of 5-HT cells were CO² stimulated (increased firing rate with hypercapnia), while 56% were unstimulated. Our results demonstrate that medullary raphé neurons are heterogeneous and clearly include a subset of 5-HT neurons that are excited by arterial hypercapnia. Together with data identifying intrinsically CO²-sensitive 5-HT neurons in vitro, these results support a role for such cells as central chemoreceptors in the intact system. [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 patterns of phrenic discharge comparable to eupnea and gasping in vivo. These ventilatory patterns differ in multiple aspects, including most prominently the rate of rise of inspiratory activity. Although we have recently demonstrated that both eupnea and gasping are similarly modulated by a Hering-Breuer expiratory-promoting reflex to tonic pulmonary stretch, it has generally been assumed that gasping was unresponsive to afferent stimuli from pulmonary stretch receptors. In the present study, we recorded eupneic and gasp-like efferent activity of the phrenic nerve in the in situ juvenile rat perfused brain stem preparation, with and without phrenic-triggered phasic pulmonary inflation. We tested the hypothesis that phasic pulmonary inflation produces reflex responses in situ akin to those in vivo and that both eupnea and gasping are similarly modulated by phasic pulmonary stretch. In eupnea, we found that phasic pulmonary inflation decreases inspiratory burst duration and the period of expiration, thus increasing burst frequency of the phrenic neurogram. Phasic pulmonary inflation also decreases the duration of expiration and increases the burst frequency during 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 respond to phasic activation of pulmonary stretch receptors in both eupnea and gasping. These findings support the homology of eupneic 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]

Sites of central CO[sub 2] chemosensitivity were investigated in isolated brain stems from Rana catesbeiana tadpoles and frogs. Respiratory neurograms were made from cranial nerve (CN) 7 and spinal nerve 2. Superfusion of the brain stem with hypercapnic artificial cerebrospinal fluid elicited increased fictive lung ventilation. The effect of focal perfusion of hypercapnic artificial cerebrospinal fluid on discrete areas of the ventral medulla was assessed. Sites of chemosensitivity, which are active continuously throughout development, were identified adjacent to CN 5 and CN 10 on the ventral surface of the medulla. In early- and middle-stage tadpoles and frogs, unilateral stimulation within either site was sufficient to elicit the hypercapnic response, but simultaneous stimulation within both sites was required in late-stage tadpoles. The chemosensitive sites were individually disrupted by unilateral application of 1 mg/ml protease, and the sensitivity to bath application or focal perfusion of hypercapnia was reassessed. Protease lesions at CN 10 abolished the entire hypercapnic response, but lesions at CN 5 affected only the hypercapnic response originating from the CN 5 site. Neurons within the chemosensitive sites were also destroyed by unilateral application of 1 mM kainic acid, and the sensitivity to bath or focal application of hypercapnia was reassessed. Kainic acid lesions within either site abolished the hypercapnic response. Using a vital dye, we determined that kainic acid destroyed neurons by only within 100 µm of the ventral medullary surface. Thus, regardless of developmental stage, neurons necessary for CO[sub 2] sensitivity are located in the ventral medulla adjacent to CN 5 and 10. [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.