The pontine Kölliker-Fuse nucleus is important for reduced post-inspiratory airflow elicited by stimulation of the ventral respiratory parafacial region.

Considering that the retrotrapezoid nucleus/respiratory parafacial region (RTN/pFRG) would be an important center in the central nervous system involved in the maintenance and modulation of respiratory activity, we hypothesized that neurons in this nucleus would also be involved in the postinspiratory phase of the respiratory cycle through a connection with the pontine Kölliker-Fuse (KF) region. Here we performed pharmacogenetic manipulation (AAV-hM3D(Gq)-mCherry or AAV-hM4D(Gi)-mCherry) in VGlut2-cre, Ai6 conscious mice to evaluate breathing parameters through whole body plethysmography under baseline conditions (normoxia: FiO2 = 0.21) or under hypercapnia or hypoxia challenges (FiCO2 = 0.07 or FiO2 = 0.08). Under normoxia, selective stimulation of RTN/pFRG resulted in a smaller increase in VE (1,272 ± 102.5, vs. RTN/pFRG stimulation: 1,878 ± 122.1 ml/kg/min), due to a smaller increase in VT (5.4 ± 0.35, vs. RTN/pFRG stimulation: 7.77 ± 0.21 ml/kg) without changing fR in a condition of KF inhibition. However, inhibition of the VGlut2 neurons in the KF did affect the TE1 produced by selective activation of RTN/pFRG (119.9 ± 2.53, vs. RTN/pFRG stimulation: 104 ± 2.46 ms). Both the hypercapnia and hypoxia ventilatory response were reduced after inhibition of VGlut2-expressing KF neurons. Therefore, consistent with anatomical projections RTN/pFRG neurons regulate lung ventilation by controlling all aspects of breathing, i.e breathing frequency, inspiration, postinspiration and active expiration. All the modulation seems to be dependent on the integrity of the glutamatergic neurons in the KF region.

Inhibitory control of active expiration by the Bötzinger complex in rats.

KEY POINTS: Contraction of abdominal muscles at the end of expiration during metabolic challenges (such as hypercapnia and hypoxia) improves pulmonary ventilation. The emergence of this active expiratory pattern requires the recruitment of the expiratory oscillator located on the ventral surface of the medulla oblongata. Here we show that an inhibitory circuitry located in the Bötzinger complex is an important source of inhibitory drive to the expiratory oscillator. This circuitry, mediated by GABAergic and glycinergic synapses, provides expiratory inhibition that restrains the expiratory oscillator under resting condition and regulates the formation of abdominal expiratory activity during active expiration. By combining experimental and modelling approaches, we propose the organization and connections within the respiratory network that control the changes in the breathing pattern associated with elevated metabolic demand. ABSTRACT: The expiratory neurons of the Bötzinger complex (BötC) provide inhibitory inputs to the respiratory network, which, during eupnoea, are critically important for respiratory phase transition and duration control. Here, we investigated how the BötC neurons interact with the expiratory oscillator located in the parafacial respiratory group (pFRG) and control the abdominal activity during active expiration. Using the decerebrated, arterially perfused in situ preparations of juvenile rats, we recorded the activity of expiratory neurons and performed pharmacological manipulations of the BötC and pFRG during hypercapnia or after the exposure to short-term sustained hypoxia - conditions that generate active expiration. The experimental data were integrated in a mathematical model to gain new insights into the inhibitory connectome within the respiratory central pattern generator. Our results indicate that the BötC neurons may establish mutual connections with the pFRG, providing expiratory inhibition during the first stage of expiration and receiving excitatory inputs during late expiration. Moreover, we found that application of GABAergic and glycinergic antagonists in the BötC caused opposing effects on abdominal expiratory activity, suggesting complex inhibitory circuitry within the BötC. Using mathematical modelling, we propose that the BötC network organization and its interactions with the pFRG restrain abdominal activity under resting conditions and contribute to abdominal expiratory pattern formation during active expiration observed during hypercapnia or after the exposure to short-term sustained hypoxia.

Centrally acting adrenomedullin in the long-term potentiation of sympathetic vasoconstrictor activity induced by intermittent hypoxia in rats.

NEW FINDINGS: What is the central question of this study? Adrenomedullin in the rostral ventrolateral medulla (RVLM) increases sympathetic activity; given that adrenomedullin is released during hypoxia, what are the effects of its agonism and antagonism in the RVLM after chronic intermitent hypoxia (CIH) exposure? What is the main finding and its importance? CIH exposure sensitizes adrenomedullin-dependent mechanisms in the RVLM, supporting its role as a sympathoexcitatory neuromodulator. A novel mechanism was identified for the generation of sympathetic overdrive and hypertension associated with hypoxia, providing potential guidance on new therapeutic approaches for controlling sympathetic hyperactivity in diseases such as sleep apnoea and neurogenic hypertension. ABSTRACT: Adrenomedullin in the rostral ventrolateral medulla (RVLM) has been shown to increase sympathetic activity whereas the antagonism of its receptors inhibited this autonomic activity lowering blood pressure in conditions of hypertension. Given that hypoxia is a stimulant for releasing adrenomedullin, we hypothesized that the presence of this peptide in the RVLM associated with chronic intermittent hypoxia (CIH) would cause sympathetic overdrive. Juvenile male rats (50-55 g) submitted to CIH (6% oxygen every 9 min, 8 h day-1 for 10 days) were studied in an arterially perfused in situ preparation where sympathetic activity was recorded. In control rats (n = 6), exogenously applied adrenomedullin in the RVLM raised baseline sympathetic activity when combined with episodic activation of peripheral chemoreceptors (KCN 0.05%, 5 times every 5 min). This sympathoexcitatory response was markedly amplified in rats previously exposed to CIH (n = 6). The antagonism of adrenomedullin receptors in the RVLM caused a significant reduction in sympathetic activity in the CIH group (n = 7), but not in controls (n = 8). The transient reflex-evoked sympathoexcitatory response to peripheral chemoreceptor stimulation was not affected by either adrenomedullin or adrenomedullin receptor antagonism in the RVLM of control and CIH rats. Our findings indicate that CIH sensitizes the sympathoexcitatory networks within the RVLM to adrenomedullin, supporting its role as an excitatory neuromodulator when intermittent hypoxia is present. These data reveal novel state-dependent mechanistic insights into the generation of sympathetic overdrive and provide potential guidance on possible unique approaches for controlling sympathetic discharge in diseases such as sleep apnoea and neurogenic hypertension.

The effect of central growth hormone action on hypoxia ventilatory response in conscious mice.

Growth hormone (GH)-responsive neurons regulate several homeostatic behaviors including metabolism, energy balance, arousal, and stress response. Therefore, it is possible that GH-responsive neurons play a role in other responses such as CO2/H+-dependent breathing behaviors. Here, we investigated whether central GH receptor (GHR) modulates respiratory activity in conscious unrestrained mice. First, we detected clusters of GH-responsive neurons in the tyrosine hydroxylase-expressing cells in the rostroventrolateral medulla (C1 region) and within the locus coeruleus (LC). No significant expression was detected in phox2b-expressing cells in the retrotrapezoid nucleus. Whole body plethysmography revealed a reduction in the tachypneic response to hypoxia (FiO2 = 0.08) without changing baseline breathing and the hypercapnic ventilatory response. Contrary to the physiological findings, we did not find significant differences in the number of fos-activated cells in the nucleus of the solitary tract (NTS), C1, LC and paraventricular nucleus of the hypothalamus (PVH). Our finding suggests a possible secondary role of central GH action in the tachypneic response to hypoxia in conscious mice.

Short-Term Sustained Hypoxia Elevates Basal and Hypoxia-Induced Ventilation but Not the Carotid Body Chemoreceptor Activity in Rats.

Exposure to chronic sustained hypoxia (SH), as experienced in high altitudes, elicits an increase in ventilation, named ventilatory acclimatization to hypoxia (VAH). We previously showed that rats exposed to short-term (24 h) SH exhibit enhanced abdominal expiratory motor activity at rest, accompanied by augmented baseline sympathetic vasoconstrictor activity. In the present study, we investigated whether the respiratory and sympathetic changes elicited by short-term SH are accompanied by carotid body chemoreceptor sensitization. Juvenile male Holtzman rats (60-80 g) were exposed to SH (10% O2 for 24 h) or normoxia (control) to examine basal and hypoxic-induced ventilatory parameters in unanesthetized conditions, as well as the sensory response of carotid body chemoreceptors in artificially perfused in situ preparations. Under resting conditions (normoxia/normocapnia), SH rats (n = 12) exhibited higher baseline respiratory frequency, tidal volume, and minute ventilation compared to controls (n = 11, P < 0.05). SH group also showed greater hypoxia ventilatory response than control group (P < 0.05). The in situ preparations of SH rats (n = 8) exhibited augmented baseline expiratory and sympathetic activities under normocapnia, with additional bursts in abdominal and thoracic sympathetic nerves during late expiratory phase that were not seen in controls (n = 8, P < 0.05). Interestingly, basal and potassium cyanide-induced afferent activity of carotid sinus nerve (CSN) was similar between SH and control rats. Our findings indicate that the maintenance of elevated resting ventilation, baseline sympathetic overactivity, and enhanced ventilatory responses to hypoxia in rats exposed to 24 h of SH are not dependent on increased basal and sensorial activity of carotid body chemoreceptors.