Inhibition of the pontine Kölliker-Fuse nucleus reduces genioglossal activity elicited by stimulation of the retrotrapezoid chemoreceptor neurons

Highlights

• Stimulation of central chemoreflex elicits an increase in genioglossal EMG activity.

• Histological evidence for glutamatergic and GABAergic inputs to the KF region from RTN chemoreceptors.

• Inhibition in KF region abolishes the GG response due to increased RTN activity.

• KF integrates and modulates breathing during chemoreceptor activation.

Abstract

The Kölliker-Fuse (KF) region, located in the dorsolateral pons, projects to several brainstem areas involved in respiratory regulation, including the chemoreceptor neurons within the retrotrapezoid nucleus (RTN). Several lines of evidence indicate that the pontine KF region plays an important role in the control of the upper airways for the maintenance of appropriate airflow to and from the lungs. Specifically, we hypothesized that the KF region is involved in mediating the response of the hypoglossal motor activity to central respiratory chemoreflex activation and to stimulation of the chemoreceptor neurons within the RTN region. To test this hypothesis, we combined immunohistochemistry and physiological experiments. We found that in the KF, the majority of biotinylated dextran amine (BDA)-labeled axonal varicosities contained detectable levels of vesicular glutamate transporter-2 (VGLUT2), but few contained glutamic acid decarboxylase-67 (GAD67). The majority of the RTN neurons that were FluorGold (FG)-immunoreactive (i.e., projected to the KF) contained hypercapnia-induced Fos, but did not express tyrosine hydroxylase. In urethane-anesthetized sino-aortic denervated and vagotomized male Wistar rats, hypercapnia (10% CO₂) or N-methyl-d-aspartate (NMDA) injection (0.1 mM) in the RTN increased diaphragm (Dia_EMG) and genioglossus muscle (GG_EMG) activities and elicited abdominal (Abd_EMG) activity. Bilateral injection of muscimol (GABA-A agonist; 2 mM) into the KF region reduced the increase in Dia_EMG and GG_EMG produced by hypercapnia or NMDA into the RTN. Our data suggest that activation of chemoreceptor neurons in the RTN produces a significant increase in the genioglossus muscle activity and the excitatory pathway is dependent on the neurons located in the dorsolateral pontine KF region.

Introduction

For millennia, the functions of breathing are well quite linked with life. The mechanics of breathing start with muscle contractions, generating essential pressure to facilitate airflow into and out of the lungs, with its regulatory role of keeping blood in an ideal physiological condition. One important component of the respiratory cycle is the laryngeal muscles, which are involved in the control of the upper airway resistance (Horner, 2012). The laryngeal abductor muscle produces a dilation of the glottis during the inspiratory phase, to make airflow to the lungs easy (Brancatisano et al., 1991). On the other hand, the laryngeal adductor muscles increase the airway resistance, to control expiration during the postinspiratory phase of the respiratory cycle (Paton and Dutschmann, 2002). The respiratory function of the airways has clinical relevance in sleep disorders, such as obstructive sleep apnea (OSA) (Dempsey et al., 2010). Patients with OSA have reduced upper airway motor tone during sleep, rendering the airway vulnerable to collapse (Stettner et al., 2013).

Considering the coordination required of the breathing muscles, the ponto-medullary region in mammals features different types of elements (neurons and glia), which operate with the goal of keeping stable breathing patterns. It is well established that the dorsal pontine region of the parabrachial complex, including the Kölliker-Fuse nucleus (KF) and the external lateral and lateral crescent subnuclei of the parabrachial complex, receives intense inputs from the ventral respiratory column (VRC), and projects directly to respiratory motorneurons in the brainstem and spinal cord (Yokota et al., 2001, Dutschmann and Herbert, 2006, Yokota et al., 2011, Dutschmann and Dick, 2012, Yokota et al., 2015, Jones et al., 2015). The lateral aspects of the parabrachial nucleus and the KF region are also activated during hypoxia and hypercapnia, and bilateral blockade of those regions elicited a reduction in the ventilatory response to chemoreceptor activation (Mizusawa et al., 1995, Damasceno et al., 2014, Damasceno et al., 2015).

The rostral aspect of the ventrolateral medulla contains a network of active neurons, with chemoreceptor properties innervating the entire VRC, as well as the KF region, and it is involved in the breathing control elicited by hypercapnia and hypoxia (Smith et al., 1989, Rosin et al., 2006, Takakura et al., 2006). That region lies under the facial motor nucleus, and was named retrotrapezoid nucleus (RTN) (Smith et al., 1989, Mulkey et al., 2004a). Using opto- and pharmacogenetic experiments, strong evidence suggests that the RTN neurons are involved in several aspects of breathing, including inspiratory rate and amplitude, and are also capable of making active expiration (Marina et al., 2010, Pagliardini et al., 2011, Abbott et al., 2013, Huckstepp et al., 2015). In addition, RTN neurons have strong modulation under high levels of CO₂, and some of those neurons develop a preinspiratory discharge (Onimaru and Homma, 2003, Guyenet and Bayliss, 2015).

Based on the fact that the pontine KF region and the RTN chemoreceptor neurons are involved in breathing regulation, in the present study, we hypothesize a possible interaction between both regions in the control of respiratory muscle output, with special attention to genioglossus muscle activity, involved in preinspiratory activity. In support to our hypothesis, activation of the orexin B receptors at the level of the KF region modulates the preinspiratory hypoglossal motor activity in rats, suggesting that KF neurons are involved in genioglossal activity, and that modulation could presumably interact with the chemoreceptor neurons in the RTN (Dutschmann et al., 2007). Consistent with that possibility, we find that the increase in breathing output, elicited by central chemoreceptor stimulation or by RTN neuronal stimulation, is dependent on the integrity of the pontine KF region. In addition, using standard anatomical technique, we showed an excitatory pathway from the chemoreceptor neurons in the RTN to the KF region. Our findings could certainly suggest a possible future application for the treatment of hypoventilation in humans.