Neural Circuitry Underlying Waking Up to Hypercapnia

Abstract

Obstructive sleep apnea is a sleep and breathing disorder, in which, patients suffer from cycles of atonia of airway dilator muscles during sleep, resulting in airway collapse, followed by brief arousals that help re-establish the airway patency. These repetitive arousals which can occur hundreds of times during the course of a night are the cause of the sleep-disruption, which in turn causes cognitive impairment as well as cardiovascular and metabolic morbidities. To prevent this potential outcome, it is important to target preventing the arousal from sleep while preserving or augmenting the increase in respiratory drive that reinitiates breathing, but will require understanding of the neural circuits that regulate the cortical and respiratory responses to apnea. The parabrachial nucleus (PB) is located in rostral pons. It receives chemosensory information from medullary nuclei that sense increase in CO2 (hypercapnia), decrease in O2 (hypoxia) and mechanosensory inputs from airway negative pressure during apneas. The PB area also exerts powerful control over cortical arousal and respiration, and therefore, is an excellent candidate for mediating the EEG arousal and restoration of the airway during sleep apneas. Using various genetic tools, we dissected the neuronal sub-types responsible for relaying the stimulus for cortical arousal to forebrain arousal circuits. The present review will focus on the circuitries that regulate waking-up from sleep in response to hypercapnia.

Keywords: arousal; calcitonin gene related peptide; hypercapnia; obstructive sleep apnea; parabrachial nucleus.

FIGURE 1

(A), Three main stimuli related to apnea converge on the parabrachial area: Increased CO₂ (Hypercapnia), Hypoxia and negative air-way pressure cause activation of both central and peripheral chemoreceptors whose signals are integrated in the nucleus of the solitary tract (NTS) and retrotrapezoid nucleus (RTN). The NTS and RTN activate neurons in the lateral parabrachial nucleus, major node in the brain stem that relay visceral sensory information to the forebrain areas. Mouse model of apnea: (B), shows the “repetitive CO2 arousal (RCA) protocol” where a mouse is recorded in the plethysmograph chamber for the EEG, EMG and breathing, while exposed to repeated bouts of CO2 (hypercapnia). Mice undergo spontaneous periods of sleep and wake, however, only trials where the mouse is in NREM sleep for at least 30 s prior to onset of the CO2 are used to examine arousal. During these trials, the arousals are judged by EEG arousal (loss of delta waves and appearance of low voltage fast EEG), which is usually accompanied by EMG activation. Scale = 45 s (C), is a schematic of the plethysmography chamber used to model apnea in mice, while they are exposed to CO2 and recorded for EEG/EMG and breathing responses, with and without laser light that is transmitted through the pre-implanted optical glass fibers. [Adapted and modified from Kaur et al. (2013)]. Kaur et al. (2013), is published under Creative Commons Attribution-Non-commercial-Share License, and therefore no permission is required reproducing this modified version.

FIGURE 2

Testing the role of glutamatergic signaling in hypercapnia induced arousal: (A,B), are the two representative trials from a control mouse (A), where cortcial EEG arousal in response to hypercapnia occurs in 15 s after onset of CO2, while the mouse with deletion of Vglut2 gene in the LPB (B), fails to wake wake up to hypercapnia. (C), Photomicrograph of the Nissl-stained coronal section of the mouse brain, showing different sub divisions of the parabrachial (PB) nucleus, Cre-immunoreactivity (brown) against a Nissl-stained background (blue) in the neurons in the lateral parabrachial (LPB) region after injection of AAV-Cre in Vglut2 ^flox/flox mice and last panel shows the shows a photomicrograph of a brain section immunostained for Neu-N, a neuronal marker after bilateral injection of AAV-DTA killed Vglut2+ neurons into the LPB. (D), Show graphs of the latency of arousal during and after a hypercapnic stimulus of 30 s in mice injected bilaterally with AAV-DTA (green) compared to the control (black, gray, and striped green) and LPB group from which Vglut2 was deleted in the LPB including the PBel (red). scp – superior cerebellar peduncle; dl – dorso-lateral; cl – centro-lateral; el – external lateral; vl – ventrolateral PB subnucleus; MPB – medial and MPB-ext – medial external-lateral parabrachial nucleus; KF – Kolliker Fuse; vsct – ventral cerebro-spinal tract; Scale = 100 μm. ^∗∗represents p < 0.01 compared to the control group (AAV-GFP) and ^#p < 0.05, compared to the AAVCreWT group. [Adapted and modified from Kaur et al. (2013)]. Kaur et al. (2013), is published under Creative Commons Attribution-Non-commercial-Share License, and therefore no permission is required reproducing this modified version.

FIGURE 3

Selective activation of the PBel^CGRP neurons, using chemogenetics (A) and optogenetics (B): The (A1,B1) represent the strategy used to first express either hM3Dq or Channel rhodopsin (ChR2) in the PBel^CGRP neurons, using the CGRP-CreER mice. Chemogenetic activation of PBel^CGRP neurons, significantly increased wakefulness for 2 h post injection of the designer ligand (CNO) that binds hM3Dq (A2). Optogenetically driving the PBel^CGRP neurons also produced very short latency arousals both at 10 and 20 Hz, trials shown in the figure are from stimulation at 20 Hz (B2). (^∗∗∗p < 0.0001; ^∗∗p < 0.001; 1-way or repeated measures ANOVA followed by Holm-Sidak for multiple comparison). [Adapted and modified from Kaur et al. (2017)]. Kaur et al. (2017) is published in a Cell Press journal “Neuron,” and no permissions needed to reproduce the modified versions of the published figure.

FIGURE 4

Selective silencing of the PBel^CGRP neurons and the terminal fields using Optogenetics: (A), is the representative recording of EEG, EMG and respiration during the 10% CO2 stimulus in a CGRP-CreER mouse with laser photo-inhibition of PBel^CGRP neurons, the mouse in this representative trial did not wake up, and had similar increase in the ventilatory-drive in response to CO2 as the control with Laser-OFF. (B), Left panel, are the graphs comparing the respiratory rate (RR) and the tidal volume (V_T) (right panel) for 3 breaths before CO2 (Pre CO2) and for 3 breaths during CO2 just prior to waking-up in Laser-OFF and then at the same time point in trials in the same animal with Laser-ON (in which the animals did not awaken). (C), Compares the effects of PBel^CGRP soma inhibition to that of PBel^CGRP terminals field inhibition (A), latency of arousal mean (SEM) during laser (593 nm) induced inhibition of the PBelCGRP neurons is compared with inhibition of the terminal fields in the BF, CeA, and LH. (B) Survival of sleep curves during and after a hypercapnic stimulus shown with and without laser. (^∗∗∗p < 0.0001; ^∗∗p < 0.001; 1-way or repeated measures ANOVA followed by Holm-Sidak for multiple comparison). [Adapted and modified from Kaur et al. (2017)]. Kaur et al. (2017) is published in a Cell Press journal “Neuron,” and no permissions needed to reproduce the modified versions of the published figure.

FIGURE 5

Neural circuitry regulating cortical arousals and respiratory efforts to hypercapnia: PBel^CGRP neurons receive CO2, O2, and airway mechanoreceptor inputs, as well as inputs via medullary nuclei, NTS (nucleus of solitary tract) and RTN (Retrotrapezoid nucleus). The PBel^CGRP neurons in turn project extensively to the lateral hypothalamus (LH), basal forebrain (BF), and central nucleus of amygdala (CeA). Based on our findings, PBel^CGRP neurons mainly cause cortical arousal by projections to the BF, which has potent waking effects, followed by CeA and LH. PBel^CGRP neurons did not contribute to respiratory component of apnea, as its inhibition did not diminish respiratory drive to CO2. The glutamatergic FoxP2 neurons (PB^FoxP2) in the lateral crescent (PBlc) and Kolliker Fuse (KF) have descending projections to the hypoglossal nucleus (tongue), and to the retroambiguus (larynx) and phrenic motor nucleus (Diaphragm); they also project extensively to the ventro-lateral and commissural (Comm) subdivisions of NTS. These projections of the PB^FoxP2 neurons may influence the respiratory efforts during apneas, either by direct projections to the motor neurons in medulla and spinal cord or by indirect projections to the premotor neurons in the ventro-lateral medulla, also known as the rostral Ventral Respiratory Group (rVRG).