Chemogenetic modulation of the parafacial respiratory group influences the recruitment of abdominal activity during REM sleep

Abstract

Current theories on respiratory control postulate that the respiratory rhythm is generated by oscillatory networks in the medulla: preBötzinger complex (preBötC) is the master oscillator responsible for generating inspiration, while parafacial respiratory group (pFRG) drives active expiration through recruitment of expiratory abdominal (ABD) muscle activity. Research addressing the role of pFRG in ventilation and rhythm generation across sleep states is limited. We recently reported the occurrence of ABD recruitment occurring despite the induction of muscle paralysis during REM sleep. This ABD recruitment was associated with increased tidal volume and regularization of the respiratory period in rats. As pFRG generates active expiration through the engagement of ABD muscles, we hypothesized that the expiratory oscillator is also responsible for the ABD recruitment observed during REM sleep. To test this hypothesis, we inhibited and activated pFRG using chemogenetics (i.e. designer receptors exclusively activated by designer drugs) while recording EEG and respiratory muscle EMG activities across sleep–wake cycles in male Sprague–Dawley rats. Our results suggest that inhibition of pFRG reduced the number of REM events expressing ABD recruitment, in addition to the intensity and prevalence of these events. Conversely, activation of pFRG resulted in an increase in the number of REM events in which ABD recruitment was observed, as well as the intensity and prevalence of ABD recruitment. Interestingly, modulation of pFRG activity did not affect ABD recruitment during NREM sleep or wakefulness. These results suggest that the occurrence of ABD recruitment during sleep is dependent on pFRG activity and is state dependent.

Mapping of the excitatory, inhibitory, and modulatory afferent projections to the anatomically defined active expiratory oscillator in adult male rats

“…The lateral parafacial region (pF L ; which encompasses the parafacial respiratory group, pFRG), is a conditional oscillator that drives active expiration when metabolic or respiratory demands are high, as occurs during exercise, sojourns to altitude (hypoxia) or with elevations in arterial CO 2 (hypercapnia) (de Britto & Moraes, 2017; Del Negro, Funk, & Feldman, 2018; Huckstepp, Cardoza, Henderson, & Feldman, 2015; Janczewski & Feldman, 2006; Janczewski, Onimaru, Homma, & Feldman, 2002; Pagliardini et al, 2011; Pisanski, Ding, Koch, & Pagliardini, 2019). During restful breathing and under anesthesia, late expiratory neurons in pF L appear to be silent, but when disinhibited or activated, they trigger late‐expiratory activity in abdominal, internal intercostal and upper airway musculature (de Britto & Moraes, 2017; Huckstepp et al, 2015; Janczewski & Feldman, 2006; Pagliardini et al, 2011).…”

“…During restful breathing and under anesthesia, late expiratory neurons in pF L appear to be silent, but when disinhibited or activated, they trigger late‐expiratory activity in abdominal, internal intercostal and upper airway musculature (de Britto & Moraes, 2017; Huckstepp et al, 2015; Janczewski & Feldman, 2006; Pagliardini et al, 2011). The recruitment of expiratory muscles facilitates air outflow, decreases functional residual capacity, and enhances the tidal volume of the following breath (Del Negro et al, 2018; Pisanski et al, 2019; Pisanski & Pagliardini, 2019).…”

“…The contribution of active expiration to ventilation changes with postnatal development and arousal state (Andrews & Pagliardini, 2015; Leirao, Silva Jr., Gargaglioni, & da Silva, 2018; Pisanski et al, 2019; Saini & Pagliardini, 2017). Our previous work has shown that throughout postnatal development active expiration is recruited during both nonREM (nonrapid eye movement) and REM sleep epochs.…”

“…Our previous work has shown that throughout postnatal development active expiration is recruited during both nonREM (nonrapid eye movement) and REM sleep epochs. However, it is only during REM sleep that this activity is associated with an increase in respiratory stability, inspiratory diaphragm (DIA) EMG activity, tidal volume, and minute ventilation (Andrews & Pagliardini, 2015; Pisanski et al, 2019).…”

“…This is due to the fact that currently the phenotype of adult pF L neurons, and the identification of their distinct anatomical and electrophysiological characteristics are still unknown, and therefore it is not currently possible to distinguish these neurons (and their overall extension) from adjacent neurons with distinct physiological function. Based on this lack of knowledge, we opted to address our research question by using previous stereotaxic coordinates (and relative histological verification) to target the pF L region that generated active expiration with release of inhibition, optogenetic and chemogenetic modulation, and cholinergic modulation in previous investigations (Boutin et al, 2017; Huckstepp et al, 2015, 2016; Pagliardini et al, 2011; Pisanski et al, 2019). Thus, the objectives of the present study were to: (a) identify brainstem regions that directly project to the putative location of the pF L expiratory oscillator using a retrograde viral approach in which herpes simplex viral particles (HSV; HSV/hEF1α‐EYFP‐IRES‐CRE; #RN411, Massachusetts Institute of Technology ‐MIT, Cambridge, USA) expressing a fluorescent reporter (enhanced yellow fluorescent protein, eYFP, driven by the human elongation factor 1α, hEF1α) was injected unilaterally into the pF L of adult male rats; and (b) define the neurochemical phenotype of these projections by combining immunofluorescent labeling for eYFP with (a) immunofluorescent labeling of somatostatin (SST), choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), paired Like Homeobox 2 (PHOX2B) and 5‐hydroxytryptamine (5HT); or (b) the RNAScope in situ hybridization assay to identify markers for glutamatergic (Vesicular Glutamate Transporter, Vglut2 ), GABAergic (Glutamate Decarboxylase 1, Gad1 ), and Glycinergic (Glycine Transporter 2, Glyt2 ) neurons.…”