Neural mechanisms underlying respiratory regulation within the preBötzinger complex of the rabbit

https://doi.org/10.1016/j.resp.2021.103736Get rights and content

Highlights

  • An overview on the role of neuroactive agents within the rabbit preBötC is provided.

  • Excitatory and inhibitory amino acids provide important contributions to respiratory pattern formation.

  • Neurokinins, somatostatin and serotonin induce excitatory respiratory effects.

  • preBötC plays a key role in the genesis of μ-opioid receptor-induced disorders.

  • Comparative studies may contribute to a better understanding of the respiratory CPG.

Abstract

The preBötzinger complex (preBötC) is a medullary area essential for normal breathing and widely recognized as necessary and sufficient to generate the inspiratory phase of respiration. It has been studied mainly in rodents. Here we report the main results of our studies revealing the characteristics of the rabbit preBötC identified by means of neuronal recordings, D,L-homocysteic acid microinjections and histological controls. A crucial role in the respiratory rhythmogenesis within this neural substrate is played by excitatory amino acids, but also GABA and glycine display important contributions. Increases in respiratory frequency are induced by microinjections of neurokinins, somatostatin as well by serotonin (5-HT) through an action on 5-HT1A and 5-HT3 receptors or the disinhibition of a GABAergic circuit. Respiratory depression is observed in response to microinjections of the μ-opioid receptor agonist DAMGO. Our results show similarities and differences with the rodent preBötC and emphasize the importance of comparative studies on the mechanisms underlying respiratory rhythmogenesis in different animal species.

Introduction

One of the most intriguing problems in neuroscience concerns the understanding of how the respiratory activity is generated and controlled. Early studies determined the location and role of respiratory-related central neural structures through lesioning, stimulation, anatomical mapping, and neuronal recordings (review by Von Euler, 1986, 1997). Subsequent studies using pharmacological and electrophysiological tools as well as genetics and molecular biology identified discrete neural substrates that operate within the ponto-medullary respiratory network (Smith et al., 2009, 2013; Pagliardini et al., 2011; Ramirez et al., 2012; Dhingra et al., 2019, 2020; Ashhad and Feldman, 2020; for review see Alheid et al., 2004; Dutschmann and Dick, 2012; Feldman et al., 2013; Jones and Dutschmann, 2016; Del Negro et al., 2018).

The motor pattern during normal breathing is generally considered to consist of three phases: inspiration, postinspiration or expiratory phase 1 and expiratory phase 2 (active expiration). Respiratory rhythmogenesis in adult mammals results from synaptic interactions between neurons located in the ventral respiratory column (VRC) in the medulla oblongata (Richter et al., 1986; Von Euler, 1986; Bianchi et al., 1995; Haji et al., 2000; McCrimmon et al., 2000; Alheid et al., 2002; Feldman and Del Negro, 2006; Alheid and McCrimmon, 2008; Doi and Ramirez, 2008; Del Negro et al., 2018). A subregion critical for inspiratory rhythm generation, the preBötzinger complex (preBötC), was initially identified in vitro in neonatal rats (Feldman et al., 1990; Smith et al., 1991) and has since been described in vivo in several animal species, including humans (e.g. Smith et al., 1991; Johnson et al., 1994, 2001; Schwarzacher et al., 1995, 2011; Ramirez et al., 1998). Other rhythmogenic circuits in the VRC may contribute to respiratory pattern formation: the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) was initially described as the main location of preinspiratory neurons (Onimaru and Homma, 1992, 2003), but has more recently been suggested as an expiratory oscillator contributing to active expiration (Janczewski and Feldman, 2006; Abdala et al., 2009; Abbott et al., 2011). Other components of the RTN/pFRG have also been characterized as responsible for central CO2 chemosensitivity (Mulkey et al., 2004; Guyenet, 2012; Guyenet et al., 2012; Wang et al., 2013; Del Negro et al., 2018). More recently, evidence has been provided in the mouse that a medullary region, named Postinspiratory Complex (PiCo), is characterized by rhythm-generating properties and is necessary and sufficient to generate postinspiratory activity (Anderson et al., 2016). A “triple oscillator model” has been proposed in which inspiration, postinspiration and active expiration are generated by three distinct oscillators, i.e. the preBötC, the PiCo and the pFRG (Anderson and Ramirez, 2017; Del Negro et al., 2018). In rats a corresponding area was identified in the intermediate reticular nucleus, however, inhibition of this area had no effect on inspiratory duration (Toor et al., 2019). Toor et al. suggested that these neurons were not involved in the inspiratory off-switch mechanism of the respiratory rhythm generator but rather serve as premotor neurons to motoneurons controlling airway patency and swallow. Similarly, local field potentials did not show highly synchronized activity in the intermediate reticular nucleus during the phase switch from inspiration to postinspiration (Dhingra et al., 2020), which supports the notion that the area does not contribute to respiratory rhythm generation.

Other regions with a respiratory function have been described. They include the dorsal respiratory group, largely corresponding to the ventrolateral nucleus of the solitary tract (NTS), and portions of the VRC such as the expiratory Bötzinger complex (BötC), the inspiratory portion of the VRG and the caudal expiratory component of the VRG. Respiration-related neurons are also present in the dorsolateral pons at the level of the parabrachial (PB) complex and Kölliker-Fuse (KF) nuclei. The main respiratory function of these nuclei is the regulation of the inspiratory-expiratory phase transition and the dynamic control of upper airway patency during the respiratory cycle (see e.g. Dutschmann and Dick, 2012; Dhingra et al., 2017; Ramirez and Baertsch, 2018). Recent studies in rodents have demonstrated that preBötC somatostatin (SST)-expressing glutamatergic neurons and neurons that express the glycine transporter GlyT2 extensively project to multiple brainstem regions involved in the control of breathing (Tan et al., 2010; Yang and Feldman, 2018). Interestingly, many brainstem regions, including the contralateral preBötC, the BötC, the PB/KF, the RTN/pFRG and the NTS, have reciprocal connections with excitatory and inhibitory preBötC neurons (Yang et al., 2020). In addition, suprapontine regions such as the superior colliculus, the dorsomedial and lateral hypothalamus, and the zona incerta also have similar reciprocal projections that may represent potential direct pathways for volitional, emotional and physiological control of breathing (Yang et al., 2020).

Despite substantial advances in the understanding of the anatomical and neurophysiological basis of respiratory rhythm and pattern generation, the underlying neural mechanisms have not yet been exhaustively defined. Since breathing is a vital function, it seems plausible that the respiratory central pattern generator (CPG) does not rely on a single neural substrate, but engages distributed neuronal populations within the different brainstem respiratory compartments and is characterized by a great deal of redundancy and degeneracy (see e.g. Von Euler, 1997; Mutolo et al., 2002; Smith et al., 2007; Jones and Dutschmann, 2016; Dhingra et al., 2019, 2020).

Our research activity has been devoted, to a large extent, to investigate the rostral VRC in the rabbit and in particular the preBötC region. Although the bulk of the basic knowledge of the mammalian respiratory network derives from experiments on cats and rodents, rabbits have also been widely used in studies of the control of breathing and of the localization of respiration-related regions (Gromysz and Karczewski, 1981; Yamamoto and Lagercrantz, 1985; Jiang and Shen, 1991; Stucke et al., 2015; for reviews see Von Euler, 1986; Bianchi et al., 1995; Hilaire and Duron, 1999). We used several criteria to identify the preBötC in the rabbit and its analogy with the preBötC described in other animal species. Extracellular recordings demonstrated predominantly expiratory neurons with prevailing augmenting discharge patterns in the BötC, inspiratory neurons with prevailing augmenting discharge patterns in the rostral VRG as well as a mix of different types of respiration-related neurons within a subregion located between the BötC and the rostral VRG, corresponding to the preBötC. This region contains expiratory neurons with augmenting discharge patterns, expiratory neurons with decrementing discharge patterns or postinspiratory neurons, inspiratory neurons with augmenting discharge patterns, and phase-spanning neurons. Some phase-spanning neurons started firing during the expiratory phase (expiratory-inspiratory) and reached a maximum rate during inspiration, others started firing during inspiration (inspiratory-expiratory) and reached a firing peak at the transition from inspiration to expiration (Fig. 1A; see e.g. Mutolo et al., 2002; Bongianni et al., 2008, 2010). These neuronal activities matched the neuronal discharge patterns described in the preBötC in other species (e.g. Connelly et al., 1992; Rekling and Feldman, 1998; Schwarzacher et al., 1995; St Jacques and St John, 1999; Sun et al., 1998).

The location of the preBötC was identified through tachypneic responses to microinjections of D,L-homocysteic acid (DLH; Fig. 1B). Postmortem histology confirmed the location in the rabbit, which is ventro-medial to the rostral portion of the nucleus ambiguus (Fig. 1C and Fig. 2D; see Mutolo et al., 2002, 2005; Bongianni et al., 2008, 2010; Iovino et al., 2019; Cinelli et al., 2020). Experiments were performed on α-chloralose-urethane anaesthetized, vagotomized, paralyzed and artificially ventilated rabbits. Bilateral microinjections were performed into the VRG regions as defined by neuronal recordings. In each experiment, recordings of neuronal activity preceded drug microinjections. To restrict the spread of the injectate and thereby the number of neurons affected, relatively small volumes (30–50 nl) of drugs were bilaterally injected. Antagonist concentrations were always supramaximal. Agonist concentrations were selected in preliminary trials and were just above the minimum effective concentration capable to produce obvious and consistent effects. All drug concentrations were in the same range as those used in in vivo preparations in previous studies. Respiratory variables, i.e. respiratory frequency (breaths/min), the inspiratory (TI) and expiratory (TE) duration, as well as the peak amplitude of the rectified, integrated phrenic nerve activity (measured in arbitrary units, normalized to control), were measured in the period immediately preceding each trial, at the time when the maximum response to drug microinjections occurred and during the recovery period (see Mutolo et al., 2002, 2005; Bongianni et al., 2008, 2010; Pantaleo et al., 2011; Iovino et al., 2019; Cinelli et al., 2020). The respiratory effects observed in response to microinjections of several neuroactive agents into this region allowed us to characterize the rabbit preBötC. We believe that a better understanding of the mammalian respiratory CPG could be enhanced by comparative studies. However, we must take into account that previous results in rodents have been obtained from much reduced preparations, largely in vitro slice preparations, and this might lead to conclusions very different from those that can be drawn from results achieved under more intact respiratory network conditions in in vivo or in situ perfused brainstem preparations.

Section snippets

Glutamatergic mechanisms

Several lines of evidence indicate that glutamatergic transmission is essential for respiratory rhythmogenesis within the preBötC (Greer et al., 1991; Wallen-Mackenzie et al., 2006, 2010; Cook-Snyder et al., 2019; for review see Del Negro et al., 2018; Ramirez et al., 2016; Ramirez and Baertsch, 2018). Different hypotheses have been suggested to explain the mechanism underlying preBötC rhythmogenesis, from models based on pacemaker neurons to simple circuits dependent on inhibition (for review

GABAergic and glycinergic mechanisms

The role of inhibitory neurotransmission in the generation of the breathing pattern was investigated by blocking GABAA, GABAB and glycine receptors within the preBötC (Bongianni et al., 2010; see also Iovino et al., 2019). Bicuculline microinjections caused a pattern of breathing characterized by an overall decrease in respiratory frequency and the presence of two alternating different levels of peak phrenic activity (Fig. 3A and B). In contrast, the blockade of GABAB receptors did not alter

Modulatory role of some neuroactive agents

Neuromodulation involved in the process of respiratory rhythm and pattern generation is very complex and comprises several neuromodulators and different areas of the central nervous system (see e.g. Doi and Ramirez, 2008; Ramirez et al., 2016; Ramirez and Baertsch, 2018). Neuromodulators are implicated in the regulation of frequency and amplitude of respiratory activity and in the adaptive control of the respiratory network during different breathing behaviours.

Concluding remarks

The preBötC is a neural network containing a heterogeneous neuronal population crucial for respiratory rhythm generation and pattern formation. Much remains to be clearly assessed about the role of different subclasses of preBötC neurons and related microcircuits. This region is characterized by the presence of multiple neuronal receptors subserving the integration of many inputs to generate and modify the breathing behaviour. The results reported in this short review provide an overview of the

Authors contributions

All Authors prepared figures, drafted manuscript, edited and revised manuscript and approved final version of the manuscript.

Acknowledgements

This study was supported by grants from the University of Florence and from the Ente Cassa di Risparmio Firenze, Italy.

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