Understanding the rhythm of breathing: so near, yet so far

Abstract

Breathing is an essential behavior that presents a unique opportunity to understand how the nervous system functions normally, how it balances inherent robustness with a highly regulated lability, how it adapts to both rapidly and slowly changing conditions, and how particular dysfunctions result in disease. We focus on recent advancements related to two essential sites for respiratory rhythmogenesis: (a) the preBötzinger Complex (preBötC) as the site for the generation of inspiratory rhythm and (b) the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) as the site for the generation of active expiration.

Figures

Figure 1. Overview of the central pattern generator for breathing

Core rhythm generating circuits appear to have two distinct brainstem oscillators, the endogenously active preBötC (red box) driving inspiratory activity by projections to various premotor populations that in turn project to inspiratory muscles that pump air, e.g., diaphragm and external intercostals, and those that modulate airflow resistance, e.g., laryngeal and tongue muscles, and the conditionally active RTN/pFRG (blue box) that have a similar functional path to expiratory muscles. There are numerous influences: i) Neuromodulatory (left). Respiratory pattern is highly labile. When you go from quiet sitting to slow walking, your O2 consumption increases ~3-fold, and if your ventilation does not increase rapidly you probably will pass out within 100 meters. There is widespread agreement that peptides, serotonin, norepinephrine and other endogenous neuromodulators can affect rhythmogenesis (Garcia et al 2011). These actions are essential for normal regulation and may go awry in diseases affecting breathing. ii) Suprapontine inputs (top) related to volition and emotion. iii) Sensory inputs (right) essential for proper regulation of blood gases and for mechanical adjustments related to posture, body mechanics and likely metabolic efficiency. This review focuses on the core oscillators.

Figure 2. preBötC is essential for breathing in adult rats

Rapid silencing of AlstR-expressing preBötC SST neurons induces persistent (>45 min when mechanically ventilated) apnea in anesthetized or awake adult rats. Traces are plethysmographic recordings. AL administered intracerebrocisternally induces a gradual decline of frequency and tidal volume until apnea develops after several minutes. After ~60 min mechanical ventilation, rats resume spontaneous breathing. From (Tan et al 2008).

Figure 3. Inspiratory burst generation: role of dendrites and properties of Dbx1+ preBötC neurons

A and B, dendritic two-photon Ca2+ imaging and somatic patch clamp. A, preBötC neuron filled with fluorescent dye from somatic whole-cell recording. Regions of interest (ROIs) shown, which correspond to B. B, dendritic Ca2+ transients (arrowheads) precede somatic bursts and XII motor output. Asterisks indicate somatic spike-driven Ca2+ transients (Del Negro et al 2011). C and D, morphology (C) and physiology (D) of a Dbx1+ preBötC neuron. Drive potentials of ~25 mV amplitude and depolarization block of spiking indicative of ICAN activation during the inspiratory burst. E, transverse view of a mouse slice (ventral) showing two Dbx1+ neurons (right) and a Dbx1− neuron (left) recorded and biocytin reconstructed. All three were inspiratory neurons. Dbx1+ neurons are commissural (axons in red). IO is inferior olive.

Figure 4. Passive expiration transformed into active expiration

Anesthetized adult rat has active inspiration, reflected in diaphragm EMG (DIA), but passive expiration, reflected in tonic abdominal EMG. Photoactivation of lateral RTN/pFRG neurons transfected with ChR2 (gray) induces active expiration. Redrawn from (Pagliardini et al, 2010).

Figure 5. Genetic organization of brainstem respiratory regions

A. Schematized description of brainstem progenitor domains for 8 dorsal (dA1–dB4) and 5 ventral progenitor populations (left) based on their relative location within brainstem progenitor region. Partial list of transcription factors expressed at some point within progenitors (italics) or post-mitotic neurons within each domain (middle). Neurotransmitter(s) identity of neurons derived from each domain. Adapted from (Gray 2008) B. Cartoon showing partial migratory path of ventral medulla (left) and caudal pons (right) neurons in embryonic mouse brainstem. Colors correspond to domains in (A). Thick arrows correspond to populations important for breathing. Note dB2 population (light green) is present only in caudal pons. C. Cartoon of developmental origin and approximate anatomical locations of respiratory-related populations in sagittal plane within ventral medulla and caudal pons. Colors correspond to developmental progenitor domain from A. Legend describes transmitter released by these neurons. Within the ventral respiratory column, nearly all respiratory-related glutamatergic neurons are Dbx1-derived. Magenta box outlines the location of preBötC SST-expressing neurons. RTN/pFRG, in contrast, contains Dbx1, Atoh1, and Phox2b glutamatergic populations. Ventral medulla also contains a large number of dB1 derived glycinergic neurons. B. PreBötC neurons are derived from Dbx1-expressing progenitors. Four color confocal image showing coexpression of NK1R (magenta), SST2aR (green), SST (cyan), and β-gal (yellow) in P0 Dbx1 β-gal mouse (adapted from (Gray et al 2010). Arrows indicate coexpression of all 4 genes. Images to right show single channel expression. Red arrowhead indicates Dbx1-derived NK1R/SST2aR-expressing neuron that lacks SST. Yellow arrowheads indicate Dbx1 derived neurons lacking coexpression. Magenta arrowhead indicates NK1R expressing nucleus ambiguus neuron. E. preBötC Dbx1 neurons are glutamatergic. Image showing β-gal (magenta) expression within the majority of VGlut2 (green) expressing preBötC neurons (adapted from (Gray et al 2010)). Inset is enlarged from central square. F. preBötC contains glycinergic neurons. Three color confocal image showing Pax2 (red) and Sst (blue) immunoreactivity with intrinsic GFP from a P0 GlyT2-GFP transgenic mouse (Morgado-Valle et al 2010). Arrows show Pax2 co-localization with Sst or GFP but no GFP expression in SST neurons. Inset is enlarged from central square. Scale bars = 200 μm. D – dorsal, L – lateral. G. RTN/pFRG Phox2b neurons express Phox2b and Atoh1. (A) Magnification of ventral respiratory column from E16.5 Math1M1GFP/M1GFP hindbrain, as indicated by the black rectangle on inset model hindbrain (black ventral region is pFRG/RTN while yellow circle indicates preBötC), showing NK1R (red), Phox2b (blue), and Math1-EGFP (green) expression. NK1R labeled both the pFRG/RTN and 17 preBötC neurons. Magnified pFRG/RTN neurons from the caudal pole of VII (solid white rectangle in A) showing co-localization of Math1EGFP with Phox2b and NK1R. (B) shows the three markers merged. Further magnification from white boxes in B is shown to right of each panel in (B′). Image from (Rose et al 2009b).