Normal breathing requires preBötzinger complex neurokinin-1 receptor-expressing neurons

Abstract

The normal breathing rhythm in mammals is hypothesized to be generated by neurokinin-1 receptor (NK1R)-expressing neurons in the preBötzinger complex (preBötC), a medullary region proposed to contain the kernel of the circuits generating respiration. If this hypothesis is correct, then complete destruction of preBötC NK1R neurons should severely perturb and perhaps even fatally arrest breathing. Here we show that specific and near complete bilateral (but not unilateral) destruction of preBötC NK1R neurons results in both an ataxic breathing pattern with markedly altered blood gases and pH, and pathological responses to challenges such as hyperoxia, hypoxia and anesthesia. Thus, these ~600 neurons seem necessary for the generation of normal breathing in rats.Breathing is an exceptionally reliable and continuous mammalian behavior that regulates blood gases and pH at rest and in response to diverse challenges such as exercise, sleep or altitude. The rhythm underlying breathing is postulated to depend critically on neurons in the preBötC1 , 2. Two recent developments allowed us to determine in awake adult rats whether this critical regulatory behavior would be affected by lesions in the preBötC. First, the extent of the preBötC can be anatomically defined by the sub-population of propriobulbar respiratory neurons within the ventrolateral respiratory column expressing NK1R3 -5 . Second, NK1R neurons can be specifically lesioned by substance P conjugated to saporin (SP-SAP) 6 , an effect that takes several days, allowing for complete recovery from surgery6. ResultsSP-SAP was effective in eliminating preBötC NK1R neurons in adult rats. Injections of 0.1-0.2 pmol of SP-SAP or 0.3 pmol each of unconjugated saporin and SP were made in the preBötC. Unilateral SP-SAP injection transiently produced sighs (large inspiratory efforts followed by prolonged expiration), consistent with the effects of injection of SP alone into the pre-BötC in vitro 7 and in vivo (P.A.G., D.R.M. and J.L.F., unpublished observations). All rats returned to normal behavior upon recovery from surgery. Two to eighteen days after injection, rats were perfused and their medullas were stained for NK1R immunoreactivity (n = 20 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript lesion extent was estimated by counting NK1R immunopositive neuronal soma in the rostral medulla inside a circle of 600-μm diameter approximating the preBötC (ventrolateral to the nucleus ambiguus) and in a rectangle (1600 × 1070 μm) outside this circle (Fig. 1, inset). Counts were estimated from four transverse sections beginning within 100 μm of the rostral border of the lateral reticular nucleus and spanning the preBötC 3,4 containing approximately 12-15% of the total preBötC volume. Uninjected controls (n = 4) had 35 ± 5.1 (mean ± s.e.m., per side) NK1R neurons within the preBötC (~600 preBötC NK1R neurons total, which we estimate represent less than 10% of all preBötC neurons) and 82 ± 9 NK1R neurons outside the preBötC. Most of the latter were i...

Cumulative lesioning of respiratory interneurons disrupts and precludes motor rhythms in vitro

“…However, such techniques do not allow cellular-level quantitative analyses of lesion effects on behavior. Similarly, the toxin saporin causes graded cell destruction over several days, but it is impossible to keep a running tally of cell destruction in vivo while monitoring behavioral changes (5,6). We developed the present system for slices that retain behaviorally relevant function and expose CPG interneurons.…”

“…The preBötzinger Complex (preBötC) of the medulla putatively contains the inspiratory CPG (3,4). Over several days in vivo, saporinmediated destruction of preBötC neurons that express neurokinin-1 receptors (NK1Rs) causes sleep-disordered breathing and fatal respiratory pathology (5,6). Similarly, acute cell-silencing of a subset of the same NK1R-expressing population of preBötC neurons stops spontaneous breathing in awake adult rats (7).…”

Large lesions in the pre-Bötzinger complex area eliminate eupneic respiratory rhythm in awake goats

“…Gray et al (6) demonstrated that Ͼ80% bilateral destruction of NK1R-expressing neurons was necessary to affect breathing and blood gases in awake rats. We previously demonstrated that 29% bilateral destruction of NK1R-expressing pre-BötzC area neurons in awake goats had minimal effect on arterial blood gases but caused transient alterations in breathing rhythm and/or pattern (26), and we reported here that 70% destruction of the pre-BötzC eliminates eupneic breathing.…”

“…In addition, in vivo studies in anesthetized or decerebrate cats or rats demonstrate that lesioning of the pre-BötzC results in transient (24) or irreversible (7,10,18) elimination of eupneic respiratory activity. Further demonstrating the importance of the pre-BötzC in control of breathing was a study showing that Ͼ80% destruction of neurokinin-1 receptor (NK1R)-expressing neurons in the pre-BötzC resulted in an ataxic breathing pattern and hypoventilation in awake rats (6).…”

A bilateral cortico-bulbar network associated with breath holding in humans, determined by functional magnetic resonance imaging

“…Direct parallels of breath holding do not exist in other human behaviours but imaging studies of response inhibition during Go/NoGo and Stop-signal tasks (Aron and Poldrack, 2006;Liddle et al, 2001;Watanabe et al, 2002) and those related to inhibiting saccadic eye reflexes (Jueptner et al, 1996), highlight the dorsolateral prefrontal cortex, basal ganglia and thalamus as having a role in inhibiting behaviours. Within the brainstem, animal models have shown that stimulation of pontine respiratory neurones can lead to apnoea, hypernoea or apneusis (Chamberlin, 2004;Fung and St John, 1994;Mutolo et al, 1998), while ablation of the preBötzinger complex (preBötC), a small region of the ventrolateral medulla, can lead to central sleep apnoea and ataxic breathing during wakefulness (Gray et al, 2001;McKay et al, 2005) We hypothesised that breath holding would be associated with an inhibitory network of subcortical structures including the pons. We were aware that our subjects would experience secondary effects, such as interoceptive awareness and an increasing desire for, and an anticipation of, the subsequent breath; therefore, we also hypothesised activity within areas associated with respiratory awareness and air hunger, such as the insula and cingulate cortices (Banzett et al, 2000b;Evans et al, 2002;Peiffer et al, 2001).…”