Locus Coeruleus as a vigilance centre for active inspiration and expiration in rats

Abstract

At rest, inspiration is an active process while expiration is passive. However, high chemical drive (hypercapnia or hypoxia) activates central and peripheral chemoreceptors triggering reflex increases in inspiration and active expiration. The Locus Coeruleus contains noradrenergic neurons (A6 neurons) that increase their firing frequency when exposed to hypercapnia and hypoxia. Using recently developed neuronal hyperpolarising technology in conscious rats, we tested the hypothesis that A6 neurons are a part of a vigilance centre for controlling breathing under high chemical drive and that this includes recruitment of active inspiration and expiration in readiness for flight or fight. Pharmacogenetic inhibition of A6 neurons was without effect on resting and on peripheral chemoreceptors-evoked inspiratory, expiratory and ventilatory responses. On the other hand, the number of sighs evoked by systemic hypoxia was reduced. In the absence of peripheral chemoreceptors, inhibition of A6 neurons during hypercapnia did not affect sighing, but reduced both the magnitude and incidence of active expiration, and the frequency and amplitude of inspiration. These changes reduced pulmonary ventilation. Our data indicated that A6 neurons exert a CO₂-dependent modulation of expiratory drive. The data also demonstrate that A6 neurons contribute to the CO₂-evoked increases in the inspiratory motor output and hypoxia-evoked sighing.

Figure 1

Selective and functional expression of AlstR in rat A6 neurons. Fifteen days after LC injection of PRSx8-AlstR-GFP-LVV, there is robust selective expression in noradrenergic neuronal somata and processes. GFP expression (green), TH immunoreactivity (red), and overlaid images (right). 4^thV: fourth ventricle. Scale bar: 50 µm. (B) One neuron juxtacellular labelled in vivo with biocytin, exhibiting GFP and TH immunofluorescence (noradrenergic LC neuron). Scale bar: 20 µm. (C) Instantaneous firing frequency (bin width 10 ms) extracellularly recorded from the same labelled A6 neuron during baseline condition and in response to activation of peripheral chemoreceptors using KCN. This neuron also increased its firing frequency to 7% CO₂. Intracerebroventricular application of Alst rapidly and reversibly abolished its firing frequency.

Figure 2

Acute inhibition of A6 neurons and effect on the baseline ventilatory, inspiratory and expiratory parameters of conscious adult rats. (A) Raw and integrated (∫) records of Dia_EMG and Abd_EMG activities and barometric respiratory movements, as well as fR, V_T and V_E data from one rat, in which the LC was transduced with PRSx8-AlstR-GFP-LVV before and after Alst application into the lateral ventricle. Note the absence of changes in baseline inspiratory and expiratory activities, in the ventilatory parameters, as well as in the number of sighs (red lines) after acute inhibition of A6 neurons. Summary of data showing the changes in the fR (B), V_T (C), VE (D) and in the number of sighs (E) after application of Alst.

Figure 3

Acute inhibition of A6 neurons and effect on the inspiratory and expiratory responses to activation of peripheral chemoreceptors of conscious adult rats. (A) Raw and integrated (∫) records of Dia_EMG and Abd_EMG activities from one representative rat in which the LC was transduced with PRSx8-AlstR-GFP-LVV. The inspiratory and expiratory responses to peripheral chemoreceptor activation using KCN, before and after Alst application into lateral ventricle, are shown. Magnification of baseline and reflex inspiratory and expiratory responses from the same rat before (Bi) and after (Bii) Alst application. Note the absence of changes in the peripheral chemoreflex-induced inspiratory and expiratory responses after acute inhibition of A6 neurons. Summary of data showing the changes in the reflex responses of fR (C), Dia_EMG amplitude (D) and Abd_EMG (E) after application of Alst.

Figure 4

Acute inhibition of A6 neurons and effect on the inspiratory and ventilatory responses to systemic hypoxic hypoxia of conscious adult rats. (A) Raw and integrated (∫) records of Dia_EMG activity from a rat in which the LC was transduced with PRSx8-AlstR-GFP-LVV, illustrating the changes in inspiration and in the number of sighs (red lines) induced by systemic hypoxic hypoxia (7% O₂) before and after Alst application into lateral ventricle. (B) Magnification of baseline and reflex inspiratory and ventilatory (barometric respiratory movements) responses from the same rat before and after Alst application. Note the absence of changes in the hypoxia-induced inspiratory and ventilatory responses, but the significant reduction in the number of sighs, after acute inhibition of A6 neurons.

Figure 5

Acute inhibition of A6 neurons and effect on the respiratory responses to systemic hypoxic hypoxia of conscious adult rats. Summary of data showing the changes in the responses of the number of sighs (A), sigh amplitude (B), fR (C), V_T (D), VE (E) and Dia_EMG amplitude (F) to systemic hypoxic hypoxia after application of Alst in rats in which the LC was transduced with PRSx8-AlstR-GFP-LVV.

Figure 6

Acute inhibition of A6 neurons and effect on the inspiratory, active expiratory and ventilatory responses to stimulation of central chemoreceptors in conscious adult rats. (A) Raw and integrated (∫) records of Dia_EMG and Abd_EMG activities from one animal in which the LC was transduced with PRSx8-AlstR-GFP-LVV. Note the changes in inspiration, expiration and in the number of sighs (red lines) induced by activation of the central chemoreceptor (7% CO₂), before and after Alst application into lateral ventricle. (B) Magnification of baseline and reflex inspiratory, expiratory and ventilatory (barometric respiratory movements) responses from the same rat before and after Alst application. Note that Alst application reduced significantly the fR, Dia_EMG amplitude, the Abd_EMG active expiration incidence (green arrow: absence of active expiration; red arrow: active expiration) and magnitude, as well as the ventilatory parameters.

Figure 7

Acute inhibition of A6 neurons and effect on respiratory responses to activation of central chemoreceptors of conscious adult rats. Summary of data showing the changes in the reflex responses of the fR (A), V_T (B), VE (C), Dia_EMG amplitude (D), number of sighs (E), sigh amplitude (F), active expiration magnitude (G) and active expiration incidence (H; number 1 means that Abd_EMG active expiration is at its maximal value established in each animal during hypercapnia – see Methods) after application of Alst in conscious adult rats in which the LC was transduced with PRSx8-AlstR-GFP-LVV. *p < 0.0001.

Figure 8

Schematic depicting proposed neural mechanisms by which A6 neurons regulate inspiratory and expiratory responses to high chemical drive. Parasagittal views of the brainstem showing the location of the medullary ventral respiratory group and C1 catecholaminergic region, as well as the pontine LC A6 neurons (red) and Facial Motor Nucleus (VII). The respiratory rhythmogenic sites of the pre-Bötzinger Complex (pre-BötC, inspiratory neurons) and parafacial Respiratory Group (pFRG, for active expiration and sigh), as well as the expiratory Bötzinger Complex (BötC), rostral Ventral Respiratory Group (rVRG; containing inspiratory bulbo-spinal premotor neurons) and caudal Ventral Respiratory Group (cVRG; containing expiratory bulbo-spinal premotor neurons) are also shown. (A) Sigh response during systemic hypoxia: A6 neurons might be directly activated by systemic hypoxia (↓ O₂)^, or by sigh-promoting bombesin-like pFRG and C1 catecholaminergic neurons to increase the frequency of sighing through adrenergic receptor activation of pre-BötC. (B) Active inspiratory and expiratory responses during hypercapnia: A6 neurons, activated by CO₂ or C1 catecholaminergic neurons, enhance inspiration and V_E through adrenergic receptor activation of pre-BötC^,. A6 neurons might also provide either tonic or expiratory-related excitatory input to the conditional expiratory oscillator located in the pFRG or directly to the cVRG, for onward relay to expiratory spinal motoneurons, enhancing active expiration.