Background
The dorsolateral periaqueductal grey (dlPAG) coordinates defensive behaviours and associated cardiorespiratory responses. Although the nucleus ambiguus (nA) contains laryngeal motoneurons responsible for upper airway patency and vocal fold control, the functional interactions between the dlPAG and nA remain poorly understood [1-4].
Aims
To describe the anatomical, electrophysiological and functional relationship between these regions in anaesthetised rats.
Methods
Experiments were performed in adult male Sprague–Dawley rats (n=36; 250–300 g) anaesthetised with sodium pentobarbitone (60 mg kg⁻¹ i.p., supplemented intravenously as required). Three complementary experimental approaches were used:
(1) Neuromorphological study: c-Fos and FoxP2 immunoreactivity were quantified throughout the rostrocaudal extent of the nA following repeated electrical stimulation of the dlPAG (30-40 μA, 100 Hz, 1 ms pulses, during 5 s).
(2) Electrophysiological study: extracellular recordings were obtained from neurones located within the nA and adjacent ventrolateral medullary regions during dlPAG stimulation (50–100 μA, 1 Hz, 0.1 ms pulses).
(3) Neuropharmacological study: an isolated glottis in situ preparation evaluated subglottic pressure during dlPAG electrical stimulation or chemical stimulation with glutamate (0,25 M, 50 nL, during 5 s), with phosphate-buffered saline microinjections (50 nL, pH 7,4 ± 0,1, during 5 s) serving as controls. Airflow, pleural pressure, arterial blood pressure, heart rate and subglottic pressure were continuously recorded.
Only experiments with histologically verified dlPAG sites were included. Paired-sample t-tests (within-animal) and one-way ANOVA (between groups) were used for statistical comparisons.
Results
dlPAG electrical stimulation significantly increased c-Fos expression within the loose formation (42.8 ± 5.8 vs 22.6 ± 4.9 cells; p=0.032) and compact formation (3.4 ± 0.8 vs 0.4 ± 0.4 cells; p=0.002) of the ipsilateral nA, confirming activation of neuronal populations associated with laryngeal motor control. FoxP2 immunoreactivity was predominantly localised within the loose and semicompact formations of the nA.
Extracellular recordings (n= 160 neurones) identified six functional neuronal populations. dlPAG stimulation modified neuronal activity in 76% of inspiratory decrementing neurones, 47% of inspiratory augmenting neurones, 67% of expiratory E1 neurones, 86% of expiratory E2 neurones, 52% of cardiovascular neurones and 46% of non-cardiorespiratory neurones, demonstrating widespread recruitment of medullary circuits associated with respiratory, autonomic and laryngeal control.
Electrical dlPAG stimulation increased respiratory frequency (p<0.01), arterial pressure (p<0.001) and heart rate (p<0.001), while reducing subglottic pressure (p<0.01). Similar responses were observed following glutamate microinjection, including a reduction in subglottic pressure (p<0.001), accompanied by significant increases in respiratory frequency (p<0.001), arterial pressure (p<0.001) and heart rate (p<0.001).
Conclusions
These findings provide new anatomical, electrophysiological and functional evidences showing that dlPAG–nA pathway appears to represent an important neural substrate linking behavioural state with laryngeal and cardiorespiratory control.
Ethical approval
All experimental protocols were performed in accordance with the recommendations of the European Union Directive 2010/63/EU for animal care and experimental procedures. The experiments were approved by the Ethical Committee for Animal Research of the University of Málaga and the Junta de Andalucía.
Keywords: dorsolateral periaqueductal grey; nucleus ambiguus; laryngeal motoneurons; subglottic pressure; defence response.