Background
The dorsomedial hypothalamic nucleus and perifornical area (DMH-PeF) are key components of the hypothalamic defence network and play an important role in autonomic and respiratory regulation. Although activation of the DMH-PeF evokes characteristic defence-like cardiorespiratory responses, its influence on laryngeal control mechanisms remains poorly understood [1–4].
Aims
To describe the anatomical, electrophysiological and functional relationship between the DMH-PeF and nucleus ambiguus (nA) circuits involved in laryngeal motor control.
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 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 DMH-PeF (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 DMH-PeF stimulation (50–100 μA, 1 Hz, 0.1 ms pulses).
(3) Neuropharmacological study: an isolated glottis in situ preparation evaluated subglottic pressure during DMH-PeF 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 DMH/PeF sites were included. Paired Student’s t-tests and the Mann–Whitney U test were used for within-animal and between-group comparisons, respectively.
Results
DMH/PeF electrical stimulation significantly increased c-Fos expression within the ipsilateral nA and nRA. The largest effects were observed in the semicompact formation, where Δ values increased in motoneurons (5.0 ± 0.7 vs 1.1 ± 0.4 cells; p<0.001) and total cells (19.0 ± 2.2 vs 13.6 ± 3.2 cells; p<0.001). Although overall FoxP2 immunoreactivity remained unchanged, c-Fos/FoxP2 co-expression increased significantly within the semicompact formation (34.7 ± 4.9 vs 19.5 ± 2.2 cells; p<0.05; Δ: 15.1 vs 5.2 cells, p=0.008).
Extracellular recordings (n=78 neurones) identified six neuronal populations. DMH/PeF stimulation modified neuronal activity in 41% of recorded neurones overall, including 51.3% of cardiovascular neurones and 40% of expiratory E1/E2 neurones. Co-stimulation of the vagus nerve and DMH/PeF facilitated responses in 37.5% of inspiratory decrementing laryngeal motoneurons, increasing discharge frequency up to four-fold.
DMH/PeF electrical stimulation increased respiratory frequency (p<0.01), arterial pressure (p<0.001) and heart rate (p<0.001), while reducing subglottic pressure (p<0.001). Similar responses followed glutamate microinjection were observed: subglottic pressure fell (p<0.01), with significant increases in respiratory frequency (p<0.001), arterial pressure (p<0.001) and heart rate (p<0.01).
Conclusions
These findings provide new anatomical, electrophysiological and functional evidence that DMH-PeF pathways modulate brainstem laryngeal circuits and contribute to the coordination of upper airway, respiratory and cardiovascular responses during the defence reaction.
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
dorsomedial hypothalamus; perifornical area; nucleus ambiguus; laryngeal motoneurons; subglottic pressure; defence response.