Background
The stimulation of the Periaqueductal Gray matter (PAG) produces an increase in sympathetic tone includiing an increase in blood pressure, heart rate and respiratory frequency1. PAG and nucleus retroambiguus (nRA) are necessary to produce vocalization2. The nRA is the perfect target to turn passive into active expiration modifying the activity of laryngeal motoneurons located in the nucleus ambiguous (nA)3.We have shown that rostral and ventral pontine structures are involved in changes of laryngeal caliber4. A high expression of FOXP2 protein (transcription factor closely related to vocalization) at mesencephalic (PAG) and pontine regions (Parabrachial complex and A5 Region) involved in cardiorespiratory control has been described5.
Objectives
The aim of this study was to characterize the possible role of the dlPAG in modulating laryngeal activity and their effects on vocalization.
Methods
Experimental studies were carried out with non-inbred male rats (n=27), SPF, Sprague-Dawley (250-300 g) housed under standard conditions. Animals were anesthetized with sodium pentobarbitone (60 mg/kg i.p., initial dose, supplemented 2mg/ kg, i.v., as necessary).
Neuromorphological study (n=6)
The pattern of staining for c-Fos and FOXP2 protein immunoreactivity (c-Fos-ir) were examinated throughout the rostrocaudal extent of the nRa/nA region during electrical stimulation of the dlPAG.
Neuropharmacological study (n=21)
A double tracheal cannulation was used to obtain an “isolated glottis in situ” and to record respiratory airflow. Subglottic pressure was recorded with an aneroid transducer (Hugo Sachs Elektronik D-7801, ±0,1 psi) by passing a stream of humidified warm medical air upwards through the larynx at a constant rate of 30-70ml/min with a thermal mass digital air flow meter controller (Bronkhorst Hi-Tec F-201CV-AGD-22-V). Thus, at constant air flow, changes in pressure indicate changes in laryngeal resistance. Bilateral parietostomy allowed access to the dlPAG. Electrical stimulations (n=7) of this region using concentric bipolar electrodes (1ms pulses, 20-40µA, 100Hz for 5s) were performed. Microinjections of PBS-Evans Blue (250nl, pH 7.4±0.1, 5-s duration) (n=7) or glutamate (0,25M, 250nl) (n=7) were performed. Respiratory flow, pleural pressure, blood pressure and heart rate were also recorded.
Only data from animals in which the histology showed that the microelectrodes were positioned within the dlPAG and the A5 region were used for statistical procedures.
Results
Activation of the dlPAG elicited a selective increase in c-Fos-ir with an ipsilateral predominance in nRA/nA somatas (p<0.01) and confirm the expression of FOXP2 bilaterally in both nuclei. dlPAG PBS-Evans Blue microinjections did not produce any significant changes in any of the cardiorespiratory variables recorded. dlPAG electrical and chemical (glutamate) stimulations evoked a decrease of laryngeal resistance (subglottal pressure) (p<0,001) accompanied with an inspiratory facilitatory response consisted of an increase in respiratory rate (p<0,001), together with a pressor (p<0,001) and tachycardic response (p<0,001).
Conclusions
Our study contributes with new data on the role of the mesencephalic neuronal circuits in the control mechanisms of subglottic pressure and laryngeal activity.
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 Malaga and the Junta de Andalucía.
Keywords
Subglottic Pressure, Laryngeal Motoneurons, dlPAG, Nucleus Ambiguus