Hypothalamic and mesencephalic regions involved in the control of laryngeal activity and subglottic pressure in spontaneously breathing anaesthetized rats

Changes of laryngeal caliber allow changes in airflow which are necessary for the vibration of the vocal folds and emission of voice. It is known that stimulation of the Periaqueductal Gray matter (PAG) and nucleus retroambiguus (nRA) produces vocalization, and lesions in PAG cause mutism in animals and humans. The nRA is the perfect target to turn passive into active expiration modifying the activity of laryngeal motoneurons located in the nucleus ambiguous (Paton and Nolan, 2000). We have shown that rostral and ventral pontine structures are involved in changes of laryngeal caliber (Lara et al., 2002). It has been also demonstrated a high expression of FOXP2 protein (transcription factor related to vocalization) at mesencephalic and pontine regions (PAG, Parabrachial complex and A5 Region) involved in cardiorespiratory control. The aim of this study was to characterize the relations between hypothalamic and mesencephalic regions involved in cardiorespiratory control and their possible role in modulating laryngeal activity. Experimental studies were carried out with non-inbred male rats (n=7), SPF, Sprague-Dawley (250-300 g) housed under standard conditions. Animals were anesthetized with sodium pentobarbitone (60 mg/kg i.p., initial dose, supplemented 2 mg/kg, i.v., as necessary). A double tracheal cannulation to develop the classical technique of the “glottis isolated in situ” and for the recording of respiratory airflow was carried out. DMH-PeF, dlPAG, and CnF stimulations evoked a significant decrease of laryngeal resistance (subglottal pressure) (p<0.01) accompanied with an inspiratory facilitatory response consisted of an increase in respiratory rate, together with a pressor and tachycardic response. The results of our study contribute with new data on the role of the hypothalamic-mesencephalic neuronal circuits in the control mechanisms of subglottic pressure and laryngeal activity.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Laryngeal effects of stimulation of the dorsomedial hypothalamic nucleus and perifornical area in spontaneously breathing anaesthetized rats

Background: Stimulation of the dorsomedial hypothalamic nucleus and perifornical area (DMH-PeF) in rats evokes a cardiorespiratory response characterised by an increase in blood pressure, heart rate and respiratory frequency. In previous studies (Díaz-Casares et al., 2012; López-González et al., 2018) we have shown a functional interaction between DMH-PeF and pontine structures (A5 region and Parabrachial Complex). We have also shown that rostral and ventral pontine structures are involved in the changes of laryngeal caliber (Lara et al., 2002). The aim of this study was to characterize the relations between hypothalamic and pontine neuronal circuits involved in laryngeal activity and its effect on vocalization. Methods: Experimental studies were carried out with non-inbred male rats (n=7). Animals were anesthetized with sodium pentobarbitone (60 mg/kg i.p., initial dose, supplemented 2 mg/ kg, i.v., as necessary). A double tracheal cannulation (upwards in direction of the glottis for the “glottis isolated in situ” technique) was done. Subglottic pressure was recorded with an aneroid transducer by passing a stream of humidified medical air upwards through the larynx at a constant rate of 30-70 ml/min with a thermal mass digital air flow meter controller. Electrical stimulation of the DMH-PeF using concentric bipolar electrodes (1 ms pulses, 20-40 µA, 100 Hz for 5 s) was performed. Respiratory flow, pleural pressure, blood pressure, heart rate and ECG activity were also recorded. Results: DMH-PeF stimulation 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 a tachycardic response (p<0,001). Conclusions: The results of our study contribute with new data on the role of the DMH-PeF in the mechanisms controlling subglottic pressure and laryngeal activity.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Design and implementation of a method to study laryngeal resistance during the stimulation of dorsolateral periaqueductal grey (dlpag) in spontaneously breathing anaesthetized rats

Stimulation of the dorsolateral periaqueductal gray matter (dl-PAG) in rats evokes a cardiorespiratory response characterised by an increase of blood pressure, heart rate and respiratory frequency. In previous studies we have demonstrated a functional interaction between dlPAG and the pontine A5 region (Peinado-Aragonés C.A., 2016). A5 region modulates the cardiorespiratory response evoked from the dlPAG and it is also involved in the changes of laryngeal caliber (Lara et al., 2002). The aim of this study was to characterise the relationship between mesencephalic-pontine neuronal circuits to understand their role in laryngeal control and its effect on vocalization. Experimental studies were carried out with non-inbred male rats (n=7), Animals were anesthetized with sodium pentobarbitone (60 mg/kg i.p., initial dose, supplemented 2 mg/ kg, i.v., as necessary). A double tracheal cannulation (upwards in direction of the glottis for the “glottis isolated in situ” technique) was performed. Subglottic pressure was recorded with an aneroid transducer by passing a stream of humidified medical air upwards through the larynx at a constant rate of 30-70 ml/min with a thermal mass digital air flow meter controller. Electrical stimulation of the dlPAG using concentric bipolar electrodes (1 ms pulses, 20-40 µA, 100 Hz for 5 s) was performed. dlPAG stimulation 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 a tachycardic response (p<0,001). Our variation of the classical technique for the recording of the“isolated glottis in situ”in rats shows good dynamic responses and can be perfectly used as an index of subglottic pressure and laryngeal activity, thus our results contribute with new data on the role of the dlPAG in the control mechanisms of subglottic pressure and laryngeal activityUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Impact of the glutamatergic neurotransmission within the A5 region on the cardiorespiratory response evoked from the midbrain dlPAG

Stimulation of the dorsolateral periaqueductal grey matter (dlPAG) in rats evokes an active defensive behaviour together with a cardiorespiratory response characterised by tachypnoea, tachycardia and hypertension. The dlPAG neurons involved in these responses are excitatory, presumably glutamatergic, due to the presence of vesicular glutamate transporter VGLUT2 within their axon terminals. Previously, our group described a functional interaction between dlPAG and the pontine A5 region. Accordingly, in the present work, in order to characterize the role of glutamate within this interaction, experiments were carried out in spontaneously breathing anaesthetized rats (sodium pentobarbitone 60 mg/kg i.p., suplemented with 20 mg/kg i.p.). The cardiorespiratory response evoked by electrical stimulation of the dlPAG (1 ms pulses, 20–50 μA, given at 100 Hz, during 5 s) was analysed before and after the microinjection, within the A5 region, of either kynurenic acid (non-specific glutamate receptor antagonist; 5–10 nmol), DAP-5 (NMDA antagonist; 1 pmol), CNQX (non-NMDA antagonist; 1 pmol) or MCPG (metabotropic antagonist; 0,1 nmol). Kynurenic acid decreased the intensity of both the tachypnoea (p < 0,001) and tachycardia (p < 0,001) induced by dl-PAG stimulation. Blockade of no-NMDA receptors reduced the increase of respiratory frequency, heart rate and pressor response to dl-PAG stimulation (p < 0,01, p < 0,001, p < 0,05 respectively). Blockade of either NMDA or metabotropic receptors reduced the dlPAG-evoked tachycardia and pressor response (p < 0,01; p < 0,05 respectively). These results suggest a neuromodulatory role for A5 region via glutamate neurotransmission of the dlPAG-evoked cardiorespiratory response, confirming the role of the ventrolateral pons in the neuronal circuits involved in respiratory and heart rate control.Funding for open access publishing: Universidad Málaga/CBUA // The study was supported by a program grant Junta de Andalucía, Group nº CTS-156, Spain

Cuneiform nucleus stimulation modifies laryngeal activity and subglottic pressure in rats

The Cuneiform nucleus (CnF) of the mesencephalon has afferent and efferent connections with different regions of the CNS involved in cardiorespiratory control, i.e. dorsolateral part of the Periaqueductal Gray matter (dlPAG) and Parabrachial complex (PBc). In recent studies (López-González et al., 2018, 2020), we have characterized functional interactions between all these hypothalamic (DMH-PeF), mesencephalic (dlPAG) and pontine regions (PBc, A5 region) that are also involved in changes of laryngeal caliber (Lara et al., 2002), due to the abduction and adduction of the vocal folds controlled by motoneurons located in the caudal portion of the nucleus Ambiguus (Amb). The aim of this study was to test the possible role of the CnF in laryngeal control and its effect on vocalization. Experimental studies were carried out with non-inbred male rats (n=14), SPF, Sprague-Dawley (250-300 g) housed under standard conditions. Animals were anesthetized with sodium pentobarbitone (60 mg/kg i.p., initial dose, supplemented 2 mg/kg, i.v., as necessary). A double up and down tracheal cannulation was performed to measure subglottic pressure and 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 with a thermal mass digital air flow meter controller (Bronkhorst Hi-Tec F-201CV-AGD-22-V). Microinjections of PBS-Evans Blue (250 nl, pH 7.4 ± 0.1, 5-s duration) or glutamate (0,25M, 250 nl) were performed. PBS-Evans Blue microinjections within CnF did not produce any significant changes in any of the cardiorespiratory variables recorded. However, glutamate microinjections within the CnF 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<0,01) and tachycardic response (p<0,001).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Cuneiform nucleus stimulation modifies laryngeal activity and subglottic pressure in spontaneously breathing anaesthetized rats

Background Abduction and adduction of the vocal folds are performed by two populations of motoneurons located in the caudal portion of the nucleus Ambiguus (nA). In the rat, the loose formation of the nA contains motoneurons innervating the laryngeal muscles. In previous studies we have demonstrated a functional interaction between hypothalamic (DMH-PeF), mesencephalic (dlPAG) and pontine nuclei (PBc, A5 region) involved in cardiorespiratory control and in changes of laryngeal caliber (López-González et al., 2020; Lara et al., 2002). The Cuneifom nucleus (CnF) of the mesencephalon has afferent and efferent connections with all these nuclei. The aim of this study was to characterize the electrophysiological relationships between the CnF and those pontine-medullary neuronal circuits to understand their role in laryngeal control and its effect on vocalization.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Dorsolateral Periaqueductal Grey Matter stimulation modifies laryngeal activity and subglottic pressure in spontaneously breathing anaesthetized rats

Abduction and adduction of the larynx allow changes in airflow necessary for the vibration of the vocal folds and emission of voice. It is known that stimulation of the Periaqueductal Gray matter (PAG) and nucleus retroambiguus (nRA) produces vocalization (Holstege et al., 1989) and lesions in PAG cause mutism in animals and humans (Esposito et al., 1999). The nRA is the perfect target to turn passive into active expiration modifying the activity of laryngeal motoneurons located in the nucleus ambiguus (Subramanian et al., 2009). We have shown that rostral and ventral pontine structures are involved in changes of laryngeal caliber (Lara et al., 2002). It has been also demonstrated a high expression of FOXP2 protein at mesencephalic and pontine regions (PAG, Parabrachial complex and A5 Region) involved in cardiorespiratory control. FOXP2 is a transcription factor required for brain and lung development and it is closely related to vocalization (Stanic et al., 2018). The aim of this study was to characterize the relations between mesencephalic regions (dlPAG) involved in cardiorespiratory control and their possible role in modulating laryngeal activity and their effects on vocalization.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Effects of stimulation of cuneiform nucleus and the dorsomedial Hypothalamic nucleus and Perifornical area on the mechanisms involved in the control of laryngeal activity and subglottic pressure in spontaneously breathing anaesthetized rats.

The dorsomedial Hypothalamic nucleus and Perifornical area (DMH-PeF) and the mesencephalic Cuneiform nucleus (CnF) have been involved in sympathetic activity due their connectivity with several nuclei involved in cardiorespiratory control. In previous studies we have demonstrated a functional interaction between hypothalamic and mesencephalic structures (DMH-PeF, dlPAG) with several pontine regions (PBc, A5) (2, 3). We have also shown that rostral and ventral pontine structures are involved in the changes of laryngeal caliber (4). The aim of this study was to characterize the relations between hypothalamic and mesencephalic regions involved in cardiorespiratory control and their possible role in modulating laryngeal activity and their possible effects on vocalization. Experimental studies were carried out with non-inbred male rats (n=42), Sprague-Dawley (250-300g). Animals were anesthetized with sodium pentobarbitone (60 mg/kg i.p., initial dose, supplemented 2mg/kg, i.v., as necessary). A double tracheal cannulation was used to obtain an “isolated glottis in situ” and to record respiratory airflow. Bilateral parietostomy allowed access to the DMH-PeF and CnF. Electrical (n=14) and chemical (n=14) stimulations of these regions using bipolar electrodes or glutamate was performed. Microinjections (n=14) of PBS-Evans Blue served as control purpose. Respiratory flow, pleural pressure, blood pressure and heart rate were also recorded. DMH-PeF and CnF PBS-Evans Blue microinjections did not produce any significant changes in any of the cardiorespiratory variables recorded. Glutamate microinjections within the DMH-PeF and CnF evoked a decrease of laryngeal resistance (subglottal pressure) (p<0,01 and p<0,001 respectively) with an inspiratory facilitatory response consisted of an increase in respiratory rate (p<0,001 in both cases), together with a pressor (p<0,001 and p<0,01 respectively) and tachycardic response (p<0,001in both cases)Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Impact of Dorsolateral Periaqueductal Grey Matter stimulation in laryngeal activity and subglottic pressure in rats

The stimulation of the dorsolateral Periaqueductal Gray matter (dlPAG) produces an increase in sympathetic tone that includes an increase in cardiorespiratory parameters (blood pressure, heart rate and respiratory frequency). PAG and nucleus retroambiguus (nRA) are necessary to produce vocalization. The nRA modifies the activity of laryngeal motoneurons located in the nucleus ambiguus. Rostral and ventral pontine structures involved in cardiorespiratory control, Parabrachial complex (PBc) and A5 Region (A5) are also involved in changes of laryngeal caliber. A high expression of FOXP2 protein, a transcription factor closely related to vocalization, at the level of the PAG, PBc and A5 has been shown. The aim of this study was to characterize the relations between mesencephalic regions (dlPAG) involved in cardiorespiratory control and their possible role in modulating laryngeal activity. Experimental studies were carried out with non-inbred male rats (n=14), SPF, Sprague-Dawley (250-300 g) housed under standard conditions. Animals were anesthetized with sodium pentobarbitone (60 mg/kg i.p., initial dose, supplemented 2 mg/kg, i.v., as necessary). A double tracheal cannulation was carried out. 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 with a thermal mass digital air flow meter controller (Bronkhorst Hi-Tec F-201CV-AGD-22-V). Microinjections of PBS-Evans Blue (250 nl, pH 7.4 ± 0.1, 5-s duration) or glutamate (0,25M, 250 nl) were performed within dlPAG. Microinjection of PBS-Evans Blue within dlPAG did not produce any significant cardiorespiratory changes. However, glutamate microinjections within the dlPAG evoked a decrease of laryngeal resistance (p<0,001) accompanied with an increase in respiratory rate (p<0,001) together with a pressor (p<0,001) and tachycardic response (p<0,001).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Cuneiform nucleus and Dorsomedial Hypothalamic nucleus and Perifornical area in the control of laryngeal activity and subglottic pressure in spontaneously breathing anaesthetized rats.

Background: The DMH-PeF and the CnF have been involved in sympathetic activity due their connectivity with several nuclei involved in cardiorespiratory control. In previous studies we have demonstrated a functional interaction between hypothalamic and mesencephalic structures with several rostral and ventral pontine structures that are involved in the changes of laryngeal caliber. The aim is to characterize the role of the DMH-PeF and CnF in modulating laryngeal activity and their effects on vocalization. Experimental studies were carried out with male Sprague-Dawley rats (n=42) (250-300g). Animals were anesthetized with sodium pentobarbitone (60 mg/kg i.p., initial dose, supplemented 2 mg/kg, i.v.). Electrical stimulations (n=14) using concentric bipolar electrodes (1ms pulses, 20-40μA, 100Hz for 5s) microinjections of PBS-Evans Blue (250nl, pH 7.4±0.1, 5-s duration) (n=14) or glutamate (0,25M, 250nl) (n=14) were performed. Respiratory flow, pleural pressure, subglottic pressure, blood pressure and heart rate were also recorded. DMH-PeF and CnF PBS-Evans Blue microinjections did not produce any significant changes in any of the cardiorespiratory variables recorded. However, electrical stimulations in both regions evoked a decrease of laryngeal resistance (subglottal pressure) (p<0,001). Glutamate microinjections within the DMH-PeF and CnF evoked a decrease of laryngeal resistance (p<0,01 and p<0,001 respectively) accompanied with an inspiratory facilitatory response consisted of an increase in respiratory rate (p<0,001 in both cases), together with a pressor (p<0,001 and p<0,01 respectively) and tachycardic response (p<0,001in both cases). Conclusions The results of our study contribute with new data on the role of the hypothalamic-mesencephalic neuronal circuits in the control mechanisms of subglottic pressure and laryngeal activity.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech