Uncovering the neural control of laryngeal activity and subglottic pressure in anaesthetized rats: insights from mesencephalic regions

Uncovering the neural control of laryngeal activity and subglottic pressure in anaesthetized rats: insights from mesencephalic regions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Uncovering the neural control of laryngeal activity and subglottic pressure in anaesthetized rats: insights from mesencephalic regions González-García Marta, Carrillo-Franco Laura, Morales-Luque Carmen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3891131/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract To assess the possible interactions between the dorsolateral Periaqueductal Gray matter (dlPAG) and the laryngeal motor neurons of the nucleus Ambiguus (nA), we have examined the pattern of double staining c-Fos/FoxP2 protein immunoreactivity (c-Fos-ir/Fox-P2-ir) and Tyrosine Hydroxylase (TH) throughout the rostro-caudal extent of nA in spontaneously breathing anaesthetised male Sprague–Dawley rats during dlPAG electrical stimulation. Activation of the dlPAG elicited a selective increase in c-Fos-ir with an ipsilateral predominance in the somatas of the loose (p<0.05) and compact formation (p<0.01) within the nA and confirm the expression of FoxP2 bilaterally in all the domains within the nA. A second group of experiments was made to examine the importance of the dlPAG in modulating the laryngeal response evoked after electrical or chemical (glutamate) dlPAG stimulations. Both electrical and chemical stmulations evoked a significant decrease of laryngeal resistance (subglottal pressure) (p<0.001) accompanied with 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 mesencephalic neuronal circuits in the control mechanisms of subglottic pressure and laryngeal activity. dorsolateral periaqueductal gray matter subglottic pressure nucleus ambiguous laryngeal motoneurons central cardiorespiratory control rat Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The larynx performs three pivotal functions: respiration, protection (cough and swallowing reflexes) and phonation. Each of these laryngeal functions necessitates distinctive movements of the vocal folds [32, 39]. The contraction and relaxation of the laryngeal muscles actively modulate the opening and closing of the vocal folds, thereby intricately regulating the egress of air and the subglottic pressure, which denotes the air pressure buildup beneath the vocal folds within the subglottic region of the trachea. Proper synchronization between subglottic pressure and glottic opening profoundly influences proficient phonation and optimum vocal quality. Vocal fold movements are characterized as adduction and abduction. Intrinsic laryngeal muscles are commonly categorized based on their specific actions, with certain muscles identified as adductors, namely the thyroarytenoid, lateral cricoarytenoid, and interarytenoid muscles. Conversely, the posterior cricoarytenoid muscle is recognized for its abductor function [31]. Therefore, during vocal fold closure, subglottic pressure escalates due to the muscular contraction of respiratory muscles and vocal fold occlusion [20]. The motor neurons that govern the laryngeal musculature primarily reside within the nA of the medulla oblongata. Axons originating from the nA merge with the vagal roots, thus constituting the vagus nerve. Emanating from it, both the superior laryngeal nerve and the recurrent laryngeal nerve dispense the afferent and efferent fibers that intricately regulate the laryngeal functionality [17]. The nA can be anatomically partitioned into three principal domains: the compact formation, housing motor neurons innervating the esophagus; the semi-compact formation, accommodating motor neurons that innervate the pharynx and the cricothyroid muscle, the latter being supplied by the superior laryngeal nerve; and the loose formation, harboring motor neurons that innervate the remaining laryngeal muscles, excluding the cricothyroid [3, 38]. In relation to the central regulation of laryngeal phonatory function, vocalization entails the engagement of diverse interconnected cerebral structures within intricate networks. Among mammals, vocalization relies upon a network originating from the laryngeal motor cortex, which projects to the mesencephalic Periaqueductal Gray Matter (PAG) [8, 22]. The PAG serves to modulate the activity of several pontomedullary structures responsible for generating the complete array of laryngeal-respiratory motor patterns imperative for vocal production [14, 48]. These pontomedullary generators exercise command over the sequential activation and intensity of respiratory, laryngeal, oropharyngeal, and craniofacial motor neurons [21, 47]. One of the most important areas connected with the PAG is the nucleus retroambiguus (nRA) [20, 53]. The nRA is positioned caudal to the pre-Bötzinger complex and contains inspiratory premotor neurons in its rostral section called rostral ventral respiratory group, and expiratory premotor neurons in its caudal section, called caudal ventral respiratory group (cVRG) [24, 34]. These nRA premotor neurons are the perfect target to convert passive breathing into active breathing to generate motor activities that produce changes in abdominal pressure, in addition to modifying the activity of the motor neurons that are located in the nA and that control the caliber of the pharynx and larynx [5, 14, 18, 48, 50]. Besides, in previous works by our research group, the activity of the laryngeal motor neurons of nA and the reflex mechanisms involved in respiratory laryngeal responses were characterized through the technique of the “isolated glottis in situ ”, suggesting that the parabrachial complex (PBc) and the A5 region (A5) have a role in modifying the activity of laryngeal motoneurones localised in the nA and, consequently, the striated laryngeal muscles of the upper airway [6, 26]. We and others have also shown functional connections between the dlPAG and these pontine structures [11, 15, 30]. Studies in animal models that communicate vocally and/or learn to speak such as mice, birds or humans, have shown that the transcription factor FoxP2 (Forkhead box protein P2) is involved in the acquisition of fine motor skills necessary for the production of species-specific vocalisations. These studies show that FoxP2 has a highly conserved role through evolution in the development of language in these species, particularly in mammals [12, 13, 42, 43]. Although FOXP2 protein is expressed in various body tissues during development (pulmonary, nervous, cardiovascular and intestinal), it has a particularly strong presence in brain regions involved in cognitive function, learning, language production and comprehension [36, 44]. A high expression of FOXP2 protein at PAG, PBc and A5 region has also been described [46]. However, the relation between PAG and nA is still unknown. Therefore, the main objective of this work was to characterize the anatomo-functional interactions between mesencephalic and medullary neuronal circuits, especially from dlPAG region that possibly control the activity of nA laryngeal motor neurons in the rat. To this end, we have analysed the pattern of double staining c-Fos or FOXP2 immunoreactivity (c-Fos-ir, FoxP2-ir) and Tyrosine Hydroxylase (TH-ir), throughout the rostro-caudal extent of the nA region of anesthetized male Sprague–Dawley rats during dlPAG electrical stimulation. Subsequently, in a second phase of our study, we have updated the "isolated glottis in situ " technique from previous work and been able to measure subglottic pressure by stimulating the dlPAG both electrically and chemically. Thus, we were able to verify how dlPAG plays a role in laryngeal control in anaesthetised animals. Materials And Methods 1. Animals and housing Experiments were conducted on 31 adult male SPF Sprague-Dawley rats weighing between 250-350 g, procured from Charles River (Barcelona, Spain). The rats were housed in groups of six per cage, residing in a climate-controlled chamber maintained at a temperature of 22-24º C, and adhering to a 12:12 hour light-dark cycle with lights on at 8:00 am. These animals were maintained in the Animal House at the University of Malaga, with unrestricted access to food and water. 2. General procedures The surgical interventions employed in this study were based on the methodologies described in previous publications [11, 29]. All surgical procedures were performed under anaesthesia induced by sodium pentobarbitone (initial dose of 60 mg kg-1 i.p., supplemented with 2 mg kg-1 i.v. as needed). Cannulation of the femoral artery and vein was carried out for the purpose of arterial blood pressure measurement and drug administration, respectively. To enable the measurement of respiratory flow using a Fleish pneumotachograph and pleural pressure utilizing an air-filled catheter, both the trachea and esophagus were cannulated. The animals spontaneously breathed a humidified mixture of oxygen-enriched room air. In order to gauge end-tidal CO2 levels during the experiment, a fast response CO2 analyser (ADC FM1, The Analytical Development Co. Ltd., Great Amwell, UK) was employed, and values ranged between 3 to 5%. Throughout the experimental procedures, measures were taken to maintain the animals' body temperature at 37ºC utilizing a heated surgical table and a servo-controlled heating pad that monitored rectal temperature, thereby mitigating the risk of hypothermia. Anaesthesia levels were continuously monitored during the procedures and throughout the duration of the experiment. This was achieved by assessing any potential alterations in cardiovascular variables under resting conditions and evaluating the absence of a significant withdrawal reflex upon paw pinching. To ensure stability and immobility, the animals' heads were securely fixed in a stereotaxic frame, with the upper incisor bar positioned 3.3 mm below the interaural line. 3. Interaction of dlPAG and nA: c-Fos/FoxP2/TH-ir experiments In a group of 10 animals, a hole was drilled into the skull to provide access to the right dlPAG. A concentric bipolar electrode (Rhodes Medical electrodes, NE-100) was positioned in the right dlPAG. The stereotaxic coordinates to locate the right dlPAG were from -4.0 mm caudal to bregma, 0.6 lateral to midline and 4.5 to 5 mm depth from the surface of the calota (approaching with an angle of 30º) based on the coordinates provided by the atlas of Paxinos and Watson [40]. The right dlPAG was stimulated once using 1 millisecond pulses, ranging from 30-50 μA, at a frequency of 100 Hertz for a duration of 5 seconds, in order to elicit the classical defense response evoked by this region. Following this, guanethidine (10 milligrams per kilogram intravenous) was administered to suppress sympathetically mediated cardiovascular responses. Following this, in one subgroup of animals (n=5), the right dlPAG was stimulated using trains of 1 millisecond pulses, between 30-50 μA, at a frequency of 100 Hertz for 5 seconds, every 60 seconds for a period of one hour. The other subgroup of animals (n=5) did not receive any stimulation and served as the control group. One hour after the completion of the experiments, the animals were deeply anesthetized and subjected to perfusion through the ascending aorta using a phosphate-buffered saline (PBS, pH 7.4) solution, followed immediately by a solution of 4% paraformaldehyde in 0.1M PBS (pH 7.4) to serve as the fixative. The brains were rapidly extracted and subjected to additional fixation by immersion in the same fixative solution for 24 hours at 4°C. Subsequently, the brains were cryoprotected using 30% phosphate-buffered sucrose for several days. The brainstem was then sectioned into 30 μm coronal sections obtained with a freezing microtome (CM 1325, Leica, Wetzlar, Germany). Sampling was performed by selecting every fifth section with a random starting point, and these sections were further processed to assess c-Fos/FOXP2/TH-ir at the level of nA. c-Fos-TH double immunohistochemistry Sections collected from control and stimulated rats were processed under identical free-floating immunohistochemical conditions. The sections were rinsed in 0.1 M PBS (pH 7.4) and pre-treated during 15 minutes in 3% H 2 0 2 to eliminate endogenous peroxidase activity. After a second round of washing in PBS saline, sections were incubated overnight at room temperature (RT), using a mouse anti-c-Fos monoclonal primary antibody (1:1000 dilution in PBS containing 0.3% Triton X-100 0.2% Azida 0.02%; Santa Cruz Biotech, ref SC-271243). A biotinylated secondary antibody (1:500 dilution; goat anti-mouse; Vector Laboratories) and ExtrAvidin Peroxidase (1:2000 dilution; Sigma-Aldrich) were used for subsequent incubations, of 1 hour at RT each. After washing in PBS (pH 7.4), staining was carried out with 3,3’-diaminobenzidine (DAB; 25mg in 100 ml Tris–HCl, pH 7.4 and 0.002% H 2 O 2 ; Sigma-Aldrich) in the presence of nickel sulphate (1%) to produce a dark reaction product. In order to investigate the presence of c-Fos-ir in TH-ir neurons, the same sections were then incubated overnight at RT with a polyclonal primary antibody against the tyrosine-hydroxylase enzyme (1:5000 dilution; anti-rabbit; Sigma-Aldrich, ref T2928), followed by a re-incubated 1h at RT in a biotonylated secondary antibody (1:500 dilution; goat anti-rabbit; Vector Laboratories). The TH-ir was revealed only by reacting DAB to produce a brown labelling. Sections were dehydrated and coverslipped with DPX mounting medium. FoxP2 -TH double inmunohistochemistry This double labelling was performed under the same protocol as for c-Fos-TH-ir but using a sheep anti-FOXP2 primary antibody (1:1000 dilution; RD Systems, ref AF5647) and a biotinylated secondary antibody (1:500 dilution; rabbit anti-sheep; Vector Laboratories). Cell counting and statistical analysis The rostrocaudal level of each brain section was determined using the Paxinos and Watson rat brain atlas [40]. The cell bodies displaying c-Fos-ir or FOXP2-ir were identified by the presence of a dark nucleus and were quantified bilaterally in the sections containing the nA using a BX61VS optical microscope (Olympus), an Olympus VS software for brightfield image acquisition and QuPath Software. All data were presented as mean ± SEM. To compare the differences between groups, a one-way analysis of variance (ANOVA) was employed for statistical analyses. The significance level was set at p<0.05. Cell quantification along the nA was executed in accordance with the categorization into three primary subdivisions ( compact, semicompact, and loose formation ). To this end, distances from Bregma, as per the Paxinos Atlas (2007), were employed to guide the selection of slices across the three domains of the nA. Specifically, the criterion for demarcating cells within the compact region of the nA spanned from Bregma -12.00 mm to -12.84 mm; the semi-compact region extended from Bregma -12.84 mm to -13.44 mm; and the loose formation region encompassed Bregma -13.44 mm to -14.16 mm. 4. Interaction of dlPAG and nA: electrical/chemical stimulation and subglottic pressure measurement In 21 animals, divided in 3 groups, the described general procedures were modified. A double tracheal cannulation was carried out to develop the classical technique of the “isolated glottis in situ ” and for the recording of respiratory airflow [26]. Subglottal pressure was recorded with an aneroid transducer (ADInstrument model FE141, ± 0,03 psi) by passing a stream of humidified warm medical air upwards through the larynx at a constant rate of 30-70 ml/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. dlPAG electrical stimulation and subglottic pressure measurement In a subset of 7 animals, a burr hole was drilled into the skull of each animal to access to the right dlPAG. The right dlPAG was stimulated with 1 ms pulses, 30-50 µA given at 100 Hz for 5 s by positioning a concentric bipolar electrode (NE-100; Rhodes Medical Electrodes, Summerland, CA, USA). dlPAG chemical stimulation and subglottic pressure measurement In 14 animals, a burr hole was drilled into the skull of each animal to access to the right dlPAG. Microinjections of a solution of PBS (50nl, pH 7.4±0.1, 5-s duration) (n=7), or glutamate (0,25M, 50nl) (n=7), through a stereotaxically positioned single glass micropipette (Hamilton 1.0µL Model 7001 Knurled Hub (KH) Neuros Syringe) were performed in the same coordinates cited above. Evans blue was used to dissolve all drugs and served to mark microinjection sites. Microinjections of PBS-Evans blue alone were used for control purposes. Microinjection volumes of 50 nl were programmed with a micropump controller (Ultra Micro Pump II, Micro 4; World Precision Instruments, Inc., Sarasota, FL, USA), driving 1 µl microsyringes attached to the micropump. Only one microinjection was delivered in each animal. Electrical lesions (250 μA DC for 20 seconds) or Evans blue serve to locate dlPAG electrical stimulation/microinjections sites, respectively. Brains were perfused with 10 % formal saline and serially sectioned (50 µm) at the level of the midbrain. The midbrain was counterstained with Neutral Red. Different physiological parameters were analyzed in each group of experiments under the following protocol: (a) Study of laryngeal and cardiorespiratory parameters at rest. (b) Study of laryngeal and cardiorespiratory changes during dlPAG stimulation (electrical/chemical). In each animal airflow, pleural pressure (as an index of inspiratory activity), subglottic pressure and arterial pressure were monitored and stored on PC. Measurements were made of instantaneous respiratory frequency, subglottic pressure, mean blood arterial pressure and instantaneous heart rate. In all experiments baseline values of the parameters were measured immediately prior to dlPAG electrical/chemical stimulation and after (4 min after electrical stimulation and 30 min after chemical stimulation). Changes in mean arterial blood pressure or heart rate were assessed by measuring the peak rise in blood pressure or heart rate observed during the 5 seconds electrical stimulation of the dlPAG or the maximum cardiovascular response in the chemical stimulation experiments. Stimulus-evoked changes in respiratory parameters were measured as the average response observed during the 5 seconds electrical stimulation of the dlPAG, or the maximum respiratory response in the chemical stimulation experiments. The parameter monitoring and the offline analysis was done using LabChartPro (PowerLab System, ADInstruments®/ LabChart Software, version 8.0, Sydney, Australia). Statistical analysis All data are expressed as mean ± SEM. For statistical comparisons, once the statistical normality (Kolmogorov-Smirnov test) and the homoscedasticity (Bartlett's test) of the data were verified, a paired-sample test was applied to compare the control with the evoked response period for each animal. One-way ANOVA was used to compare different groups of animals. At each time point, p<0.05 was regarded as significant. Only data from animals in which the histology showed that the microelectrodes were positioned within the dlPAG were used for statistical procedures. Results 1. Interaction of dlPAG and nA: c-Fos/FoxP2/TH-ir experiments Distribution of FoxP2-ir Control animals In non-stimulated animals no significant differences were observed regarding the number of FOXP2-ir profiles between ipsilateral and contralateral sides in any of the three principal domains of the nA (Table 1, Fig 2). Electrically stimulated animals Both, loose formation (p<0,001) and semicompact formation (p<0,05) presented a significant increase in FOXP2-ir profiles in the ipsilateral side compared with the contralateral side in electrically stimulated animals (Table 1, Figs 1, 2). Control vs stimulated animals No differences were found between control and electrically stimulated animals when comparing the amplitude of the differences between the number and distribution of ipsilateral or contralateral FOXP2-ir neurons within any of the domains in the nA nuclei (Table 1, Figs 1, 2). Distribution of c-Fos-ir Control animals A higher amount of c-Fos-ir profiles were observed in both the loose formation and semicompact formation in the ipsilateral side compared with the contralateral side (p<0.01) in non-stimulated animals (Table 1, Fig 2). Electrically stimulated animals An significant ipsilateral predominance for c-Fos-ir staining in all the three domains of nA were observed in electrically stimulated animals: loose formation (p<0,001), semicompact formation (p<0,001) and compact formation (p<0,05) (Table 1, Figs 1, 2). Control vs stimulated animals When comparing the amplitude of the differences of the c-Fos-ir expression in ipsilateral and contralateral sides in control and stimulated groups, a significant higher increase was observed in both the loose formation (p=0.032) and the compact formation (p=0.002) (Table 1, Figs 1, 2). 2. Interaction of dlPAG and nA: electrical/chemical stimulation and subglottic pressure measurement dlPAG electrical stimulation and subglottic pressure measurement In all animals (n=7), the dlPAG electrical stimulation elicited a cardiorespiratory response characterized by tachypnoea (p<0,01), a decrease in laryngeal resistance (subglottal pressure) (p<0,01) and a pressor response (p<0,001) accompanied with tachycardia (p<0,001) (Table 2, Fig 4). dlPAG PBS microinjections and subglottic pressure measurement Microinjections of PBS within the dlPAG (n=7) did not produce changes in any of the resting cardiorespiratory parameters (Table 2, Figs 3, 5). dlPAG Glutamate microinjections and subglottic pressure measurement Glutamate microinjections within the dlPAG (n=7) 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) (Table 2, Figs 3, 4, 5). dlPAG PBS vs Glutamate microinjections and subglottic pressure measurement When comparing the amplitude of the differences at the laryngeal and cardiorespiratory responses between PBS and Glutamate microinjected animals, higher values in delta heart rate (0,29 ± 2,5 to 45,57 ± 3,9; p = 0,001), delta blood pressure (0,00 ± 1,0 to 10,57 ± 0,82; p = 0,001), delta subglottic pressure (0,05 ± 0,05 to -4,60 ± 0,24; p = 0,001), and delta respiratory rate (1 ± 0,69 to 29,57 ± 8,7; p= 0,001) were observed in stimulated animals (Fig 5). Discussion The results of our study demonstrated, using neuromorphological and electroneurophysiological techniques, the existence of functional interactions between the dlPAG, a mesencephalic region known for its involvement in cardiorespiratory regulation, and the nA, a medullary region containing the majority of the motor neurons that govern the laryngeal musculature. Firstly, we demonstrated that electrical stimulation of dlPAG induces a significant increase in cFos-ir expression in the loose formation and compact formation domains of the nA. Secondly, we show that the loose formation and semicompact formation regions are the nA subdivisions with the highest expression of FOXP2-ir, while the compact region shows the lowest expression. No significant changes in FOXP2 expression occur after dlPAG stimulation in either region. Finally, using the "isolated glottis in situ " technique together with classical electrophysiological techniques, we demonstrate for the first time how dlPAG is involved in the control of laryngeal resistance. All these results suggest that, in our experimental conditions, the dlPAG modulates the activity of laryngeal motoneurons located within loose formation of the nA, thereby influencing the striated laryngeal muscles of the upper airway, during the concomitant cardiorespiratory changes evoked by dlPAG electrical or chemical stimulation. Interaction of dlPAG and nA: c-Fos and FOXP2 expression The neuronal cell bodies responsible for innervating the intrinsic muscles of the larynx are situated within nA. This cell-column comprises neurons oriented in a rostrocaudal direction and is positioned in the ventrolateral region of the medulla oblongata. Its spatial extent ranges from the motor nucleus of the facial nerve to at least the level of the pyramidal decussation [3]. We could divide the nA into three main parts or domains: the compact formation , with motor neurons innervating the oesophagus; the semi-compact formation , with motor neurons innervating the pharynx and the cricothyroid muscle of the larynx, i.e. that which is innervated by the superior laryngeal nerve; and the loose formation , with motor neurons innervating the laryngeal muscles except the cricothyroid. It is therefore accepted that laryngeal neurons are located the caudal part of the nA ( semi-compact and loose formation ). Thus, the nA, in addition to innervating the laryngeal muscles, also provides motor innervation to the oesophagus and pharynx [16, 38, 54]. In the present study, the guanethidine, a sympatholytic agent, was administered to prevent secondary c-Fos expression resulting from alterations in arterial blood pressure. Thus, despite the blockage of the cardiovascular changes, the nA presents a significant increase of ipsilateral c-Fos-ir within the three principal domains of the nA after electrical stimulation of the dlPAG. When comparing the differences between stimulated and control animals in the increase of cFos-ir between ipsilateral and contralateral side, only the loose formation and compact formation presents significant changes in their activity Therefore, both populations of neurons of the nA seem likely to be activated directly or indirectly from the dlPAG and not secondarily to blood pressure changes evoked from the dlPAG during electrical stimulation. Thus, this data suggests a possible role of the dlPAG in the glottal opening/closing movements regulating the laryngeal resistance during innate vocalizations or during the respiratory responses developed in the classical defence response vehiculated by this mesencephalic region. When examining the data of FOXP2 nuclei, we confirmed a high level of expression at the level of semicompact formation (controls the vocal fold tension) and loose formation (regulates the subglottic pressure during vibration) [33], giving a role to the neurons of these domains in the production of a laryngeal-respiratory motor patterns necessaries for the correct production of species-specific vocalisations. It should be noticed that FOXP2 expression levels were not modified by electrical stimulation of the dlPAG. Regarding these quantitative data from FOXP2-ir, it should be noted that they are in line with previous studies in which numerical approximations were made within these domains [38], confirming that the expression of this transcription factor is a characteristic rather than an expression of cellular activity. Interaction of dlPAG and nA: electrical/chemical stimulation experiments and subglottic pressure . Chemical microstimulation of the PAG reveals that different columns of the PAG coordinate different types of responses depending on the stimulus [1, 4, 10, 28], allowing the modulation of an autonomic response dependent on the type of stress and the individual's subjective perception of a threat or stressful stimulus. For this purpose, the PAG presents many afferences and efferences projections. The most important afferences have their origin from the prefrontal cortex, amygdala and hypothalamus [9, 23, 27, 45, 51, 52]. The PAG, in turn, projects to the pontomedullary cardiorespiratory nuclei involved in cardiorespiratory rhythmogenesis that allows the development of different patterns of cardiorespiratory and motor responses depending on the type of stimulus [10, 11, 24, 30]. Specifically, the dlPAG column controls flight/flight, or coping/fighting behavioural responses, triggering a defence response that is haemodynamically characterised by hypertension, tachycardia and redistribution of blood flow to the skeletal muscles of the extremities from the abdominal and visceral area. In addition, the response is accompanied by mydriasis, tachypnoea, increased tidal volume and vocalisations. This allows the animal to cope with environmental stress situations that require a rapid response [30, 35, 52]. Other functions of PAG include thermoregulation, participation in wakefulness and sleep mechanisms, or modulation of neuropathic pain or micturition [7, 19, 25]. At clinical level, its activity is modified in different neurodegenerative processes such as Alzheimer's and multisystemic atrophy [2, 37]. Moreover, the PAG assumes a pivotal role in the regulation of vocalizations [21, 47]. This is facilitated by its robust connectivity with the cVRG within the nRA, an exclusive cerebral region uniquely dedicated to directly influencing all requisite motor areas associated with vocalization [20]. Regarding this role, neurochemical microstimulation within the PAG produces vocalisations in cats [49], monkey [22], birds [41] and humans [20] corresponding with the sound part of human speech. We have previously demonstrated, using the “isolated glottis in situ” technique, that glottic caliber is modulated by pontine regions (cPB and A5 region) [26]. This technique has some advantages, as it separates the lower airways from the larynx, facilitates the study of modifications in neuro-muscular tone and abolishes the influence of variations of the airflow activating laryngeal reflexes. In that sense, there have been few developments in subglottic pressure measurement studies. This is possibly attributed to the intricacy of the surgical technique and the challenge of maintaining a stable recording of subglottic pressure. Hence, it is of particular interest to reapply this technique to measure changes in subglottic pressure associated with the cardiorespiratory response evoked from dlPAG stimulation. For this purpose, we have maintained the original design, updating the means of obtaining the subglottic pressure variable thanks to new models of both aneroid transducer (ADInstrument model FE141, ± 0.03 psi) and a digital thermal mass air flowmeter controller (Bronkhorst Hi-Tec F-201CV-AGD-22-V). In addition, we have upgraded both the data processing software (LabChartPro) and the signal acquisition hardware (PowerLab 16/30). Thanks to these updates we have been able to improve the sensitivity and stability of the subglottic pressure signal. In fact, in our study, we have included for statistical analysis only data from animals in which stable recordings were observed before, during and after dlPAG stimulation, providing with new data on the laryngeal effects induced by the dlPAG. Therefore, electrical stimulation was used primarily to locate dlPAG and to study the laryngeal calibre through the measure of the subglottal pressure. In a second group of animals, we have developed chemical stimulation with glutamate microinjections within dlPAG. While the effects of electrical stimulation are very difficult to interpret, the effects of glutamate microinjection suggested that the elicited laryngeal and cardiorespiratory responses can be attributed to the activation of cell bodies located in these dlPAG region but not to the activation of axons of passage. Finally, we can conclude that the chemical stimulation of the dlPAG with glutamate produced a sympathoexcitatory response characterized by hypertension, tachycardia and tachypnoea. All these changes were accompanied with a reduction in subglottal pressure. The glottal dilatation during the inspiratory respiratory response facilitates respiratory movements because of the decrease of the upper airway resistance. These findings suggest the involvement of this mesencephalic region in the regulation of laryngeal activity. The magnitude of the changes in subglottal pressure and cardiorespiratory variables were similar, except the arterial pressure, using both methods of stimulation. Thus, our present observations suggest that the dlPAG has a role in modifying the activity of laryngeal motoneurons localised in the nA and accordingly the striated laryngeal muscles of the upper airway. Declarations Compliance with Ethical Standards Conflicts of interest : The authors declare that they have no conflict of interest. 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. Every attempt was made to reduce animal suffering, discomfort and the total number of animals needed to obtain reliable results. Consent for publication Not applicable Availability of supporting data The data included in the study are available in a generalist data repository provided by the University of Málaga (https://riuma.uma.es/xmlui/ ) Competing interest The authors declare no commercial or financial conflicts of interest in association with the present study. Funding Information Funding for open access publishing: Universidad Málaga/CBUA. The study was supported by a program grant Junta de Andalucía, Groups nº CTS-156, CTS-160 and UMA20-FEDERJA-122, Spain. Part of the final study was supported with a grant from the Own Funds Program of the University of Málaga. Authors Contributions MVLG and MSDM conceived and designed the research. MGG and LCF performed the experiments. MGG, LCF and CML analysed the data. MVLG, MGG and MSDM interpreted the results of the experiments. MGG, LCF, MPV and BG made the immunohistochemistry and interpreted the data. MVLG, LCF and MGG drafted manuscript. BG and MSDM edited and revised manuscript. All authors provided critical and intellectual contributions on previous version of the manuscript and approved the final version. The authors declare that all data were generated in-house and that no paper mill was used. Acknowledgements Not applicable Authors' information Marta González-García 1,2,3 * # Laura Carrillo-Franco 1,3 # Carmen Morales-Luque 1 Marina Ponce-Velasco ,3,4 Belén Gago 1,3 Marc Stefan Dawid-Milner 1,2,3 † Manuel Víctor López-González 1,2,3 * † (1) Department of Human Physiology, Faculty of Medicine, University of Malaga, Malaga (Spain) (2) Unit of Neurophysiology of the Autonomic Nervous System (CIMES), University of Malaga, Málaga (Spain) (3) IBIMA Plataforma BIONAND, Málaga, Spain (4) Department of Cell Biology, University of Málaga, Málaga, Spain (#) M. González-García and L. Carrillo-Franco both contributed equally to this study and must be considered first authors . (†) M.V. López-González and M.S. Dawid-Milner both had equally responsibility and must be considered last authors . References Bandler R, Keay KA, Floyd N, Price J (2000). Central circuits mediating patterned autonomic activity during active vs. passive emotional coping. 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Ann Otol Rhinol Laryngol 120:761-8. https://doi.org/10.1177/000348941112001 Tables Table 1 Relative quantification and distribution of c-Fos and FOXP2 inmunoreactive cells (cFos-ir and FOXP2-ir) within the nA in not stimulated animals (control) and dlPAG electrically stimulated animals (stimulated). Both groups are spontaneously breathing rats in which sympathetically mediated cardiovascular changes were abolished with guanethidine. The nA has been divided into its 3 classical domains (loose, semi-compact and compact) and ipsilateral and contralateral sides were also analysed. NEUROMORPHOLOGICAL STUDY CONTROL (n=5) STIMULATED (n=5) Ipsilateral Contralateral Ipsilateral Contralateral LOOSE FORMATION c-Fos-ir cells 29.6 ± 5.1** 18.2 ± 2.7 42.8 ± 5.8*** 22.6 ± 4.9 FOXP2-ir cells 115 ± 18.9 93.4 ± 14.8 115.4 ± 15.5*** 88.8 ± 17.6 Delta c-Fos-ir 11.4 ± 2.4 20.2 ± 2.4 † Delta FOXP2-ir 21.6 ± 8.1 26.6 ± 2.1 SEMICOMPACT FORMATION c-Fos-ir cells 23.6 ± 3.8** 14.6 ± 3.3 31 ± 3.3*** 16.8 ± 2.4 FOXP2-ir cells 105 ± 34.8 75.3 ± 26.6 104.5 ± 26.2* 81.5 ± 24.0 Delta c-Fos-ir 9 ± 1.7 14.2 ± 1.8 Delta FOXP2-ir 29.8 ± 12.6 23 ± 5.4 COMPACT FORMATION c-Fos-ir cells 1.6 ± 0.2 1.4 ± 0.2 3.4 ± 0.8* 0.4 ± 0.4 FOXP2-ir cells 10.8 ± 2.3 4.5 ± 0.9 10 ± 4.7 6.5 ± 3.7 Delta c-Fos-ir 0.2 ± 0.2 3 ± 0.8 † Delta FOXP2-ir 6.3 ± 1.8 3.5 ± 1.2 Data are expressed as mean ± SEM. Asterisks show differences between ipsilateral and contralateral sides in control or stimulated animals. (* p0.01; **p<0.001). Daggers show differences between the delta differences between control and stimulated groups ( † p< 0.05). Table 2 Laryngeal and cardiorespiratory parameters measured before electrical or chemical stimulation of dlPAG ( Rest ), during electrical or chemical stimulation (microinjections of PBS or Glutamate) within the dlPAG ( Stimulation ) and 4 minutes after electrical or 30 minutes after chemical stimulation of dlPAG ( Recovery ). NEUROPHARMACOLOGICAL STUDY ELECTRICAL Rest Stimulation Recovery dlPAG ( n=7) RR (rpm) 104 ± 9 145 ± 15 ** 105 ± 9 TE (s -1 ) 0.343 ± 0.028 0.233 ± 0.021 ** 0.341 ± 0.025 TI (s -1 ) 0.261 ± 0.021 0.209 ± 0.026 * 0.259 ± 0.019 SGP (cmH 2 O) 8.1 ± 1.3 3.3 ± 0.3 ** 8.1 ± 1.4 BP (mmHg) 106 ± 3 148 ± 6 *** 104 ± 4 HR (bpm) 408 ± 10 435 ± 10 *** 410 ± 7 PBS Rest Stimulation Recovery dlPAG ( n=7) RR (rpm) 85 ± 7 86 ± 7 85 ± 7 TE (s -1 ) 0.394 ± 0.001 0.385 ± 0.002 0.393 ± 0.001 TI (s -1 ) 0.311 ± 0.001 0.312 ± 0.001 0.312 ± 0.001 SGP (cmH 2 O) 10.4 ± 0.6 10.5 ± 0.6 10.4 ± 0.6 BP (mmHg) 103 ± 3 104 ± 2 103 ± 2 HR (bpm) 394 ± 8 395 ± 7 393 ± 10 GLUTAMATE Rest Stimulation Recovery dlPAG ( n=7) RR (rpm) 81 ± 5 111 ± 13 *** 83 ± 6 TE (s -1 ) 0.474 ± 0.038 0.346 ± 0.037 *** 0.472 ± 0.035 TI (s -1 ) 0.287 ± 0.009 0.244 ± 0.023 * 0.286 ± 0.008 SGP (cmH 2 O) 9.4 ± 0.4 4.6 ± 0.4 *** 9.8 ± 0.6 BP (mmHg) 105 ± 3 116 ± 4 *** 107 ± 4 HR (bpm) 391 ± 8 436 ± 8 *** 393 ± 10 Data are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 Stimulation vs Rest. Abbreviations: RR, Respiratory Rate; TE, Espiratory Time; TI, Inspiratory Time; SGP, Subglottic Pressure; BP, Blood Pressure; HR, Heart Rate. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 11 Mar, 2024 Reviews received at journal 05 Feb, 2024 Reviewers agreed at journal 25 Jan, 2024 Reviewers invited by journal 25 Jan, 2024 Submission checks completed at journal 25 Jan, 2024 Editor assigned by journal 25 Jan, 2024 First submitted to journal 23 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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16:33:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1483917,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"FIGURE5PBSGLUTAMATO.png","url":"https://assets-eu.researchsquare.com/files/rs-3891131/v1/775699b3221b01d2f42c6fb9.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Uncovering the neural control of laryngeal activity and subglottic pressure in anaesthetized rats: insights from mesencephalic regions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe larynx performs three pivotal functions: respiration, protection (cough and swallowing reflexes) and phonation. Each of these laryngeal functions necessitates distinctive movements of the vocal folds [32, 39]. The contraction and relaxation of the laryngeal muscles actively modulate the opening and closing of the vocal folds, thereby intricately regulating the egress of air and the subglottic pressure, which denotes the air pressure buildup beneath the vocal folds within the subglottic region of the trachea. Proper synchronization between subglottic pressure and glottic opening profoundly influences proficient phonation and optimum vocal quality. Vocal fold movements are characterized as adduction and abduction. Intrinsic laryngeal muscles are commonly categorized based on their specific actions, with certain muscles identified as adductors, namely the thyroarytenoid, lateral cricoarytenoid, and interarytenoid muscles. Conversely, the posterior cricoarytenoid muscle is recognized for its abductor function [31]. Therefore, during vocal fold closure, subglottic pressure escalates due to the muscular contraction of respiratory muscles and vocal fold occlusion [20].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe motor neurons that govern the laryngeal musculature primarily reside within the nA of the medulla oblongata. Axons originating from the nA merge with the vagal roots, thus constituting the vagus nerve. Emanating from it, both the superior laryngeal nerve and the recurrent laryngeal nerve dispense the afferent and efferent fibers that intricately regulate the laryngeal functionality [17]. The nA can be anatomically partitioned into three principal domains: the compact formation, housing motor neurons innervating the esophagus; the semi-compact formation, accommodating motor neurons that innervate the pharynx and the cricothyroid muscle, the latter being supplied by the superior laryngeal nerve; and the loose formation, harboring motor neurons that innervate the remaining laryngeal muscles, excluding the cricothyroid [3, 38].\u003c/p\u003e\n\u003cp\u003eIn relation to the central regulation of laryngeal phonatory function, vocalization entails the engagement of diverse interconnected cerebral structures within intricate networks. Among mammals, vocalization relies upon a network originating from the laryngeal motor cortex, which projects to the mesencephalic Periaqueductal Gray Matter (PAG) [8, 22]. The PAG serves to modulate the activity of several pontomedullary structures responsible for generating the complete array of laryngeal-respiratory motor patterns imperative for vocal production [14, 48]. These pontomedullary generators exercise command over the sequential activation and intensity of respiratory, laryngeal, oropharyngeal, and craniofacial motor neurons [21, 47]. One of the most important areas connected with the PAG is the nucleus retroambiguus (nRA) [20, 53]. \u0026nbsp;The nRA is positioned caudal to the pre-B\u0026ouml;tzinger complex and contains inspiratory premotor neurons in its rostral section called rostral ventral respiratory group, and expiratory premotor neurons in its caudal section, called caudal ventral respiratory group (cVRG) [24, 34]. These nRA premotor neurons are the perfect target to convert passive breathing into active breathing to generate motor activities that produce changes in abdominal pressure, in addition to modifying the activity of the motor neurons that are located in the nA and that control the caliber of the pharynx and larynx [5, 14, 18, 48, 50].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBesides, in previous works by our research group, the activity of the laryngeal motor neurons of nA and the reflex mechanisms involved in respiratory laryngeal responses were characterized through the technique of the \u0026ldquo;isolated glottis \u003cem\u003ein situ\u003c/em\u003e\u0026rdquo;, suggesting that the parabrachial complex (PBc) and the A5 region (A5) have a role in modifying the activity of laryngeal motoneurones localised in the nA and, consequently, the striated laryngeal muscles of the upper airway [6, 26]. We and others have also shown functional connections between the dlPAG and these pontine structures [11, 15, 30].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStudies in animal models that communicate vocally and/or learn to speak such as mice, birds or humans, have shown that the transcription factor FoxP2 (Forkhead box protein P2) is involved in the acquisition of fine motor skills necessary for the production of species-specific vocalisations. These studies show that FoxP2 has a highly conserved role through evolution in the development of language in these species, particularly in mammals [12, 13, 42, 43]. Although FOXP2 protein is expressed in various body tissues during development (pulmonary, nervous, cardiovascular and intestinal), it has a particularly strong presence in brain regions involved in cognitive function, learning, language production and comprehension [36, 44]. A high expression of FOXP2 protein at PAG, PBc and A5 region has also been described [46].\u003c/p\u003e\n\u003cp\u003eHowever, the relation between PAG and nA is still unknown. Therefore, the main objective of this work was to characterize the anatomo-functional interactions between mesencephalic and medullary neuronal circuits, especially from dlPAG region that possibly control the activity of nA laryngeal motor neurons in the rat. To this end, we have analysed the pattern of double staining c-Fos or FOXP2 immunoreactivity (c-Fos-ir, FoxP2-ir) and Tyrosine Hydroxylase (TH-ir), throughout the rostro-caudal extent of the nA region of anesthetized male Sprague\u0026ndash;Dawley rats during dlPAG electrical stimulation. Subsequently, in a second phase of our study, we have updated the \u0026quot;isolated glottis \u003cem\u003ein situ\u003c/em\u003e\u0026quot; technique from previous work and been able to measure subglottic pressure by stimulating the dlPAG both electrically and chemically. Thus, we were able to verify how dlPAG plays a role in laryngeal control in anaesthetised animals.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003e1. Animals and housing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiments were conducted on 31 adult male SPF Sprague-Dawley rats weighing between 250-350 g, procured from Charles River (Barcelona, Spain). The rats were housed in groups of six per cage, residing in a climate-controlled chamber maintained at a temperature of 22-24\u0026ordm; C, and adhering to a 12:12 hour light-dark cycle with lights on at 8:00 am. These animals were maintained in the Animal House at the University of Malaga, with unrestricted access to food and water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. General procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe surgical interventions employed in this study were based on the methodologies described in previous publications [11, 29].\u003c/p\u003e\n\u003cp\u003eAll surgical procedures were performed under anaesthesia induced by sodium pentobarbitone (initial dose of 60 mg kg-1 i.p., supplemented with 2 mg kg-1 i.v. as needed). Cannulation of the femoral artery and vein was carried out for the purpose of arterial blood pressure measurement and drug administration, respectively. To enable the measurement of respiratory flow using a Fleish pneumotachograph and pleural pressure utilizing an air-filled catheter, both the trachea and esophagus were cannulated. The animals spontaneously breathed a humidified mixture of oxygen-enriched room air. In order to gauge end-tidal CO2 levels during the experiment, a fast response CO2 analyser (ADC FM1, The Analytical Development Co. Ltd., Great Amwell, UK) was employed, and values ranged between 3 to 5%. Throughout the experimental procedures, measures were taken to maintain the animals\u0026apos; body temperature at 37\u0026ordm;C utilizing a heated surgical table and a servo-controlled heating pad that monitored rectal temperature, thereby mitigating the risk of hypothermia. Anaesthesia levels were continuously monitored during the procedures and throughout the duration of the experiment. This was achieved by assessing any potential alterations in cardiovascular variables under resting conditions and evaluating the absence of a significant withdrawal reflex upon paw pinching. To ensure stability and immobility, the animals\u0026apos; heads were securely fixed in a stereotaxic frame, with the upper incisor bar positioned 3.3 mm below the interaural line.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Interaction of dlPAG and nA: c-Fos/FoxP2/TH-ir experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn a group of 10 animals, a hole was drilled into the skull to provide access to the right dlPAG. A concentric bipolar electrode (Rhodes Medical electrodes, NE-100) was positioned in the right dlPAG.\u0026nbsp;The stereotaxic coordinates to locate the right dlPAG were from -4.0 mm caudal to bregma, 0.6 lateral to midline and 4.5 to 5 mm depth from the surface of the calota (approaching with an angle of 30\u0026ordm;)\u0026nbsp;based on the coordinates provided by the atlas of Paxinos and Watson [40].\u0026nbsp;The right dlPAG was stimulated once using 1 millisecond pulses, ranging from 30-50\u0026nbsp;\u0026mu;A, at a frequency of 100 Hertz for a duration of 5 seconds, in order to elicit the classical defense response evoked by this region. Following this, guanethidine (10 milligrams per kilogram intravenous) was administered to suppress sympathetically mediated cardiovascular responses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFollowing this, in one subgroup of animals (n=5), the right dlPAG was stimulated using trains of 1 millisecond pulses, between 30-50\u0026nbsp;\u0026mu;A, at a frequency of 100 Hertz for 5 seconds, every 60 seconds for a period of one hour. The other subgroup of animals (n=5) did not receive any stimulation and served as the control group.\u003c/p\u003e\n\u003cp\u003eOne hour after the completion of the experiments, the animals were deeply anesthetized and subjected to perfusion through the ascending aorta using a phosphate-buffered saline (PBS, pH 7.4) solution, followed immediately by a solution of 4% paraformaldehyde in 0.1M PBS (pH 7.4) to serve as the fixative. The brains were rapidly extracted and subjected to additional fixation by immersion in the same fixative solution for 24 hours at 4\u0026deg;C. Subsequently, the brains were cryoprotected using 30% phosphate-buffered sucrose for several days. The brainstem was then sectioned into 30 \u0026mu;m coronal sections obtained with a freezing microtome (CM 1325, Leica, Wetzlar, Germany). Sampling was performed by selecting every fifth section with a random starting point, and these sections were further processed to assess c-Fos/FOXP2/TH-ir at the level of nA.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003ec-Fos-TH double immunohistochemistry\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSections collected from control and stimulated rats were processed under identical \u003cem\u003efree-floating\u003c/em\u003e immunohistochemical conditions. The sections were rinsed in 0.1 M PBS (pH 7.4) and pre-treated during 15 minutes in 3% H\u003csub\u003e2\u003c/sub\u003e0\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eto eliminate endogenous peroxidase activity. After a second round of washing in PBS saline, sections were incubated overnight at room temperature (RT), using a mouse anti-c-Fos monoclonal primary antibody (1:1000 dilution in PBS containing 0.3% Triton X-100 0.2% Azida 0.02%;\u0026nbsp;Santa Cruz Biotech, ref SC-271243). A biotinylated secondary antibody (1:500 dilution; goat anti-mouse; Vector Laboratories) and ExtrAvidin Peroxidase (1:2000 dilution; Sigma-Aldrich) were used for subsequent incubations, of 1 hour at RT each. After washing in PBS (pH 7.4), staining was carried out with 3,3\u0026rsquo;-diaminobenzidine (DAB; 25mg in 100 ml Tris\u0026ndash;HCl, pH 7.4 and 0.002% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e; Sigma-Aldrich) in the presence of nickel sulphate (1%) to produce a dark reaction product. In order to investigate the presence of\u0026nbsp;c-Fos-ir in TH-ir neurons, the same sections were then incubated overnight at RT with a polyclonal primary antibody against the tyrosine-hydroxylase enzyme (1:5000 dilution; anti-rabbit;\u0026nbsp;Sigma-Aldrich, ref T2928), followed by a re-incubated 1h at RT in a biotonylated secondary antibody (1:500 dilution; goat anti-rabbit; Vector Laboratories). The TH-ir was revealed only by reacting DAB to produce a brown labelling. Sections were dehydrated and coverslipped with DPX mounting medium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eFoxP2 -TH double inmunohistochemistry\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis double labelling was performed under the same protocol as for c-Fos-TH-ir but using a sheep anti-FOXP2 primary antibody (1:1000 dilution; RD Systems, ref AF5647) and a biotinylated secondary antibody (1:500 dilution; rabbit anti-sheep; Vector Laboratories).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eCell counting and statistical analysis\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe rostrocaudal level of each brain section was determined using the Paxinos and Watson rat brain atlas [40]. The cell bodies displaying c-Fos-ir or FOXP2-ir were identified by the presence of a dark nucleus and were quantified bilaterally in the sections containing the nA using a BX61VS optical microscope (Olympus), an Olympus VS software for brightfield image acquisition and QuPath Software. All data were presented as mean \u0026plusmn; SEM. To compare the differences between groups, a one-way analysis of variance (ANOVA) was employed for statistical analyses. The significance level was set at p\u0026lt;0.05.\u003c/p\u003e\n\u003cp\u003eCell quantification along the nA was executed in accordance with the categorization into three primary subdivisions (\u003cem\u003ecompact, semicompact, and loose formation\u003c/em\u003e). To this end, distances from Bregma, as per the Paxinos Atlas (2007), were employed to guide the selection of slices across the three domains of the nA. Specifically, the criterion for demarcating cells within the \u003cem\u003ecompact region\u003c/em\u003e of the nA spanned from Bregma -12.00 mm to -12.84 mm; the \u003cem\u003esemi-compact region\u003c/em\u003e extended from Bregma -12.84 mm to -13.44 mm; and the \u003cem\u003eloose formation\u003c/em\u003e region encompassed Bregma -13.44 mm to -14.16 mm.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eInteraction of dlPAG and nA: electrical/chemical stimulation and subglottic pressure measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn 21 animals, divided in 3 groups, the described general procedures were modified. A double tracheal cannulation was carried out to develop the classical technique of the \u0026ldquo;isolated glottis\u0026nbsp;\u003cem\u003ein situ\u003c/em\u003e\u0026rdquo; and for the recording of respiratory airflow [26]. Subglottal pressure was recorded with an aneroid transducer (ADInstrument model FE141, \u0026plusmn; 0,03 psi) by passing a stream of humidified warm medical air upwards through the larynx at a constant rate of 30-70 ml/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.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003edlPAG electrical stimulation and subglottic pressure measurement\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn a subset of 7 animals, a burr hole was drilled into the skull of each animal to access to the right dlPAG. The right dlPAG was stimulated with 1 ms pulses, 30-50 \u0026micro;A given at 100 Hz for 5 s by positioning a concentric bipolar electrode (NE-100; Rhodes Medical Electrodes, Summerland, CA, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003edlPAG chemical stimulation and subglottic pressure measurement\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn 14 animals, a burr hole was drilled into the skull of each animal to access to the right dlPAG. Microinjections of a solution of PBS (50nl, pH 7.4\u0026plusmn;0.1, 5-s duration) (n=7), or glutamate (0,25M, 50nl) (n=7), through a stereotaxically positioned single glass micropipette (Hamilton 1.0\u0026micro;L Model 7001 Knurled Hub (KH) Neuros Syringe) were performed in the same coordinates cited above. Evans blue was used to dissolve all drugs and served to mark microinjection sites. Microinjections of PBS-Evans blue alone were used for control purposes. Microinjection volumes of 50 nl were programmed with a micropump controller (Ultra Micro Pump II, Micro 4; World Precision Instruments, Inc., Sarasota, FL, USA), driving 1 \u0026micro;l microsyringes attached to the micropump. Only one microinjection was delivered in each animal.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eElectrical lesions (250\u0026nbsp;\u0026mu;A\u0026nbsp;DC for 20 seconds) or Evans blue serve to locate dlPAG electrical stimulation/microinjections sites, respectively. Brains were perfused with 10 % formal saline and serially sectioned (50 \u0026micro;m) at the level of the midbrain. The midbrain was counterstained with Neutral Red.\u003c/p\u003e\n\u003cp\u003eDifferent physiological parameters were analyzed in each group of experiments under the following protocol:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(a)\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Study of laryngeal and cardiorespiratory parameters at rest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(b)\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Study of laryngeal and cardiorespiratory changes during dlPAG stimulation (electrical/chemical).\u003c/p\u003e\n\u003cp\u003eIn each animal airflow, pleural pressure (as an index of inspiratory activity), subglottic pressure and arterial pressure were monitored and stored on PC. Measurements were made of instantaneous respiratory frequency, subglottic pressure, mean blood arterial pressure and instantaneous heart rate. In all experiments baseline values of the parameters were measured immediately prior to dlPAG electrical/chemical stimulation\u0026nbsp;and after (4 min after electrical stimulation and 30 min after chemical stimulation). Changes in mean arterial blood pressure or heart rate were assessed by measuring the peak rise in blood pressure or heart rate observed during the 5 seconds electrical stimulation of the dlPAG or the maximum cardiovascular response in the chemical stimulation experiments. Stimulus-evoked changes in respiratory parameters were measured as the average response observed during the 5 seconds electrical stimulation of the dlPAG, or the maximum respiratory response in the chemical stimulation experiments. The parameter monitoring and the offline analysis was done using LabChartPro (PowerLab System, ADInstruments\u0026reg;/ LabChart Software, version 8.0, Sydney, Australia).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eStatistical analysis\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll data are expressed as mean \u0026plusmn; SEM. For statistical comparisons, once the statistical normality (Kolmogorov-Smirnov test) and the homoscedasticity (Bartlett\u0026apos;s test) of the data were verified, a paired-sample test was applied to compare the control with the evoked response period for each animal. One-way ANOVA was used to compare different groups of animals. At each time point, p\u0026lt;0.05 was regarded as significant. Only data from animals in which the histology showed that the microelectrodes were positioned within the dlPAG were used for statistical procedures.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. Interaction of dlPAG and nA: c-Fos/FoxP2/TH-ir experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eDistribution of FoxP2-ir\u0026nbsp;\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eControl animals\u003c/p\u003e\n\u003cp\u003eIn non-stimulated animals no significant differences were observed regarding the number of FOXP2-ir profiles between ipsilateral and contralateral sides in any of the three principal domains of the nA (Table 1, Fig 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eElectrically stimulated animals\u003c/p\u003e\n\u003cp\u003eBoth, loose formation (p\u0026lt;0,001) and semicompact formation (p\u0026lt;0,05) presented a significant increase in FOXP2-ir profiles in the ipsilateral side compared with the contralateral side in electrically stimulated animals (Table 1, Figs 1, 2).\u003c/p\u003e\n\u003cp\u003eControl vs stimulated animals\u003c/p\u003e\n\u003cp\u003eNo differences were found between control and electrically stimulated animals when comparing the amplitude of the differences between the number and distribution of ipsilateral or contralateral FOXP2-ir neurons within any of the domains in the nA nuclei (Table 1, Figs 1, 2).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eDistribution of c-Fos-ir\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eControl animals\u003c/p\u003e\n\u003cp\u003eA higher amount of c-Fos-ir profiles were observed in both the loose formation and semicompact formation in the ipsilateral side compared with the contralateral side (p\u0026lt;0.01) in non-stimulated animals (Table 1, Fig 2).\u003c/p\u003e\n\u003cp\u003eElectrically stimulated animals\u003c/p\u003e\n\u003cp\u003eAn significant ipsilateral predominance for c-Fos-ir staining in all the three domains of nA were observed in electrically stimulated animals: loose formation (p\u0026lt;0,001), semicompact formation (p\u0026lt;0,001) and compact formation (p\u0026lt;0,05) (Table 1, Figs 1, 2).\u003c/p\u003e\n\u003cp\u003eControl vs stimulated animals\u003c/p\u003e\n\u003cp\u003eWhen comparing the amplitude of the differences of the c-Fos-ir expression in ipsilateral and contralateral sides in control and stimulated groups, a significant higher increase was observed in both the loose formation (p=0.032) and the compact formation (p=0.002) (Table 1, Figs 1, 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Interaction of dlPAG and nA: electrical/chemical stimulation and subglottic pressure measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003edlPAG electrical stimulation and subglottic pressure measurement\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn all animals (n=7), the dlPAG electrical stimulation elicited a cardiorespiratory response characterized by tachypnoea (p\u0026lt;0,01), a decrease in laryngeal resistance (subglottal pressure) (p\u0026lt;0,01) and a pressor response (p\u0026lt;0,001) accompanied with tachycardia (p\u0026lt;0,001) (Table 2, Fig 4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003edlPAG PBS microinjections and subglottic pressure measurement\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMicroinjections of PBS within the dlPAG (n=7) did not produce changes in any of the resting cardiorespiratory parameters (Table 2, Figs 3, 5).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003edlPAG Glutamate microinjections and subglottic pressure measurement\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGlutamate microinjections within the dlPAG (n=7) evoked a decrease of laryngeal resistance (subglottal pressure) (p\u0026lt;0,001) accompanied with an inspiratory facilitatory response consisted of an increase in respiratory rate (p\u0026lt;0,001), together with a pressor (p\u0026lt;0,001) and tachycardic response (p\u0026lt;0,001) (Table 2, Figs 3, 4, 5).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003edlPAG PBS vs Glutamate microinjections and subglottic pressure measurement\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWhen comparing the amplitude of the differences at the laryngeal and cardiorespiratory responses between PBS and Glutamate microinjected animals, higher values in delta heart rate (0,29 \u0026plusmn; 2,5 to 45,57 \u0026plusmn; 3,9; p = 0,001), delta blood pressure (0,00 \u0026plusmn; 1,0 to 10,57 \u0026plusmn; 0,82; p = 0,001), delta subglottic pressure (0,05 \u0026plusmn; 0,05 to -4,60 \u0026plusmn; 0,24; p = 0,001), and delta respiratory rate (1 \u0026plusmn; 0,69 to 29,57 \u0026plusmn; 8,7; p= 0,001) were observed in stimulated animals (Fig 5).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of our study demonstrated, using neuromorphological and electroneurophysiological techniques, the existence of functional interactions between the dlPAG, a mesencephalic region known for its involvement in cardiorespiratory regulation, and the nA, a medullary region containing the majority of the motor neurons that govern the laryngeal musculature. Firstly, we demonstrated that electrical stimulation of dlPAG induces a significant increase in cFos-ir expression in the \u003cem\u003eloose formation\u003c/em\u003e and \u003cem\u003ecompact formation\u003c/em\u003e domains of the nA. Secondly, we show that the \u003cem\u003eloose formation\u0026nbsp;\u003c/em\u003eand \u003cem\u003esemicompact formation\u003c/em\u003e regions are the nA subdivisions with the highest expression of FOXP2-ir, while the \u003cem\u003ecompact\u003c/em\u003e region shows the lowest expression. No significant changes in FOXP2 expression occur after dlPAG stimulation in either region. Finally, using the \u0026quot;isolated glottis\u003cem\u003e\u0026nbsp;in situ\u003c/em\u003e\u0026quot; technique together with classical electrophysiological techniques, we demonstrate for the first time how dlPAG is involved in the control of laryngeal resistance.\u003c/p\u003e\n\u003cp\u003eAll these results suggest that, in our experimental conditions, the dlPAG modulates the activity of laryngeal motoneurons located within loose formation of the nA, thereby influencing the striated laryngeal muscles of the upper airway, during the concomitant cardiorespiratory changes evoked by dlPAG electrical or chemical stimulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eInteraction of dlPAG and nA: c-Fos and FOXP2 expression\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe neuronal cell bodies responsible for innervating the intrinsic muscles of the larynx are situated within nA. This cell-column comprises neurons oriented in a rostrocaudal direction and is positioned in the ventrolateral region of the medulla oblongata. Its spatial extent ranges from the motor nucleus of the facial nerve to at least the level of the pyramidal decussation [3]. We could divide the nA into three main parts or domains: \u003cem\u003ethe compact formation\u003c/em\u003e, with motor neurons innervating the oesophagus; the \u003cem\u003esemi-compact formation\u003c/em\u003e, with motor neurons innervating the pharynx and the cricothyroid muscle of the larynx, i.e. that which is innervated by the superior laryngeal nerve; and the \u003cem\u003eloose formation\u003c/em\u003e, with motor neurons innervating the laryngeal muscles except the cricothyroid. It is therefore accepted that laryngeal neurons are located the caudal part of the nA (\u003cem\u003esemi-compact and loose formation\u003c/em\u003e). Thus, the nA, in addition to innervating the laryngeal muscles, also provides motor innervation to the oesophagus and pharynx [16, 38, 54].\u003c/p\u003e\n\u003cp\u003eIn the present study, the guanethidine, a sympatholytic agent, was administered to prevent secondary c-Fos expression resulting from alterations in arterial blood pressure. Thus, despite the blockage of the cardiovascular changes, the nA presents a significant increase of ipsilateral c-Fos-ir within the three principal domains of the nA after electrical stimulation of the dlPAG. When comparing the differences between stimulated and control animals in the increase of cFos-ir between ipsilateral and contralateral side, only the \u003cem\u003eloose formation\u003c/em\u003e and \u003cem\u003ecompact formation\u003c/em\u003e presents significant changes in their activity\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTherefore, both populations of neurons of the nA seem likely to be activated directly or indirectly from the dlPAG and not secondarily to blood pressure changes evoked from the dlPAG during electrical stimulation. Thus, this data suggests a possible role of the dlPAG in the glottal opening/closing movements regulating the laryngeal resistance during innate vocalizations or during the respiratory responses developed in the classical defence response vehiculated by this mesencephalic region.\u003c/p\u003e\n\u003cp\u003eWhen examining the data of FOXP2 nuclei, we confirmed a high level of expression at the level of \u003cem\u003esemicompact formation\u003c/em\u003e (controls the vocal fold tension) and \u003cem\u003eloose formation\u003c/em\u003e (regulates the subglottic pressure during vibration) [33], giving a role to the neurons of these domains in the production of a laryngeal-respiratory motor patterns necessaries for the correct production of species-specific vocalisations. It should be noticed that FOXP2 expression levels were not modified by electrical stimulation of the dlPAG. Regarding these quantitative data from FOXP2-ir, it should be noted that they are in line with previous studies in which numerical approximations were made within these domains [38], confirming that the expression of this transcription factor is a characteristic rather than an expression of cellular activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInteraction of dlPAG and nA: electrical/chemical stimulation experiments and subglottic pressure\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eChemical microstimulation of the PAG reveals that different columns of the PAG coordinate different types of responses depending on the stimulus [1, 4, 10, 28], allowing the modulation of an autonomic response dependent on the type of stress and the individual\u0026apos;s subjective perception of a threat or stressful stimulus. For this purpose, the PAG presents many afferences and efferences projections. The most important afferences have their origin from the prefrontal cortex, amygdala and hypothalamus [9, 23, 27, 45, 51, 52]. The PAG, in turn, projects to the pontomedullary cardiorespiratory nuclei involved in cardiorespiratory rhythmogenesis that allows the development of different patterns of cardiorespiratory and motor responses depending on the type of stimulus [10, 11, 24, 30]. Specifically, the dlPAG column controls flight/flight, or coping/fighting behavioural responses, triggering a defence response that is haemodynamically characterised by hypertension, tachycardia and redistribution of blood flow to the skeletal muscles of the extremities from the abdominal and visceral area. In addition, the response is accompanied by mydriasis, tachypnoea, increased tidal volume and vocalisations. This allows the animal to cope with environmental stress situations that require a rapid response [30, 35, 52].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOther functions of PAG include thermoregulation, participation in wakefulness and sleep mechanisms, or modulation of neuropathic pain or micturition [7, 19, 25]. At clinical level, its activity is modified in different neurodegenerative processes such as Alzheimer\u0026apos;s and multisystemic atrophy [2, 37].\u003c/p\u003e\n\u003cp\u003eMoreover, the PAG assumes a pivotal role in the regulation of vocalizations [21, 47]. This is facilitated by its robust connectivity with the cVRG within the nRA, an exclusive cerebral region uniquely dedicated to directly influencing all requisite motor areas associated with vocalization [20]. Regarding this role, neurochemical microstimulation within the PAG produces vocalisations in cats [49], monkey [22], birds [41] and humans [20] corresponding with the sound part of human speech.\u003c/p\u003e\n\u003cp\u003eWe have previously demonstrated, using the \u0026ldquo;isolated glottis \u003cem\u003ein situ\u0026rdquo;\u003c/em\u003e technique, that glottic caliber is modulated by pontine regions (cPB and A5 region) [26].\u0026nbsp;This technique has some advantages, as it separates the lower airways from the larynx, facilitates the study of modifications in neuro-muscular tone and abolishes the influence of variations of the airflow activating laryngeal reflexes.\u0026nbsp;In that sense, there have been few developments in subglottic pressure measurement studies. This is possibly attributed to the intricacy of the surgical technique and the challenge of maintaining a stable recording of subglottic pressure.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHence, it is of particular interest to reapply this technique to measure changes in subglottic pressure associated with the cardiorespiratory response evoked from dlPAG stimulation. For this purpose, we have maintained the original design, updating the means of obtaining the subglottic pressure variable thanks to new models of both aneroid transducer (ADInstrument model FE141, \u0026plusmn; 0.03 psi) and a digital thermal mass air flowmeter controller (Bronkhorst Hi-Tec F-201CV-AGD-22-V). In addition, we have upgraded both the data processing software (LabChartPro) and the signal acquisition hardware (PowerLab 16/30). Thanks to these updates we have been able to improve the sensitivity and stability of the subglottic pressure signal. In fact, in our study, we have included for statistical analysis only data from animals in which stable recordings were observed before, during and after dlPAG stimulation, providing\u0026nbsp;with new data on the laryngeal effects induced by the dlPAG.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTherefore, electrical stimulation was used primarily to locate dlPAG and to study the laryngeal calibre through the measure of the subglottal pressure. In a second group of animals, we have developed chemical stimulation with glutamate microinjections within dlPAG. While the effects of electrical stimulation are very difficult to interpret, the effects of glutamate microinjection suggested that the elicited laryngeal and cardiorespiratory responses can be attributed to the activation of cell bodies located in these dlPAG region but not to the activation of axons of passage.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, we can conclude that the chemical stimulation of the dlPAG with glutamate produced a sympathoexcitatory response characterized by hypertension, tachycardia and tachypnoea. All these changes were accompanied with a reduction in subglottal pressure. The glottal dilatation during the inspiratory respiratory response facilitates respiratory movements because of the decrease of the upper airway resistance. These findings suggest the involvement of this mesencephalic region in the regulation of laryngeal activity. The magnitude of the changes in subglottal pressure and cardiorespiratory variables were similar, except the arterial pressure, using both methods of stimulation. Thus, our present observations suggest that the dlPAG has a role in modifying the activity of laryngeal motoneurons localised in the nA and accordingly the striated laryngeal muscles of the upper airway.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConflicts of interest\u003c/em\u003e\u003c/strong\u003e: The authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthical approval\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e:\u003c/em\u003e 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 Andaluc\u0026iacute;a. Every attempt was made to reduce animal suffering, discomfort and the total number of animals needed to obtain reliable results.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of supporting data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data included in the study are available in a generalist data repository provided by the University of M\u0026aacute;laga (https://riuma.uma.es/xmlui/ )\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no commercial or financial conflicts of interest in association with the present study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding for open access publishing: Universidad M\u0026aacute;laga/CBUA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe study was supported by a program grant Junta de Andaluc\u0026iacute;a, Groups n\u0026ordm; CTS-156, CTS-160 and\u0026nbsp;UMA20-FEDERJA-122,\u0026nbsp;Spain.\u003c/p\u003e\n\u003cp\u003ePart of the final study was supported with a grant from the Own Funds Program of the University of M\u0026aacute;laga.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMVLG and MSDM\u0026nbsp;conceived and designed the research. MGG and LCF performed the experiments. \u0026nbsp;MGG, LCF and CML analysed the data.\u0026nbsp;MVLG, MGG and MSDM interpreted the results of the experiments. MGG, LCF, MPV and BG made the immunohistochemistry and interpreted the data.\u0026nbsp;MVLG, LCF and MGG drafted manuscript. BG and MSDM edited and revised manuscript.\u0026nbsp;All authors provided critical and intellectual contributions on previous version of the manuscript and approved the final version. The authors declare that all data were generated in-house and that no paper mill was used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMarta Gonz\u0026aacute;lez-Garc\u0026iacute;a\u003csup\u003e1,2,3\u0026nbsp;\u003c/sup\u003e* \u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eLaura Carrillo-Franco\u003csup\u003e1,3 #\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eCarmen Morales-Luque\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eMarina Ponce-Velasco\u003csup\u003e,3,4\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eBel\u0026eacute;n Gago\u003csup\u003e1,3\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eMarc Stefan Dawid-Milner\u003csup\u003e1,2,3 \u0026dagger;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eManuel V\u0026iacute;ctor L\u0026oacute;pez-Gonz\u0026aacute;lez\u003csup\u003e1,2,3\u003c/sup\u003e*\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e(1)\u003c/strong\u003e Department of Human Physiology, Faculty of Medicine, University of Malaga, Malaga (Spain)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(2)\u003c/strong\u003e Unit of Neurophysiology of the Autonomic Nervous System (CIMES), University of Malaga, M\u0026aacute;laga (Spain)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(3)\u003c/strong\u003e IBIMA Plataforma BIONAND, M\u0026aacute;laga, Spain\u003c/p\u003e\n\u003cp\u003e(4) Department of Cell Biology, University of M\u0026aacute;laga, M\u0026aacute;laga, Spain\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e(#)\u003c/strong\u003e M. Gonz\u0026aacute;lez-Garc\u0026iacute;a and L. Carrillo-Franco both contributed equally to this study and must be considered \u003cstrong\u003efirst authors\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(\u0026dagger;)\u003c/strong\u003e M.V. L\u0026oacute;pez-Gonz\u0026aacute;lez and M.S. Dawid-Milner both had equally responsibility and must be considered \u003cstrong\u003elast authors\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBandler R, Keay KA, Floyd N, Price J (2000). Central circuits mediating patterned autonomic activity during active vs. passive emotional coping. Brain Res Bull 53:95\u0026ndash;104. https://doi.org/10.1016/S0361-9230(00)00313-0 \u003c/li\u003e\n\u003cli\u003eBenarroch EE, Schmeichel AM, Low PA, Parisi JE (2010). Differential involvement of the periaqueductal gray in multiple system atrophy. 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Ann Neurol 48:344-53. https://doi.org/10.1002/1531-8249(200009)48:3\u0026lt;344::AID-ANA9\u0026gt;3.0.CO;2-S\u003c/li\u003e\n\u003cli\u003ePascual-Font A, Hern\u0026aacute;ndez-Morato I, McHanwell S, V\u0026aacute;zquez T, Maranillo E, Sa\u0026ntilde;udo J, Valderrama-Canales FJ (2011). The central projections of the laryngeal nerves in the rat. J Anat 219:217\u0026ndash;228. https://doi.org/10.1111/j.1469-7580.2011.01390.x\u003c/li\u003e\n\u003cli\u003ePathak S, Slovarp L, Clary MS, Jett\u0026eacute; ME (2020). Laryngeal Chemoreflex in Health and Disease: A Review. Chem Senses 45:823-831. https://doi.org/10.1093/chemse/bjaa069\u003c/li\u003e\n\u003cli\u003ePaxinos G, Watson C (2007). The Rat Brain in Stereotaxic Coordinates, 6th Ed. San Diego, Elsevier Academic Press.\u003c/li\u003e\n\u003cli\u003eSchmidt MF, Wild JM (2014) The respiratory-vocal system of songbirds: anatomy, physiology, and neural control. Prog Brain Res 212:297-335. https://doi.org/10.1016/B978-0-444-63488-7.00015-X\u003c/li\u003e\n\u003cli\u003eSchulz SB, Haesler S, Scharff C, Rochefort C (2010). Knockdown of FoxP2 alters spine density in Area X of the zebra finch. Genes Brain Behav 9:732-40. https://doi.org/10.1111/j.1601-183X.2010.00607.x\u003c/li\u003e\n\u003cli\u003eShu W et al (2005). Altered ultrasonic vocalization in mice with a disruption in the Foxp2 gene. Proc Natl Acad Sci USA 102:9643\u0026ndash;9648. https://doi.org/10.1073/pnas.0503739102\u003c/li\u003e\n\u003cli\u003eShu W, Yang H, Zhang L, Lu MM, Morrisey EE (2001). Characterization of a new subfamily of winged-helix/forkhead (Fox) genes that are expressed in the lung and act as transcriptional repressors. J Biol Chem 276:27488\u0026ndash;27497. https://doi.org/10.1074/jbc.M100636200\u003c/li\u003e\n\u003cli\u003eSingh U et al (2022). Neuroanatomical organization and functional roles of PVN MC4R pathways in physiological and behavioral regulations. \u003cem\u003eMol Metab\u003c/em\u003e\u003cem\u003e55\u003c/em\u003e:101401. https://doi.org/10.1016/j.molmet.2021.101401\u003c/li\u003e\n\u003cli\u003eStanić D, Dhingra RR, Dutschmann M (2018). Expression of the transcription factor FOXP2 in brainstem respiratory circuits of adult rat is restricted to upper-airway pre-motor areas. Respir Physiol Neurobiol 250:14-18. https://doi.org/10.1016/j.resp.2018.01.014\u003c/li\u003e\n\u003cli\u003eSubramanian HH, Balnave RJ, Holstege G (2021). Microstimulation in Different Parts of the Periaqueductal Gray Generates Different Types of Vocalizations in the Cat. J Voice 35:804.e9\u0026ndash;804.e25. https://doi.org/10.1016/j.jvoice.2020.01.022\u003c/li\u003e\n\u003cli\u003eSubramanian HH, Holstege G (2009). The nucleus retroambiguus control of respiration. J Neurosci 29:3824\u0026ndash;3832. https://doi.org/10.1523/JNEUROSCI.0607-09.2009\u003c/li\u003e\n\u003cli\u003eSubramanian HH, Holstege G (2014). The midbrain periaqueductal gray changes the eupneic respiratory rhythm into a breathing pattern necessary for survival of the individual and of the species. Prog Brain Res 212:351-84. https://doi.org/10.1016/B978-0-444-63488-7.00017-3\u003c/li\u003e\n\u003cli\u003eSubramanian HH, Huang ZG, Silburn PA, Balnave RJ, Holstege G (2018). The physiological motor patterns produced by neurons in the nucleus retroambiguus in the rat and their modulation by vagal, peripheral chemosensory, and nociceptive stimulation. J Comp Neurol 526:229-242. https://doi.org/10.1002/cne.24318\u003c/li\u003e\n\u003cli\u003eTovote P (2016). Midbrain circuits for defensive behaviour. Nature 534:206-12. doi:10.1038/nature17996\u003c/li\u003e\n\u003cli\u003eTrevizan-Ba\u0026uacute; P, Dhingra RR, Furuya WI, Stanić D, Mazzone SB, Dutschmann M (2021). Forebrain projection neurons target functionally diverse respiratory control areas in the midbrain, pons, and medulla oblongata. J Comp Neurol \u003cem\u003e529\u003c/em\u003e:2243\u0026ndash;2264. https://doi.org/10.1002/cne.25091\u003c/li\u003e\n\u003cli\u003eVanderhorst VG, Terasawa E, Ralston HJ, Holstege G (2000). Monosynaptic projections from the lateral periaqueductal gray to the nucleus retroambiguus in the rhesus monkey: implications for vocalization and reproductive behavior. J Comp Neurol 424:251-268. https://doi.org/10.1002/1096-9861(20000821)424:2%3C251::AID-CNE5%3E3.0.CO;2-D\u003c/li\u003e\n\u003cli\u003eWeissbrod P, Pitman MJ, Sharma S, Bender A, Schaefer SD. Quantity and three-dimensional position of the recurrent and superior laryngeal nerve lower motor neurons in a rat model. Ann Otol Rhinol Laryngol 120:761-8. https://doi.org/10.1177/000348941112001\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRelative quantification and distribution of c-Fos and FOXP2 inmunoreactive cells (cFos-ir and FOXP2-ir) within the nA in not stimulated animals (control) and dlPAG electrically stimulated animals (stimulated). Both groups are spontaneously breathing rats in which sympathetically mediated cardiovascular changes were abolished with guanethidine. The nA has been divided into its 3 classical domains (loose, semi-compact and compact) and ipsilateral and contralateral sides were also analysed.\u0026nbsp;\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"690\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.3768115942029%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"83.6231884057971%\" colspan=\"5\"\u003e\n \u003cp\u003e\u003cstrong\u003eNEUROMORPHOLOGICAL STUDY\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.7536231884058%\" colspan=\"2\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.89855072463768%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eCONTROL (n=5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"34.34782608695652%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eSTIMULATED (n=5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.406047516198704%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIpsilateral\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.406047516198704%\"\u003e\n \u003cp\u003e\u003cstrong\u003eContralateral\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.406047516198704%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIpsilateral\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.781857451403887%\"\u003e\n \u003cp\u003e\u003cstrong\u003eContralateral\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.400580551523948%\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eLOOSE FORMATION\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.400580551523948%\"\u003e\n \u003cp\u003e\u003cstrong\u003ec-Fos-ir cells\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.400580551523948%\"\u003e\n \u003cp\u003e29.6 \u0026plusmn; 5.1**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.400580551523948%\"\u003e\n \u003cp\u003e18.2 \u0026plusmn; 2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.400580551523948%\"\u003e\n \u003cp\u003e42.8 \u0026plusmn; 5.8***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.99709724238026%\"\u003e\n \u003cp\u003e22.6 \u0026plusmn; 4.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.618055555555557%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFOXP2-ir cells\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.618055555555557%\"\u003e\n \u003cp\u003e115 \u0026plusmn; 18.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.618055555555557%\"\u003e\n \u003cp\u003e93.4 \u0026plusmn; 14.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.618055555555557%\"\u003e\n \u003cp\u003e115.4 \u0026plusmn; 15.5***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.52777777777778%\"\u003e\n \u003cp\u003e88.8 \u0026plusmn; 17.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.584055459272097%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDelta c-Fos-ir\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.341421143847484%\" colspan=\"2\"\u003e\n \u003cp\u003e11.4 \u0026plusmn; 2.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"41.074523396880416%\" colspan=\"2\"\u003e\n \u003cp\u003e20.2 \u0026plusmn; 2.4\u003cstrong\u003e\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.584055459272097%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDelta FOXP2-ir\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.341421143847484%\" colspan=\"2\"\u003e\n \u003cp\u003e21.6 \u0026plusmn; 8.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"41.074523396880416%\" colspan=\"2\"\u003e\n \u003cp\u003e26.6 \u0026plusmn; 2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.400580551523948%\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eSEMICOMPACT\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFORMATION\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.400580551523948%\"\u003e\n \u003cp\u003e\u003cstrong\u003ec-Fos-ir cells\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.400580551523948%\"\u003e\n \u003cp\u003e23.6 \u0026plusmn; 3.8**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.400580551523948%\"\u003e\n \u003cp\u003e14.6 \u0026plusmn; 3.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.400580551523948%\"\u003e\n \u003cp\u003e31 \u0026plusmn; 3.3***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.99709724238026%\"\u003e\n \u003cp\u003e16.8 \u0026plusmn; 2.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.618055555555557%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFOXP2-ir cells\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.618055555555557%\"\u003e\n \u003cp\u003e105 \u0026plusmn; 34.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.618055555555557%\"\u003e\n \u003cp\u003e75.3 \u0026plusmn; 26.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.618055555555557%\"\u003e\n \u003cp\u003e104.5 \u0026plusmn; 26.2*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.52777777777778%\"\u003e\n \u003cp\u003e81.5 \u0026plusmn; 24.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.584055459272097%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDelta c-Fos-ir\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.341421143847484%\" colspan=\"2\"\u003e\n \u003cp\u003e9 \u0026plusmn; 1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"41.074523396880416%\" colspan=\"2\"\u003e\n \u003cp\u003e14.2 \u0026plusmn; 1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.584055459272097%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDelta FOXP2-ir\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.341421143847484%\" colspan=\"2\"\u003e\n \u003cp\u003e29.8 \u0026plusmn; 12.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"41.074523396880416%\" colspan=\"2\"\u003e\n \u003cp\u003e23 \u0026plusmn; 5.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.400580551523948%\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eCOMPACT FORMATION\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.400580551523948%\"\u003e\n \u003cp\u003e\u003cstrong\u003ec-Fos-ir cells\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.400580551523948%\"\u003e\n \u003cp\u003e1.6 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.400580551523948%\"\u003e\n \u003cp\u003e1.4 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.400580551523948%\"\u003e\n \u003cp\u003e3.4 \u0026plusmn; 0.8*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.99709724238026%\"\u003e\n \u003cp\u003e0.4 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.618055555555557%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFOXP2-ir cells\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.618055555555557%\"\u003e\n \u003cp\u003e10.8 \u0026plusmn; 2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.618055555555557%\"\u003e\n \u003cp\u003e4.5 \u0026plusmn; 0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.618055555555557%\"\u003e\n \u003cp\u003e10 \u0026plusmn; 4.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.52777777777778%\"\u003e\n \u003cp\u003e6.5 \u0026plusmn; 3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.584055459272097%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDelta c-Fos-ir\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.341421143847484%\" colspan=\"2\"\u003e\n \u003cp\u003e0.2 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"41.074523396880416%\" colspan=\"2\"\u003e\n \u003cp\u003e3 \u0026plusmn; 0.8\u003cstrong\u003e\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"19.584055459272097%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDelta FOXP2-ir\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.341421143847484%\" colspan=\"2\"\u003e\n \u003cp\u003e6.3 \u0026plusmn; 1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"41.074523396880416%\" colspan=\"2\"\u003e\n \u003cp\u003e3.5 \u0026plusmn; 1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eData are expressed as mean \u0026plusmn; SEM. Asterisks show differences between ipsilateral and contralateral sides in control or stimulated animals. (* p\u0026lt;0.05; ** p\u0026gt;0.01; **p\u0026lt;0.001). Daggers show differences between the delta differences between control and stimulated groups (\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003ep\u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLaryngeal and cardiorespiratory parameters measured \u003cstrong\u003ebefore\u003c/strong\u003e electrical or chemical stimulation of dlPAG (\u003cstrong\u003eRest\u003c/strong\u003e), \u003cstrong\u003eduring\u003c/strong\u003e electrical or chemical stimulation (microinjections of PBS or Glutamate) within the dlPAG (\u003cstrong\u003eStimulation\u003c/strong\u003e) and 4 minutes \u003cstrong\u003eafter\u003c/strong\u003e electrical or 30 minutes \u003cstrong\u003eafter\u003c/strong\u003e chemical stimulation of dlPAG (\u003cstrong\u003eRecovery\u003c/strong\u003e). \u0026nbsp;\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.139705882352942%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"56.61764705882353%\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eNEUROPHARMACOLOGICAL STUDY\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.24264705882353%\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.139705882352942%\"\u003e\n \u003cp\u003e\u003cstrong\u003eELECTRICAL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.63970588235294%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eRest\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.977941176470587%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eStimulation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.24264705882353%\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eRecovery\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003edlPAG (\u003cem\u003en=7)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.63970588235294%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.977941176470587%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.24264705882353%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003eRR (rpm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.63970588235294%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e104\u0026nbsp;\u0026plusmn;\u0026nbsp;9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.977941176470587%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e145\u0026nbsp;\u0026plusmn;\u0026nbsp;15\u003cstrong\u003e**\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.24264705882353%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e105\u0026nbsp;\u0026plusmn;\u0026nbsp;9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003eTE (s\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.63970588235294%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.343\u0026nbsp;\u0026plusmn;\u0026nbsp;0.028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.977941176470587%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.233\u0026nbsp;\u0026plusmn;\u0026nbsp;0.021\u003cstrong\u003e**\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.24264705882353%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e0.341\u0026nbsp;\u0026plusmn;\u0026nbsp;0.025\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003eTI (s\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.63970588235294%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.261\u0026nbsp;\u0026plusmn;\u0026nbsp;0.021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.977941176470587%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.209\u0026nbsp;\u0026plusmn;\u0026nbsp;0.026\u003cstrong\u003e*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.24264705882353%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e0.259\u0026nbsp;\u0026plusmn;\u0026nbsp;0.019\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" colspan=\"2\"\u003e\n \u003cp\u003eSGP (cmH\u003csub\u003e2\u003c/sub\u003eO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.169117647058826%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e8.1 \u0026plusmn; 1.3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e3.3\u0026nbsp;\u0026plusmn;\u0026nbsp;0.3\u003cstrong\u003e**\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e8.1\u0026nbsp;\u0026plusmn;\u0026nbsp;1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5514705882352942%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003eBP (mmHg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.63970588235294%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e106\u0026nbsp;\u0026plusmn;\u0026nbsp;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.977941176470587%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e148\u0026nbsp;\u0026plusmn;\u0026nbsp;6\u003cstrong\u003e***\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.24264705882353%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e104\u0026nbsp;\u0026plusmn;\u0026nbsp;4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003eHR (bpm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.63970588235294%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e408\u0026nbsp;\u0026plusmn;\u0026nbsp;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.977941176470587%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e435\u0026nbsp;\u0026plusmn;\u0026nbsp;10\u003cstrong\u003e***\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.24264705882353%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e410\u0026nbsp;\u0026plusmn;\u0026nbsp;7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePBS\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.169117647058826%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eRest\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eStimulation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRecovery\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5514705882352942%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003edlPAG (\u003cem\u003en=7)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.169117647058826%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5514705882352942%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" colspan=\"2\"\u003e\n \u003cp\u003eRR (rpm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.169117647058826%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e85\u0026nbsp;\u0026plusmn;\u0026nbsp;7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e86\u0026nbsp;\u0026plusmn;\u0026nbsp;7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e85\u0026nbsp;\u0026plusmn;\u0026nbsp;7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5514705882352942%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eTE (s\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.169117647058826%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.394\u0026nbsp;\u0026plusmn;\u0026nbsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.385\u0026nbsp;\u0026plusmn;\u0026nbsp;0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e0.393\u0026nbsp;\u0026plusmn;\u0026nbsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5514705882352942%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eTI (s\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.169117647058826%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.311\u0026nbsp;\u0026plusmn;\u0026nbsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.312\u0026nbsp;\u0026plusmn;\u0026nbsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e0.312\u0026nbsp;\u0026plusmn;\u0026nbsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5514705882352942%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" colspan=\"2\"\u003e\n \u003cp\u003eSGP (cmH\u003csub\u003e2\u003c/sub\u003eO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.169117647058826%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e10.4 \u0026plusmn; 0.6\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e10.5\u0026nbsp;\u0026plusmn;\u0026nbsp;0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e10.4\u0026nbsp;\u0026plusmn;\u0026nbsp;0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5514705882352942%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" colspan=\"2\"\u003e\n \u003cp\u003eBP (mmHg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.169117647058826%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e103\u0026nbsp;\u0026plusmn;\u0026nbsp;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e104\u0026nbsp;\u0026plusmn;\u0026nbsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e103 \u0026plusmn; 2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5514705882352942%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" colspan=\"2\"\u003e\n \u003cp\u003eHR (bpm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.169117647058826%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e394\u0026nbsp;\u0026plusmn;\u0026nbsp;8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e395\u0026nbsp;\u0026plusmn;\u0026nbsp;7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e393\u0026nbsp;\u0026plusmn;\u0026nbsp;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5514705882352942%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGLUTAMATE\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.169117647058826%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eRest\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eStimulation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRecovery\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5514705882352942%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003edlPAG (\u003cem\u003en=7)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.169117647058826%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5514705882352942%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" colspan=\"2\"\u003e\n \u003cp\u003eRR (rpm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.169117647058826%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e81\u0026nbsp;\u0026plusmn;\u0026nbsp;5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e111\u0026nbsp;\u0026plusmn;\u0026nbsp;13\u003cstrong\u003e***\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e83\u0026nbsp;\u0026plusmn;\u0026nbsp;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5514705882352942%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eTE (s\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.169117647058826%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.474\u0026nbsp;\u0026plusmn;\u0026nbsp;0.038\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.346\u0026nbsp;\u0026plusmn;\u0026nbsp;0.037\u003cstrong\u003e***\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e0.472\u0026nbsp;\u0026plusmn;\u0026nbsp;0.035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5514705882352942%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eTI (s\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.169117647058826%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.287\u0026nbsp;\u0026plusmn;\u0026nbsp;0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e0.244\u0026nbsp;\u0026plusmn;\u0026nbsp;0.023\u003cstrong\u003e*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e0.286\u0026nbsp;\u0026plusmn;\u0026nbsp;0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5514705882352942%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" colspan=\"2\"\u003e\n \u003cp\u003eSGP (cmH\u003csub\u003e2\u003c/sub\u003eO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.169117647058826%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e9.4 \u0026plusmn; 0.4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e4.6\u0026nbsp;\u0026plusmn;\u0026nbsp;0.4\u003cstrong\u003e***\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e9.8\u0026nbsp;\u0026plusmn;\u0026nbsp;0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5514705882352942%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" colspan=\"2\"\u003e\n \u003cp\u003eBP (mmHg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.169117647058826%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e105\u0026nbsp;\u0026plusmn;\u0026nbsp;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e116\u0026nbsp;\u0026plusmn;\u0026nbsp;4\u003cstrong\u003e***\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e107 \u0026plusmn; 4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5514705882352942%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" colspan=\"2\"\u003e\n \u003cp\u003eHR (bpm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.169117647058826%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e391\u0026nbsp;\u0026plusmn;\u0026nbsp;8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e436\u0026nbsp;\u0026plusmn;\u0026nbsp;8\u003cstrong\u003e***\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.139705882352942%\" valign=\"top\"\u003e\n \u003cp\u003e393\u0026nbsp;\u0026plusmn;\u0026nbsp;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0.5514705882352942%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;Data are expressed as mean \u0026plusmn; SEM. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 Stimulation vs Rest. Abbreviations: RR,\u003c/p\u003e\n\u003cp\u003eRespiratory Rate; TE, Espiratory Time; TI, Inspiratory Time; SGP, Subglottic Pressure; BP, Blood Pressure;\u003c/p\u003e\n\u003cp\u003eHR, Heart Rate.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"pflugers-archiv-european-journal-of-physiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paej","sideBox":"Learn more about [Pflügers Archiv - European Journal of Physiology](http://link.springer.com/journal/424)","snPcode":"424","submissionUrl":"https://submission.nature.com/new-submission/424/3","title":"Pflügers Archiv - European Journal of Physiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"dorsolateral periaqueductal gray matter, subglottic pressure, nucleus ambiguous, laryngeal motoneurons, central cardiorespiratory control, rat ","lastPublishedDoi":"10.21203/rs.3.rs-3891131/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3891131/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"To assess the possible interactions between the dorsolateral Periaqueductal Gray matter (dlPAG) and the laryngeal motor neurons of the nucleus Ambiguus (nA), we have examined the pattern of double staining c-Fos/FoxP2 protein immunoreactivity (c-Fos-ir/Fox-P2-ir) and Tyrosine Hydroxylase (TH) throughout the rostro-caudal extent of nA in spontaneously breathing anaesthetised male Sprague–Dawley rats during dlPAG electrical stimulation. Activation of the dlPAG elicited a selective increase in c-Fos-ir with an ipsilateral predominance in the somatas of the loose (p\u003c0.05) and compact formation (p\u003c0.01) within the nA and confirm the expression of FoxP2 bilaterally in all the domains within the nA. A second group of experiments was made to examine the importance of the dlPAG in modulating the laryngeal response evoked after electrical or chemical (glutamate) dlPAG stimulations. Both electrical and chemical stmulations evoked a significant decrease of laryngeal resistance (subglottal pressure) (p\u003c0.001) accompanied with 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 mesencephalic neuronal circuits in the control mechanisms of subglottic pressure and laryngeal activity.","manuscriptTitle":"Uncovering the neural control of laryngeal activity and subglottic pressure in anaesthetized rats: insights from mesencephalic regions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-29 16:33:23","doi":"10.21203/rs.3.rs-3891131/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-11T08:54:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-05T06:04:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"153e6ec5-d5ff-438c-b73b-a2b8f1e9cd97","date":"2024-01-26T03:49:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-25T15:57:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-25T06:22:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-25T06:22:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Pflügers Archiv - European Journal of Physiology","date":"2024-01-23T12:56:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"pflugers-archiv-european-journal-of-physiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paej","sideBox":"Learn more about [Pflügers Archiv - European Journal of Physiology](http://link.springer.com/journal/424)","snPcode":"424","submissionUrl":"https://submission.nature.com/new-submission/424/3","title":"Pflügers Archiv - European Journal of Physiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d75aaea9-2929-4df5-93dd-c001787fffc5","owner":[],"postedDate":"January 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-21T20:38:27+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-29 16:33:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3891131","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3891131","identity":"rs-3891131","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}