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% H202 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% H2O2; 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.