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J Appl Physiol 93: 440-449, 2002. First published February 15, 2002; doi:10.1152/japplphysiol.00752.2001
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Vol. 93, Issue 2, 440-449, August 2002

Activation of the midbrain periaqueductal gray induces airway smooth muscle relaxation

Musa A. Haxhiu1,2,4, Bryan K. Yamamoto3, Ismail A. Dreshaj4, and Donald G. Ferguson2

1 Department of Physiology and Biophysics, College of Medicine Howard University and Specialized Neuroscience Research Program of Howard University, Washington, DC 20059; Departments of 2 Anatomy, 3 Neuroscience, 4 Pediatrics, and 5 Psychiatry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we examined effects of chemical stimulation of the ventrolateral region of the midbrain periaqueductal gray (vl PAG) on airway smooth muscle tone. We observed that in anesthetized, paralyzed, and artificially ventilated ferrets, vl PAG stimulation elicited airway smooth muscle relaxation. To clarify the mechanisms underlying this observation, we examined the GABA-GABAA receptor signaling pathway by 1) examining the expression of GABAA receptors on airway-related vagal preganglionic neurons (AVPNs) located in the rostral nucleus ambiguus region (rNA), by use of receptor immunochemistry and confocal microscopy; 2) measuring GABA release within the rNA by using microdialysis; and 3) performing physiological experiments to determine the effects of selective blockade of GABAA receptors expressed by AVPNs in the rNA region on vl PAG-induced airway relaxation, thereby defining the role of the GABAA receptor subtype in this process. We observed that AVPNs located in the rNA region do express the GABAA receptor beta -subtype. In addition, we demonstrated that activation of vl PAG induced GABA release within the rNA region, and this release was associated with airway smooth muscle relaxation. Blockade of the GABAA receptor subtype expressed by AVPNs in the rNA by bicuculline diminished the inhibitory effects of vl PAG stimulation on airway smooth muscle tone. These data indicate, for the first time, that activation of vl PAG dilates the airways by a release of GABA and activation of GABAA receptors expressed by AVPNs.

central control of airway smooth muscle tone; airway dilation; ventrolateral periaqueductal gray; nucleus ambiguus; airway-related vagal preganglionic neurons; parasympathetic nervous system; microdialysis; GABA; GABAA receptors; bicuculline


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PARASYMPATHETIC OUTFLOW to the airways depends primarily on inputs arising from afferent receptors of the respiratory tract that are transmitted via vagal afferents to the nucleus of the tractus solitarius (NTS). In the NTS, signals are processed and then sent to airway-related vagal preganglionic neurons (AVPNs), located in the most rostral parts of the dorsal vagal nucleus and in the rostral nucleus ambiguus (rNA) (15, 19). From these preganglionic neurons, cholinergic outflow is wired to the tracheobronchial effector systems, i.e., the airway vasculature, submucosal glands, and smooth muscle via efferent descending fibers and airway intramural ganglia (6).

The responses of airway smooth muscle tone to sensory information, including inputs from the peripheral and central chemoreceptors, can be reduced or augmented by descending projections to the AVPNs. Signals from these regions may affect cholinergic outflow to the airways in normal subjects and in patients with bronchopulmonary disorders characterized with airway hyperresponsiveness. Asthmatic subjects often experience reduced airway conductivity during sleep (44), and exacerbations in asthma have been linked temporally to periods of fear and heightened emotionality (29). Although the role of neural mechanisms in airway disorders is well recognized (25), the pathways through which behavioral changes and emotional reactions may affect cholinergic outflow to the airways have yet to be established.

Recently, using conventional and transneuronal labeling techniques, our laboratory has localized the brain stem and suprapontine regions that project to the AVPNs (16). These studies demonstrated that AVPNs receive projections from multiple sites along the neuraxis, including the amygdaloid central nucleus, lateral hypothalamic neurons, and the midbrain ventrolateral (vl) periaqueductal gray column (PAG), the structures that are involved in the expression of the complex autonomic and somatomotor responses to changes in behavioral state, emotion, and fear (1, 2, 4, 8, 41). Previous neuroanatomic evidence suggests that the PAG, organized in longitudinal columns, plays a critical role in the motor and the autonomic nervous system responses to different sets of environmental demands (for review, see Refs. 1 and 2). However, the role of the PAG neurons in the regulation of parasympathetic outflow to the airway smooth muscle is not known.

Hence the present study was carried out to provide insight into the network that regulates airway smooth muscle responses to changes in behavioral state and to emotional stress. The PAG is a part of this network and offers a useful site for studies related to the regulation of cholinergic outflow of the airways by cell groups that constitute the central circuits mediating distinct autonomic and somatomotor responses to changes in behavioral state and stressful environmental conditions.

We hypothesized that activation of vl PAG neurons would cause inhibition of AVPNs within the rNA and withdrawal of cholinergic outflow to the airways via the release of gamma -aminobutyric acid (GABA) and activation of GABAA receptors expressed by these cells. This assumption was based on previous findings demonstrating that both GABA and benzodiazepines acting via GABAA receptors in the rNA inhibit cholinergic outflow to the airways (17, 18). Furthermore, stimulation of somatosensory receptors by skeletal muscle contractions that activate the PAG neurons (31) inhibit airway smooth muscle tone (26, 27, 46). This hypothesis was tested by examining the effects of activation of vl PAG neurons on the airway smooth muscle tone and the possible involvement of GABAA receptors activated by endogenously released GABA in the transmission of inputs from the vl PAG to the AVPNs in the rNA. We observed, for the first time, that stimulation of vl PAG caused release of GABA within the rNA region, activation of GABAA receptors expressed by AVPNs in the rNA, and airway smooth muscle relaxation.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The studies were performed with the use of a total of 24 male European ferrets, Mustella putorius furo (0.6-0.8 kg). Nineteen animals were used for physiological experiments, and five were used to examine the expression of GABAA receptors on AVPNs within the rNA.

Instrumentation of ferrets. Physiological experiments were performed in 19 ferrets anesthetized with alpha -chloralose (70 mg/kg ip). In 3 of the 19 animals, cervical spinalectomy was performed at the level of C7 to eliminate effects of changes in sympathetic outflow on airway responses to vl-PAG stimulation. In these ferrets, a tracheostomy tube was inserted through the tracheal window placed in the caudal portion of the cervical trachea and connected to a Harvard ventilator. Animals were mechanically ventilated with 100% O2 at a constant volume of 7 ml/kg delivered at a frequency of 30-35 breaths/min. Body temperature was continuously monitored through an esophageal probe and maintained at 38-39°C by means of a heating pad.

After instrumentation, ferrets were placed in a prone position in a head-out plethysmograph. A latex collar was fastened to the proximal opening of the plethysmograph to ensure that an adequate seal was achieved, and airway pressure was measured by attaching a pressure transducer to a side port of the tracheal cannula. Flow was measured by placing a pneumotachograph in the distal part of the body box. These signals were used to obtain total lung resistance (RL) by using Buxco software (Buxco Electronics, Troy, NY). The values of RL obtained in this fashion include both airway resistance and tissue viscance. To obtain RL, five respiratory cycles were analyzed and averaged during a control period and at peak response after PAG stimulation (14).

For chemical stimulation of the vl PAG region, the head was fixed in a stereotaxic apparatus with the upper incisor bar 12 mm below the level of the interaural line. The occipital bone was removed, and the atlanto-occipital membrane was opened. The calamus scriptorius was visualized by incising the overlying dura; this landmark served as a stereotaxic zero for rostrocaudal and lateral coordinates, and its surface was used as the dorsoventral zero. Ferrets were paralyzed with gallamine triethiodide (4 mg/kg iv) to reduce the changes in breathing pattern and chest wall afferent activity that might influence airway smooth muscle tone.

In six ferrets with intact spinal cords, the respiratory drive responses after stimulation of the vl PAG were studied by recording changes in phrenic nerve activity. Briefly, the phrenic nerve was exposed, separated, cut, and desheathed, and the central end was placed on a bipolar hook electrode immersed in a mixture of petroleum jelly and mineral oil. The electrode was positioned to minimize the possibility of electrode drift during the experiment. The electrical signal was amplified (Grass, Quincy, MA), band-pass filtered (0.3-3 kHz), and processed by a Paynter filter (CWE, Ardmore, PA), with a time constant of 100 ms to obtain a moving time average signal.

Airway smooth muscle responses evoked by stimulation of the vl PAG were measured as changes in tracheal smooth muscle tone and RL. Tracheal smooth muscle tone was assessed indirectly by measuring the changes in pressure (in cmH2O) in a balloon placed in a bypassed rostral segment of the cervical trachea, as previously described (17). In bypassing the extrathoracic tracheal segment, care was taken not to damage the recurrent and superior laryngeal nerves and the plexus of ganglia on the posterior wall or to interrupt the blood supply. The balloon in the extrathoracic trachea was distended with 0.8-1.2 ml of saline.

Initial measurements were performed to ensure that the efferent transmission of cholinergic outflow to the airways was not affected by the surgery. This was achieved by demonstrating that the reflex responses of tracheal smooth muscle tone to hyperoxic hypocapnia and lung deflation were intact. To determine basal tracheal tone, the pressure in the balloon (Ptseg) was measured after withdrawal of cholinergic outflow to the airways induced by hyperoxic hypocapnia. The hyperoxic hypocapnia was produced by gradually increasing the rate of the ventilator to lower arterial PCO2 (PaCO2). We observed that tracheal tone diminished to 10 ± 1 cmH2O as hyperoxic hypocapnic apnea (PaCO2 = 26.8 ± 1 Torr) occurred. This value was considered to be basal tracheal tone and was close to that recorded after intravenous administration of atropine, as was previously described in cats (34). After the minimum level of cholinergic activity was established, a single airway reflex response was determined by turning off the ventilator during the deflation phase for a period of 30 s and measuring peak response. This procedure caused an airway smooth muscle contraction (Ptseg increased from 10 ± 1 to 24 ± 2 cmH2O; P < 0.001). After hyperventilation-induced hypocapnia and lung deflation for 30 s, the rate of the ventilator was decreased to elevate PaCO2 above apneic threshold (between 37 and 45 Torr) and to restore airway smooth muscle tone. Once a steady state was reached, arterial blood was withdrawn for blood gas measurements. On average, the arterial PO2 was 354 ± 26 Torr, PaCO2 was 41 ± 1 Torr, and pH was 7.326 ± 0.013 units.

Measurements of GABA release. In 6 of the 19 ferrets, GABA release from the rNA region was measured before and after chemical stimulation of vl PAG. Figure 1 shows the injection sites in the vl PAG and the sites within the rNA region from which microdialysates were obtained to measure GABA release (19). A concentric-shaped microdialysis probe with a tip diameter of 0.21 mm was constructed as previously described (10, 50). The dialysis membrane (SpectraPor, 13,000 molecular weight cutoff) was 1.5 mm in length. The probe was inserted unilaterally into the rostral ventrolateral portion of the medulla oblongata, 3.5-3.8 mm rostral to the calamus scriptorius, 3.0 mm lateral to the midline, and 1 mm dorsal to the ventral medullary surface (19). In this region of the ferret brainstem, we have previously observed airway-related preganglionic neurons that project to the extrathoracic trachea. After placement of the microdialysis probes, Dulbecco's PBS (containing, in mM, 138 NaCl, 2.7 KCl, 0.5 MgCl2, 1.5 KH2PO4, 8.1 Na2HPO4, 1.2 CaCl2, and 0.5 D-glucose, pH 7.4) was perfused through the probes. The flow rate was maintained at 2.5 µl/min by using a microinjection pump (Harvard Instruments, South Natick, MA). A minimum of 2 h was allowed for equilibration of the dialysis probes, and then three 20-min baseline samples were collected on ice, frozen, and stored. Unilateral stimulation of the vl PAG was then induced by pressure microinjection of glutamate (1 and 4 nmol/80 nl per site), by using a glass micropipette with a 40-µm tip diameter placed into the vl PAG, 14.5-15.2 mm rostral to the calamus scriptorius, 0.8 mm lateral to the midline, and 5.5 mm dorsal to ventral surface. Microinjections of glutamate were performed every 5 min for a period of 15 min. All samples were collected on ice, frozen, and stored for subsequent measurements of the GABA content of the dialysate. At the end of each experiment, 80 nl of fast green dye was injected through the second barrel of the micropipette into the vl PAG microdialysis probe into the rNA region to aid in histological identification. The areas with greatest dye density were considered to be the injection or microperfusion sites.


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  		Fig. 1.   Line drawings illustrating the sites in which probes were placed for stimulation of neurons within the ventrolateral periaqueductal gray matter (vl PAG; left) and the sites in the region of the rostral ventrolateral medulla oblongata in which microdialysis probes were placed (right). , Regions of greatest dye density; Aq, central aqueduct.

Each dialysate sample (20 µl) was assayed for GABA by HPLC with electrochemical detection as previously described (10, 50) with some modifications. Briefly, the dialysate sample was derivatized with o-phthaldialdehyde. The derivation reagent was prepared by dissolving 22 mg of o-phthaldialdehyde in 0.5 ml of 100% ethanol, 0.5 ml of 1 M sodium sulfite, and 9 ml of 0.4 M boric acid (pH 10.4). The reagent (2 µl) was automatically added to each dialysate sample (20 µl) by an autosampler, mixed, and allowed to react for exactly 5 min before injection into the HPLC system. Amino acids were separated on a C18 reverse-phase column (2 × 100 mm; 3-µm particle size; Phenomenex, Torrance, CA) with a 0.1 M NaH2PO4 (pH 4.50) buffer containing 10% methanol and 40 mg/l EDTA. Detection was performed by use of a glassy carbon electrode maintained at 0.8 V by an LC4C amperometric detector. Flow rate was 0.25 ml/min, and the column temperature was maintained at 34°C. GABA was expressed in picograms per 20 µl.

At the end of each experiment, heparin (1,000 units in 1 ml of saline) was administered and arterial blood was withdrawn from the carotid artery until the mean pressure decreased to 40 mmHg. Subsequently, the right atrial cannula was opened and the animal was perfused with 150 ml of 10% formalin via a catheter advanced into the aorta, and 12 h later the brain was removed, postfixed in formalin, and processed for identification of the injection and microdialysis sites.

Expression of GABAA receptors on AVPNs that innervate extrathoracic trachea. In five ferrets, cholera toxin beta  subunit (CT-b) was microinjected into the wall of the extrathoracic trachea as earlier described (19). After 5 days of survival, ferrets were deeply anesthetized, and perfused brains were removed, and 50-µm sections of the rostral medulla oblongata were cut; sequential immunohistochemistry was then performed to determine whether GABAA receptors are expressed by identified AVPNs located in rNA (14, 30). Briefly, in the first step, free floating sections were washed in PBS containing 0.3% Triton X-100 and then transferred for 30 min to PBS-Triton solution containing 5% normal goat serum to block nonspecific binding sites. After a second 30-min wash, the sections were incubated overnight at room temperature in the blocking solution containing a goat anti-CT-b antibody (1:750, List Biological Laboratories, Campbell, CA). The sections then were rinsed and incubated with donkey anti-goat (1:200, Jackson ImunnoResearch, West Grove, PA) conjugated to rhodamine. In the second step, colabeling for GABAA receptors was performed. The sections were washed in PBS containing 0.3% Triton X-100 and then incubated in PBS-0.3% Triton X-100 containing 1% bovine serum albumin (BSA) for 1 h. Subsequently, sections were incubated overnight at room temperature in PBS-Triton-BSA containing a GABAA receptor (beta  subunit) mouse monoclonal antibody (1:200). The sections were then washed in PBS-Triton-BSA and incubated with goat anti-mouse IgG2a secondary antibodies (1:100, Organon Teknika, CAPPEL Research Products, Durham, NC) conjugated to fluorescein (FITC). Finally, the sections were rinsed in PBS, mounted, and coverslipped with the use of 100% glycerol containing 0.025% p-phenyldiamine, 0.25% 1,4-diazabicyclo[2,2,2]octane (DABCO), and 5% n-propylgallate, as earlier described (30). In control experiments, sections were stained with all possible combinations of primary and secondary antibodies in which a single immunoprobe was omitted.

Sections were examined by using a Zeiss 400 LSM laser scanning confocal microscope, and digitized images were collected with the use of LSM software (14).

Effects of blockade of the ionotropic GABAA receptors in the rostral ventrolateral medulla in mediating airway dilation. To define the potential role of GABAA receptors in mediating the airway responses to stimulation of vl PAG, changes in the airway smooth muscle tone were examined before and after bilateral microperfusion of bicuculline (1 mM solution, 2.5 µl · min-1 · 30 min-1), a GABAA receptor antagonist. In 9 of the 19 ferrets, bicuculline was administered via microdialysis probes that were stereotaxically advanced into the rNA regions in which airway-related parasympathetic preganglionic neurons are located. During microperfusion of Dulbecco's PBS, or 15 to 30 min after microdialysis of bicuculline dissolved in Dulbecco's PBS, the vl PAG was stimulated by microinjection of 1 or 4 nmol of L-glutamate. After the effects of bicuculline on the AVPNs response to vl PAG stimulation were tested, the dialysate was switched to Dulbecco's PBS containing fast green for 30 min.

Data collection and analysis. Two to three sections from the rNA region of each animal were used to examine GABAA receptor expression on the AVPNs. GABA concentrations were expressed in picograms per 20 µl. Records from physiological experiments were analyzed to determine the airway responses to vl PAG stimulation before and after interventions. Average values of all ferrets are presented as means ± SE for each variable. Statistical comparisons were made by using the Student's t-test, and a two-way analysis of variance was used for comparison of results obtained before and after bicuculline administration. The criterion for statistical significance was P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of stimulation of vl PAG on airway smooth muscle tone, phrenic nerve discharge, arterial pressure, and heart rate. In spinalectomized ferrets (n = 3) and animals with intact spinal cords (n = 16), activation of vl PAG neurons by microinjection of glutamate caused a decrease in tracheal tone. Tracheal pressure started to decline within 5 s, and maximal depressive effects were noted between 30 and 60 s after vl PAG stimulation. Tracheal pressure returned to prestimulation values in less than 5 min. In general, changes in tracheal tone were associated with a decrease in RL, an increase in peak phrenic nerve activity, a decline in arterial pressure, and slowing of the heart rate. An example of the response to unilateral injection of 4 nmol of glutamate into the vl PAG region is presented in Fig. 2. We observed that tracheal pressure decreased from 25.5 ± 1.8 to 12.3 ± 1.4 cmH2O (P < 0.05) and RL fell from 0.165 ± 0.008 to 0.146 ± 0.008 cmH2O · ml-1 · s (n = 14; P < 0.05). However, vl PAG stimulation raised the amplitude of phrenic nerve activity from 17.2 ± 2 to 28.1 ± 4 units (P < 0.05) and slightly reduced phrenic frequency discharge from 27 ± 2 to 23 ± 3 bursts/min (P > 0.05). In addition, vl PAG stimulation caused a fall in mean arterial pressure from 124 ± 7 to 118 ± 8 mmHg (P < 0.05) and slowing of the heart rate from 350 ± 12 to 335 ± 11 beats/min (P > 0.05). In 6 of the 19 ferrets studied, vl PAG stimulation induced a transient, slight increase in arterial pressure, as shown in Fig. 2.


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  		Fig. 2.   Example of the effect of chemical stimulation of vl PAG by microinjection of 4 nmol of L-glutamate (L-Glut, which avoids activating fibers of passage) on tracheal segment pressure (Ptseg), integrated phrenic nerve activity (Phr ENG), and mean arterial blood pressure (ABP) in a paralyzed ferret mechanically ventilated with O2 at arterial CO2 pressure (PaCO2) of 38 Torr.

GABA release within the AVPNs region after vl PAG stimulation. In six ferrets, we tested the effects of vl PAG stimulation on GABA release within the rNA region in which the AVPNs are located (19). Repeated stimulation within the vl PAG at 5-min intervals elicited a large increase in GABA release within the AVPNs region that was paralleled by a decrease in tracheal smooth muscle tone. Typical HPLC chromatograms of microdialysates collected from the rNA in a control state and after repeated vl PAG stimulation are shown in the top and the average results are presented in the bottom of Fig. 3. After equilibration of the dialysis probe, before stimulation of vl PAG region, the average concentration of GABA was 29.3 ± 7.1 pg/20 µl. After vl PAG stimulation, GABA concentration increased to 61.4 ± 22.1 pg/20 µl. In the poststimulation recovery period, GABA returned to control levels. Differences between baseline concentrations of GABA and those after stimulation of vl PAG region were statistically significant (P < 0.05).


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  		Fig. 3.   Top: typical HPLC chromatograms obtained from microdialysates collected from the airway-related vagal preganglionic neurons (AVPNs) within the rostral ventrolateral medulla in a control state (Baseline) and during repeated excitation of vl PAG neurons (Stimulated). Bottom: average results (mean ± SE; n = 6) of GABA in the control state (Baseline) and after vl PAG stimulation (PAG stim). *P < 0.05.

Expression of GABAA receptors on the AVPNs. To demonstrate that GABAA receptors are expressed by the AVPNs innervating extrathoracic trachea, sections from ferret brain stem were stained by using a mouse monoclonal GABAA beta -subunit-specific antibody. In the rostral ventrolateral medulla, ventral to the compact portion of the nucleus ambiguus, we observed retrogradely labeled AVPNs (red), after CT-b injection into the wall of the extrathoracic trachea (Fig. 4A). In the same region, we observed punctate GABAA receptor-specific staining (green). This is likely associated with the cell membranes, because it surrounds empty spaces in which neuron cell bodies lie (Fig. 4B). There were also profiles of GABAA receptor-specific staining in between the neuron cell bodies. These presumably identify receptors located in presynaptic terminals that contact the processes of the motoneurons. In double- labeling studies, we observed that within the rNA region the CT-b-labeled neurons contain GABAA beta - subunit. The GABAA beta -subunit staining (green) was observed as punctate green staining around the membrane of the perikaryon as well as on dendrites (Fig. 4C). In control experiments, there was no apparent cross-reactivity of the secondary antibodies (data not shown).


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  		Fig. 4.   Example of a confocal microscope image of GABAA beta  subunit receptor expression by the AVPNs innervating the extrathoracic trachea. In the rostral ventrolateral medulla, ventral to the compact portion of the nucleus ambiguus, we observed retrogradely labeled AVPNs after cholera toxin beta  subunit (CT-b) injection into the wall of the extrathoracic trachea (A). In the same region, we observed punctate GABAA receptor-specific staining (green) that is likely associated with the cell membranes, because it surrounds empty spaces (N) in which neuron cell bodies lie (B). There were also profiles of GABAA receptor-specific staining in between the neuron cell bodies. These presumably identify receptors located in presynaptic terminals that contact processes of the motor neurons. In double-labeling studies we observed that, within the rostral ventrolateral medulla, the CT-b-labeled neurons contain GABAA beta  subunit. The GABAA beta  subunit staining (green) was observed as punctate green staining on the membrane of the perikaryon (arrows) as well as on dendrites (C, *). In control experiments, there was no apparent cross-reactivity of the secondary antibodies. Bar = 50 µm (A), 30 µm (B), 15 µm (C).

Effects of GABAA receptor blockade on airway responses to stimulation of vl PAG. The effects of GABAA receptor blockade on airway smooth muscle tone in response to vl PAG stimulation were studied in 9 of 19 ferrets. Blockade of GABAA receptors by bilateral microperfusion of bicuculline caused an average increase in intratracheal pressure of 4.2 ± 1.8 cmH2O in the bypass segment (Ptseg) and elevated RL on average by 0.017 ± 0.007 cmH2O · ml-1 · s (P > 0.05). Bicuculline increased mean arterial pressure from 120 ± 9 to 131 ± 10 mmHg (P > 0.05) and decreased heart rate from 330 ± 11 to 326 ± 14 beats/min (P > 0.05). In addition, blockade of GABAA receptor in the rNA region almost completely abolished the decrease in tracheal smooth muscle tone elicited by activation of vl PAG neurons (Fig. 5). In control periods after vl PAG stimulation, Ptseg decreased from 25 ± 1.5 to 11.3 ± 2 cmH2O (P < 0.05). The effects of vl PAG stimulation were considerably diminished by prior application of bicuculline. During bicuculline microperfusion, the tracheal pressure in response to vl PAG stimulation declined only from 29.6 ± 0.2 to 27.5 ± 1.0 cmH2O (P > 0.05). The difference of tracheal tone response to vl PAG stimulation before and after blockade of GABAA receptors was significant (P < 0.05). After bicuculline administration, vl PAG stimulation had no effect on RL, mean arterial pressure, or heart rate (data not shown).


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  		Fig. 5.   Top: tracings from a paralyzed and oxygen-ventilated ferret. In a control period (Control), activation of vl PAG neurons by L-glutamate (1 and 4 nmol/80 nl) induced a concentration-dependent decrease in tracheal tone, expressed as a decrease of Ptseg. Bilateral microperfusion of bicuculline into the AVPN region (after bicuculline) abolished the response of airway smooth muscle tone to vl PAG stimulation. Bottom: average data (mean ± SE; n = 9) of the effect of vl PAG stimulation on tracheal smooth muscle tone in response to L-glutamate-induced activation of vl PAG before and after bicuculline microperfusion into the AVPN region.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

These studies demonstrate for the first time that activation of neurons within the vl PAG results in dilation of the airways. This response is primarily mediated via endogenously released GABA and the activation of GABAA receptors expressed by AVPNs located within rNA. Furthermore, these findings indicate that preganglionic neurons of the dorsal vagal motor nucleus do not appear to play a role in the control of airway caliber in response to vl PAG stimulation.

An alternate explanation is that stimulation of the vl PAG may dilate the airways by increasing sympathetic outflow and subsequent noradrenaline release from efferent nerve terminals. In ferrets, stimulation of sympathetic axons of the vagosympathetic trunk has been observed to inhibit the trachealis muscle tone (32). However, vl PAG stimulation in C7 spinalectomized ferrets had similar effects as vl PAG stimulation in animals with intact spinal cords, excluding the involvement of sympathetic inhibitory pathways in this response. Furthermore, vl PAG stimulation decreases rather then increases sympathetic discharge (1, 2), and a nonadrenergic, noncholinergic inhibitory system does not appear to play an important role in the ferret airways (32). Taken together, the previous observations and the findings of the present study indicate that stimulation of vl PAG dilates the airways by eliciting a release of GABA within the rNA region that acts on GABAA receptors expressed on AVPNs, causing withdrawal of cholinergic outflow. Furthermore, because bicuculline almost completely abolished the effects of Vl PAG stimulation, our findings suggest that the effects we have observed are mediated mainly through postsynaptic and less via presynaptic mechanisms.

After completion of instrumentation, we verified that the efferent transmission of cholinergic outflow to the airways was intact by demonstrating that the responses of tracheal smooth muscle tone to hyperoxic hypercapnia and lung deflation were present. The reflex tracheomotor responses triggered by turning off the ventilator for a period of 30 s most likely are initiated by withdrawal of inhibitory stretch receptor inputs to AVPNs and activation of rapidly adapting airway receptors. Conceivably, when a ferret is ventilated with 100% oxygen, suspending mechanical ventilation during the "expiratory" phase for 30 s could cause an incremental increase in arterial CO2 and a gradual decrease in arterial PO2. Thus inputs from central and peripheral chemoreceptors could amplify the central reflex mechanisms (33). Whether this has any significant effect on the observed GABAergic-mediated changes in cholinergic outflow to the airways after vl PAG stimulation in the postdeflation period needs to be investigated.

Stimulation of the vl PAG elicited a release of GABA within the rNA region, as measured by microdialysis and HPLC. This approach allows for a high specificity but lacks temporal resolution, because of long sampling times and low flow rates of perfusate through the probe. Another possible limitation is that the size of the probe reduces the anatomic specificity of the field from which the dialysate is collected. However, the majority of parasympathetic preganglionic neurons innervating the airways are located in the external part of the nucleus ambiguus. This so-called loose portion of the nucleus ambiguus occupies a larger field than the compact portion (19) and is larger than the size of the microdialysis probe (0.21 mm). However, this does not exclude the possibility that the perfusate collected for analysis may partially originate from the surrounding tissue.

Although our results do demonstrate that there is an inhibitory pathway from the vl PAG to AVPNs, our studies do not indicate whether the pathway is direct or indirect. GABA-immunoreactive neurons are present in the PAG (40), and discrete subregions of the midbrain periaqueductal gray matter project to the nucleus ambiguus and the periambigual region (5, 11, 13, 47). Furthermore, transneuronal labeling studies have shown that cells within the vl PAG do innervate the AVPNs (16). Hence, GABAergic neurons within vl PAG may contribute to descending inhibitory inputs to the AVPNs. However, the number of projecting neurons was relatively small (1-3 cells per section), much smaller than the number of the raphe magnus and gigantocellular cells that project to the AVPNs (16, 20). The majority of the GABA-containing neurons of the raphe magnus and gigantocellular nuclei, subpopulations of which coexpress serotoninergic traits (45), receive dense innervation from the vl PAG (22, 40, 41). Hence, it could be assumed that withdrawal of parasympathetic outflow to the airways elicited by chemical stimulation of the vl PAG region may be mediated through GABA-containing raphe magnus and gigantocellular neurons that project to AVPNs. In addition, this effect may be mediated in part via other GABA-expressing neurons, including those within the parabrachial nucleus because it was recently demonstrated that neurons within the vl PAG project to parabrachial cell groups (28), activation of which dilates the airways in cats (37). Thus a variety of cell groups may potentially serve as a neural link between the vl PAG and the AVPNs.

Effective GABA-mediated synaptic inhibition requires that GABAA receptors are expressed at appropriate multiple postsynaptic sites, in close proximity to GABA-releasing nerve terminals (35). We observed that clusters of the beta -isoform of GABAA receptor subunits were present on the cell bodies and nerve processes of the AVPNs. The GABAA receptor subunits can be divided into seven families with multiple isoforms: alpha  (alpha 1-6), beta  (beta 1-3), gamma  (gamma 1-3), delta , epsilon , theta , and pi  (one isoform each) (35). Coexpression of alpha - and beta -subunits produces GABA-gated ion channels, but the gamma -subunit is required to produce receptors that mediate the effects of benzodiazepines (39). The exact composition of the GABAA receptor subtype expressed by AVPNs that mediate airway changes induced by vl PAG stimulation remains to be characterized. However, one would expect that the receptors consist of alpha -, beta -, and gamma -subunits. This assumption is based on our previous studies showing that benzodiazepines topically applied or microinjected into the rostral ventrolateral medulla cause withdrawal of cholinergic outflow to the airways and airway smooth muscle relaxation, similar to that observed with GABA (18).

From the findings of the present study, we cannot be certain how endogenously released GABA reaches the GABAA receptors expressed on the AVPNs because the relationship between the percentage of the cell surface covered by GABAA receptors and the surface of these neurons to which synaptic terminals are apposed is not known. However, fast and effective GABA-mediated synaptic inhibition requires that GABAA receptors are expressed and concentrated at appropriate multiple postsynaptic sites in apposition to GABA-releasing nerve terminals (35). In the present study, the speed and the specificity of the response that we observed, i.e., a decrease in tracheal smooth muscle tone, suggest that GABA was acting on neurons expressing the GABAA receptors at a distance from its site of release. In the ferret, as in other species, however, airway smooth muscle constriction or relaxation in response to an increase or withdrawal of cholinergic input is slow and gradual compared with changes in neuronal discharge (32). In addition, in ferrets, descending efferent fibers are nonmyelinated and signal transmission via these channels is slower than through myelinated nerves (32). Hence, the latency of 2-5 s does not mean that AVPNs were affected by GABA that had diffused a considerable distance from its site of release through the process of "volume transmission."

Previous studies have indicated that the PAG exerts a strong modulation on respiratory drive (38). Most emotional reactions and behavioral states are associated with changes in breathing pattern and vocalization that are dependent on an intact midbrain, specifically the caudal PAG (9), which controls the activity of the premotoneurons in the nucleus retroambigualis (23, 51) and influences the activity of bulbospinal neurons that project to the phrenic nuclei (38). Hence, the vl PAG may play an important role in producing changes in breathing during vocalization and speech. Many stimuli that increase respiratory drive also elevate cholinergic outflow to the airways. Furthermore, the parasympathetic postganglionic nerve fibers, which innervate the trachealis smooth muscle, fire in phase with phrenic nerve discharge. These observations have led to the hypothesis that cholinergic motor outflow to the airways is under the control of the same pattern generators that drive the phrenic and intercostal motoneurons (34). However, the qualitatively different responses in airway smooth muscle tone and phrenic nerve discharge that we observed in this study suggest that descending neuronal circuits may exert qualitatively different influences on respiratory output and the cholinergic outflow to the airways.

The PAG neurons are organized in longitudinal columns and are components of the neuronal networks involved in behavioral state control (42) and cardiovascular responses to aversive stimuli (1, 2). Apart from local connections between the columns that could provide pathways for intrinsic neuromodulation (24), chemical or electrical stimulation of the PAG columns elicits a variety of autonomic and motor responses that depend on the site of stimulation. For example, activation of the PAG neurons located dorsolateral and lateral to the cerebral aqueduct elicits potent increases in arterial blood pressure, heart rate, and lumbar, and splanchnic sympathetic nerve discharge, whereas stimulation of the PAG ventrolateral to the aqueduct causes sympathoinhibition and a decrease in arterial blood pressure and heart rate (1, 2). Conceivably, the response of tracheal tone to stimulation of the vl PAG could be due to modulation of sympathetic outflow and changes in arterial pressure, which may result in a reciprocal modulation of airway tone by a baroreceptor reflex. However, in the present studies, stimulation of vl PAG tended to cause a decrease in arterial pressure and heart rate, probably acting through the caudal brainstem depressor regions (22), including the midline neurons that inhibit sympathoexcitatory medullary cells (48) and the AVPNs (20). Furthermore, spinalectomy at C7 had no effect on airway smooth muscle response to vl PAG stimulation. Therefore, it seems unlikely that changes in sympathetic outflow and vasomotor activity explain the vl PAG stimulation-induced airway smooth muscle relaxation, because anything that lowers the arterial blood pressure would cause airway smooth muscle contraction rather than relaxation (43, 49).

Stimulation of somatosensory fibers activates the midbrain PAG neurons (31) and elicits a decrease in bronchomotor tone (26, 27, 46). The neural mechanisms responsible for the exercise-induced airway dilation are not well understood, but it was thought that the mesencephalic and the hypothalamic locomotor regions that comprise the neuroanatomic substrate for central command play a role in the airway smooth muscle relaxation response to exercise. However, recent studies provided no support that these "central command" structures contribute to the tracheobronchial dilation evoked by dynamic exercise. Stimulation of the hypothalamic (3) or mesencephalic locomotor regions (36) constricts, rather than relaxes, the airway smooth muscle. It is possible that the vl PAG neurons could be involved in the exercise-induced airway smooth muscle relaxation reflex, because static muscle contraction stimulates vl PAG cells partly by the arterial baroreceptor reflex (31) and, as we observe in this study, their activation inhibits cholinergic outflow to the airways. Hence, airway smooth muscle relaxation after activation of vl PAG may be part of a negative feedback reflex that acts to reduce airway resistance and the work of breathing during exercise-induced hyperventilation.

Neuroanatomic studies indicate that the vl PAG receives projections from a variety of different CNS cell groups, including central nucleus of amygdala (7, 21, 41), the region that projects to the AVPNs (16). This circuitry can be implicated in the expression of respiratory and airway changes associated with a central fear state. Somatic and autonomic expression of fear and responses induced by the introduction of conditioned stimuli seem to be processed through the central fear system (4, 8) and mediated via the PAG regions (9, 23).

In summary, this work addressed the physiological role of the vl PAG neurons in regulating cholinergic outflow to the airways. The results of our study indicate that the activity of the AVPNs can be influenced by descending inputs from the vl PAG region, the same area that is involved in a generalized central autonomic and behavioral control of cardiovascular and respiratory function. Such parallel control originating from a common region is not surprising in light of the coordination required between circulation and breathing, ventilation, and airway caliber. Further studies are required to determine the importance of a GABAergically mediated inhibition of cholinergic outflow induced by stimulation of vl PAG neurons. However, it could be assumed that, because the vl PAG is part of the descending pathways, it is an important site that integrates airway responses with changes in respiration and cardiovascular functions. Decrease in cholinergic outflow during exercise could be mediated, at least in part, through the neural circuitry that includes the vl PAG and the AVPNs. The bronchodilation and withdrawal of cholinergic outflow that were observed after administration of morphine or morphinelike substances (12) may also be evoked by way of the vl PAG- and GABA-containing neurons that project to AVPNs. Alteration in this pathway may be an important central component of exercise or emotional stress-related deterioration of airway functions, particularly in asthmatic subjects. Further investigations are required to determine the role of this pathway in the motor and affective dimensions of the chronic bronchoconstrictive states that are often associated with anxiety, depression, and inadequate coping strategies.


    ACKNOWLEDGEMENTS

We wish to thank Illona Gillette-Ferguson for helpful advice on data presentation and for creating the diagrams presented as Fig. 1.


    FOOTNOTES

This work was supported by the National Heart, Lung, and Blood Institute HL-50527 and National Institute of Neurological Disorders and Stroke Grant 1U54 NS-39407.

Address for reprint requests and other correspondence: D. G. Ferguson, Dept. of Anatomy, 10900 Euclid Ave., Cleveland, Ohio 44106, Mail Stop 3940 (E-mail: dgf4{at}po.cwru.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

February 15, 2002;10.1152/japplphysiol.00752.2001

Received 18 July 2001; accepted in final form 7 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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