Download Brain stem excitatory and inhibitory signaling pathways regulating

J Appl Physiol 98: 1961–1982, 2005;
doi:10.1152/japplphysiol.01340.2004.
Invited Review
Brain stem excitatory and inhibitory signaling pathways regulating
bronchoconstrictive responses
Musa A. Haxhiu,1,3,4 Prabha Kc,1 Constance T. Moore,1 Sandra S. Acquah,1
Christopher G. Wilson,3 Syed I. Zaidi,1 V. John Massari,1,2 and Donald G. Ferguson4
Specialized Neuroscience Research Program, Departments of 1Physiology and Biophysics and
2
Pharmacology, Howard University College of Medicine, Washington, District of Columbia;
and Departments of 3Pediatric and 4Anatomy, Case Western Reserve University, Cleveland, Ohio
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Haxhiu, Musa A., Prabha Kc, Constance T. Moore, Sandra S. Acquah, Christopher G. Wilson, Syed I. Zaidi, V. John Massari, and Donald G. Ferguson. Brain
stem excitatory and inhibitory signaling pathways regulating bronchoconstrictive responses. J Appl Physiol 98: 1961–1982, 2005; doi:10.1152/japplphysiol.01340.2004.—
This review summarizes recent work on two basic processes of central nervous system
(CNS) control of cholinergic outflow to the airways: 1) transmission of bronchoconstrictive signals from the airways to the airway-related vagal preganglionic
neurons (AVPNs) and 2) regulation of AVPN responses to excitatory inputs by
central GABAergic inhibitory pathways. In addition, the autocrine-paracrine modulation of AVPNs is briefly discussed. CNS influences on the tracheobronchopulmonary system are transmitted via AVPNs, whose discharge depends on the
balance between excitatory and inhibitory impulses that they receive. Alterations in
this equilibrium may lead to dramatic functional changes. Recent findings indicate
that excitatory signals arising from bronchopulmonary afferents and/or the peripheral chemosensory system activate second-order neurons within the nucleus of the
solitary tract (NTS), via a glutamate-AMPA signaling pathway. These neurons,
using the same neurotransmitter-receptor unit, transmit information to the AVPNs,
which in turn convey the central command to airway effector organs: smooth
muscle, submucosal secretory glands, and the vasculature, through intramural
ganglionic neurons. The strength and duration of reflex-induced bronchoconstriction is modulated by GABAergic-inhibitory inputs and autocrine-paracrine controlling mechanisms. Downregulation of GABAergic inhibitory influences may
result in a shift from inhibitory to excitatory drive that may lead to increased
excitability of AVPNs, heightened airway responsiveness, and sustained narrowing
of the airways. Hence a better understanding of these normal and altered central
neural circuits and mechanisms could potentially improve the design of therapeutic
interventions and the treatment of airway obstructive diseases.
airways; airway reflex responses; autonomic control; nucleus of the solitary tract;
glutamatergic pathways: glutamate; AMPA receptors; NMDA receptors; GABAergic microcircuitry; GAD; GABAA receptors; synaptic transmission; volume transmission; vagal preganglionic neurons
CHRONIC AIRWAY DISEASES such as bronchial asthma and chronic
obstructive bronchitis share the salient features of inflammation, hyperresponsiveness to various inhalants, increased cholinergic outflow to the airways, and sustained airway narrowing. Although these two conditions result in enormous morbidity and the neural mechanisms are thought to be play an
important role (13, 203), the central mechanisms involved in
airway hyperreactivity remain poorly understood.
Airway bronchoconstrictive responses initiated by inhaled
agents or psychogenic factors suggest the existence of bidirectional central nervous system (CNS)-airway communication
that serves to protect the respiratory system. Repeated exposure to environmental pollutants (like ozone, cigarette smoke,
allergens), through parallel or convergent inflow (68), may
Address for reprint requests and other correspondence: M. A. Haxhiu, Dept.
of Physiology and Biophysics, Howard Univ. College of Medicine, 520 W St.
NW, Washington, DC 20059 (E-mail: [email protected]).
http://www. jap.org
modulate afferent airway sensory pathways (38, 108, 175,
200 –202, 216), setting the stage for airway hyperresponsiveness. Under these conditions, the excitability of airway-related
vagal preganglionic neurons (AVPNs) is increased and weak
stimuli may trigger responses that last for hours, suggesting
that the CNS is involved in causing airway constrictive
changes.
A better understanding of the role that CNS pathways play
in regulating airway functions in normal and diseased states
undoubtedly will provide novel therapeutic approaches in the
treatment of diseases whose clinical, biochemical, and pharmacological features indicate a pathophysiological link with
the CNS. Therefore, this review is focused on the central
determinants of airway control and adds to several recent
excellent editorials and reviews on sensory neuroplasticity,
autonomic regulation of airway functions, and associated pharmacological implications (42, 43, 55, 111, 113, 137, 176,
201–203, 213, 214).
8750-7587/05 $8.00 Copyright © 2005 the American Physiological Society
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In this article, we briefly discuss recent studies that clarify
two of the basic processes in the central control of the vagal
preganglionic neurons that provide cholinergic outflow to the
airways: 1) glutamatergic signaling pathways involved in
transmission and processing of bronchoconstrictive signals
from airway sensory receptors, via the nucleus of the solitary
tract (NTS) to AVPNs and 2) GABAergic inhibitory network
that downregulates excitability of the AVPNs, consequently
decreasing cholinergic outflow to the tracheobronchial system.
An imbalance between excitatory and inhibitory inputs to
AVPNs could be of considerable importance.
GENERAL CONSIDERATIONS OF THE NEURAL CONTROL OF
THE AIRWAYS
Fig. 1. General scheme illustrating the organization of autonomic parasympathetic control of airway functions. Central nervous system (CNS) cell groups
(level 4) regulate the activity of airway-related vagal preganglionic neurons
(AVPNs; level 3). Axons of the AVPNs, as the final common pathway out of
the medulla oblongata, synapse on intrinsic ganglionic neurons within airway
walls (level 2). These ganglia give rise to postganglionic fibers that control the
function of specific effector targets (i.e., airway smooth muscle, mucous
glands, and blood vessels). Sensory feedback for these systems occurs via
sensory fibers originating from sensory ganglia (nodose and jugular ganglionic
neurons). These fibers innervate sensory receptors and transmit information
from the airways to the CNS. They modulate the activity of AVPNs through
central multisynaptic pathways and they may affect function of effector organs
via 2 ill-defined local networks that include axon reflex responses (level 1) and
sensory innervation of intrinsic airway ganglia (level 2).
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CNS control of airway functions (Fig. 1, level 4) involves
integrated networks along the neural axis that funnel information to tracheobronchopulmonary effector units via the AVPNs
in the medulla oblongata. The AVPNs are the final common
pathway from the brain to the airways and transmit signals to
the intrinsic tracheobronchial ganglia that are part of the
network for automatic feedback control (level 3). Each ganglion, located in close proximity to effector systems, possesses
a relatively large number of neurons (11, 40, 54, 131, 155) that
can be considered as an expanded parasympathetic, preganglionic efferent motor system (level 2). However, intrinsic airway
neurons contain neurochemicals other than ACh, including
vasoactive intestinal peptide (VIP) and neuronal nitric oxide
synthase [nNOS, an enzyme involved in generation of nitric
oxide (NO); 54, 219] that are considered to mediate nonadrenergic neuronal airway smooth muscle relaxation (21, 56, 78).
Signals transmitted through the preganglionic nerves are
relayed, integrated, filtered, and modulated by intrinsic ganglionic neurons before reaching the airway neuroeffector sites
through postganglionic axons. This structural organization
could explain the strong effects of a relatively small number of
vagal efferent fibers on coordinated reflex changes in airway
smooth muscle tone, submucosal gland secretion, and blood
flow along the tracheobronchial tree. A similar arrangement
has been described in the parasympathetic control of the enteric
tract (215). This unified concept does not exclude the probability that some of the vagal preganglionic neurons also provide, to a lesser degree, direct innervation of airway epithelial
cells and the alveolar interstitium of the lung, because ganglia
are absent altogether from the bronchial subepithelial space
and the most distal gas exchanging units (168). These observations suggest that AVPNs may be involved in regulating the
release of the biologically active molecules from epithelial
cells, i.e., activation of NOS and release of NO that might
oppose excessive cholinergically mediated airway contractile
responses at both central and peripheral sites (107, 120).
Furthermore, cholinergic mechanisms are involved in controlling the conductivity of the most distal airways (166) and tissue
resistance (120, 132), by influencing smooth muscle tone, lung
interstitium pericytes, and alveolar myofibroblasts (116).
However, it is not clear whether individual AVPNs provide
parallel innervation to airway smooth muscle, submucosal
glands, and local blood vessels. A relative simultaneity of
airway effector cell responses to stimulation of bronchoconstrictive vagal afferent fibers, such as reflex elevation of
smooth muscle tone, increase in submucosal gland secretion,
and changes in blood flow, is consistent with the assumption
that the same cell bodies of AVPNs that cause smooth muscle
constriction also elicit activation of smooth muscle glands or
changes in blood flow supplying regional airway structures.
Alternatively, it is also possible that groups of functionally
selective AVPNs may exert a highly coordinated control over
multiple airway functions by central mechanisms that synchronize their output to individual effectors. This assumption is
analogous to evidence obtained relevant to coordination of
multiple cardiac functions by vagal preganglionic neurons,
where functionally distinct, but closely integrated, vagal
preganglionic neurons in the nucleus ambiguus (NA) mediate
the coordinated control of cardiac rate, atrioventricular conduction, and left ventricular contractility (65, 133, 134). Recent
preliminary data in ferrets suggest that presumptive gap junctions between identified AVPNs (Blinder K, Karibi-Ikiriko A,
Massari VJ, Haxhiu MA, unpublished data) may serve to
synchronize their electrical activity in the rostral nucleus ambiguus (rNA). Alterations in central mechanisms that mediate
such synchronization could cause regional differences in airway reflex responses, expressed as inhomogeneity in ventilation. For example, in an asthma attack or induced bronchoconstriction, some branches of the airway may totally close while
others remain normal (181). Future studies should address the
question of the central mechanisms and origin of synchronization and asynchrony of bronchoconstrictive reflex responses.
An extensive network of vagal afferent fibers of sensory
ganglia innervates the bronchopulmonary sensory receptors
that are specialized for detecting changes in chemical, mechanical, or thermal stimuli. The bipolar airway vagal afferent
neurons are located in the nodose and jugular ganglia and
participate in reflex events. Furthermore, the afferent nerve
endings (i.e., C fibers) are also believed to be responsible for
mediating local axon reflexes (level 1) and neurogenic inflammation via release of neuropeptides such as substance P (141),
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BRAIN STEM EXCITATORY AND INHIBITORY SIGNALING PATHWAYS
contact of primary bronchopulmonary slowly adapting receptors (49). Activation of these receptors causes a reflex airway
smooth muscle relaxation (212). Similarly, peripheral chemoreceptors and baroreceptors acting centrally can readily affect
cholinergic outflow to the airways. Although stimulation of the
carotid bodies reflexly elicits bronchoconstriction (157) and
submucosal gland secretion (50) and facilitates bronchoconstrictive responses (205), the activation of baroreceptors leads
to opposite changes (157). In this article, signaling mechanisms
involved in these and other possible reflex interactions that can
centrally influence bronchoconstrictive responses are not considered.
Recently, using conventional and transneuronal labeling
techniques and ultrastructural, molecular, and physiological
approaches, we identified the central excitatory (82– 84, 91, 93)
as well as inhibitory pathways and neurotransmitters (82, 92,
94, 96) that regulate the excitability and firing rate of AVPNs.
These processes occur via both wiring (synaptic) and volume
(nonsynaptic) transmission. We hypothesize that downregulation of central inhibitory influences upon AVPNs result in a
shift from inhibitory to excitatory transmission, leading to a
hyperexcitable state of the AVPNs and, consequently, cholinergic hyperresponsiveness that can predispose to and worsen
bronchial asthma (13, 57).
VAGAL PREGANGLIONIC NEURONS INNERVATING
THE AIRWAYS
Vagal preganglionic neurons that generate cholinergic outflow to the airways can be viewed as the central integrators of
multiple excitatory and inhibitory inputs that connect the brain
with the bronchopulmonary effector system. The critical circuits that regulate these processes include both central glutamatergic and GABAergic pathways controlling cholinergic
outflow to the airways (Fig. 2).
Location of AVPNs
Studies using retrograde tracer techniques showed that in
mammals (Fig. 3), the cholinergic preganglionic motor neurons
innervating the airways arise from the rNA and from the rostral
portion of the dorsal motor nucleus of the vagus (DMV).
Furthermore, the findings imply that the majority of AVPNs
are involved in the innervation of multiple airway segments,
thereby assuring the symmetry and simultaneity of bronchomo-
Fig. 2. Central glutamatergic excitatory and GABAergic
inhibitory pathways regulating cholinergic outflow to the
airways. In this oversimplified schematic illustration are
presented 2 main neural pathways that determine the activity of AVPNs. Excitatory signaling pathway uses glutamate
(Glu) as a neurotransmitter in conveying bronchoconstrictive signals from airway sensory receptors to the nucleus of
the solitary tract (NTS) and from the NTS to the AVPNs.
Inhibitory projections employ GABA as a signaling molecule to downregulate excitability of the AVPNs and cholinergic outflow to the tracheobronchial system transmitted
through ACh release. These 2 signaling pathways are emphasized because they comprise the central theme of this
review.
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which can be modulated by alterations in activity of neutral
endopeptidases (156). In addition, endogenous substance P
facilitates synaptic transmission in airway parasympathetic
ganglia (34, 154), similar to the effect observed with cyclooxygenase activation and the release of prostaglandins in antigeninduced lung injury (112). The central fibers of these sensory
neurons ascend in the vagus nerve and enter the brain stem
through the solitary tract (26, 88, 89, 114, 123), making their
first synapse with the NTS second-order neurons that are
required for full expression of the pulmonary C fiber reflex (26)
and bronchoconstrictive airway reflex responses (94).
In the NTS, the afferent vagal terminals responsible for
transmitting information from the airway bronchoconstrictive
receptors form a distinct wiring diagram with specific secondorder neurons that project to AVPNs (74, 88, 89, 167). However, a variety of inputs from afferent receptors is transmitted
to the NTS, including those from cough receptors that are
activated by stimuli that may also elicit reflex bronchoconstriction, submucosal gland secretion, and increased blood flow.
Cough receptors are preferentially located within the larynx,
trachea, and large intrapulmonary airways (25, 35, 213). As is
the case with vagal efferents, it is not clear whether the same
or different vagal afferent neurons transmit different components of airway defensive reflex responses.
More recently, however, it is reported that a subpopulation
of myelinated, polymodal A␦-fibers that arise from the nodose
ganglia respond to punctate mechanical stimulation and acid
but are unresponsive to capsaicin, bradykinin, histamine,
smooth muscle contraction, longitudinal or transverse stretching of the airways, or distension. Unlike these afferent neurons,
considered as the putative cough receptors, the majority of
capsaicin-sensitive afferents (both A␦- and C-fibers) innervating the rostral trachea and larynx have their cell bodies in the
jugular ganglia and project to the airways via the superior
laryngeal nerves (35). These findings suggest the possibility of
presynaptic or postsynaptic interactions of afferent nerves
originating from cough and non-cough receptor neurons at the
NTS level. (25, 35). For example, in conscious guinea pigs, C
fiber-dependent cough may require coactivation of bronchoconstrictive airway afferent nerves for the full expression of the
cough reflex.
Bronchoconstrictive inputs can be modulated by increasing
or decreasing the afferent signals from slowly adapting receptors to NTS second-order neurons, the first site of synaptic
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tor responses (20, 74, 80, 88, 99, 114, 139, 167, 168, 204). In
addition, some preganglionic neurons may provide direct innervation to the airway tissues and lung parenchyma, without
the interposition of intrinsic neurons (74, 168). This somewhat
atypical organization of parasympathetic circuits has been
demonstrated in other effector systems, for example, ciliary
muscle, which receives dual parasympathetic innervation directly from preganglionic neurons and via ganglionic cells
(210), indicating the possible functional importance of direct
projections that bypass intrinsic cholinergic ganglia, an assumption that was not supported by recent physiological experiments (119).
Functionally, the AVPNs within the rNA play a greater role
in generating the cholinergic outflow to airway smooth muscle
than the preganglionic cells of the DMV (80). On the basis of
this observation, it was suggested that DMV neurons projecting to the airways might innervate tracheobronchial secretory
glands and blood vessels. However, more recent studies have
indicated that AVPNs within the rNA also mediate reflex
increases in submucosal gland secretion and blood flow (93);
supporting the notion that cholinergic innervation arising from
AVPNs lacks target specificity.
Ultrastructural Characteristics of AVPNs
It has been suggested that the viscerotropic representation,
ultrastructure, and synaptology of the different divisions of the
NA innervating the alimentary system may be associated with
specific physiological functions (22, 104, 183). To define
ultrastructural characteristics of AVPNs, recently, brain stems
from ferrets, in which cholera toxin ␤-subunit (CT-b) conjugated to horseradish peroxidase had been used as a retrograde
cell body tracer, were examined. Electron microscopy was
employed to determine ultrastructural characteristics of these
J Appl Physiol • VOL
tracheal AVPNs (135). Retrogradely labeled AVPNs in the
rNA were readily detectable in the electron microscope. The
cell bodies of labeled AVPNs were observed to be 32 ⫾ 1 ⫻
23.0 ⫾ 1.3 ␮m (means ⫾ SE) in size, with abundant cytoplasm
and intracellular organelles. The cell bodies had round uninvaginated nuclei that occasionally contained a prominent nucleolus and displayed both somatic and dendritic spines (Fig.
4). Somatosomatic appositions or somatodendritic appositions
without intervening glial processes and dendritic bundling of
the tracheal AVPNs were not observed. The axons of these
neurons were seldom labeled (Fig. 5).
The localization and the ultrastructural features of the
AVPNs differed from neurons innervating the alimentary system that have been examined in the dorsal and ventral columns
of the NA (104, 183). By comparison, esophageal motoneurons, despite some similarities, can be distinguished from
AVPNs located within the rNA by the presence of extensive
somatosomatic and somatodendritic appositions. Furthermore,
they also display finger- and leaf-like somatic protrusions that
partially envelop longitudinally oriented dendrites and axons,
and dendritic bundling is prominent (104, 183). These latter
characteristics appear to be unique to the compact formation of
the NA and clearly differentiate these neurons from tracheal
VPNs and vagal preganglionic neurons innervating the heart
(133, 134).
In summary, at the ultrastructural level, AVPNs innervating
the extrathoracic trachea are clearly distinguished from pharyngeal, laryngeal, or esophageal motoneurons in other subdivisions of the NA. These data are consistent with the hypothesis that differences in the ultrastructure and synaptology of the
different divisions of the NA may be associated with specific
physiological functions (104, 133, 134, 183).
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Fig. 3. Location of AVPNs innervating airways of the
ferret. A: schematic presentation of a coronal section of the
medulla oblongata showing that in ferrets, as in other
mammals, the preganglionic motor neurons innervating the
airways arise from the rostral nucleus ambiguus (rNA) and
from the rostral portion of the dorsal motor nucleus of the
vagus (DMV). MeV, medical vestibular nucleus; LV, lateral
vestibular nucleus. B: confocal photomicrographs showing
retrogradely labeled AVPNs within the rNA 5 days after
cholera toxin ␤-subunit (CT-b) injections into the lung. C:
same section immunostained for choline acetyl transferase
(ChAT). D: higher power of the M image of CT-b and
ChAT traits of the region marked by a quadrangle in A.
Arrow indicates a representative neuron that is immunolabeled with CT-b and ChAT. *ChAT-positive neuron that
does not contain CT-b. Scale bar ⫽ 50 ␮m for B and C and
35 ␮m for D. Data are from Ref. 119.
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molecules in AVPNs (119). In addition, recent studies demonstrated that brain-derived neurotrophic factor (BDNF) mRNA
and its protein transcript are expressed by a subpopulation of
AVPNs, suggesting that active BDNF synthesis occurs in the
cells within the rNA that innervate the airways (Zaidi SI, Jafri
A, Doggett T, Haxhiu MA, unpublished data). The possible
role of the ACh, VIP, and BDNF-tyrosine kinase (Trk) B
signaling pathways in the autocrine/paracrine regulation of
AVPNs responses to afferent inputs is briefly mentioned at the
end of the article.
CNS Innervation of AVPNs
Fig. 4. Electron microscope image of AVPN in the rNA. This neuron (N) is
retrogradely labeled from the trachea and is identified ultrastructurally by the
presence of tetramethylbenzidine tungstate (TMB) crystalline reaction product
(large arrows). Note the round nucleus and a prominent nucleolus. Area inside
the box is enlarged in the inset and shows an example of a substance
P-immunoreactive nerve terminal (T), indicated by amorphous diaminobenzidine reaction product, forming an asymmetric axosomatic synapse with the
perikaryon. Calibration bars ⫽ 2 ␮m in the larger panel and 500 nm in the
inset. Modified from Ref. 135.
Chemical Profile of AVPNs
Studies using a double-labeling method that combines immunolocalization of the retrograde tracer CT-b and immunohistochemistry for choline acetyl transferase (ChAT), the enzyme catalyzing the biosynthesis of ACh, indicate that in
ferrets, AVPNs innervating the trachea and the intrapulmonary
airways are cholinergic in nature (Fig. 3) and use ACh as a
neurotransmitter to convey signals to the airway motor systems
(119). These neurochemical findings are in agreement with the
results of physiological studies showing that chemical stimulation of the vagal preganglionic neurons (80, 82– 84) or
efferent fibers of the vagus nerve originating from AVPNs
produces pronounced contraction of the airway smooth muscle
and lung parenchyma that is solely mediated via cholinergic
parasympathetic nerves and mechanisms (120, 132, 143, 166).
Furthermore, reflexly (42, 43, 171, 218) and centrally (86, 87)
induced increases in airway secretion are mediated mainly via
cholinergic mechanisms.
Virtually all vagal preganglionic neurons innervating the
trachea and intrapulmonary airways coexpress VIP, but not
NOS, indicating that ACh and VIP are coexisting messenger
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Fig. 5. Somatic, dendritic, and axonic profiles of retrogradely labeled AVPNs.
A: this perikaryon contains abundant rough endoplasmic reticulum (RER) and
somatic spines forms axosomatic synaptic contacts (small arrows) with SP-ir
terminal. B: a dendrite illustrating dendritic spines forming axodendritic
synapses (small arrows) with SP-ir terminals. C: axons of AVPNs are myelinated (M). Unlabeled myelinated axons (m) are indicated for comparison.
Calibration bar ⫽ 500 nm for all panels. Modified from Ref. 135.
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Activity of AVPNs depends on afferent inputs, although
they possess an ability to express synchronous electrical oscillations, unveiled by stimulation of NMDA receptors (83) or
blockade of GABAA receptors (150). Recently, it has been
shown using conventional and transneuronal labeling techniques that the innervation of vagal preganglionic neurons
regulating parasympathetic outflow to the airways arises from
cell groups located in the brain stem and from several higher
brain regions. By comparing the CNS inputs to the vagal
preganglionic neurons that innervate intrapulmonary airways
with those controlling extrathoracic trachea, common patterns
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drawal of inhibitory inputs to AVPNs and facilitation of
cholinergic outflow to the airways (92, 95, 98), suggesting that
an association of OSA and bronchoconstriction during sleep
could be expected more often than it is recognized.
PATHWAYS AND NEUROTRANSMITTERS INVOLVED IN
CONVEYING BRONCHOCONSTRICTIVE INFORMATION FROM
THE AIRWAYS TO THE NTS
Bronchopulmonary sensory receptors, innervated by small
diameter (A␦) myelinated vagal afferents known as rapidly
adapting receptors (RARs), and nonmyelinated C fibers (C
fiber receptors), are characterized by their well-defined sensitivity to chemical stimuli in the airways, most of which affect
both RARs and C fiber receptors (42). The net airway motor
response results from the interaction of sensory signals within
the NTS, including those from cough and slowly adapting
pulmonary receptors (25, 35, 212, 213).
Recently, the NTS second-order neurons activated by afferent inputs from the airways were identified using the expression of the nuclear protein (c-Fos), encoded by the protooncogene c-fos. The c-fos gene, a member of the immediate early
genes, and its product, c-Fos, are expressed in restricted numbers of CNS neurons in response to strong stimuli applied to
the airway receptors. Thus c-Fos is considered to be an inducible and high-resolution marker of neurons activated by sensory inputs and extracellular stimuli (63).
The advantage of using c-fos expression as a functionally
oriented anatomical approach is that the activity of a large
number of cells can readily be identified under conditions that
do not affect reflex responses such as anesthesia. Using this
approach, we found that after stimulation of pulmonary and
bronchial C fibers by capsaicin or stimulation of rapidly adapting receptors and bronchial C fiber receptors by histamine
aerosol (41), a significant number of NTS neurons within the
commissural, medial, and the ventrolateral NTS subregions
expressed c-Fos protein (63, 94), indicating that they had been
activated by stimulation of pulmonary C fiber and rapidly
adapting receptors (26, 94). In ferrets, the labeling pattern
correlated well with studies that defined retrograde tracings of
NTS subregions in which afferent fibers from the airways
terminate (89). The same subregions contained cell body labeling after pseudorabies virus (PRV) injections into the tracheal wall or into the most distal airways (74, 88, 167),
suggesting that the NTS activated neurons are those that
transmit information from the airways to the AVPNs. Multiple
neurotransmitters, including ACh and/or glutamate, are expressed by the primary sensory neurons that may participate in
sensory transmission (17, 99, 160).
Cholinergic Transmission
It was predicted that afferent inputs could be conveyed to the
NTS by ACh acting on nicotinic ACh receptors. This postulate
was based on findings that cholinergic neurons are present in
nodose ganglia (160, 163), whereas 125I-labeled ␣-bungarotoxin binding sites, indicating the presence of nicotinic ACh
receptors (nAChRs) that mediate nicotinic cholinergic transmission, are observed within the NTS (9). Furthermore, nodose
ganglionectomy decreases ChAT activity and causes a reduction in nAChR binding sites in NTS (99). The dissociated rat
NTS cells were observed to express nicotinic but not musca-
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of innervation are seen (74, 88, 167). AVPNs receive inputs
from cell groups located in the ventral aspect of the medulla
oblongata; NTS; pons; ventrolateral part of periaqueductal gray
(PAG) cell group; dorsal, lateral, and paraventricular hypothalamus; and the central nucleus of amygdala (74). Hence, the
parasympathetic preganglionic neurons innervating the airways are controlled by networks of brain stem and suprapontine cell groups that lie in regions known to be involved in the
central control of autonomic functions (129). Recent studies
suggest that some respiratory and airway responses could be
produced by single neurons capable of affecting multiple
neural pathways, rather than by a complex set of heterogeneous
cells regulating individual systems (90, 98).
The AVPNs provide the final common pathway for vagal
control of the airways. Although we emphasized the importance of medullary circuits that control the reflex output of
these AVPNs, functions of the airways are also powerfully
regulated by information from higher CNS centers that are
relayed caudally to the AVPNs. These forebrain circuits are
critical for expression of behavioral state control and the
emotional dimensions of airway disorders. For example, the
prefrontal cortex innervates the interconnected amygdaloid
complex, the central nucleus of which project subsequently
to multiple targets regulating autonomic functions, including the dorsomedial and ventrolateral PAG, the NTS, and
the NA (74, 172).
The projections from the amygdala to the PAG are of
particular importance because the PAG neurons coordinate
functions of multiple visceral organs involved in responses to
stress. Active coping responses (fight or flight reactions) are
evoked by activation of either the dorsolateral or lateral columns of the PAG; whereas passive coping strategies (e.g.,
quiescence, immobility, decreased responsiveness to the environment) are usually elicited by activation of the ventrolateral
part of the PAG (118). The phenotypic traits of some patients
experiencing severe or near-fatal asthma, such as psychosocial
barriers, blunted respiratory, and other sensory responses that
cannot be explained by different medication regimens (16),
resemble the expression of passive coping response seen after
stimulation of the ventrolateral PAG region in animals (118).
In humans, little is known about CNS innervation of
AVPNs. More recently, in conscious subjects, the CNS sites
subserving the experience of loaded breathing and air hunger
were studied using the positron emission tomography (PET)
and functional nuclear magnetic resonance imaging (fMRI; 71,
77, 165). For example, compared with the unloaded control
condition, a moderate inspiratory resistive load is associated
with an increased fMRI signal intensity in discrete brain
regions, including areas of the dorsal pons that correspond to
the locus ceruleus (LC) and parabrachial nucleus (PBN). In
animals, stimulation of the LC noradrenergic cell group (97)
and activation of PBN (153) induces centrally mediated airway
smooth muscle relaxation, a compensatory response that tends
to decrease the resistive load. In addition, activity associated
with perceived intensity of respiratory discomfort induced by
loaded breathing was found within the right posterior cingulate
cortex and amygdaloid complex, demonstrating laterality to the
challenge (165). Of particular interest are recent findings in
obstructive sleep apnea (OSA) showing lower signals in medullary and midbrain areas (77), sites that innervate AVPNs (74,
88). A decrease in activity of these sites may lead to with-
Invited Review
BRAIN STEM EXCITATORY AND INHIBITORY SIGNALING PATHWAYS
1967
rinic acetylcholine receptors (199). In addition, a subset of
second-order NTS neurons expresses ChAT in perikarya and
dendrites (6).
Experiments demonstrated that activation of airway sensory
receptors (RARs and C fiber receptors) induced c-Fos expression in a subset of NTS neurons that also expressed the
␣3-subtype nicotinic ACh receptor (nAChR). Furthermore,
activation of nAChR within the commissural NTS subnucleus
by nicotine increased cholinergic outflow to the airways. These
effects were diminished by prior administration of hexamethonium (nAChR antagonist) within the commissural NTS cell
group. However, hexamethonium had no significant effects on
airway reflex constrictions induced by lung deflation. These
findings indicated that endogenously released ACh within the
NTS and activation of nAChR are not required for transmission
of bronchoconstrictive stimuli from the airways to the NTS
(63), suggesting involvement of other excitatory molecules
such as glutamate.
L-Glutamate, a naturally occurring excitatory amino acid, is
a main sensory neurotransmitter (47) mediating communication between neurons within neuronal networks coordinating
motor outputs (for review and references see 72, 209). It is
present in vagal afferents in the NTS (179, 180, 195, 206) and
is required for transmission of fast signals from sensory nerve
endings to second-order neurons (5, 94, 211), including baroreceptor inputs (125, 126, 197).
Recently, we studied the role of glutamate in conveying
bronchoconstrictive inputs from the airways to the NTS and
from the NTS to the AVPNs (91, 93, 94). Glutamate release
within the NTS after stimulation of airway sensory receptors
was also examined. These studies indicated that stimulating
airway sensory receptors increased the spontaneous efflux of
glutamate in the commissural NTS subnucleus (94), the site
where airway afferent fibers terminate in ferrets (89), and that
the release was correlated with airway smooth muscle contraction (94). Typical HPLC chromatograms of microdialysates
were collected from the commissural NTS subnucleus in a
control state, during repeated excitation of bronchopulmonary
sensory receptors, and in the poststimulation period. After
airway sensory stimulation, glutamate concentration increased
significantly from 42 ⫹ 11 to 109 ⫹ 17 pg/␮l (average of 3
trials for each ferret), and pressure in a bypassed tracheal
segment (Ptseg) increased from 7.4 ⫹ 1.4 to 26.1 ⫹ 4.6
cmH2O (P ⬍ 0.01). In the poststimulation recovery period,
L-glutamate and Ptseg returned to control levels. Removal of
the vagal efferent and afferent innervations of the tracheobronchial tree eliminated the reflex increase in glutamate release
induced by lung deflation (Fig. 6).
Conceivably, the release of endogenous glutamate could be
due to baroreceptor loading (125, 126, 197) and/or activation
of peripheral chemoreceptors by oxygen deprivation (147).
However, in reported experiments, lung deflation had insignificant effects on arterial pressure and ferrets were ventilated
with O2, likely excluding the possibility that lung deflation
induced release of glutamate by triggering of chemosensory
reflex responses. Furthermore, denervation of the airways and
the lungs completely blocked the glutamate release induced by
lung deflation, suggesting that the release of endogenous glu-
J Appl Physiol • VOL
Fig. 6. Reflex-induced glutamate release within the NTS. Top: typical HPLC
chromatograms of microdialysates collected from the commissural subnucleus
of the NTS in a control state (baseline), during repeated excitation of bronchopulmonary sensory receptors (stimulation), and in a poststimulation period
(recovery). Middle: average concentrations (means ⫾ SE) of L-glutamate,
expressed in pg/␮l, under 3 different experimental conditions. Filled bars:
animals with intact innervation of the airways and lungs (vagus intact; n ⫽ 5).
Open bars: ferrets after afferent and efferent denervation of the airways and
lungs (vagotomized and superior laryngeal nerves cut; n ⫽ 3). Bottom: tracheal
smooth muscle tone measured as pressure in a bypassed tracheal segment
(Ptseg) before, during, and after stimulation in ferrets with intact innervation of
the airways. *P ⬍ 0.05. Modified from Ref. 94.
tamate was related to sensory inputs from the bronchopulmonary system.
Over the last two decades, extensive research has revealed a
host of glutamate receptor subtypes belonging to the ionotropic
(iGluR) and metabotropic glutamate receptor (mGluR) classes,
which subserve excitatory synaptic transmission and neurotransmitter release.
iGluRs. iGluRs are ligand-gated ion channels consisting of
three known functional receptor subtypes: the AMPA, kainate,
and NMDA receptors, which respond to glutamate and glutamate analogs by the opening of a cation channel (4, 10, 27, 44,
138). Quisqualate, AMPA, glutamate, and kainate are potent
agonists of the AMPA receptor subunits (GluR1-GluR4) when
applied alone or in combination. The quinoxalinediones, such
as 6,7-dinitroquinoxaline-2, dione (DNQX) and 6-cyano-7nitroquinoxaline-2,3-dione (CNQX), are the most potent antagonists of the AMPA receptors. This class of antagonists also
blocks the kainate receptors but does not block NMDA receptors. Hence, it can be used to differentiate AMPA receptormediated effects from those of the NMDA signaling pathway.
The kainate receptors, composed of five subunits, GluR5GluR7 and KA1 and KA2, contribute to excitatory postsynaptic currents in many regions of the central and peripheral
nervous systems, including the hippocampus, cortex, spinal
cord, and retina. In some cases, postsynaptic kainate receptors
are codistributed with AMPA and NMDA receptors, but there
are also synapses where transmission is mediated exclusively
by postsynaptic kainate receptors. Recent analysis of knockout
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Glutamatergic Transmission
Invited Review
1968
BRAIN STEM EXCITATORY AND INHIBITORY SIGNALING PATHWAYS
J Appl Physiol • VOL
Fig. 7. Coexpression of c-Fos and GluR2 AMPA receptor subunit in secondorder nTS neurons after stimulation of excitatory bronchopulmonary receptors.
Top: confocal microscope image of the commissural region of the NTS. c-Fos
staining (red) was observed only within nuclei. These were visualized as round
or oval structures, depending on their orientation within the plane of the
section. GluR2 receptor staining (green) was observed to be punctate and
uniformly distributed on the membrane of the perikarya of neurons with
unstained nuclei (GluR2) and on neurons with c-Fos-stained nuclei (c-Fos and
GluR2). Middle: numbers of neurons within the commissural region of the
NTS that express c-Fos, GluR2, and c-Fos-positive neurons that coexpress
GluR2. Bottom: superimposed tracings from a paralyzed ferret ventilated with
oxygen. In a control period, lung deflation induced an increase in tracheal tone,
expressed by elevation of Ptseg that was not affected by bilateral microinjection of vehicle into the commissural subnucleus of the NTS (A). Administration of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) into the same region in
a dose-dependent fashion decreased the response (B: 1 nmol; C: 4 nmol/site
CNQX). Modified from Ref. 94.
mGluR1 and mGluR5 that are found mainly on postsynaptic
terminals and are positively coupled to phospholipase C.
Group II mGluRs (mGluR2 and mGluR3) are observed on both
pre- and postsynaptic terminals, whereas group III mGluRs
(mGluR4, mGluR6, mGluR7, and mGluR8) are predominantly
located in or near presynaptic zones. In general, group I
mGluRs increase neuronal excitability, whereas group II and
group III modulate frequency dependence of synaptic vesicle
exocytosis, inhibiting signal transmission (33, 36, 128, 158,
161, 164, 184).
It is accepted that released glutamate acts locally on postsynaptic receptors and is cleared from the synaptic cleft within a
few milliseconds by diffusion and by specific reuptake mechanisms. This rapid clearance restricts the spread of neurotransmitter and, combined with the low affinities of many ionotropic
receptors, ensures that synaptic transmission occurs in a pointto-point fashion. However, when glutamate release is enhanced
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mice lacking one or more of the subunits that contribute to
kainite receptors, as well as studies with subunit-selective
agonists and antagonists, have revealed the important roles that
kainate receptors play in short- and long-term synaptic plasticity (27, 106). Because AMPA receptors are more abundant
than kainate receptors and exhibit faster signaling kinetics than
NMDA receptors (70, 169), the AMPA receptors are uniquely
suited in mediating cardiopulmonary reflexes (5, 69, 211).
Furthermore, synaptic activation of AMPA receptors may elicit
not only postsynaptic excitation but also presynaptic inhibition
of GABAergic transmission. By suppressing the inhibitory
inputs, the activation of AMPA receptors could facilitate bronchoconstrictive inputs to the NTS second-order neurons and
from these neurons to the AVPNs. This possibility is supported
by findings showing that stimulation of AMPA receptors induces presynaptic inhibition at cerebellar GABAergic synapses
(182) but increases ACh release in the hippocampus of young
and aged rats (177).
Experiments to determine whether specific AMPA receptors
are expressed by the activated NTS neurons were performed in
ferrets by using a double-staining technique and confocal
microcopy (94). Employing GluR2 mouse monoclonal antibody, it was observed that this AMPA receptor subtype is
present on the subpopulation of the NTS neurons activated by
sensory inputs from the airways (Fig. 7). In addition, the role
of the glutamate-AMPA receptor signaling pathway in transmission of bronchoconstrictive signals from the airways to the
NTS was studied using physiological experiments. Blockade of
AMPA receptors by bilateral microinjections of CNQX into
the commissural subnucleus elicited a significant decrease in
reflex tracheal smooth muscle response, markedly slowed the
rate of rise of tracheal tone, decreased the peak response, and
enhanced the decline of the response after cessation of lung
deflation (Fig. 7, bottom). Therefore, bronchoconstrictive inputs from the airways to NTS neurons are transmitted primarily
by a glutamate-AMPA receptor signaling pathway.
In addition, studies suggest that NMDA receptors expressed
by the NTS neurons that receive afferent inputs from the
airways enhance the glutamate-AMPA receptor-mediated responses. These receptors are present in vagal afferents and
their dendritic targets in the NTS. Hence they may play a role
in autoregulation of the presynaptic release and postsynaptic
responses to glutamate at the level of the first central synapse
in the NTS (2).
Therefore, it seems that the involvement of AMPA receptors
in airway reflex responses is determined by their kinetics,
which is much faster than those of the NMDA or kainate
receptors. Hence the AMPA receptors are more appropriate for
transmission of fast signals, whereas NMDA and kainate
receptors may contribute to primary visceral afferent transmission in the NTS, mediating tonic influences.
G protein-coupled metabotropic glutamate receptors. G protein-coupled metabotropic glutamate receptors are expressed in
autonomic cell groups of the medulla oblongata (79), including
NTS second-order neurons (136, 158, 162). Coactivation of
iGluRs and G protein-coupled metabotropic glutamate receptors (mGluRs) may affect glutamatergic transmission through
regulation of neurotransmitter release. On the basis of amino
acid sequence homology, pharmacology, and signal transduction mechanisms, these subtypes have been classified into three
groups (103, 136, 170, 196). Group I mGluRs consist of
Invited Review
BRAIN STEM EXCITATORY AND INHIBITORY SIGNALING PATHWAYS
from the airways to the NTS, where signals are processed,
modulated, and relayed to the vagal parasympathetic neurons
innervating the airways.
GLUTAMATERGIC PATHWAYS TRANSMITTING
BRONCHOCONSTRICTIVE INPUTS FROM THE NTS TO
THE AVPNS
Until recently, signaling mechanisms involved in transmitting bronchoconstrictive signals from the NTS to the AVPNs
were not established. However, it was assumed that the glutamate-AMPA receptor signaling pathway may play an important role. This assumption was based on findings that a majority of NTS neurons contain glutamate (121) and information
from these neurons is transmitted to AVPNs mainly via hardwired synaptic pathways (74, 88, 167).
Recent work has shown that the excitatory glutamatergic
neurons express different types of vesicular transporter proteins, named VGLUT1, VGLUT2, and VGLUT3 (102, 115).
VGLUT2, a protein that localizes to synaptic vesicles, functions as an important vesicular glutamate transporter (19, 64,
102, 194). Our unpublished studies in ferrets, using a doublelabeling technique, demonstrate heavy glutamatergic innervation of the AVPNs (Acquah S, Kc P, Massari VJ, Haxhiu MA,
unpublished results), forming the morphological basis for the
powerful influence of excitatory amino acids on the activity of
AVPNs (Fig. 8). Effects of glutamate on AVPNs are mediated
through different subtypes of glutamate receptors.
Ionotropic Glutamate Receptors and Transmission of
Excitatory Signals From NTS to AVPNs
Neurochemical studies indicate the presence of AMPA,
NMDA, and kainate receptors in vagal preganglionic neurons
(45). Neurophysiological investigations have shown that
iGluRs play an important role in transmission of excitatory
inputs to the AVPNs regulating airway smooth muscle tone.
For example, NMDA receptors are expressed by AVPNs and,
when activated with a specific NMDA receptor agonist
(NMDA), cause airway constriction that is blocked by selective NMDA antagonists (83). The possibility that NMDA
receptors may also play a role in reflex airway constriction has
been studied by inducing blockade of NMDA receptors with
2-aminophosphonovalerate. Topical application or microinjection of this NMDA antagonist within the rNA, the area in
which the AVPNs are located, only slightly affected reflex
changes in tracheal tone. However, administration of selective
antagonists for the AMPA/kainite subtype of glutamate recep-
Fig. 8. Glutamatergic axon terminals innervating identified AVPNs. A: confocal
microscopic image of AVPNs immunolabeled for CT-b with a green fluoresceinconjugated secondary antibody (green) and
glutamate transporter 2 (VGLUT2) with
Texas Red-conjugated secondary antibody
(red). B: higher magnification image of
neurons identified in A (quadrangle), illustrating VGLUT2-like immunoreactive puncta
targeting AVPNs and their proximal dendrites.
Scale bar ⫽ 20 ␮m for A and 10 ␮m for B.
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at synapses, the concentration increases and glutamate escapes
the synaptic cleft and, via volume transmission, may activate
presynaptic inhibitory mGluRs. Hence, at higher frequency
stimulation, when the released amount of glutamate exceeds
the uptake quanta, presynaptic mGluRs of groups II and III
become activated, leading to a rapid inhibition of neurotransmitter release (58, 184) and, consequently, presynaptic depression of synaptic transmission (33) that might have neuroprotective effects (61).
In the rat, activation of mGluRs affects frequency dependence response and depresses vagal and aortic baroreceptor
signal transmission in the NTS (66, 127, 128, 144) via activation of one or more phosphoprotein phosphatases such as
phosphoprotein phosphatase 2 and/or calcineurin (67). Furthermore, recent findings, determining presynaptic vs. postsynaptic
effects, indicate that glutamate released at the first central
baroreceptor synapses cannot only regulate its own signaling
but can further shape signal transmission by suppressing
GABA release via activation of heterosynaptic group II
mGluRs (39). The results highlight the complexity of mGluRs
functions in modulation of reflex responses within the NTS and
suggest the importance of differentiation of presynaptic vs.
postsynaptic effects of drugs tested.
In ferrets, recent neuroanatomical and physiological studies
showed that subtype 1 of the group I mGluRs is rarely
expressed by activated NTS neurons after stimulation of bronchopulmonary receptors. Blockade of the group I mGluRs
using a specific antagonist had no significant inhibitory effects
on airway reflex responses, when administered into the NTS.
Furthermore, blockade of groups II/III mGluRs within the NTS
had no significant enhancing effect on reflex airway smooth
muscle contraction (Ferguson DG, Haxhiu MA, unpublished
data). However, these results can be considered only as preliminary, because microinjection of the drug into the rNA, as in
any other circumscribed region, is not very selective. Namely,
administered antagonist of group II or III mGluRs may have
effects on presynaptic as well as on postsynaptic sites.
Future studies, separating presynaptic from postsynaptic
influences, are needed to define whether under pathological
conditions, such as animal model of bronchial asthma and
experimentally induced non-allergic airway hyperreactivity,
upregulation of group I and/or downregulation of group
II/III of mGluRs may facilitate reflex airway constriction
and hyperresponsiveness.
In summary, neuroanatomical and physiological studies further highlight the key role of the glutamate-AMPA receptor
signaling pathways in transmitting bronchoconstrictive inputs
1969
Invited Review
1970
BRAIN STEM EXCITATORY AND INHIBITORY SIGNALING PATHWAYS
afferent and efferent vagal pathways but preserves local mechanisms (including axon reflex pathways). This treatment diminishes more than two-thirds of the reflex vasodilatation; thus
a large portion of the vasodilatation in dogs, whether caused by
activation of sensory C fiber receptors and/or RARs, is due to
centrally mediated neural reflexes that include afferent and
efferent pathways in the vagus nerves (171). In addition,
stimulation of bronchial and pulmonary C fibers or RARs
evokes a reflex increase in secretion by tracheal submucosal
glands. The responses are abolished on interruption of the
afferent and efferent transmissions by cutting the vagus nerves
or cooling them to 0°C (42, 218). Furthermore, focal cooling of
the rostral ventrolateral medulla between 20°and 15°C significantly decreases the secretion rates produced by capsaicininduced stimulation of pulmonary C fiber receptors and by
mechanical stimulation of the carina and larynx (87).
The involvement of glutamate and glutamate receptors in the
transmission of excitatory inputs from the airway sensory
receptors to the NTS and from this site to the AVPNs was
studied (91). Stimulation of airway sensory fibers by lung
deflation induced reflex increases of tracheal blood flow (Fig.
9) and submucosal gland secretion (Fig. 10). These responses
were diminished by prior administration of an AMPA/kainate
receptor antagonist, CNQX, into the fourth ventricle, or microinjection of AMPA/kainate receptor blockers into the external formation of the rNA, where the AVPNs are located.
These findings indicate that the transmission of excitatory
inputs from the NTS to the AVPNs is mediated mainly via the
Fig. 9. Glutamatergic control of reflex-induced changes in airway blood flow. Top:
schematic presentation of the extrathoracic
tracheal segment that can be used for simultaneous recording of submucosal blood flow,
gland secretion, and airway smooth muscle
tone. Tracheal segment preparation used to
measure trachealis smooth muscle tension
was developed by Brown et al. (29), whereas
video camera method of measuring mucous
secretion was developed by Davis et al. (50).
Bottom: tracings from decerebrate, paralyzed, and mechanically ventilated dog after
administration of vehicle into the IV ventricle. Vehicle had no effects on resting tracheal circulation. Lung deflation induced an
increase in submucosal blood flow (Q̇t; left)
and slightly elevated systemic blood pressure
(BP; mmHg). Administration of CNQX, indicated by the arrow, tended to decrease the
basal blood flow and abolished the response to
lung deflation (right). V̇ (l/s), air flow. Modified from Ref. 93.
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tor to the same site caused a dose-dependent decrease in reflex
response of tracheal tone induced by 1) lung deflation, 2)
stimulation of laryngeal cold receptors, and 3) activation of
peripheral or central chemoreceptors (91). These reflexes are
known to cause centrally mediated increases in cholinergic
tone (42, 51, 52). Inhibitory effects of AMPA receptor blockade were potentiated by prior administration of an NMDA
receptor antagonist. Thus an increase in central cholinergic
outflow to the airways by a variety of reflex excitatory inputs
is mediated mainly via glutamate-AMPA receptors that in turn
activate the NMDA receptor signaling pathway.
Physiological studies indicate that increases in airway blood
flow and submucosal gland secretion are integral components
of pulmonary defensive reflex responses (42, 43, 171, 218).
Neural regulation of the bronchial vasculature differs from that
of the general systemic circulation in that vasodilator reflexes
play a major part in determining blood flow (43). These
reflexes originate in the upper or lower airways, in carotid
chemoreceptors, or in cardiac chemosensitive nerves. Those
arising from the lower airways are the most potent and may
increase bronchial blood flow several-fold and cause swelling
of the airway mucosa. In addition, neuropeptides released from
C fiber terminals provide a local mechanism for vasodilation
independent of central reflex control. This so-called axon reflex
plays a major role in bronchial vasodilation in rodents but
makes only a small contribution in larger animals. In dogs, a
centrally mediated vagal reflex vasodilator pathway appears
more important. Cooling the cervical vagus nerves eliminates
Invited Review
BRAIN STEM EXCITATORY AND INHIBITORY SIGNALING PATHWAYS
1971
release of glutamate and activation of the AMPA/kainate
subtype of glutamate receptors. Therefore, it is likely that the
airway sensory stimulation-evoked airway smooth muscle contraction, vasodilation, and hypersecretion are mediated mainly
via cholinergic mechanisms, using the same, glutamate-AMPA
signaling pathway, that in turn activate NMDA receptors, as
summarized in Fig. 11.
Metabotropic Glutamate Receptors and Transmission of
Excitatory Signals From the NTS to AVPNs
In ferrets, mGluRs belonging to group I are expressed by
AVPNs (Ferguson DG, Haxhiu MA, unpublished data). Their
role in glutamatergic transmission of bronchoconstrictive inputs was investigated using a selective antagonist. Blockade of
these receptors within the rNA region had no significant effect
on airway basal smooth muscle tone or on reflex bronchoconstriction. Similarly, inhibition of group II/III mGluRs had no
J Appl Physiol • VOL
demonstrable facilitatory effect on reflex increases in unit
activity of the AVPNs (Kc P, Haxhiu MA, unpublished data),
suggesting that the physiological relevance of the excitatory
and/or inhibitory mGluRs is, at best, limited. However, these
findings do not exclude the possibility that these receptors may
operate at higher frequency afferent inputs, enhancing glutamatergic responses (group I mGluRs), or suppressing excessive
signal transmission at AVPN synapses (groups II and III
mGluRs), consequently modulating the transmission of bronchoconstrictive inputs from the NTS to the AVPNs within the rNA.
CENTRAL GABAERGIC CONTROL OF CHOLINERGIC
OUTFLOW TO THE AIRWAYS
Processing of central afferent excitatory signals by the
AVPNs and conveying integrated output to the airways are
highly dependent on synaptic GABAergic inhibitory inputs
(152). In general, GABA release and activation of postsynaptic
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Fig. 10. Glutamatergic control of reflex
changes in submucosal secretion. An example of the effect of CNQX administration
into the IV ventricle on secretory response of
tracheal submucosal glands to lung deflation.
Prior administration of CNQX abolished the
response of submucosal glands to lung deflation. A and C: tracheal epithelium 1 min
after spraying with tantalum (an inert metal
dust that prevents spread of secreted fluid),
baselines. B and D: tracheal epithelium 1
min after lung deflation after vehicle (B) or
after CNQX administration into the IV ventricle (D). Similar effects were observed
when CNQX was bilaterally microinjected
into rNA. Modified from Ref. 93.
Invited Review
1972
BRAIN STEM EXCITATORY AND INHIBITORY SIGNALING PATHWAYS
Fig. 11. Summary diagram of glutamatergic
signal transmission pathways mediating airway constrictive reflex responses. Glutamate-AMPA receptor signaling pathways
play a major role in transmission of excitatory sensory information from the airways
(sensor, afferent nerve, sensory neuron) to
the second-order NTS neurons and consequently from these neurons (terminal of presynaptic neuron) to the AVPNs. Activation
of AMPA receptors via increase in Na⫹
influx elicits excitatory postsynaptic potential (EPSP) that in turn activates NMDA
receptors leading to Ca2⫹ entry. Signals
from the stimulated AVPNs are transmitted
to the airways using ACh as a neurotransmitter.
GABAergic Microcircuitry Regulating AVPNs
GABA, the main inhibitory neurotransmitter in mammalian
brain, is synthesized mainly via decarboxylation of glutamic
acid by two specialized enzymes, the glutamic acid decarboxylases (GADs), designated GAD65 and GAD67 according to
their molecular masses (65 and 67 kDa). These enzymes differ
in their affinities for their cofactor pyridoxal-5⬘-phosphate and
are encoded by two different genes located on separate chromosomes (32, 60). Both enzymes are often colocalized in the
same GABAergic neurons but sometimes differ in their subcellular distribution. GAD67, although also detected in axon
terminals, is thought to be preferentially localized to cell
bodies, whereas GAD65 tends to be associated with synaptic
vesicles in nerve terminals (62, 100). However, more detailed
studies revealed a more complex situation, showing that both
GAD65 and GAD67 provide important reserve pools of GABA
for the regulation of inhibitory neurotransmission, and a deficiency of either isoform of GAD can have significant physiological sequelae. For example, GAD67-deficient animals are
born with a cleft palate and die within the first day of life,
apparently from respiratory failure (8). By contrast, GAD65deficient mice appear normal at birth, but the pyridoxal-5⬘phosphate-inducible apoenzyme reservoir is significantly decreased, leading to an increased susceptibility to epileptigenic
stimuli (7). Hence both isoforms of GAD appear to be physiologically important in the dynamic regulation of neural network excitability.
Recent ultrastructural studies have shown that retrogradely
labeled AVPNs receive a significant GABAergic innervation
(Fig. 12). Out of a pooled total of 3,161 synaptic contacts with
retrogradely labeled somatic and dendritic profiles, 20.2% are
GAD-immunoreactive (IR), forming significantly more axosomatic symmetric synaptic specializations than axodendritic
synapses (P ⬍ 0.02). These ultrastructural findings indicate
that central GABAergic modulation of cholinergic outflow is
mediated in large part via classically defined inhibitory axoso-
Fig. 12. GABAergic innervation of AVPNs. A: a GABA
immunoreactive nerve terminal (T) containing a large
dense-core vesicle as well as multiple small pleomorphic
vesicles forms an axodendritic synapse (small arrowhead)
with the proximal dendrite (D) of a retrogradely labeled
(large arrowheads) tracheal vagal preganglionic neuron.
Unlabeled nerve terminal (t) forms an asymmetric axodendritic synapse (small arrowheads) with a dendrite (D). B: in
the same field, note that a GABA immunoreactive nerve
terminal (T) forms a symmetric axodendritic synapse (small
arrowheads) with an unlabeled dendrite (d). Calibration bar
equals 200 nm. Modified from Ref. 150.
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GABAA receptors play an important role in controlling neuronal excitability in adult mammalians. This occurs by increasing
membrane conductance to chloride ions, thereby causing a
potent inhibition of neuronal activity that in turn diminishes the
depolarizing effects of excitatory synaptic signals (208). The
action of released GABA within or in close vicinity to the
synaptic cleft is terminated by its rapid uptake into surrounding
neurons and astrocytes by selective GABA transporters (53,
81, 186).
Invited Review
BRAIN STEM EXCITATORY AND INHIBITORY SIGNALING PATHWAYS
1973
GABA Levels Within the rNA
Recently, baseline levels of GABA within the rNA region
have been measured by microdialysis and HPLC. After equilibration of the dialysis probe, the average measured concentration of GABA was 29.3 ⫾ 7.1 pg/20 ␮l during a steady-state
condition. After stimulation of the ventrolateral region of the vl
PAG, GABA concentration significantly increased (61.4 ⫾
22.1 pg/20 ␮l; Fig. 13, top). Although the increase in GABA
levels after chemical stimulation is of an exocytotic origin, the
basal levels of extracellular GABA could be derived in part
from nonexocytotic source, as in other brain regions (53, 198).
Extracellular levels of GABA within the rNA, measured by
microdialysis and HPLC techniques, can originate from various sources and depend upon release, diffusion, and uptake
mechanisms. GABA released from GABAergic nerve endings
can spill over from the synaptic cleft to perisynaptic or extrasynaptic regions (76, 149, 178) and activate extrasynaptic
receptors, including those on the edge between effective synapses and uninnervated zones, modulating the gain and maintaining inhibitory tone (148, 187). GABA levels within the
synaptic cleft and at extrasynaptic regions depend on the
activity of high-affinity GABA transporters (i.e., GAT-1) located in axon terminals around the synaptic cleft and/or expressed by surrounding astrocytes (81, 186). GABA uptake
mechanisms play a critical role in termination of both synaptic
and extrasynaptic GABAergic inhibitory signaling. To a lesser
degree, however, GABA spillover through activation of
GABAB receptors on nerve terminals may control neurotransmitter release at the target site (3, 185).
J Appl Physiol • VOL
Fig. 13. GABA release within rNA and GABAA receptors expressed by
AVPNs. Top: GABA levels (means ⫾ SE; n ⫽ 6) obtained from microdialysates collected from the rNA region in a control state (Baseline) and during
repeated excitation of ventrolateral periaqueductal gray (vl PAG) neurons
(PAG stim). *P ⬍ 0.05. Bottom: example of a confocal microscope image of
GABAA ␤2-subunit receptor expression by the AVPNs innervating the extrathoracic trachea. A: CT-b-labeled AVPNs (red). B: in the same region,
punctate GABAA receptor-specific staining (green) is observed that is likely
associated with the cell membranes, because it surrounds empty spaces (N) in
which neuron cell bodies lie. There were also profiles of GABAA receptorspecific staining in between the neuron cell bodies (*). These presumably
identify receptors located in presynaptic terminals that contact processes of the
motor neurons. C: in double-labeling studies it is clearly observed that the
CT-b-labeled neurons contain GABAA ␤2-subunit (green) expressed as punctate green staining on the membrane of the perikaryon (arrows) as well as on
dendrites. Bar ⫽ 50 ␮m (A), 30 ␮m (B), 15 ␮m (C). Modified from Ref. 96.
GABA Receptor-Mediated Signaling
GABA receptors are categorized in two distinct types: the
ionotropic GABAA and GABAC and the metabotropic GABAB
receptors.
GABAA receptors. Fast GABA-mediated synaptic inhibitory
neurotransmission requires that GABAA receptors are expressed and assembled at appropriate postsynaptic sites facing
GABA-releasing nerve terminals (124, 130, 149). These chloride ion channel-associated, ligand-gated receptors are heteropentamers that can be assembled from a number of subunit
classes that include: ␣ (1– 6), ␤ (1– 4), ␥ (1–3), ␦, ⑀, ␪, ␲ (1
isoform each), and ␳ (1–3). The majority of these receptors are
composed of two ␣-, two ␤-, and one ␥2-subtype. The coexpression of a ␥2-subunit with ␣- and ␤-subunits produces
GABAA receptors displaying high-affinity binding for central
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matic synapses and to a lesser degree through axodendritic
synaptic transmission. A dense population of GABAergic synaptic contacts on AVPNs provides a morphological basis for
the potent physiological effects of GABA on the excitability of
AVPNs (150).
Both axosomatic and axodendritic GABAergic innervation
may exert modulatory tonic influences. For example, tonic
GABAergic signaling regulates the activity of cerebellar granule cells (28, 76). Similarly, the activity and the dischargefrequency patterns of medullary respiratory premotor neurons
are subject to potent tonic GABAergic gain modulation (140,
220). The axodendritic synaptic GABAergic modulation of
AVPNs structurally distant from the soma/spike initiation zone
and proximal dendrites may exert less impact on the resting
membrane potential of these neurons than do axosomatic
synapses close to the axon hillock or proximal axodendritic
GABAergic synapses. However, quantitative assessments of
the relative distribution of GABAergic synapses on various
parts of individual AVPNs must necessarily be somewhat
imprecise because the distal parts of the AVPN dendrites were
not as robustly retrogradely labeled with CT-b-horseradish
peroxidase as the more proximal portions of the dendrites and
the perikarya and therefore these distal synaptic interactions
would be seen less frequently. Nonetheless, the ultrastructural
data clearly demonstrate that a central GABAergic inhibitory
microcircuit uses both axosomatic and proximal axodendritic
synapses on AVPNs to modulate cholinergic drive to the
tracheobronchial system (150).
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1974
BRAIN STEM EXCITATORY AND INHIBITORY SIGNALING PATHWAYS
J Appl Physiol • VOL
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benzodiazepine ligands and anesthetics (15, 23, 149, 174, 189,
190). Changes in GABAA receptor subtype expression induce
plasticity in fast synaptic inhibition. For example, a decrease in
the ␣1:␣2-subunit mRNA ratio is associated with a decrease in
potentiation and increase in decay time constant of GABAA
receptor-mediated inhibitory postsynaptic potentials (30).
The exact composition of the GABAA receptor subtype
expressed by the AVPNs that mediate airway changes induced
by GABA remains to be characterized. However, our most
recent study demonstrated that clusters of the ␤2-isoform of
GABAA receptor subunit are present on the cell bodies and
nerve processes of the AVPNs (96) as shown in Fig. 13,
bottom. This subunit plays an important role in determining the
pharmacology of the GABAA receptors (75). Furthermore,
physiological and pharmacological studies indicated that benzodiazepines, topically applied or microinjected into the rNA
region, cause withdrawal of cholinergic outflow to the airways
and airway smooth muscle relaxation (85), indicating that this
receptor subtype coexpresses a ␥2-subunit (189).
GABAB receptors. In the mammalian CNS, GABAB receptors exist as a functional GABAB1/GABAB2 heterodimer (46).
It was thought that the GABAB receptors function predominantly presynaptically and that they are activated only when
GABA is released in large amounts, e.g., on simultaneous
activation of multiple GABAergic neurons. Activation of
GABAB receptors produces a presynaptic inhibition and a
decrease of neurotransmitter release, including glutamate and
ACh (3, 185, 188). Inhibition is mediated either by blocking
the action potential invasion into nerve terminals or by reducing the amplitude of the propagated signal. This may occur via
multiple mechanisms, including depression of Ca2⫹ channels
(3). GABAB receptors are also expressed by effector cells and
can produce hyperpolarization in postsynaptic membranes
(46). Furthermore, our preliminary studies demonstrated that
GABAB receptors are expressed by axon terminals innervating
the AVPNs and by cell bodies of AVPNs (Kc P, Haxhiu MA,
unpublished data) that, in part, may mediate pre- and postsynaptic
modulatory effects of GABA on glutamatergic transmission.
GABAC receptors. GABAC receptors are less abundant and
less widely distributed in the CNS compared with GABAA or
GABAB receptors. These receptors, described in the rodent
retina, the optic tectum, hippocampus, pituitary gland, and
spinal cord, gate a Cl⫺ channel similar to GABAA receptors
but exhibit a different pharmacological profile. The main
difference between the GABAC receptors and conventional
GABAA receptors is their bicuculline and barbiturate resistances. Furthermore, GABAC, in contrast to GABAA receptors,
do not desensitize, rendering them particularly well suited for
processing graded potentials (110, 173). Separate lines of
recent evidence suggest that the effects of GABA in central
neurons can be mediated by coassembly of GABAA and
GABAC receptor subunits (146). The role of this receptor
subtype in mediating GABAergic modulation of AVPNs and
cholinergic outflow to the airways needs to be studied.
Tonic GABAergic influences. GABAA receptor signaling
may exert tonic and phasic inhibitory effects (148), thereby
modulating the gain and the firing threshold of AVPNs. Recently, Moore et al. (150) reported that under baseline conditions, GABAA-receptor-mediated tonic inhibitory currents are
important determinants of AVPN firing rate. Microinjection of
a low concentration of the GABAA receptor blocker bicucul-
Fig. 14. Summary diagram of GABAergic inhibitory influences on AVPNs.
GABAergic microcircuit uses synaptic (axosomatic and axodendritic) and
nonsynaptic transmission (activation of GABAAR at and just outside of the
synaptic edge) to downregulate excitability of AVPNs. GABAergic gain
modulation of AVPNs may also occur via axoaxonic synapses between
GABA-containing terminals and glutamatergic axons innervating AVPNs.
Release of GABA and activation of GABABR at these sites decreases glutamate release and consequently reduces AVPNs discharge and cholinergic
outflow to the tracheobronchial system.
line into the rNA region significantly increased the frequency
of spontaneous discharge of the AVPNs (Fig. 14) and led to an
increase in airway smooth muscle tone. These responses were
not due to bicuculline-induced blockade of apamine-sensitive
Ca2⫹-activated currents (109), indicating that tonically active
GABAA receptors are present and play a role in the control of
bronchomotor tone. The tonic GABAergic inhibition could be
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BRAIN STEM EXCITATORY AND INHIBITORY SIGNALING PATHWAYS
J Appl Physiol • VOL
Fig. 15. Tonic GABAergic inhibition of the AVPNs. Top: after picospritzing of
bicuculline (Bic) (0.6 nmol) into the rNA, multiple putative AVPN units increased
their firing rate and exhibited phasic burstlike output (top). The rate histogram is
binned in 10-ms intervals (bottom). Middle: in 7 putative AVPNs, deflation and
Bic significantly increased the firing rate over baseline control conditions (*P ⬍
0.05). Data are reported as spikes per second (frequency) for each treatment.
Bottom: blockade of GABAA receptors within rNA induced an increase in tracheal
tone, expressed as an elevation of the Ptseg. B: average Ptseg values (means ⫾ SE)
in a control state (A); average change in Ptseg induced by blockade of GABAA
receptors (n ⫽ 6; B); average change in Ptseg induced by blockade of GABAA
receptors after microperfusion of 2% lidocaine in the rNA (n ⫽ 3; C); and average
change in Ptseg induced by blockade of GABAA receptors after systemic administration of atropine methylnitrate (n ⫽ 3; D). Modified from Ref. 150.
In summary, the functional relevance of the GABAergic
microcircuit in the central regulation of cholinergic outflow to
the airways is presented in Fig. 15. Abnormalities of GABAergic function caused by either genetic or acquired alterations of
GABAA receptors or ion channelopathies may result in a shift
from inhibitory to excitatory neurotransmission. These changes
may lead to a hyperexcitable state of AVPNs and to airway
hyperreactivity. Hence enhancement of GABA-induced neuronal inhibition may be useful in treating disorders associated
with an increased cholinergic outflow to the airways (i.e.,
obstructive bronchitis and bronchial asthma).
PARACRINE-AUTOCRINE REGULATION OF AVPNS
Activity of the vagal preganglionic neurons innervating the
airways is extrinsically regulated by the neurotransmitters or
neuromodulators that are released from the nerve terminals that
innervate them. In addition, these inputs may selectively modulate intrinsic oscillatory processes, unrevealed by activation
of NMDA receptors (83) or inhibition of GABAergic inputs of
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mediated via nonsynaptic transmission (1, 53), through
GABAA receptors with higher affinity then those needed for
synaptic GABAergic signaling, and/or synapses close to the
soma (187, 192, 193, 217).
Tonic GABAA receptor-mediated inhibitory currents can be
influenced by changes in GABA release, enhanced GABA
uptake, and alterations in the assembly of GABAA receptor
isoforms that affect the proportion of high- and low-affinity
GABAA receptor subunits. These modifications may either
augment or decrease the neuronal excitable state. Recently it
was shown that the tonic GABAA receptor-mediated inhibition
can be enhanced by the action of steroid anesthetics (14) or by
neurosteroids synthesized by glia and neurons (18) that are
involved in ethanol action, tolerance, and dependence (151).
An increase in neurosteroid concentration would produce a
local, positive modulation of the effect of GABA on GABAA
receptor gain and thereby can contribute in maintaining inhibitory tone (187).
The GABA modulatory actions of the neurosteroids are
dependent on the GABAA receptor’s subunit composition (18).
GABA-evoked responses mediated by ␣1␤1␥2- and ␣3␤1␥2receptors are significantly potentiated by a relatively low concentration of neurosteroids, whereas ␣4␤1␥2-receptors are relatively insensitive to these compounds. The isoform of the
␤-subunit (1–3) was observed to have little influence on the
GABA modulatory actions of the pregnane steroids. Whereas
the presence of a ␥2-subunit within the GABAA receptor
complex is essential for a robust benzodiazepine effect at
submicromolar concentrations (190), replacement of the ␥-subunit by the ␦-subunit enhances steroid sensitivity of the receptor (18). Expression of the ␦-subunit in the brain is relatively
restricted, region selective, and with an extrasynaptic location,
being the major contributor of tonic GABAergic nonsynaptic
inhibition. As shown by Moore et al. (150), tonic inhibitory
currents may exert a considerable influence on neuronal signaling; therefore, these receptors may be expressed by AVPNs
at extrasynaptic sites, a short distance from the subregion
innervated by GABA-containing axon terminals (Fig. 15).
Hormonal changes have been suspected to be a cause of
increases in airway dysfunction in a subgroup of female asthmatic patients. In these subjects, episodic and potentially fatal
increases in the severity of asthma typically occurs during the
few days before their menses in the absence of other trigger
factors and are associated with a rapid fall in serum progesterone (59). The major metabolite of progesterone, 3␣-OH-dihydroprogesterone (3␣-OH-DHP), is the most potent endogenous
positive modulator of CNS GABAA receptor function and has
an action similar to that observed for neurosteroids. Hence it
can be postulated that in female patients with severe premenstrual worsening of bronchial asthma (59) as well as in postmenopausal asthma (122), beneficial effects of hormone replacement therapy in reducing the severity of airway dysfunctions could be partly mediated by enhanced central GABAergic
influences on the AVPNs, which cause withdrawal of cholinergic outflow to the airways. However, it could be expected
that long-lasting exposure of GABAA receptors to steroids and
to other modulators, as well as their withdrawal, may induce
marked effects on receptor structure and function. Possible
synergic action between endogenous steroids and GABAergic
pathways in modulating the functional activity of AVPNs
needs to be studied.
1975
Invited Review
1976
BRAIN STEM EXCITATORY AND INHIBITORY SIGNALING PATHWAYS
FUNCTIONAL RELEVANCE OF THE CENTRAL CONTROL OF
CHOLINERGIC OUTFLOW TO THE AIRWAYS
Under normal conditions, an increase or a decrease in
chemical drive induces parallel changes in airway smooth
muscle tone and breathing pattern, revealing a link between the
neuronal networks that control the resistance of the tracheobronchial conduits and the respiratory drive to chest wall
pumping muscles (83, 145). Recently, the neuroanatomical
basis for this integration has been studied by combining a
retrograde tracer technique and a transneuronal labeling
method (90). It was shown that a subset of bulbospinal cells
that project to the phrenic nuclei also innervate AVPNs,
coupling inspiratory activity and parasympathetic outflow to
the airways.
Physiological findings indicate that, during normal breathing
and changes in chemical drive, cholinergic outflow to the
airways changes in parallel with the inspiratory drive of the
phrenic nerve (51, 52, 132, 145). However, responses of the
phrenic nerve and AVPNs need not parallel each other. For
example, chemical stimulation of pulmonary C fiber receptors,
or the aspiration reflex, causes an inhibition of inspiratory
activity, but induces an increase in cholinergic outflow to the
J Appl Physiol • VOL
airways that leads to an elevation of airway smooth muscle
tone (42). In contrast, activation of the vl PAG induces a
release of GABA within the rNA region and airway smooth
muscle relaxation, but increases phrenic nerve activity (96).
Furthermore, excitation of somatosensory fibers elicits an augmentation in respiratory output but a decrease in bronchomotor
tone that cannot be explained by adrenergic or nonadrenergic
noncholinergic (NANC) inhibitory influences (117, 191).
Taken together, these results indicate that AVPNs within the
rNA form a distinct and identifiable cell group, the activity of
which is regulated by multiple CNS nuclei using common
and/or specific neuronal pathways.
During normal breathing and after changes in chemical
drive, airway smooth muscle tone and airway resistance manifest rhythmic fluctuations that are superimposed on baseline
levels (82, 145). After hypocapnic apnea, in which there is a
complete cessation of phrenic nerve discharge, the gradual
increase in arterial PCO2, or step decrease in inspired O2, causes
a progressive increase in airway smooth muscle tone (51, 52)
that commences before the reappearance of phrenic nerve
discharge. A similar response is observed after stimulation of
AVPNs by activation of the NMDA receptors within the rNA
region. With the onset of rhythmic phrenic nerve firing, rhythmic oscillations of airway smooth muscle tone and airway
resistance can be observed (83). Within the respiratory cycle,
the peak airway smooth muscle tone and airway resistance
appear early in expiration, in phase with the postinspiration
inspiratory activity of the phrenic nerve (82). As a result of the
elevation of airway smooth muscle tone, the airway and tissue
components of total lung resistance reduce the dead space and
oppose distortion of the airways and the most distal ventilatory
units. Phasic oscillation, with the peak in early expiration, may
serve to optimize gas exchange by reducing large fluctuations
in the functional residual capacity and protect the airway from
collapsing. Therefore, the central coordination of cholinergic
outflow to the airways with changes in the respiratory drive and
fluctuations of smooth muscle tone may help in maintaining the
airway wall stability, optimizing gas exchange and the work of
breathing, on a breath-by-breath basis throughout the respiratory cycle. Loss of central cholinergic outflow may cause
significant alterations in respiration and smooth muscle functions.
It has been shown that unilateral infarct involving the medullary reticular formation and NA is sufficient to generate
Ondine’s curse, a loss of automatic respiratory control and
hypoventilation during sleep (24). Furthermore, congenital
failure of autonomic control could be expressed with an abnormal brain stem integration of central and peripheral chemoreceptor inputs and the primary hypoventilation. These disorders are often associated with other neurocristopathies, especially with Hirschsprung’s disease (loss of the gastrointestinal
motility), an association known as Haddad syndrome (73). We
assume that diminished cholinergic outflow to the airways may
contribute to alterations in airway function and ventilation in
these disorders. This assumption is supported by recent experiments showing that interruption of the nerve supply to the
lungs (for instance after lung transplantation) abolishes the
integration of bronchomotor and ventilatory activities, and, by
increasing airway deformation due to loss of airway smooth
muscle tone, initiates the fibroproliferative responses in the
airway walls. This causes structural changes that are analogous
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AVPNs blocking GABAA receptors (150). Intrinsic synchronization of the activity of AVPNs may provide coordinated
motor commands to the airway effector systems.
Excitability of AVPNs may also be enhanced via paracrineautocrine mechanisms and through suppression of inhibition.
Although direct evidence for the activity-dependent release of
ACh, VIP, or BDNF from AVPNs into surrounding environment is lacking, it has been shown in other cell groups that
these molecules can be released from postsynaptic neurons and
their dendritic vesicle clusters, supporting the concept that they
can act as messengers, changing basal and stimulated neuronal
discharge (12, 101). Furthermore, BDNF could be required and
sufficient for long-term facilitation of excitatory inputs, as
convincingly shown for spinal respiratory plasticity after intermittent hypoxia (12).
Neutrophins and neutrophin receptors play an important role
in the pathophysiology of allergic bronchial asthma (159).
Apart of peripheral effects, neurotrophins such as BDNF may
affect dendritic growth (105) and facilitate glutamatergic synaptic transmission, partly via suppression of inhibitory inputs.
The site of action of BDNF was found to be predominantly
postsynaptic, caused by acute downregulation of GABAA receptor surface expression (31) and decreasing the efficacy of
inhibitory transmission by acute postsynaptic downregulation
of Cl⫺ transport (207). Hence, BDNF through increasing
excitability of AVPNs may contribute to airway hyperresponsiveness in asthmatic patients.
In summary, recent studies provide a wealth of new information about paracrine-autocrine regulation of AVPN responses and on the interactions between excitatory and inhibitory signaling pathways that may set the stage for airway
hyperreactivity. Elucidating processes that affect the balance
between the excitatory and inhibitory inputs to the AVPNs will
contribute considerably to better understanding mechanisms
underlying enhanced AVPN excitability, increased airway responsiveness, sustained obstruction of the airways, and altered
smooth muscle relaxation.
Invited Review
BRAIN STEM EXCITATORY AND INHIBITORY SIGNALING PATHWAYS
to those observed in bronchiolitis obliterans after lung transplantation, or in vagally denervated rats (37). Certainly, if the
protective nature of centrally mediated vagal tone to the airways is overexpressed and sustained it may cause significant
functional changes.
ALTERATIONS IN CENTRAL CONTROL OF CHOLINERGIC
OUTFLOW AND AIRWAY DISORDERS
CONCLUSIONS AND FUTURE STUDIES
In conclusion, the integration and processing of bronchoconstrictive inputs by the AVPNs and the level of cholinergic
output to the airways is highly dependent on synaptic and
perisynaptic glutamatergic excitatory and GABAergic inhibitory inputs to the AVPNs. Conceivably, the outcome of these
competing regulatory processes could be influenced by a number of other pathways (95, 98) using different neurotransmitters and/or neuromodulators, suggesting the complexity of the
CNS control. Understandably, this expands greatly the scheme
presented in Fig. 1, leading to a conceptual framework that
includes behavioral state control (sleep-waking cycle), psychological dimensions of airway regulatory mechanisms (fear,
anxiety, mood), and possibly reentrant loops. Hence the central
control of AVPNs is a complex system of obviously interrelated networks that shape the final AVPN output. The degree
and nature of the relationship between different pathways is
imperfectly known, a fact that should elevate the enthusiasm
for future studies in central regulation of airway functions.
J Appl Physiol • VOL
Much clinically useful information may be derived from the
studies on CNS control of airway functions. For example,
studies on a molecular network and signaling pathways, which
lead ultimately to central hyperresponsiveness of AVPNs, will
provide a better understanding of the mechanisms through
which the lung injury sets the stage for centrally mediated
airway hyperreactivity. Furthermore, elucidation of the CNS
determinants of sleep-related airway instability and worsening
of airway functions during sleep (i.e., nocturnal asthma) are
critical and interrelated areas that offer exciting and useful
findings. Similarly, identification of hidden links between phenotypic changes in central neural processing and emotional
perturbations in asthmatic patients, will allow us to better
characterize the interplay among biological, cognitive, genetic
and social factors that contribute to airway hyperresponsiveness, sustainability of bronchoconstrictive changes, and altered
emotional coping. The progress made in the CNS control of the
airways will contribute to the construction of a more complete
conceptual framework that will provide deeper insight into the
pathophysiology of airway disorders, reorganize our thinking,
and allow us to switch effortlessly to more complete and
effective treatment of patients with airway dysfunctions.
GRANTS
This work was supported by the National Heart, Lung, and Blood Institute
Grant HL-50527 (to M. A. Haxhiu), National Institute of Neurological Disorders and Stroke and National Center for Research and Resources Grant 1U54
NS-39407 (to M. A. Haxhiu), and National Institute of Mental Health Grant
R24 MH-067627 (to V. J. Massari).
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