55
Respiration Physiology, 90 (1992) 55-65 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0034-5687/92/$05.00 RESP 01945
Afferent innervation and receptors of the canine extrathoracic trachea Bu-Pian Leeb, Giuseppe Sant'Ambrogio a and Franca B. Sant'Ambrogio a "Department of Physiology and Biophysics. U~iversity oj Texas Medical Branch, Galveston. TX, USA and bDepartment of Physiology. National Yang-Ming Medical College, Taipei. Taiwan (Accepted 25 June 1992) Abstract. The aim of this study was to establish the cranio-caudal distribution of slowly (SAR) and rapidly (RAR) adapting receptors of the extrathoracic trachea (ETT) as well as their innervation and response to water solutions of different compositions. Experiments were carried out on anesthetized dogs breathing spontaneously through a low cervical tracheostomy. Eighty percent of SARs and 76% of RARs with fibers in the superior laryngeal nerve (SLN) were found in the cranial third of the ETT. Fifty-seven percent of SARs and 45% of RARs with fibers in the cervical vagus and/or recurrent laryngeal nerve (RLN) were localized in the caudal third of the ETT. Instillation of water into the tracheal lumen had no effect on the activity of any SAR tested, but stimulated 41~/0 of the RARs with fibers in the SLN and 23~o of the RARs with fibers in the cervical vagus. Some of the RARs with fibers in the SLN (24°/0), but none of those with fibers in the cervical vagus/RLN, responded also to iso-osmotic dextrose solutions. Trachealis muscle contraction failed to stimulate the RARs tested. The blocking temperature for SAR and RAR fibers was similar and well within the range of myelinated fibers. We conclude that the SLN provides the innervation of the cranial ETT while the RLN has fibers for the caudal ETT with some overlap in the middle. The responses to water solutions indicate that tracheal RARs constitute a more heterogeneous group than laryngeal RARs.
Innervation, trachea, mechanoreceptors; Mammals, dog; Receptors, mechano, extrathoracic trachea; Trachea, extrathoracic, mechanoreceptors
The extrathoracic segment of the trachea has special characteristics compared to the rest of the tracheobronchial tree. Changes in transmural pressure during the breathing cycle and various respiratory maneuvers differ from those of the intrathoracic airways, therefore the neural signals that transduce these changes relate to the central nervous system a distinct type of information. An afferent activity originating from the extrathoracic trachea and elicited by changes in transmural pressure, with a lower threshold with positive than negative pressure, has been demonstrated by Traxel et aL (1976). These investigators also found that electrical stimulation of the central cut-ends of 'individual upper tracheal nerves' elicited a slowing of respiration, bradycardia and changes in arterial blood pressure.
Correspondence to: G. Sant'Ambrogio, Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX 77555-0641, USA.
56
B.-P. LEE et ai.
The activity of slowly adapting stretch receptors (SAR) in the extrathoracic trachea declines during inspiration and augments during expiration, a pattern opposite to that of intrathoracic airway stretch receptors (Sant'Ambrogio, G. and Mortola, 1977). Reflex responses compatible with the behavior of these receptors have been described and considered to play a role in the compensatory mechanisms to external loading of respiration (Sant'Ambrogio, F. et al., 1979). While we know that the vagal innervation of this segment of the trachea follows several routes (superior and inferior laryngeal nerves as well as the pararecurrent nerves), we still do not have a detailed knowledge on the distribution of SARs along the length of the extrathoracic trachea and the corresponding innervation. Previous studies suggest a prevailing innervation through the superior laryngeal nerve (SLN) for the cranial portion of the extrathoracic trachea and through the recurrent laryngeal nerve (RLN) for its caudal segment (Sant'Ambrogio, G. et al., 1977; Vidruk, 1983). We do have precise knowledge on the intramuscular location of stretch receptors within the membranous posterior wall of the trachea (Bartlett et al., 1976) and of their relative deep location within the tracheal wall (Camporesi et aL, 1979), but still do not know whether the activity of these endings could be modified by the characteristics of the surface mucosal liquid (osmolality a.nd ionic composition), a possible important factor for the transducing function of mechanoreceptors (Anderson et al., 1990, 1991). These questions concerning SARs are among the objectives of this study. Our knowledge on the ,apidly adapting 'irritant' receptors (RAR) of the extrathoracic trachea is essentially nil. Only a few such endings have been identified in this region (Sant'Ambrogio, F. B. et aL, 1988; Sant'Ambrogio, G. and F. B. Sant'Ambrogio, 1980). In this study we intend to establish the cranio-caudal distribution of extrathoracic RARs together with their innervation, the extent and shape of their receptive field to mechanical probing, responsiveness to changes in osmolality and ionic composition of the airway surface liquid, and the effect of smooth muscle contraction on their activity. Due to location and ease of access, the slowly and rapidly adapting extrathoracic tracheal endings offer a unique opportunity to evaluate the general properties of tracheobronchial receptors and, at the same time, to understand the role of the extrathotacit trachea in the control of breathing.
Methods Fourteen dogs of either sex weighing between 12 and 16 kg were used in this study. Animals were anesthetized with an intramuscular injection of ketamine (10 mg.kg -~) followed by an intravenous injection of a mixture of chloralose (50 mg.kg -l) and urethane (500 mg.kg -I) and placed supine on the operating table. A constant level of anesthesia was maintained through the experiment by a continuous infusion of a mixture of chloralose (2-10 mg.kg-l.min -l) and urethane (20-100 mg.kg-l.min -!) through a femoral venous catheter. The femoral artery was cannulated for recording
TRACHEAL INNERVATION
57
arterial blood pressure. A polyethylene tube (i.d. = 8 mm) was inserted through the mouth and positioned with the tip f'acing the opening of the larynx. The tube was then secured by closing the mandible and tying an umbilical tape around the dog's muzzle. Esophageal pressure was monitored through a saline-filled catheter placed in the midesophagus. A longitudinal incision was made on the ventral aspect of the cervical trachea between the 10th and the 22nd ring and an L-shaped cannula was inserted just above the thoracic inlet. Since the full extension of the extrathoracic trachea comprises 23-24 cartilaginous rings, this preparation included a caudal segment (1 lth-22nd ring) in which the mucosa was exposed and a closed cranial segment (1 st-10th ring)in which the normal configuration was preserved. A first group of 10 dogs was used for studying SARs and RARs with fibers in the SLN, and a second group of 4 dogs was used to study SARs and RARs with fibers in the RLN and/or the cervical vagus. In the first group of dogs the internal branch of both SLNs was isolated and cut; in 3 dogs of the second group the right vagosympathetic nerve was isolated and cut at mid-cervical level, while in the remaining 1 the right RLN was similarly prepared. The peripheral cut-end of the nerve under study was placed on a dissecting platform, covered with mineral oil and desheathed. Single unit isolation and recording was carried out by separating thin bundles from the nerve with the aid of a microscope, iridectomy scissors and watchmaker forceps. The electrical activity was amplified and filtered through an AC preamplifier; the action potentials were displayed on a Tektronix oscilloscope connected in parallel with a loudspeaker. Action potentials, arterial blood pressure and esophageal pressure were recorded on a Gould electrostatic recorder (ES 1000). The temperature at which the nervous conduction of the fibers from either SARs or RARs was blocked, was determined as follows: an ethylene glycol coolant solution from a refrigerated bath was circulated through a copper tube ( o . d . - 6 mm, i.d. = 4.5 mm) soldered to the dissecting platform at the point of entrance of the nerve. A thermocouple (time constant = 5 msec) was glued to the copper tube beneath the nerve to measure the temperature (Sant'Ambrogio, F.B. et aL, 1988). The nerve segment actually in contact with the cooling thermode was about 4 mm in length. In 5 experiments the effect of trachealis muscle contraction on the activity of RARs was also tested. Contraction of trachealis smooth muscle was elicited by occluding the tracheal cannula at end-expiratory volume and maintaining the occlusion for up to 7-8 breaths. It must be noted that, even though in this preparation the nerve from which receptor activity was recorded was cut, the motor supply to ETT smooth muscle was still sufficient to elicit a sizable contraction. Previous work has shown that such an asphyxia causes a marked activation of the tracheal smooth muscle that develops to a maximum level within 5-7 sec and invariably leads to an increase of SAR activity (Sant'Ambrogio, F.B. et al., 1988).
Experimental protocol All fibers, whether active or silent, were tested. SARs located in the cranial section of the extrathoracic trachea, in which the cartilaginous rings were intact, were in most
58
B.-P. LEE et al.
cases active at a low rate (10-20 imp/sec) and did not have any manifest respiratory modulation. SARs located in the caudal segment, where the mucosai surface had been exposed by a longitudinal section of the ventral wall, were in most cases silent (Mortola and Sant'Ambrogio, 1979). SARs were identified by their characteristic tonic discharge pattern and on the basis of a slowly adapting response to a maintained distension of the portion of posterior wall containing them. Distension was performed either by inflating the cuff of a Foley catheter in the closed tracheal segment or by exerting an outward pull on the cut cartilages in an open tracheal segment. RARs located either in the closed cranial or in the open caudal segment, when unchallenged, were in most cases silent or had a scant ( < 1 imp/sec) and irregular discharge pattern. They could be recruited by a gentle probing of the mucosal surface with a cotton applicator soaked in warm saline. They showed a burst of action potentials that could not be maintained by a steady mechanical stimulation (rapid adaptation). Once a receptor, either SAR or RAR, was identified and localized, it was challenged with water as follows: 4 ml of distilled water, at a temperature of 37 ° C, was sprayed over the receptor field with a catheter having multiple side holes over the distal 3 cm. After each trial, the trachea was flushed with warm (37°C) saline that was then removed by suction. If the receptor was stimulated by water, it was also tested with an iso-osmotic solution of dextrose. At least 3 min were allowed to elapse between the end of suc.tioning and a new trial. Warm saline (37 °C) challenges were used as controis. Whereas a response to both water and iso-osmotic dextrose is indicative of an activation due to lack of chloride ions, a response to water only indicates an activation due to hypo-osmolality (Anderson et al., 1990; Harding et al., 1977). This study was performed in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals, the NIH Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Act (7 U.S.C. et seq.); the animal use protocol was approved by the Institutional Animal Care and Use Committee (IACUC protocol No. 89-09-203) of the University of Texas Medical Branch at Galveston.
Results
Extmthoracic tracheal receptors with fibers in the superior laryngeal nerve We have recorded from SLN fibers the single unit action potentials of 126 receptors localized in the extrathoracic trachea. Ninety-seven of them were identified as SARs and 29 as RARs. Both SARs and RARs were more frequently found within the more cranial regions of the extrathoracic trachea, none of them was localized below the 12th cartilaginous ring. The longitudinal distribution of these receptors is represented graphically in Fig. I. Whereas SARs could be activated by any maneuver leading to a transversal stretching of the membranous posterior wall of the trachea site of the ending (e.g., an outward pull exerted on the cartilages, inflation of a cuff inside a closed tracheal segment, etc.), RARs were best activated by probing with a cotton applicator the mucosal sur-
59
TRACHEAL INNERVATION
SAR (n
RAR (r~ =
= 97)
50
50
45
45
29)
ffJ
f/J
tn
40
0 CL O> ..
35
1,,_
o.) I,-
40
f//
I/I
f//
fll
//I
. i ~. I . .f./. /
30
25
.H---
25
20
~//,Y/, ~/# !/i
i i i
ffi /// /// /// fJJ
"/Z
30
',.I-.
0
H~
/ 1 / ! / / i / /
i l l
2o
15 10
lo
5 0 0
i
.
3
6
!
9
!
~2 15 18 21
0
0
3
6
9
12 15 18 21
cartilGgineous rings Fig. 1. Distribution along the extrathoracic trachea of slowly (SAR) and rapidly (RAR) adapting receptors with fibers in the internal branch of the SLN. Abscissae: the sequential number of tracheal cartilagineous rings starting from the most cranial one. Ordinates: receptor number as percent of the total SARs or RARs. Each vertical bar represents receptors localized within a segment comprising 3 cartilages.
face of the trachea. No preferential directionality was ever found for the activation of RARs: circumferential probing being as effective as longitudinal probing. Gross mechanical distortion of the trachea, either longitudinally or transversally oriented, proved to be a less effective stimulus. Rapidly adapting receptor fields covered areas extending longitudinally over 2-4 cartilaginous rings and circumferentially over 45-90 degrees. Extrathomcic tracheal receptors with fibers in the cervical vagus We recorded from fibers of the cervical vagus (RLN in one experiment) the unit activity of 50 receptors localized in the extrathoracic trachea. Twenty-eight were identified as SARs and 22 as RARs. Most of these receptors were localized in the more caudal regions of the extrathoracic trachea as represented graphically in Fig. 2. None of these endings was found craniad to the 3rd cartilaginous ring. Response to water solutions None of the 22 SARs tested (with fibers in the SLN) modified its activity or, if silent, became active when water was instilled on the mucosa overlying the receptive field. A total of 51 extrathoracic tracheal RARs were challenged with water and isoosmotic solutions of glucose: 29 had fibers in the SLN and 22 in the cervical vagus/ RLN. Seventeen of the 29 (59%) RARs with fibers in the SLN did not respond to water; seven (24%) were stimulated by both instilled water and iso-osmotic dextrose and 5 (17 %) only by water. Examples of the two types ofresponses (hypo-osmolality and lack of Cl- ) are depicted in Figs. 3 and 4. Figure 5 (left panel) represents graphically the types of responses found for the RAR with fibers in the SLN.
60
B.-P. LEE et al.
RAR (n = 22)
SAR (n --- 28) 50
50
45
45
U'J L.
40
40
0
35
35
30
30
25
25
20
20
15
15
10
10
L) q) kN--
o
5 0
,
i
i
w
u
u
0
3
6
9
12
15
i
w
0
0
18 21
cartilagineous
3
w
I
6
9
12
w
w
•
15
18
21
rings
Fig. 2. Distribution along the extrathoracic trachea of SARs and RARs with fibers in the cervical vagus nerve. Abscissae and ordinates as in Fig. !.
Of the 22 RARs with fibers in the vagus nerve/RLN, 5 (23%) were stimulated by instilled water but not by iso-osmotic dextrose. The behavior of these receptors is represented graphically in Fig. 5 (right panel). WATER L L~, l':h""
A.P.
~I, ~
~
~
1
I
ijilll:i I, , Lll! l]llllNiaUil lllllJlll [lllIlllllll![l II illJ L
| ~"}=. ~ = ~ ; ~ ; ' ~ ' ~ ~'~; } 2 ' ~
2S; ;;;~=~* 'i,; ;;; :'!; 7" %=. . . . . a " V ' r rlT'n rl'lml,qv,~ri'qvl.~rl,mlllTl'rHiqmm'wP'qll~TVlSl'illllql~511rlVel
T 1ST 11 I" v r v r l l l ' ; - "
I"
(kPll)
-0.8
DEX11:iOSE A.P.
,, . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . ~,~ . . . . . . -:
" ~ ~: . ' ~ v v. . . . .
F•Ill (kPll)
-0.5
3~
SALINE
,
l
A~
~] L 11
I
]
~8 (kPa)
-0,5
Fig. 3. RAR with fiber in the SLN activated by H20 (4 mi) instilled over the receptor field, but not by an equal volume of an isotonic solution of dextrose and 0.9% saline. In each record, tracings represent action potentials (A.P.) and esophageal pressure (Pes in kPa). Horizontal bars indicate the time of instillation. This receptor, being activated by H20, but not by an isotonic solution of dextrose, behaves as an osmoreceptor.
61
TRACHEAL INNERVATION
WATER A.P.
Pes
,A
","v~vV'-'*''-~," ~
~
~ w.
(kPa) - 1 J-
"
.I
[
DEXTROSE A,P.
Pe8 (kPa) -1
~/
I
48ec
I
SALINE AEPI
~B (kP8) Fig. 4. RAR with fiber in the SLN activated by water and isotonic dextrose, but not by 0.9% saline. This receptor is activated by solutions lacking CI-. Symbols and tracings as in Fig. 3.
Contraction of the trachealis muscle and activity of RARs None of the 13 RARs (2 with fibers in the SLN and 11 with fibers in the vagus nerve/RLN), evaluated during contraction of the tracheaiis muscle elicited by asphyxia, showed any change in its baseline activity.
62
B.-P. L E E et ai.
r - - - i no water response water and dextrose water only
u} 0
,,4-,/
Q. tD 0
80
80
70
70
60
60
50
50
i
t,-
40
40
0
30
30
20
20
10
I0
0
[l
0 SLN
(n =
29)
vagus/RLN (. = 22~
Fig. 5. RARs, as percent oftotal, with fibers in the SLN (left panel) and the cervical vagus nerve/RLN (right panel) not activated by H20 (open columns), activated by both H20 and isotonic dextrose (hatched column) and by H20 only (crosshatched columns).
Cold blockade of nervous conduction of slowly and rapidly adapting receptors We cold blocked either the vagus nerve or the SLN while recording the unit activity of 4 SARs and 12 RARs. The temperature at which all activity evoked by tracheal distension (SARs) and mechanical probing (RARs) ceased, ranged for the SARs from 5.0 °C to 9.0 °C (7.1 + 0.8 °C; mean + SE) and for the RARs from 5.0 °C to 8.5 °C (6.5 _ 0.4 °C).
Discussion The results pertaining to the cranio-caudal distribution of SARs along the extrathoracic trachea confirm and substantiate previous observations (Sant'Ambrogio, G. et aL, 1977; Vidruk, 1983). Extrathoracic tracheal receptors with fibers in the SLN are distributed mostly to the cranial portions of this segment of the airways and gradually decrease in number toward the mid-extrathoracic trachea. We did not find any of these receptors below the 12th cartilage, a result quite similar to that of Vidruk (1983). Moreover, the findings of Vidruk (1983) indicate that the fibers innervating the extrathoracic tracheal stretch receptors join the pararecurrent nerves and then reach the internal branch of the SLN through the ramus anastomoticus. Slowly adapting stretch receptors recorded from fibers in the cervical vagus were instead found more frequently in the caudal portion of the ¢xtrathoracic trachea, their number tapering off toward the first few tracheal rings. This finding is also consistent with, and further substantiates, previous observations on the same species (Sant'Ambrogio, G. et al., 1977). Previous results (Sant'Ambrogio, G. et al., 1977) have shown that ¢xtrathoracic
TRACHEAL INNERVATION
63
tracheal afferents reach the cervical vagus nerves mostly through the recurrent laryngeal nerves. A distribution and innervation similar to that described for SARs has been found for RARs. Presumably the two pathways to the SLN and the cervical vagus nerves are similar for both types of receptors. The longitudinal and circumferential dimensions of the receptor field of RARs in the ETT do not differ from those described for intrathoracic tracheal receptors (Sant'Ambrogio, G. et al., 1978). Although we could identify RARs less frequently than SARs we could always find at least one in the course of an experiment. The fact that, at variance with Vidruk (1983), we did identify several of these endings, could be attributed to the different methodologies employed in the two studies. We relied mostly on a mechanical probing of the tracheal mucosa, while Vidruk (1983) used a step-wise hyperinflation of the tracheal segment. Presumably, local probing is more effective than inflation for attaini~g the deformation required to stimulate these endings. Indeed, we found, in this as well as in a previous study (Sant'Ambrogio, G. and F.B. Sant'Ambrogio, 1980), that probing the mucosal surface was a more effective stimulus than gross deformation of the airway. The response to water and water solutions of extrathoracic tracheal receptors, with fibers either in the SLN or the vagus nerve, are in general very different from those of laryngeal receptors. Most (79~o) of the rapidly adi~pting laryngeal receptors, which respond to local probing with discharge patterns similar to those of tracheobronchial irritant receptors, are stimulated by both water and iso-osmotic dextrose solutions (Anderson et al., 1990). They are therefore characterized as 'lack of C I - ' receptors (Anderson et al., 1990). On the other hand, 2/3 of the extrathoracic tracheal receptors, with fibers in either SLN or vagus/RLN, did not respo~:~d to either water or iso-osmotic dextrose. Only 14~o of them responded to both water and iso-osmotic dextrose (lack of C I - ) and the rest (20%)to water alone (osmorecep~ors). This behavior differentiates these endings from their laryngeal counterpart, but makes them more similar to bronchial rapidly adapting receptors (Pisarri et al., 1990). These results suggest that coughing elicited by inhalation of water and iso-osmotic solutions of dextrose (Escherlbacker et al., 1984) is mostly attributable to RARs ir~ the larynx and the uppermost portion of the trachea. Our observations also indicate :hat the composition of the surface mucosal liquid is capable of modifying receptors behavior in a different way in various airway segments. Furthermore, they demonstrate the,~ heterogeneous nature of a type of endings that should not therefore be considered to constitute a well defined afferent pathway subserving stereotyped reflex responses. The lack of effect of water instillation on the tracheal stret,:h receptors is probably due to the location of these endings within the trachealis musc!le (Bartlett et al., 1976): this explains also the lesser susceptibility of stretch receptors compared to rapidly adapting receptors to topical anesthesia of the tracheal mucosa (Camporesi et aE, " 1979). These endings are therefore expected to be independent, in their transducing properties, of the composition of the surface liquid overlying the airway mucosa.
64
B.-P. LEE et ai.
Contrary to the observations reported for intrapulmonary rapidly adapting irritant receptors in dogs as well as in other species (Mills et al., !969; Sampson and Vidruk, 1975) we were unable to demonstrate any effect of smooth muscle contraction on R A R function. Perhaps the different arrangement of the tracheal smooth muscle that runs transversely between the tips of the cartilaginous rings minimizes any deformation at the receptor site. On the contrary, stimulation of the trachealis muscle obtained by a similar procedure was found to be consistently effective in stimulating tracheal stretch receptors (Sant'Ambrogio, F.B. et al., 1988). The blocking temperatures of fibers from both SARs and R A R s indicate their myelinated nature. The present data, even when results from previous observations on extrathoracic tracheal S A R (Sant'Ambrogio, F.B. et al., 1988) are considered, do not disclose any significant difference between the two types of fibers. Fibers of extrathoratio tracheal SARs and R A R s seem therefore to have similar blocking temperature than corresponding intrapulmonary receptors (Pisarri et al., 1986). However, a previous work by Sampson and Vidruk (1975)in which the conduction velocity of these two types of fibers from intrapulmonary receptors was measured found a significantly lower value for RARs than for SARs, although the values showed a considerable degree of overlap.
Acknowledgements.This study was supported by grants from the National Institute of Health HL-20122 and the National Science Council of the Republic of China (NSC-78-0412-B010-42).
References Anderson, J.W., F.B. Sant'Ambrogio, O.P. Mathew and G. Sant'Ambrogio (1990). Water-responsive laryngeal receptors in the dog are not specialized endings. Respir. Physiol. 79: 33-44. Anderson, J.W., F.B. Sant'Ambrogio and G. Sant'Ambrogio (1991). Changes in osmolality modify the pressure response of laryngeal receptors. FASEB J. 5: AI 119. Bartlett, D. Jr., P. Jeffery, G. Sant'Ambrogio and J. C. M. Wise (1976). Location of stretch receptors in the trachea and bronchi of the dog. J. Physiol. (London) 258: 409-420. Camporesi, E.M., J. P, Mortola, F. Sant'Ambrogio and G. Sant'Ambrogio (1979). Topical anesthesia of tracheal receptors. J. Appl. Physiol. 47:1123-1126. Eschenbacker, W. L., H.A. Boushey and D. Sheppard (1984). Alteration in osmolarity of inhaled aerosols causes bronchoconstriction and cough, but absence of a permeant anion causes cough alone. Am. Rev. Respir. Dis. 129:211-215. Harding, R., P. Johnson and M. E. McCleiland (1977), Liquid-sensitivelaryngealreceptors in the developing sheep, cat and monkey. J. Physiol. (London) 277: 409-422. Mills, J., H. Sellick and J.G. Widdicombe (1969). The role of lung irritant receptors in respiratory responses to multiple pulmonary embolism, anaphylaxis and histamine induced bronchoconstriction. J. Physiol. (London) 203: 337-357. Mortola, J.P. and G. Sant'Ambrogio (1979). Mechanics of the trachea and behavior of its slowly adapting stretch receptors. J. Physiol. (London) 286: 577-590. Pisarri, T. E., J. Yu, H.M. Coleridge and J. C. G. Coleridge (1986). Background activity in pulmonary vagal C-fiber and its effects on breathing. Respir. Physiol. 64: 29-43.
TRACHEAL INNERVATION
65
Pisarri, T. E., A. Jonzon, H.M. Coleridge and J. C.G. Coleridge (1990). Aspiration of hypotonic or hypertonic sodium chloride solution stimulates pulmonary vagal afferents in dogs. FASEB J. 4: A715. Sampson, S.R. and E.H. Vidruk (1975). Properties of'irritant' receptors in canine lung. Respir. Physiol. 25: 9-22. Sant'Ambrogio, F., G. Sant'Ambrogio and J.P. Mortola (1979). Reflex influences from the ¢xtrathoracic trachea during airway occlusion. Respir. Physiol. 36: 327-336. Sant'Ambrogio, F.B., G. Sant'Ambrogio, O.P. Mathew and H. Tsubon¢ (1988). Contraction of trachealis muscle and activity of tracheal stretch receptors. Respir. Physiol. 71: 343-354. Sant'Ambrogio, G., D. Bartlett, Jr. and J. Mortola (1977). lnnervation of stretch receptors in the extrathoracic trachea. Respir. Physiol. 29: 93-99. Sant'Ambrogio, G. and J.P. Mortola (1977). Behavior of slowly adapting stretch receptors in the extrathoracic trachea of the dog. Respir. Physiol. 31: 377-385. Sant'Ambrogio, G., J.E. Remmers, W.J. De Groot, G. Callas and J.P. Mortola (1978). Localization of rapidly adapting receptors in the trachea and main stem bronchus of the dog. Respir. Physiol. 33: 359-366. Sant'Ambrogio, G. and F.B. Sant'Ambrogio (1980). Tracheal rapidly adapting mechanoreceptors. Fed. Proc. 39: 836. Traxel, R. M., W. F. Prudlow, J. P. Kampine, R. L. Coon and E.J. Zuperku (1976). Tracheal afferent nerves. Ann. Otol. 85: 664-669. Vidruk, E.H. (1983). Extravagal innervation of canine tracheal stretch receptors. J. Physiol. (London) 338: 11-20.
Respiration Physiology, 90 (1992) 55-65 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0034-5687/92/$05.00 RESP 01945
Afferent innervation and receptors of the canine extrathoracic trachea Bu-Pian Leeb, Giuseppe Sant'Ambrogio a and Franca B. Sant'Ambrogio a "Department of Physiology and Biophysics. U~iversity oj Texas Medical Branch, Galveston. TX, USA and bDepartment of Physiology. National Yang-Ming Medical College, Taipei. Taiwan (Accepted 25 June 1992) Abstract. The aim of this study was to establish the cranio-caudal distribution of slowly (SAR) and rapidly (RAR) adapting receptors of the extrathoracic trachea (ETT) as well as their innervation and response to water solutions of different compositions. Experiments were carried out on anesthetized dogs breathing spontaneously through a low cervical tracheostomy. Eighty percent of SARs and 76% of RARs with fibers in the superior laryngeal nerve (SLN) were found in the cranial third of the ETT. Fifty-seven percent of SARs and 45% of RARs with fibers in the cervical vagus and/or recurrent laryngeal nerve (RLN) were localized in the caudal third of the ETT. Instillation of water into the tracheal lumen had no effect on the activity of any SAR tested, but stimulated 41~/0 of the RARs with fibers in the SLN and 23~o of the RARs with fibers in the cervical vagus. Some of the RARs with fibers in the SLN (24°/0), but none of those with fibers in the cervical vagus/RLN, responded also to iso-osmotic dextrose solutions. Trachealis muscle contraction failed to stimulate the RARs tested. The blocking temperature for SAR and RAR fibers was similar and well within the range of myelinated fibers. We conclude that the SLN provides the innervation of the cranial ETT while the RLN has fibers for the caudal ETT with some overlap in the middle. The responses to water solutions indicate that tracheal RARs constitute a more heterogeneous group than laryngeal RARs.
Innervation, trachea, mechanoreceptors; Mammals, dog; Receptors, mechano, extrathoracic trachea; Trachea, extrathoracic, mechanoreceptors
The extrathoracic segment of the trachea has special characteristics compared to the rest of the tracheobronchial tree. Changes in transmural pressure during the breathing cycle and various respiratory maneuvers differ from those of the intrathoracic airways, therefore the neural signals that transduce these changes relate to the central nervous system a distinct type of information. An afferent activity originating from the extrathoracic trachea and elicited by changes in transmural pressure, with a lower threshold with positive than negative pressure, has been demonstrated by Traxel et aL (1976). These investigators also found that electrical stimulation of the central cut-ends of 'individual upper tracheal nerves' elicited a slowing of respiration, bradycardia and changes in arterial blood pressure.
Correspondence to: G. Sant'Ambrogio, Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX 77555-0641, USA.
56
B.-P. LEE et ai.
The activity of slowly adapting stretch receptors (SAR) in the extrathoracic trachea declines during inspiration and augments during expiration, a pattern opposite to that of intrathoracic airway stretch receptors (Sant'Ambrogio, G. and Mortola, 1977). Reflex responses compatible with the behavior of these receptors have been described and considered to play a role in the compensatory mechanisms to external loading of respiration (Sant'Ambrogio, F. et al., 1979). While we know that the vagal innervation of this segment of the trachea follows several routes (superior and inferior laryngeal nerves as well as the pararecurrent nerves), we still do not have a detailed knowledge on the distribution of SARs along the length of the extrathoracic trachea and the corresponding innervation. Previous studies suggest a prevailing innervation through the superior laryngeal nerve (SLN) for the cranial portion of the extrathoracic trachea and through the recurrent laryngeal nerve (RLN) for its caudal segment (Sant'Ambrogio, G. et al., 1977; Vidruk, 1983). We do have precise knowledge on the intramuscular location of stretch receptors within the membranous posterior wall of the trachea (Bartlett et al., 1976) and of their relative deep location within the tracheal wall (Camporesi et aL, 1979), but still do not know whether the activity of these endings could be modified by the characteristics of the surface mucosal liquid (osmolality a.nd ionic composition), a possible important factor for the transducing function of mechanoreceptors (Anderson et al., 1990, 1991). These questions concerning SARs are among the objectives of this study. Our knowledge on the ,apidly adapting 'irritant' receptors (RAR) of the extrathoracic trachea is essentially nil. Only a few such endings have been identified in this region (Sant'Ambrogio, F. B. et aL, 1988; Sant'Ambrogio, G. and F. B. Sant'Ambrogio, 1980). In this study we intend to establish the cranio-caudal distribution of extrathoracic RARs together with their innervation, the extent and shape of their receptive field to mechanical probing, responsiveness to changes in osmolality and ionic composition of the airway surface liquid, and the effect of smooth muscle contraction on their activity. Due to location and ease of access, the slowly and rapidly adapting extrathoracic tracheal endings offer a unique opportunity to evaluate the general properties of tracheobronchial receptors and, at the same time, to understand the role of the extrathotacit trachea in the control of breathing.
Methods Fourteen dogs of either sex weighing between 12 and 16 kg were used in this study. Animals were anesthetized with an intramuscular injection of ketamine (10 mg.kg -~) followed by an intravenous injection of a mixture of chloralose (50 mg.kg -l) and urethane (500 mg.kg -I) and placed supine on the operating table. A constant level of anesthesia was maintained through the experiment by a continuous infusion of a mixture of chloralose (2-10 mg.kg-l.min -l) and urethane (20-100 mg.kg-l.min -!) through a femoral venous catheter. The femoral artery was cannulated for recording
TRACHEAL INNERVATION
57
arterial blood pressure. A polyethylene tube (i.d. = 8 mm) was inserted through the mouth and positioned with the tip f'acing the opening of the larynx. The tube was then secured by closing the mandible and tying an umbilical tape around the dog's muzzle. Esophageal pressure was monitored through a saline-filled catheter placed in the midesophagus. A longitudinal incision was made on the ventral aspect of the cervical trachea between the 10th and the 22nd ring and an L-shaped cannula was inserted just above the thoracic inlet. Since the full extension of the extrathoracic trachea comprises 23-24 cartilaginous rings, this preparation included a caudal segment (1 lth-22nd ring) in which the mucosa was exposed and a closed cranial segment (1 st-10th ring)in which the normal configuration was preserved. A first group of 10 dogs was used for studying SARs and RARs with fibers in the SLN, and a second group of 4 dogs was used to study SARs and RARs with fibers in the RLN and/or the cervical vagus. In the first group of dogs the internal branch of both SLNs was isolated and cut; in 3 dogs of the second group the right vagosympathetic nerve was isolated and cut at mid-cervical level, while in the remaining 1 the right RLN was similarly prepared. The peripheral cut-end of the nerve under study was placed on a dissecting platform, covered with mineral oil and desheathed. Single unit isolation and recording was carried out by separating thin bundles from the nerve with the aid of a microscope, iridectomy scissors and watchmaker forceps. The electrical activity was amplified and filtered through an AC preamplifier; the action potentials were displayed on a Tektronix oscilloscope connected in parallel with a loudspeaker. Action potentials, arterial blood pressure and esophageal pressure were recorded on a Gould electrostatic recorder (ES 1000). The temperature at which the nervous conduction of the fibers from either SARs or RARs was blocked, was determined as follows: an ethylene glycol coolant solution from a refrigerated bath was circulated through a copper tube ( o . d . - 6 mm, i.d. = 4.5 mm) soldered to the dissecting platform at the point of entrance of the nerve. A thermocouple (time constant = 5 msec) was glued to the copper tube beneath the nerve to measure the temperature (Sant'Ambrogio, F.B. et aL, 1988). The nerve segment actually in contact with the cooling thermode was about 4 mm in length. In 5 experiments the effect of trachealis muscle contraction on the activity of RARs was also tested. Contraction of trachealis smooth muscle was elicited by occluding the tracheal cannula at end-expiratory volume and maintaining the occlusion for up to 7-8 breaths. It must be noted that, even though in this preparation the nerve from which receptor activity was recorded was cut, the motor supply to ETT smooth muscle was still sufficient to elicit a sizable contraction. Previous work has shown that such an asphyxia causes a marked activation of the tracheal smooth muscle that develops to a maximum level within 5-7 sec and invariably leads to an increase of SAR activity (Sant'Ambrogio, F.B. et al., 1988).
Experimental protocol All fibers, whether active or silent, were tested. SARs located in the cranial section of the extrathoracic trachea, in which the cartilaginous rings were intact, were in most
58
B.-P. LEE et al.
cases active at a low rate (10-20 imp/sec) and did not have any manifest respiratory modulation. SARs located in the caudal segment, where the mucosai surface had been exposed by a longitudinal section of the ventral wall, were in most cases silent (Mortola and Sant'Ambrogio, 1979). SARs were identified by their characteristic tonic discharge pattern and on the basis of a slowly adapting response to a maintained distension of the portion of posterior wall containing them. Distension was performed either by inflating the cuff of a Foley catheter in the closed tracheal segment or by exerting an outward pull on the cut cartilages in an open tracheal segment. RARs located either in the closed cranial or in the open caudal segment, when unchallenged, were in most cases silent or had a scant ( < 1 imp/sec) and irregular discharge pattern. They could be recruited by a gentle probing of the mucosal surface with a cotton applicator soaked in warm saline. They showed a burst of action potentials that could not be maintained by a steady mechanical stimulation (rapid adaptation). Once a receptor, either SAR or RAR, was identified and localized, it was challenged with water as follows: 4 ml of distilled water, at a temperature of 37 ° C, was sprayed over the receptor field with a catheter having multiple side holes over the distal 3 cm. After each trial, the trachea was flushed with warm (37°C) saline that was then removed by suction. If the receptor was stimulated by water, it was also tested with an iso-osmotic solution of dextrose. At least 3 min were allowed to elapse between the end of suc.tioning and a new trial. Warm saline (37 °C) challenges were used as controis. Whereas a response to both water and iso-osmotic dextrose is indicative of an activation due to lack of chloride ions, a response to water only indicates an activation due to hypo-osmolality (Anderson et al., 1990; Harding et al., 1977). This study was performed in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals, the NIH Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Act (7 U.S.C. et seq.); the animal use protocol was approved by the Institutional Animal Care and Use Committee (IACUC protocol No. 89-09-203) of the University of Texas Medical Branch at Galveston.
Results
Extmthoracic tracheal receptors with fibers in the superior laryngeal nerve We have recorded from SLN fibers the single unit action potentials of 126 receptors localized in the extrathoracic trachea. Ninety-seven of them were identified as SARs and 29 as RARs. Both SARs and RARs were more frequently found within the more cranial regions of the extrathoracic trachea, none of them was localized below the 12th cartilaginous ring. The longitudinal distribution of these receptors is represented graphically in Fig. I. Whereas SARs could be activated by any maneuver leading to a transversal stretching of the membranous posterior wall of the trachea site of the ending (e.g., an outward pull exerted on the cartilages, inflation of a cuff inside a closed tracheal segment, etc.), RARs were best activated by probing with a cotton applicator the mucosal sur-
59
TRACHEAL INNERVATION
SAR (n
RAR (r~ =
= 97)
50
50
45
45
29)
ffJ
f/J
tn
40
0 CL O> ..
35
1,,_
o.) I,-
40
f//
I/I
f//
fll
//I
. i ~. I . .f./. /
30
25
.H---
25
20
~//,Y/, ~/# !/i
i i i
ffi /// /// /// fJJ
"/Z
30
',.I-.
0
H~
/ 1 / ! / / i / /
i l l
2o
15 10
lo
5 0 0
i
.
3
6
!
9
!
~2 15 18 21
0
0
3
6
9
12 15 18 21
cartilGgineous rings Fig. 1. Distribution along the extrathoracic trachea of slowly (SAR) and rapidly (RAR) adapting receptors with fibers in the internal branch of the SLN. Abscissae: the sequential number of tracheal cartilagineous rings starting from the most cranial one. Ordinates: receptor number as percent of the total SARs or RARs. Each vertical bar represents receptors localized within a segment comprising 3 cartilages.
face of the trachea. No preferential directionality was ever found for the activation of RARs: circumferential probing being as effective as longitudinal probing. Gross mechanical distortion of the trachea, either longitudinally or transversally oriented, proved to be a less effective stimulus. Rapidly adapting receptor fields covered areas extending longitudinally over 2-4 cartilaginous rings and circumferentially over 45-90 degrees. Extrathomcic tracheal receptors with fibers in the cervical vagus We recorded from fibers of the cervical vagus (RLN in one experiment) the unit activity of 50 receptors localized in the extrathoracic trachea. Twenty-eight were identified as SARs and 22 as RARs. Most of these receptors were localized in the more caudal regions of the extrathoracic trachea as represented graphically in Fig. 2. None of these endings was found craniad to the 3rd cartilaginous ring. Response to water solutions None of the 22 SARs tested (with fibers in the SLN) modified its activity or, if silent, became active when water was instilled on the mucosa overlying the receptive field. A total of 51 extrathoracic tracheal RARs were challenged with water and isoosmotic solutions of glucose: 29 had fibers in the SLN and 22 in the cervical vagus/ RLN. Seventeen of the 29 (59%) RARs with fibers in the SLN did not respond to water; seven (24%) were stimulated by both instilled water and iso-osmotic dextrose and 5 (17 %) only by water. Examples of the two types ofresponses (hypo-osmolality and lack of Cl- ) are depicted in Figs. 3 and 4. Figure 5 (left panel) represents graphically the types of responses found for the RAR with fibers in the SLN.
60
B.-P. LEE et al.
RAR (n = 22)
SAR (n --- 28) 50
50
45
45
U'J L.
40
40
0
35
35
30
30
25
25
20
20
15
15
10
10
L) q) kN--
o
5 0
,
i
i
w
u
u
0
3
6
9
12
15
i
w
0
0
18 21
cartilagineous
3
w
I
6
9
12
w
w
•
15
18
21
rings
Fig. 2. Distribution along the extrathoracic trachea of SARs and RARs with fibers in the cervical vagus nerve. Abscissae and ordinates as in Fig. !.
Of the 22 RARs with fibers in the vagus nerve/RLN, 5 (23%) were stimulated by instilled water but not by iso-osmotic dextrose. The behavior of these receptors is represented graphically in Fig. 5 (right panel). WATER L L~, l':h""
A.P.
~I, ~
~
~
1
I
ijilll:i I, , Lll! l]llllNiaUil lllllJlll [lllIlllllll![l II illJ L
| ~"}=. ~ = ~ ; ~ ; ' ~ ' ~ ~'~; } 2 ' ~
2S; ;;;~=~* 'i,; ;;; :'!; 7" %=. . . . . a " V ' r rlT'n rl'lml,qv,~ri'qvl.~rl,mlllTl'rHiqmm'wP'qll~TVlSl'illllql~511rlVel
T 1ST 11 I" v r v r l l l ' ; - "
I"
(kPll)
-0.8
DEX11:iOSE A.P.
,, . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . ~,~ . . . . . . -:
" ~ ~: . ' ~ v v. . . . .
F•Ill (kPll)
-0.5
3~
SALINE
,
l
A~
~] L 11
I
]
~8 (kPa)
-0,5
Fig. 3. RAR with fiber in the SLN activated by H20 (4 mi) instilled over the receptor field, but not by an equal volume of an isotonic solution of dextrose and 0.9% saline. In each record, tracings represent action potentials (A.P.) and esophageal pressure (Pes in kPa). Horizontal bars indicate the time of instillation. This receptor, being activated by H20, but not by an isotonic solution of dextrose, behaves as an osmoreceptor.
61
TRACHEAL INNERVATION
WATER A.P.
Pes
,A
","v~vV'-'*''-~," ~
~
~ w.
(kPa) - 1 J-
"
.I
[
DEXTROSE A,P.
Pe8 (kPa) -1
~/
I
48ec
I
SALINE AEPI
~B (kP8) Fig. 4. RAR with fiber in the SLN activated by water and isotonic dextrose, but not by 0.9% saline. This receptor is activated by solutions lacking CI-. Symbols and tracings as in Fig. 3.
Contraction of the trachealis muscle and activity of RARs None of the 13 RARs (2 with fibers in the SLN and 11 with fibers in the vagus nerve/RLN), evaluated during contraction of the tracheaiis muscle elicited by asphyxia, showed any change in its baseline activity.
62
B.-P. L E E et ai.
r - - - i no water response water and dextrose water only
u} 0
,,4-,/
Q. tD 0
80
80
70
70
60
60
50
50
i
t,-
40
40
0
30
30
20
20
10
I0
0
[l
0 SLN
(n =
29)
vagus/RLN (. = 22~
Fig. 5. RARs, as percent oftotal, with fibers in the SLN (left panel) and the cervical vagus nerve/RLN (right panel) not activated by H20 (open columns), activated by both H20 and isotonic dextrose (hatched column) and by H20 only (crosshatched columns).
Cold blockade of nervous conduction of slowly and rapidly adapting receptors We cold blocked either the vagus nerve or the SLN while recording the unit activity of 4 SARs and 12 RARs. The temperature at which all activity evoked by tracheal distension (SARs) and mechanical probing (RARs) ceased, ranged for the SARs from 5.0 °C to 9.0 °C (7.1 + 0.8 °C; mean + SE) and for the RARs from 5.0 °C to 8.5 °C (6.5 _ 0.4 °C).
Discussion The results pertaining to the cranio-caudal distribution of SARs along the extrathoracic trachea confirm and substantiate previous observations (Sant'Ambrogio, G. et aL, 1977; Vidruk, 1983). Extrathoracic tracheal receptors with fibers in the SLN are distributed mostly to the cranial portions of this segment of the airways and gradually decrease in number toward the mid-extrathoracic trachea. We did not find any of these receptors below the 12th cartilage, a result quite similar to that of Vidruk (1983). Moreover, the findings of Vidruk (1983) indicate that the fibers innervating the extrathoracic tracheal stretch receptors join the pararecurrent nerves and then reach the internal branch of the SLN through the ramus anastomoticus. Slowly adapting stretch receptors recorded from fibers in the cervical vagus were instead found more frequently in the caudal portion of the ¢xtrathoracic trachea, their number tapering off toward the first few tracheal rings. This finding is also consistent with, and further substantiates, previous observations on the same species (Sant'Ambrogio, G. et al., 1977). Previous results (Sant'Ambrogio, G. et al., 1977) have shown that ¢xtrathoracic
TRACHEAL INNERVATION
63
tracheal afferents reach the cervical vagus nerves mostly through the recurrent laryngeal nerves. A distribution and innervation similar to that described for SARs has been found for RARs. Presumably the two pathways to the SLN and the cervical vagus nerves are similar for both types of receptors. The longitudinal and circumferential dimensions of the receptor field of RARs in the ETT do not differ from those described for intrathoracic tracheal receptors (Sant'Ambrogio, G. et al., 1978). Although we could identify RARs less frequently than SARs we could always find at least one in the course of an experiment. The fact that, at variance with Vidruk (1983), we did identify several of these endings, could be attributed to the different methodologies employed in the two studies. We relied mostly on a mechanical probing of the tracheal mucosa, while Vidruk (1983) used a step-wise hyperinflation of the tracheal segment. Presumably, local probing is more effective than inflation for attaini~g the deformation required to stimulate these endings. Indeed, we found, in this as well as in a previous study (Sant'Ambrogio, G. and F.B. Sant'Ambrogio, 1980), that probing the mucosal surface was a more effective stimulus than gross deformation of the airway. The response to water and water solutions of extrathoracic tracheal receptors, with fibers either in the SLN or the vagus nerve, are in general very different from those of laryngeal receptors. Most (79~o) of the rapidly adi~pting laryngeal receptors, which respond to local probing with discharge patterns similar to those of tracheobronchial irritant receptors, are stimulated by both water and iso-osmotic dextrose solutions (Anderson et al., 1990). They are therefore characterized as 'lack of C I - ' receptors (Anderson et al., 1990). On the other hand, 2/3 of the extrathoracic tracheal receptors, with fibers in either SLN or vagus/RLN, did not respo~:~d to either water or iso-osmotic dextrose. Only 14~o of them responded to both water and iso-osmotic dextrose (lack of C I - ) and the rest (20%)to water alone (osmorecep~ors). This behavior differentiates these endings from their laryngeal counterpart, but makes them more similar to bronchial rapidly adapting receptors (Pisarri et al., 1990). These results suggest that coughing elicited by inhalation of water and iso-osmotic solutions of dextrose (Escherlbacker et al., 1984) is mostly attributable to RARs ir~ the larynx and the uppermost portion of the trachea. Our observations also indicate :hat the composition of the surface mucosal liquid is capable of modifying receptors behavior in a different way in various airway segments. Furthermore, they demonstrate the,~ heterogeneous nature of a type of endings that should not therefore be considered to constitute a well defined afferent pathway subserving stereotyped reflex responses. The lack of effect of water instillation on the tracheal stret,:h receptors is probably due to the location of these endings within the trachealis musc!le (Bartlett et al., 1976): this explains also the lesser susceptibility of stretch receptors compared to rapidly adapting receptors to topical anesthesia of the tracheal mucosa (Camporesi et aE, " 1979). These endings are therefore expected to be independent, in their transducing properties, of the composition of the surface liquid overlying the airway mucosa.
64
B.-P. LEE et ai.
Contrary to the observations reported for intrapulmonary rapidly adapting irritant receptors in dogs as well as in other species (Mills et al., !969; Sampson and Vidruk, 1975) we were unable to demonstrate any effect of smooth muscle contraction on R A R function. Perhaps the different arrangement of the tracheal smooth muscle that runs transversely between the tips of the cartilaginous rings minimizes any deformation at the receptor site. On the contrary, stimulation of the trachealis muscle obtained by a similar procedure was found to be consistently effective in stimulating tracheal stretch receptors (Sant'Ambrogio, F.B. et al., 1988). The blocking temperatures of fibers from both SARs and R A R s indicate their myelinated nature. The present data, even when results from previous observations on extrathoracic tracheal S A R (Sant'Ambrogio, F.B. et al., 1988) are considered, do not disclose any significant difference between the two types of fibers. Fibers of extrathoratio tracheal SARs and R A R s seem therefore to have similar blocking temperature than corresponding intrapulmonary receptors (Pisarri et al., 1986). However, a previous work by Sampson and Vidruk (1975)in which the conduction velocity of these two types of fibers from intrapulmonary receptors was measured found a significantly lower value for RARs than for SARs, although the values showed a considerable degree of overlap.
Acknowledgements.This study was supported by grants from the National Institute of Health HL-20122 and the National Science Council of the Republic of China (NSC-78-0412-B010-42).
References Anderson, J.W., F.B. Sant'Ambrogio, O.P. Mathew and G. Sant'Ambrogio (1990). Water-responsive laryngeal receptors in the dog are not specialized endings. Respir. Physiol. 79: 33-44. Anderson, J.W., F.B. Sant'Ambrogio and G. Sant'Ambrogio (1991). Changes in osmolality modify the pressure response of laryngeal receptors. FASEB J. 5: AI 119. Bartlett, D. Jr., P. Jeffery, G. Sant'Ambrogio and J. C. M. Wise (1976). Location of stretch receptors in the trachea and bronchi of the dog. J. Physiol. (London) 258: 409-420. Camporesi, E.M., J. P, Mortola, F. Sant'Ambrogio and G. Sant'Ambrogio (1979). Topical anesthesia of tracheal receptors. J. Appl. Physiol. 47:1123-1126. Eschenbacker, W. L., H.A. Boushey and D. Sheppard (1984). Alteration in osmolarity of inhaled aerosols causes bronchoconstriction and cough, but absence of a permeant anion causes cough alone. Am. Rev. Respir. Dis. 129:211-215. Harding, R., P. Johnson and M. E. McCleiland (1977), Liquid-sensitivelaryngealreceptors in the developing sheep, cat and monkey. J. Physiol. (London) 277: 409-422. Mills, J., H. Sellick and J.G. Widdicombe (1969). The role of lung irritant receptors in respiratory responses to multiple pulmonary embolism, anaphylaxis and histamine induced bronchoconstriction. J. Physiol. (London) 203: 337-357. Mortola, J.P. and G. Sant'Ambrogio (1979). Mechanics of the trachea and behavior of its slowly adapting stretch receptors. J. Physiol. (London) 286: 577-590. Pisarri, T. E., J. Yu, H.M. Coleridge and J. C. G. Coleridge (1986). Background activity in pulmonary vagal C-fiber and its effects on breathing. Respir. Physiol. 64: 29-43.
TRACHEAL INNERVATION
65
Pisarri, T. E., A. Jonzon, H.M. Coleridge and J. C.G. Coleridge (1990). Aspiration of hypotonic or hypertonic sodium chloride solution stimulates pulmonary vagal afferents in dogs. FASEB J. 4: A715. Sampson, S.R. and E.H. Vidruk (1975). Properties of'irritant' receptors in canine lung. Respir. Physiol. 25: 9-22. Sant'Ambrogio, F., G. Sant'Ambrogio and J.P. Mortola (1979). Reflex influences from the ¢xtrathoracic trachea during airway occlusion. Respir. Physiol. 36: 327-336. Sant'Ambrogio, F.B., G. Sant'Ambrogio, O.P. Mathew and H. Tsubon¢ (1988). Contraction of trachealis muscle and activity of tracheal stretch receptors. Respir. Physiol. 71: 343-354. Sant'Ambrogio, G., D. Bartlett, Jr. and J. Mortola (1977). lnnervation of stretch receptors in the extrathoracic trachea. Respir. Physiol. 29: 93-99. Sant'Ambrogio, G. and J.P. Mortola (1977). Behavior of slowly adapting stretch receptors in the extrathoracic trachea of the dog. Respir. Physiol. 31: 377-385. Sant'Ambrogio, G., J.E. Remmers, W.J. De Groot, G. Callas and J.P. Mortola (1978). Localization of rapidly adapting receptors in the trachea and main stem bronchus of the dog. Respir. Physiol. 33: 359-366. Sant'Ambrogio, G. and F.B. Sant'Ambrogio (1980). Tracheal rapidly adapting mechanoreceptors. Fed. Proc. 39: 836. Traxel, R. M., W. F. Prudlow, J. P. Kampine, R. L. Coon and E.J. Zuperku (1976). Tracheal afferent nerves. Ann. Otol. 85: 664-669. Vidruk, E.H. (1983). Extravagal innervation of canine tracheal stretch receptors. J. Physiol. (London) 338: 11-20.
