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Laryngeal cold receptors
35
Respiration Physiology (1985) 59, 35-44 Elsevier
LARYNGEAL COLD RECEPTORS
G. SANT'AMBROGIO, O.P. MATHEW, F.B. SANT'AMBROGIO and J. T. FISHER Department of Physiology and Biophysics and Department of Pediatrics, University of Texas Medical Branch, Galveston, TX 77550, U.S.A.
Abstract. We have previously demonstrated the presence of specific laryngeal 'flow' receptors activated
independently oftransmural pressure. This study considers the operational characteristics of these endings. In 15 anesthetized dogs we recorded single unit action potentials from the peripheral cut end of the internal branch of the superior laryngeal nerve. All the 30 laryngeal 'flow' receptors studied showed an inspiratory modulation when the dog was breathing room air at 26 ° C and 55 ~o relative humidity (laryngeal temperature 34 °C) through the upper airway. All the receptors became silent when the temperature of the inspired air was raised to 36-40 °C, 100~ relative humidity (laryngeal temperature between 35 and 38 °C) and increased their activity when the temperature in the larynx was decreased either by lowering the temperature or the humidity of the inspired air. Fourteen laryngeal 'flow' receptors were tested with a steady flow of air, directed through the isolated in vivo larynx, at different temperatures and saturated with water vapor. Their discharge rate was found to be inversely related to laryngeal temperature (from 35 to 25 ° C) and independent of airflow. Their rate of adaptation indicates a high dynamic sensitivity, In the isolated larynx preparation these receptors were activated by airflow in both inspiratory and expiratory directions provided that laryngeal temperature was lower than 35 ° C. We conclude that the previously described laryngeal 'flow' receptors operate as thermoreceptors activated by cooling. Airway receptors Cold receptors Larynx
Superior laryngeal nerve Temperature Upper airway
In a recent study we demonstrated the presence of laryngeal receptors responding to inspiratory airflow independently of changes in transmural pressure (Sant'Ambrogio et al., 1983). This is a distinct feature of laryngeal 'flow' endings, at variance with the flow detection of both slowly and rapidly adapting receptors of the tracheobronchial tree which is entirely dependent on changes in transmural pressure (Knowlton and Larrabee, 1946; Widdicombe, 1954; Davis etal., 1956; Bartlett etal., 1976; Sant'Ambrogio, Accepted for publication 25 August 1984 0034-5687/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
36
G. SANT'AMBROG10 el a/.
1982; Pack and Delaney, 1983). A possible mechanism for the inspiratory activation of such receptors is the decrease in laryngeal temperature due to inspiratory airflow. Indeed, receptors affected by the local changes in temperature have been described in the larynx (Sampson and Eyzaguirre, 1964; Storey, 1968; Boushey e t a l . , 1974). In this study we have tested this hypothesis and provide evidence that the previously described laryngeal 'flow' receptors operate as thermoreceptors sensitive to cooling.
Methods Fifteen mongrel dogs of either sex, weighing between 9.0 and 13.0 kg were anesthetized with a mixture of chloralose and urethane (0.1 g/kg chloralose and 1.0 g/kg urethane, injected intravenously) and placed on an operating table in the supine position. A femoral vein was cannulated for further injection of the anesthetic mixture. A polyethylene cannula was inserted into a femoral artery and connected to a pressure transducer to monitor blood pressure. The cervical trachea was exposed in its entire length and the ventral aspect of 8-10 cartilaginous rings was longitudinally cut to allow the introduction of both ends of a cannula having three side arms (fig. 1). A saline-filled polyethylene catheter (internal diameter 2 ram) was placed in the mid portion of the esophagus and connected to a pressure transducer for recording esophageal pressure to indicate the respiratory phases. A large polyethylene tube was inserted through the mouth and positioned with the aid of a laryngoscope just below the epiglottis, facing the opening of the larynx. The tube was then secured by closing the mandible and constructing a face mask around the mouth and nose with quick setting epoxy (fig. 1). Laryngeal air temperature was measured by placing a thermistor (time constant = 100 msec) or a thermocouple probe (time constant = 20 msec), through a side arm of the tracheal cannula, with its tip floating just below the vocal cords. Airflow was measured with a pneumotachograph (Fleisch no. 1) connected to the oral cannula.
des
Fig. 1. Schematicrepresentation of the experimentalsetup. Mouth and nares are sealed around tube A with quick setting epoxy.The tracheal cannula with three side arms permits the diversion of breathing from the tracheostomy (C1 open; A closed) to the upper airway (A open; Cl, C2 and C3 closed) and occlusion of the airway either below (at B) or above (all outlets closed) the larynx for identifyinga 'flow' (cold) receptor. With this setup air can also be passed through the upper airway while the dog breathes through the tracheostomy (C, open, tracheal cannula occluded at D).
LARYNGEAL COLD RECEPTORS
37
The internal branch of one superior laryngeal nerve, most often the right, was identified and isolated peripherally to its entrance into the thyroid cartilage and cut 2 cm from the larynx. The peripheral cut end was then placed on a dissecting tray, covered with mineral oil, desheathed and separated into several thin bundles using a pair of iridectomy scissors and watchmaker forceps. The activity present in each small bundle could be monitored by putting the filament across a pair of platinum electrodes connected to an A.C., preamplifier and an oscilloscope in parallel with a loudspeaker amplifier. By further dissection, the action potentials originating from a 'single' fiber could be identified. The action potentials were displayed on an oscilloscope and recorded on a Gould electrostatic recorder (ES 1000) together with esophageal pressure, laryngeal temperature and airflow. The laryngeal ending was identified as a 'flow' receptor if it showed an inspiratory modulation only during upper airway breathing and was not activated during tracheostomy breathing and airway occlusions performed either above or below the larynx (fig. 2; Sant'Ambrogio et aL, 1983).
EXPERIMENTAL PROTOCOLS
Air at different temperature and humidity during upper airway breathing. Air temperature within the larynx was varied by changing temperature and/or humidity of the inhaled air. The air temperature was reduced (down to + 8 ° C) by flowing air, from a compressed air source, through a copper coil immersed in an icy brine solution at - 5 ° C. An increase in air temperature, up to 40 ° C, was obtained by bubbling air from the compressed air source through a flask partially t'filed with warm water (60 ° C). When room air had to be saturated, with minimal changes in temperature, it was bubbled through a flask partially filled with water at room temperature. Temperature and
A.P.
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.
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Pes (kPa)
LARYNX 3 5 TEMP. (°C) 33~
]
~
Tracheal breathing
3
3
35
I
35
~
33 [
Upper
Upper
Upper
airway
airway
airway
breathing
breathing
occlusion
Tracheal Tracheal breathing occlusion
Fig. 2. Characterization of a laryngeal 'flow' (cold) receptor. A.P. = action potentials; Pes = esophageal pressure; kPa = kiloPascal.Note that the receptor has an inspiratorymodulation only when air flows into the larynx and the laryngealtemperature is lowered.
38
G. SANT'AMBROGIOet al.
humidity of the inhaled air was altered by passing air, prepared as described above, through a T-tube attached to the oral cannula. Airflow at different rates and temperatures through the isolated upper airway. While the dog was breathing through the tracheostomy (C1 of fig. 1) the upper airway could be isolated by inflating the cuff of a Foley catheter, introduced through C2, at D (fig. 1). Air at different flow rates, temperature and humidity was passed through the upper airway either in the inspiratory (from A to C3) or expiratory direction (from C 3 to A). Flow rates between 0.15 and 0.45 L/sec were used.
Results
Effects of air at different temperature and humidity during upper airway breathing. We have recorded from 30 laryngeal endings responding to inspiratory airflow while the dog was breathing spontaneously through the upper airway air at 26 °C with a relative humidity of 55 ~o. The inspiratory activation of these receptors was always associated with a simultaneous decrease (1.0-1.5 °C) in laryngeal temperature (fig. 2). Fifteen of these receptors remained active, although to a reduced extent, during tracheostomy breathing provided that the upper airway was kept open (A and C open, fig. 1) constituting a passage with a higher resistance 'in parallel' with the tracheostomy. Since in this condition airflow through the upper airway is relatively low, the activation of these endings is indicative of a low threshold (Sant'Ambrogio et al., 1983). All the 30 'flow' receptors tested became silent as the animal inhaled air saturated with water vapor at a temperature of 36-40 ° C which raised the laryngeal temperature during inspiration to 35-38 °C (fig. 3). All the receptors tested increased their activity when the temperature in the laryngeal lumen was decreased either by lowering the temperature or the humidity of the inhaled air (fig. 4). Effect of air at different temperature and flow rate through the isolated upper airway. Fourteen of the 30 receptors studied during spontaneous breathing through the upper airway were also challenged with airflows at different rates and temperatures in the isolated upper airway, while the dog was breathing spontaneously through the tracheostomy. Figure 5 illustrates the response of a laryngeal 'flow' receptor to three challenges in which airflow was held constant, while the temperature in the laryngeal lumen was varied by changing air temperature. By changing either temperature or flow of the air passing through the isolated upper airway the activity of these receptors could be quantitated as a function of either laryngeal temperature or airflow. For all the 14 receptors there was an inverse relationship between laryngeal temperature (steady-state value) and discharge rate, independent of airflow. Receptor activity ceased at temperatures close to 37 °C and increased to an average of 38.7 impulses/sec at a laryngeal temperature of about 24 ° C. In contrast, the
39
LARYNGEAL COLD RECEPTORS
A.P.
(kPa)
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v
t
(I/s) 0.5
Lar. T e m p .
(*C)
34 ] 36
~ ~
~
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r
-
-
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Fig. 3. Behavior of a laryngeal 'flow' (cold) receptor during inhalation of warm air. The dog is spontaneously breathing through the upper airway. A.P. = action potentials; P=, = esophageal pressure; kPa = kiloPascal; V (l/s) = airflow in liter/second; Lar. Temp. = laryngeal temperature. Note that the inspiratory modulation of this receptor disappears when laryngeal temperature during inspiration is raised to the expiratory level by inhalation of warm air (between arrows).
A.P. Pes (kPa)
o°I
Larynx 35 Temp. (°C) 33I
~ t i
q
2s Fig. 4. Behavior of a laryngeal 'flow' (cold) receptor when laryngeal temperature is varied by changing the relative humidity of the inhaled air. The dog is breathing spontaneously through the upper airway. Symbols as in previous figures. At the arrow the relative humidity of inhaled room air is changed from 100 % to 55 %. An increase in inspiratory discharge is associated with the greater decrease in laryngeal temperature during inspiration.
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G SANT'AMBROGIO et al A.P.
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Fig. 5. Response of a laryngeal 'flow' (cold) receptor to constant airflow at different temperatures. Sym-bols as in previous figures. The discharge of the receptor increases when laryngeal temperature decreases. When the laryngeal temperature approaches body temperature even the random activity of the ending disappears.
discharge rate of these endings was not found to be related to airflow Figure 6 illustrates the two relationships for one of the receptors studied The receptor discharge peaks at the onset of the flow challenge and declines thereafter (fig 5) The discharge rate of 9 receptors, for which we have data at comparable temperatures, decreased by 512 + 54~o SE (ranging from 31~o to 75~o) after five seconds The time course of adaptation of one receptor challenged with a constant flow at three different temperatures is illustrated in figure 7 At each temperature the decline in discharge rate is more pronounced within the first second and the difference in activity between temperatures is greater at the beginning of the application of airflow (fig 7) All the laryngeal 'flow' receptors tested were activated by airflow in both inspiratory and expiratory directions provided that laryngeal temperature was lowered below 35 ° C
Discussion
The inspiratory modulation of laryngeal 'flow' receptors can be fully explained considering the decrease in laryngeal temperature during inspiration. The lack of an
LARYNGEAL COLD RECEPTORS 50
• .38 I/s
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41
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10
020
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~'~o
'~'2
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0'.3
"-'~4
Laryngeal Airflow (I/s)
Fig. 6. Relationshipbetweenlaryngealtemperature vs receptor discharge(left panel) and laryngealairflow vs receptordischarge(rightpanel)for one ofthe endings studied. Receptordischargeis measured as number of impulses in the first secondof the challenge.An inverserelationshipbetween laryngealtemperature and receptor dischargeis clearlyvisiblein the range between 37 and 25 °C (left panel). There is no relationship between receptor discharge and flowrate (right panel).
!,o, 60
50
"O
t•\
LaryngealTemp. • 24.0 °C o 31,7 °C e33.2 °C
\
~ ao
10 i
Time (seconds)
Fig. 7. Time course of the discharge of a laryngeal 'flow' (cold) receptor challenged with a constant airflow (0.38 L/sec) at three temperatures.
expiratory activation during spontaneous breathing through the upper airway cannot be ascribed to a mechanical factor related to the direction of airflow, but rather reflects the warmer laryngeal temperature in expiration. In fact, even the inspiratory activation was abolished, without any change in tidal airflow, whenever the laryngeal temperature during inspiration approached that normally present in expiration. The fact that these endings were also activated by airflow through the isolated upper airway in the expiratory direction, whenever laryngeal temperature was lower than 35 °C, and that a constant airflow at three different temperatures (fig. 5) resulted in three distinct discharge rates provide further evidence that they function as cold sensors. Experiments in which both temperature and flow were varied in the isolated upper airway allow a separate evaluation of flow and temperature as factors in the stimulation of 'flow' receptors. It is apparent that flow p e r s e is not a stimulus for these endings,
42
G. SANT'AMBROGIO
et al.
but can only contribute to the extent that it lowers laryngeal temperature. In fact (fig. 6, left panel), the relationship between laryngeal temperature and discharge rate at different flows is similar and moreover, at any particular flow (fig. 5; fig. 6, right panel) the activity of the receptor varies considerably depending on the temperature. Thus it seems appropriate to refer to these endings as laryngeal cold receptors. It is quite apparent from figs. 5 and 7 that these receptors adapt considerably and that their temperature discrimination is best at the onset of stimulation. These properties make them particularly suitable to detect the transient changes in temperature related to airflow present during breathing. A thermal sensitivity has been previously reported for different types of laryngeal mechanoreceptors (Sampson and Eyzaguirre, 1964; Storey, 1968; Boushey etal., 1974). Cold was most frequently found to inhibit them except for endings described as tactile receptors (Sampson and Eyzaguirre, 1964) which had an optimal temperature for their activation with any departure from this value in either direction decreasing their discharge. Although it is difficult to compare the cold receptors of this study with the cold sensing mechanoreceptors of previous studies, it would seem reasonable to suggest that the receptors described here constitute a different population since they do not appear to have any mechanosensitivity as indicated by the fact that flow stimulates them only when it lowers laryngeal temperature below their threshold. This view is corroborated by the observation that an instrument probing the exposed laryngeal mucosa could stimulate these endings only when its temperature was lower than 36-37 °C (Sant'Ambrogio et aL, 1984). It is indeed possible that endings, previously described as tactile (Sampson and Eyzaguirre, 1964) for their response to local probing, were in fact responding to a cold stimulus introduced by the probing instrument having initially a low temperature. The most extensively investigated cold receptors are those present in the skin (Iggo, 1969; Iggo and Young, 1975; Hensel, 1973, 1981). To what extent laryngeal cold receptors are similar to cutaneous cold receptors is difficult to say at present, but they do share some common characteristics. Both have a static discharge at constant temperature, show a dynamic response to changes in temperature, are not excited by mechanical stimuli and operate within a similar temperature range. Cutaneous cold receptors typically have a bell-shaped curve that describes the relationship between temperature and their discharge rate: their activity increases when temperature is lowered, reaching a plateau and decreasing with further decreases in temperature (Iggo, 1969; Iggo and Young, 1975; Hensel, 1981). At present a similar pattern cannot be recognized for laryngeal cold receptors since we did not test them with temperatures below 22 ° C; however, the response of few receptors tested at temperatures below 25 ° C showed a tendency to plateau (fig. 6, left panel). The relatively smaller amplitude of cold receptors action potentials, as compared to that of other types of laryngeal units recorded simultaneously, might indicate a smaller size of their fibers. Moreover, the greater difficulty to separate 'single' fibers together with the tendency of these units to occur in clusters suggests an unmyelinated nature. Only measurements of conduction velocities will permit a definitive identification.
LARYNGEAL COLD RECEPTORS
43
Breathing cold air has been shown to alter both the pattern of breathing and airway resistance. Cold air in the upper airway (above the larynx) has been found to have an inhibitory effect on respiration attributed to a stimulation of some unidentified cold receptors (Burgess and Whitelaw, 1984). Bronchoconstriction has been described in both humans and experimental animals during cold air breathing (Wells et al., 1960; Deal et al., 1979; O'Cain et al., 1980; Jammes et al., 1983). This effect may be of reflex nature and may originate from the upper and/or the lower airways. The involvement of the larynx, as an afferent source, has been convincingly demonstrated by Jammes et al. (1983) in anesthetized cats. Laryngeal cold receptors, such as those described in this study, could very well mediate these responses. In our preparation the dog was breathing through a cannula that bypassed the upper airway except the larynx, i.e., the dog was neither a mouth nor a nose breather. Therefore, we do not have any direct information on the behavior of laryngeal cold receptors in more natural circumstances. However, from the available data in humans (Cole, 1954; Proctor, 1977) the laryngeal temperature in inspiration, even during nose breathing at a comfortable ambient temperature, appears to be within the operational range of laryngeal cold receptors described in the present study. The activity of these endings is expected to increase during mouth breathing, hyperventilation and at low ambient temperature.
Acknowledgements This study was supported by the National Institutes of Health Grant HL-20122. O.P. Mathew is the recipient of an N.I.H. Clinical Investigator Award (HL-01156). Parts of this study were presented at the F.A.S.E.B. Meeting in St. Louis, Missouri (April, 1984) at a symposium on 'Contemporary Sensory Neurobiology' in Galveston, Texas (May, 1984) and at the Spring Meeting of the Italian Physiological Society in Florence (May, 1984). The authors wish to thank Ms. Lynette Morgan for her secretarial help.
References Bartlett, D., Jr., G. S ant'Ambrogio and J. C.M. Wise (1976). Transduction properties of tracheal stretch receptors. J. Physiol. (London) 258: 421-432. Boushey, H.A., P.S. Richardson, J.G. Widdicombe and J. C. M. Wise (1974). The response of laryngeal afferent fibres to mechanical and chemical stimuli. J. Physiol. (London). 240: 153-175. Burgess, K.R. and W.A. Whitelaw (1984). Reducing ventilatory response to carbon dioxide by breathing cold air. Am. Rev. Respir. Dis. 129: 687-690. Cole, P. (1954). Recordings of respiratory air temperature. J. Laryngol. Otol. 68: 295-307. Davis, H. L., W. S. Fowler and E.H. Lambert (1956). Effect of volume and rate of inflation and deflation on transpulmonary pressure and response of pulmonary stretch receptors. Am. J. Physiol. 187: 558-566. Deal, E., E.R. Chandler, E.R. McFadden, R.H. Ingram and J.J. Jaeger (1979). Hyperpnea and heat flux: initial reaction sequence in exercise-induced asthma. J. Appl. Physiol. 46: 476-483.
44
G. SANT'AMBROGIO et al.
Hensel, H. (1973). Cutaneous thermoreceptors. In: Handbook of Sensory Physiology. Vol. I1. Berlin Heidelberg - New York, Springer-Verlag, pp. 79-110. Hensel, H. (1981). Cutaneous thermoreceptors. In: Thermoreception and Temperature Regulation. New York, Academic Press, pp. 33-63. Iggo, A. (1969). Cutaneous thermoreceptors in primates and sub-primates. J. Physiol. (London). 200: 403-430. Iggo, A. and D.W. Young (1975). Cutaneous thermoreceptors and thermal nociceptors. In: The Somatosensory System, edited by H.H. Kornhuber. Stuttgart, Thieme, pp. 5-22. Jammes, Y., P. Barthelemy and S. Delpierre (1983). Respiratory effects of cold air breathing in anesthetized cats. Respir. Physiol. 54: 41-54. Knowlton, G.C. and M.G. Larrabee (1946). A unitary analysis of pulmonary volume receptors. Am. J. Physiol. 147:100-114. O'Cain, C.F., N.B. Dowling, A.S. Slutsky, M.J. Hensley, K.P. Strohl, E.R. McFadden, Jr., and R. H. Ingram, Jr. (1980). Airway effects of respiratory heat loss in normal subjects. J. Appl. Physiol. 49: 875-880. Pack, A.I. and R.B. Delaney (1983). Response of pulmonary rapidly adapting receptors during lung inflation. J. AppL Physiol. 55: 955-963. Proctor, F. P. (1977). The upper airways. I. Nasal physiology and defense of the lungs. Am. Rev. Respir. D#. 115: 97-129. Sampson, S. and C. Eyzaguirre (1964). Some functional characteristics of mechanoreceptors in the larynx of the cat. J. Neurophysiol. 27: 464-480. Sant'Ambrogio, G. (1982). Information arising from the tracheobronchial tree of mammals. Physiol. Rev. 62: 531-569. Sant'Ambrogio, G., O.P. Mathew, J.T. Fisher and F.B. Sant'Ambrogio (1983). Laryngeal receptors responding to transmural pressure, airflow and local muscle activity. Respir. Physiol. 54:317-330. Sant'Ambrogio, G., O. P. Mathew and F. B. Sant'Ambrogio (1984). Characteristics of'flow' receptors in the larynx. Physiologist, 27: 234. Storey, A.T. (1968). A functional analysis of sensory units innervating epiglottis and larynx. Exp. Neurol. 20: 366-383. Wells, R.E., J. E. C. Walker and R.B. Hickler (1960). Effects of cold air on respiratory airflow resistance in patients with respiratory tract diseases. New Engl. J. Med. 263: 268-273. Widdicombe, J. G. (1954). Receptors in the trachea and bronchi of the cat. J. Physiol. (London) 123:71-104.
Respiration Physiology (1985) 59, 35-44 Elsevier
LARYNGEAL COLD RECEPTORS
G. SANT'AMBROGIO, O.P. MATHEW, F.B. SANT'AMBROGIO and J. T. FISHER Department of Physiology and Biophysics and Department of Pediatrics, University of Texas Medical Branch, Galveston, TX 77550, U.S.A.
Abstract. We have previously demonstrated the presence of specific laryngeal 'flow' receptors activated
independently oftransmural pressure. This study considers the operational characteristics of these endings. In 15 anesthetized dogs we recorded single unit action potentials from the peripheral cut end of the internal branch of the superior laryngeal nerve. All the 30 laryngeal 'flow' receptors studied showed an inspiratory modulation when the dog was breathing room air at 26 ° C and 55 ~o relative humidity (laryngeal temperature 34 °C) through the upper airway. All the receptors became silent when the temperature of the inspired air was raised to 36-40 °C, 100~ relative humidity (laryngeal temperature between 35 and 38 °C) and increased their activity when the temperature in the larynx was decreased either by lowering the temperature or the humidity of the inspired air. Fourteen laryngeal 'flow' receptors were tested with a steady flow of air, directed through the isolated in vivo larynx, at different temperatures and saturated with water vapor. Their discharge rate was found to be inversely related to laryngeal temperature (from 35 to 25 ° C) and independent of airflow. Their rate of adaptation indicates a high dynamic sensitivity, In the isolated larynx preparation these receptors were activated by airflow in both inspiratory and expiratory directions provided that laryngeal temperature was lower than 35 ° C. We conclude that the previously described laryngeal 'flow' receptors operate as thermoreceptors activated by cooling. Airway receptors Cold receptors Larynx
Superior laryngeal nerve Temperature Upper airway
In a recent study we demonstrated the presence of laryngeal receptors responding to inspiratory airflow independently of changes in transmural pressure (Sant'Ambrogio et al., 1983). This is a distinct feature of laryngeal 'flow' endings, at variance with the flow detection of both slowly and rapidly adapting receptors of the tracheobronchial tree which is entirely dependent on changes in transmural pressure (Knowlton and Larrabee, 1946; Widdicombe, 1954; Davis etal., 1956; Bartlett etal., 1976; Sant'Ambrogio, Accepted for publication 25 August 1984 0034-5687/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
36
G. SANT'AMBROG10 el a/.
1982; Pack and Delaney, 1983). A possible mechanism for the inspiratory activation of such receptors is the decrease in laryngeal temperature due to inspiratory airflow. Indeed, receptors affected by the local changes in temperature have been described in the larynx (Sampson and Eyzaguirre, 1964; Storey, 1968; Boushey e t a l . , 1974). In this study we have tested this hypothesis and provide evidence that the previously described laryngeal 'flow' receptors operate as thermoreceptors sensitive to cooling.
Methods Fifteen mongrel dogs of either sex, weighing between 9.0 and 13.0 kg were anesthetized with a mixture of chloralose and urethane (0.1 g/kg chloralose and 1.0 g/kg urethane, injected intravenously) and placed on an operating table in the supine position. A femoral vein was cannulated for further injection of the anesthetic mixture. A polyethylene cannula was inserted into a femoral artery and connected to a pressure transducer to monitor blood pressure. The cervical trachea was exposed in its entire length and the ventral aspect of 8-10 cartilaginous rings was longitudinally cut to allow the introduction of both ends of a cannula having three side arms (fig. 1). A saline-filled polyethylene catheter (internal diameter 2 ram) was placed in the mid portion of the esophagus and connected to a pressure transducer for recording esophageal pressure to indicate the respiratory phases. A large polyethylene tube was inserted through the mouth and positioned with the aid of a laryngoscope just below the epiglottis, facing the opening of the larynx. The tube was then secured by closing the mandible and constructing a face mask around the mouth and nose with quick setting epoxy (fig. 1). Laryngeal air temperature was measured by placing a thermistor (time constant = 100 msec) or a thermocouple probe (time constant = 20 msec), through a side arm of the tracheal cannula, with its tip floating just below the vocal cords. Airflow was measured with a pneumotachograph (Fleisch no. 1) connected to the oral cannula.
des
Fig. 1. Schematicrepresentation of the experimentalsetup. Mouth and nares are sealed around tube A with quick setting epoxy.The tracheal cannula with three side arms permits the diversion of breathing from the tracheostomy (C1 open; A closed) to the upper airway (A open; Cl, C2 and C3 closed) and occlusion of the airway either below (at B) or above (all outlets closed) the larynx for identifyinga 'flow' (cold) receptor. With this setup air can also be passed through the upper airway while the dog breathes through the tracheostomy (C, open, tracheal cannula occluded at D).
LARYNGEAL COLD RECEPTORS
37
The internal branch of one superior laryngeal nerve, most often the right, was identified and isolated peripherally to its entrance into the thyroid cartilage and cut 2 cm from the larynx. The peripheral cut end was then placed on a dissecting tray, covered with mineral oil, desheathed and separated into several thin bundles using a pair of iridectomy scissors and watchmaker forceps. The activity present in each small bundle could be monitored by putting the filament across a pair of platinum electrodes connected to an A.C., preamplifier and an oscilloscope in parallel with a loudspeaker amplifier. By further dissection, the action potentials originating from a 'single' fiber could be identified. The action potentials were displayed on an oscilloscope and recorded on a Gould electrostatic recorder (ES 1000) together with esophageal pressure, laryngeal temperature and airflow. The laryngeal ending was identified as a 'flow' receptor if it showed an inspiratory modulation only during upper airway breathing and was not activated during tracheostomy breathing and airway occlusions performed either above or below the larynx (fig. 2; Sant'Ambrogio et aL, 1983).
EXPERIMENTAL PROTOCOLS
Air at different temperature and humidity during upper airway breathing. Air temperature within the larynx was varied by changing temperature and/or humidity of the inhaled air. The air temperature was reduced (down to + 8 ° C) by flowing air, from a compressed air source, through a copper coil immersed in an icy brine solution at - 5 ° C. An increase in air temperature, up to 40 ° C, was obtained by bubbling air from the compressed air source through a flask partially t'filed with warm water (60 ° C). When room air had to be saturated, with minimal changes in temperature, it was bubbled through a flask partially filled with water at room temperature. Temperature and
A.P.
' I
.
]1 '
]
I
I r
IN
'I
~
'~i' ' l l " l u
I
'i
:!
:
Pes (kPa)
LARYNX 3 5 TEMP. (°C) 33~
]
~
Tracheal breathing
3
3
35
I
35
~
33 [
Upper
Upper
Upper
airway
airway
airway
breathing
breathing
occlusion
Tracheal Tracheal breathing occlusion
Fig. 2. Characterization of a laryngeal 'flow' (cold) receptor. A.P. = action potentials; Pes = esophageal pressure; kPa = kiloPascal.Note that the receptor has an inspiratorymodulation only when air flows into the larynx and the laryngealtemperature is lowered.
38
G. SANT'AMBROGIOet al.
humidity of the inhaled air was altered by passing air, prepared as described above, through a T-tube attached to the oral cannula. Airflow at different rates and temperatures through the isolated upper airway. While the dog was breathing through the tracheostomy (C1 of fig. 1) the upper airway could be isolated by inflating the cuff of a Foley catheter, introduced through C2, at D (fig. 1). Air at different flow rates, temperature and humidity was passed through the upper airway either in the inspiratory (from A to C3) or expiratory direction (from C 3 to A). Flow rates between 0.15 and 0.45 L/sec were used.
Results
Effects of air at different temperature and humidity during upper airway breathing. We have recorded from 30 laryngeal endings responding to inspiratory airflow while the dog was breathing spontaneously through the upper airway air at 26 °C with a relative humidity of 55 ~o. The inspiratory activation of these receptors was always associated with a simultaneous decrease (1.0-1.5 °C) in laryngeal temperature (fig. 2). Fifteen of these receptors remained active, although to a reduced extent, during tracheostomy breathing provided that the upper airway was kept open (A and C open, fig. 1) constituting a passage with a higher resistance 'in parallel' with the tracheostomy. Since in this condition airflow through the upper airway is relatively low, the activation of these endings is indicative of a low threshold (Sant'Ambrogio et al., 1983). All the 30 'flow' receptors tested became silent as the animal inhaled air saturated with water vapor at a temperature of 36-40 ° C which raised the laryngeal temperature during inspiration to 35-38 °C (fig. 3). All the receptors tested increased their activity when the temperature in the laryngeal lumen was decreased either by lowering the temperature or the humidity of the inhaled air (fig. 4). Effect of air at different temperature and flow rate through the isolated upper airway. Fourteen of the 30 receptors studied during spontaneous breathing through the upper airway were also challenged with airflows at different rates and temperatures in the isolated upper airway, while the dog was breathing spontaneously through the tracheostomy. Figure 5 illustrates the response of a laryngeal 'flow' receptor to three challenges in which airflow was held constant, while the temperature in the laryngeal lumen was varied by changing air temperature. By changing either temperature or flow of the air passing through the isolated upper airway the activity of these receptors could be quantitated as a function of either laryngeal temperature or airflow. For all the 14 receptors there was an inverse relationship between laryngeal temperature (steady-state value) and discharge rate, independent of airflow. Receptor activity ceased at temperatures close to 37 °C and increased to an average of 38.7 impulses/sec at a laryngeal temperature of about 24 ° C. In contrast, the
39
LARYNGEAL COLD RECEPTORS
A.P.
(kPa)
°l
-1.5
v
t
(I/s) 0.5
Lar. T e m p .
(*C)
34 ] 36
~ ~
~
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r
-
-
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3s
Fig. 3. Behavior of a laryngeal 'flow' (cold) receptor during inhalation of warm air. The dog is spontaneously breathing through the upper airway. A.P. = action potentials; P=, = esophageal pressure; kPa = kiloPascal; V (l/s) = airflow in liter/second; Lar. Temp. = laryngeal temperature. Note that the inspiratory modulation of this receptor disappears when laryngeal temperature during inspiration is raised to the expiratory level by inhalation of warm air (between arrows).
A.P. Pes (kPa)
o°I
Larynx 35 Temp. (°C) 33I
~ t i
q
2s Fig. 4. Behavior of a laryngeal 'flow' (cold) receptor when laryngeal temperature is varied by changing the relative humidity of the inhaled air. The dog is breathing spontaneously through the upper airway. Symbols as in previous figures. At the arrow the relative humidity of inhaled room air is changed from 100 % to 55 %. An increase in inspiratory discharge is associated with the greater decrease in laryngeal temperature during inspiration.
4O
G SANT'AMBROGIO et al A.P.
r
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......... "
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ill
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L
,
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all
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Fig. 5. Response of a laryngeal 'flow' (cold) receptor to constant airflow at different temperatures. Sym-bols as in previous figures. The discharge of the receptor increases when laryngeal temperature decreases. When the laryngeal temperature approaches body temperature even the random activity of the ending disappears.
discharge rate of these endings was not found to be related to airflow Figure 6 illustrates the two relationships for one of the receptors studied The receptor discharge peaks at the onset of the flow challenge and declines thereafter (fig 5) The discharge rate of 9 receptors, for which we have data at comparable temperatures, decreased by 512 + 54~o SE (ranging from 31~o to 75~o) after five seconds The time course of adaptation of one receptor challenged with a constant flow at three different temperatures is illustrated in figure 7 At each temperature the decline in discharge rate is more pronounced within the first second and the difference in activity between temperatures is greater at the beginning of the application of airflow (fig 7) All the laryngeal 'flow' receptors tested were activated by airflow in both inspiratory and expiratory directions provided that laryngeal temperature was lowered below 35 ° C
Discussion
The inspiratory modulation of laryngeal 'flow' receptors can be fully explained considering the decrease in laryngeal temperature during inspiration. The lack of an
LARYNGEAL COLD RECEPTORS 50
• .38 I/s
o~ IDOl ) O
•
40
•
g
30
"5
20
50 II •
o .36 I/s © .20 I/s
41
|
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30 20
d~
10
10
020
3'0
0
~'~o
'~'2
Laryngeal Temperature (°C)
0'.3
"-'~4
Laryngeal Airflow (I/s)
Fig. 6. Relationshipbetweenlaryngealtemperature vs receptor discharge(left panel) and laryngealairflow vs receptordischarge(rightpanel)for one ofthe endings studied. Receptordischargeis measured as number of impulses in the first secondof the challenge.An inverserelationshipbetween laryngealtemperature and receptor dischargeis clearlyvisiblein the range between 37 and 25 °C (left panel). There is no relationship between receptor discharge and flowrate (right panel).
!,o, 60
50
"O
t•\
LaryngealTemp. • 24.0 °C o 31,7 °C e33.2 °C
\
~ ao
10 i
Time (seconds)
Fig. 7. Time course of the discharge of a laryngeal 'flow' (cold) receptor challenged with a constant airflow (0.38 L/sec) at three temperatures.
expiratory activation during spontaneous breathing through the upper airway cannot be ascribed to a mechanical factor related to the direction of airflow, but rather reflects the warmer laryngeal temperature in expiration. In fact, even the inspiratory activation was abolished, without any change in tidal airflow, whenever the laryngeal temperature during inspiration approached that normally present in expiration. The fact that these endings were also activated by airflow through the isolated upper airway in the expiratory direction, whenever laryngeal temperature was lower than 35 °C, and that a constant airflow at three different temperatures (fig. 5) resulted in three distinct discharge rates provide further evidence that they function as cold sensors. Experiments in which both temperature and flow were varied in the isolated upper airway allow a separate evaluation of flow and temperature as factors in the stimulation of 'flow' receptors. It is apparent that flow p e r s e is not a stimulus for these endings,
42
G. SANT'AMBROGIO
et al.
but can only contribute to the extent that it lowers laryngeal temperature. In fact (fig. 6, left panel), the relationship between laryngeal temperature and discharge rate at different flows is similar and moreover, at any particular flow (fig. 5; fig. 6, right panel) the activity of the receptor varies considerably depending on the temperature. Thus it seems appropriate to refer to these endings as laryngeal cold receptors. It is quite apparent from figs. 5 and 7 that these receptors adapt considerably and that their temperature discrimination is best at the onset of stimulation. These properties make them particularly suitable to detect the transient changes in temperature related to airflow present during breathing. A thermal sensitivity has been previously reported for different types of laryngeal mechanoreceptors (Sampson and Eyzaguirre, 1964; Storey, 1968; Boushey etal., 1974). Cold was most frequently found to inhibit them except for endings described as tactile receptors (Sampson and Eyzaguirre, 1964) which had an optimal temperature for their activation with any departure from this value in either direction decreasing their discharge. Although it is difficult to compare the cold receptors of this study with the cold sensing mechanoreceptors of previous studies, it would seem reasonable to suggest that the receptors described here constitute a different population since they do not appear to have any mechanosensitivity as indicated by the fact that flow stimulates them only when it lowers laryngeal temperature below their threshold. This view is corroborated by the observation that an instrument probing the exposed laryngeal mucosa could stimulate these endings only when its temperature was lower than 36-37 °C (Sant'Ambrogio et aL, 1984). It is indeed possible that endings, previously described as tactile (Sampson and Eyzaguirre, 1964) for their response to local probing, were in fact responding to a cold stimulus introduced by the probing instrument having initially a low temperature. The most extensively investigated cold receptors are those present in the skin (Iggo, 1969; Iggo and Young, 1975; Hensel, 1973, 1981). To what extent laryngeal cold receptors are similar to cutaneous cold receptors is difficult to say at present, but they do share some common characteristics. Both have a static discharge at constant temperature, show a dynamic response to changes in temperature, are not excited by mechanical stimuli and operate within a similar temperature range. Cutaneous cold receptors typically have a bell-shaped curve that describes the relationship between temperature and their discharge rate: their activity increases when temperature is lowered, reaching a plateau and decreasing with further decreases in temperature (Iggo, 1969; Iggo and Young, 1975; Hensel, 1981). At present a similar pattern cannot be recognized for laryngeal cold receptors since we did not test them with temperatures below 22 ° C; however, the response of few receptors tested at temperatures below 25 ° C showed a tendency to plateau (fig. 6, left panel). The relatively smaller amplitude of cold receptors action potentials, as compared to that of other types of laryngeal units recorded simultaneously, might indicate a smaller size of their fibers. Moreover, the greater difficulty to separate 'single' fibers together with the tendency of these units to occur in clusters suggests an unmyelinated nature. Only measurements of conduction velocities will permit a definitive identification.
LARYNGEAL COLD RECEPTORS
43
Breathing cold air has been shown to alter both the pattern of breathing and airway resistance. Cold air in the upper airway (above the larynx) has been found to have an inhibitory effect on respiration attributed to a stimulation of some unidentified cold receptors (Burgess and Whitelaw, 1984). Bronchoconstriction has been described in both humans and experimental animals during cold air breathing (Wells et al., 1960; Deal et al., 1979; O'Cain et al., 1980; Jammes et al., 1983). This effect may be of reflex nature and may originate from the upper and/or the lower airways. The involvement of the larynx, as an afferent source, has been convincingly demonstrated by Jammes et al. (1983) in anesthetized cats. Laryngeal cold receptors, such as those described in this study, could very well mediate these responses. In our preparation the dog was breathing through a cannula that bypassed the upper airway except the larynx, i.e., the dog was neither a mouth nor a nose breather. Therefore, we do not have any direct information on the behavior of laryngeal cold receptors in more natural circumstances. However, from the available data in humans (Cole, 1954; Proctor, 1977) the laryngeal temperature in inspiration, even during nose breathing at a comfortable ambient temperature, appears to be within the operational range of laryngeal cold receptors described in the present study. The activity of these endings is expected to increase during mouth breathing, hyperventilation and at low ambient temperature.
Acknowledgements This study was supported by the National Institutes of Health Grant HL-20122. O.P. Mathew is the recipient of an N.I.H. Clinical Investigator Award (HL-01156). Parts of this study were presented at the F.A.S.E.B. Meeting in St. Louis, Missouri (April, 1984) at a symposium on 'Contemporary Sensory Neurobiology' in Galveston, Texas (May, 1984) and at the Spring Meeting of the Italian Physiological Society in Florence (May, 1984). The authors wish to thank Ms. Lynette Morgan for her secretarial help.
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