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Attenuation of pulmonary afferent input by vagal cooling in dogs
Respiration Physiology (1988) 72, 19-34 Elsevier
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RSP 01389
Attenuation of pulmonary afferent input by vagal cooling in dogs A. Jonzon, T.E. Pisarri, A . M . Roberts, J. C. G. Coleridge and H . M . Coleridge Cardiovascular Research Institute and Department of Physiology, University of California San Francisco, San Francisco, CA 94143-0130, U.S.A. (Accepted for publication 16 November 1987) Abstract. In open chest, artificially ventilated, anesthetized dogs, we examined the effect of vagal cooling
on the pulmonary afferent input evoked by hyperinflating the lungs to 3 VT, recording the activity of slowly adapting pulmonary stretch receptors (PSRs), rapidly adapting receptors (RARs) and pulmonary C fibers rostral to the cooling platform. At 15 ° C and below, input in all three types of fiber was significantly reduced, attenuation being least marked in C fibers. Between 12°C and 7°C, attenuation of RAR input was significantly less than that of PSRs. At 7°C, virtually none of the hyperinflation-evoked increase in PSR activity and only 10~o of that in RARs passed the cooling platform - indeed RAR input was less than during normal ventilation at 37 ° C; by contrast, 40 ~o of the hyperinflation-evoked increase in C fiber activity was still transmitted. Cooling had similar effects on C fiber input evoked by capsaicin. If reflexes are attenuated in proportion to the attenuation of afferent input, our results suggest that a hyperinflation-evoked reflex that survives vagal cooling below 6°C is almost certainly triggered by C fibers.
Cold block of lung afferents; Conduction velocities of lung afferents; Lung C fibers; Pulmonary C fibers and capsaicin; Pulmonary stretch receptors; Rapidly adapting receptors
Cooling the vagus nerves is often used to investigate reflexes of lower airway origin. However, attempts to identify the afferents responsible for a given reflex by comparing the blocking temperature of the various pulmonary afferents with the blocking temperature of the reflex itself continue to arouse controversy (Coleridge and Coleridge, 1984, 1986). Much of what is known about the attenuating effects of cooling on conduction in single myelinated (A) and non-myelinated (C) fibers has been derived from experiments in which nerve trunks were stimulated electrically at constant frequency, beginning with trains of pulses delivered at the maximal frequency the individual axon could follow and continuing with progressively lower frequencies as the nerve was cooled (Paintal, 1965b; Franz and Iggo, 1968; Linden et al., 1981). Experiments of this type have established that C fibers continue to conduct impulses at temperatures that Correspondence address: Dr. H.M. Coleridge, Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA 94143-0130, U.S.A. 0034-5687/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
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A. J O N Z O N et al.
block conduction in A fibers. They have also shown that, regardless of fiber type, the effects of cooling are frequency dependent, the higher the firing frequency the greater the attenuation produced at a given vagal temperature. In most reflex studies, however, pulmonary afferent input to the medullary centers is evoked not by stimulating nerve fibers electrically at constant frequency but by applying a mechanical or chemical stimulus to the lower respiratory tract to excite the afferent terminals themselves. The various types of pulmonary ending respond in different ways to a given stimulus, the pattern and frequency of the evoked discharge varying with the threshold, sensitivity and rate of adaptation of the ending. Consequently, when attempts are made to correlate the effect ofvagal cooling on pulmonary afferent input with that on a particular reflex response, it is essential to stimulate the endings themselves in both cases, and to use the same stimulus. However, there is little detailed information about the attenuating effects of progressive vagal cooling on naturally evoked activity in the various types of pulmonary afferent. In a previous study in this laboratory, Pisarri et al. (1986) described the effect of vagal cooling on the baseline activity in pulmonary afferent fibers when the lungs were ventilated at normal tidal volume. We have extended these observations, examining the attenuating effects of cooling on input from a large number of slowly adapting pulmonary stretch receptors, rapidly adapting receptors and pulmonary C fibers in dogs, both during artificial ventilation at normal tidal volumes and during lung distension (3 VT). Distension of the lungs is known to stimulate all three types of pulmonary afferent, and was shown by Head (1889) in rabbits to have reflex effects on breathing that could be reversed as the vagus nerves were cooled (Head's 'paradoxical' reflex). In a reflex study in dogs, described in a companion paper (Roberts et al., 1988), vagal cooling was also found to reverse the effects of lung distension on airway smooth muscle tone, distension evoking tracheal relaxation when the vagus nerves were at body temperature but contraction when the nerves were cooled below 8-7 ° C. The present experiments shed light on the afferents responsible for this reversal of the tracheal response. In addition, because capsaicin stimulates lower airway C fibers, and is often used to evoke lower airway reflexes, we examined the effects of vagal cooling on capsaicin-evoked input in pulmonary and bronchial C fibers.
Methods General. Eighteen dogs (14-27 kg) were given promazine HC1 (Sparine, Wyeth, 50 mg i.m.); 30 min later they were anesthetized with 0.25 ml/kg i.v. of a 1 : 1 mixture of solutions of Dial Compound (allobarbital 100 mg/ml, urethane 400 mg/ml, Ciba) and sodium pentobarbital (50 mg/ml). Supplemental doses were given as required to maintain surgical anesthesia. The trachea was cannulated low in the neck and the chest was opened in the midsternal line. The lungs were ventilated with 50~o 02 in air by a Harvard respirator (model 613) whose expiratory outlet was placed under 3-5 cm water. Tidal CO 2 was
COLD-BLOCKADE OF PULMONARY A F F E R E N T INPUT
21
monitored by a Beckman LB-1 gas analyzer. Tidal volume was set at approximately 15 ml/kg, and ventilator frequency (10-16 cycles/rain) was adjusted to maintain endtidal Pco2 at approximately 35 mm Hg. Arterial Po2, Pco~, pH and base excess were measured at regular intervals with an automatic analyzer (Coming 175 blood gas/pH analyzer). Any metabolic acidosis was corrected by administration o f N a H C O 3 solution i.v.
Tracheal pressure was recorded from a sidearm of the tracheal cannula, and systemic arterial blood pressure was recorded from a femoral artery. Pressures were recorded with Statham P23Gb strain gauges. An electrocardiogram (lead II) was recorded. Afferent vagal impulses were recorded and were counted by ratemeters (see below). The signals representing tidal CO2, pressures, ratemeter counts of impulse frequency, and vagal temperature (see below) were recorded by a Grass polygraph. Vagal action potentials, ratemeter counts and other selected variables were also recorded by an ultraviolet light recorder (SE Laboratories) or by a Gould (ES 1000) electrostatic recorder.
Dissection of neck. The anterior muscles of the neck were dissected on the left side of the trachea and retracted to form two separate pools. In the rostral pool, 3-4 cm of the left cervical vagus nerve was freed from the carotid sheath and placed upon a small platform (1 cm wide). After the sheath had been removed from the vagus nerve on the platform, the rostral pool was filled with mineral oil, and fine vagal strands were dissected for the recording of afferent impulses (see below). In the caudal pool, 7 cm of the left vagus nerve was freed from the carotid sheath, and the nerve was placed upon a cooling platform (see below). Rostral to the cooling platform, the nerve was placed upon stimulating electrodes for measurement of fiber conduction velocities.
Pulmonary afferent activity. Using conventional techniques, we recorded impulses from afferent vagal fibers arising from the left lung. Slowly adapting pulmonary stretch receptors (PSRs), rapidly adapting receptors (RARs) and pulmonary and bronchial C fibers were identified by their characteristic patterns of discharge, by their responses to lung distension (3 VT) and to injection of capsaicin (10 #g/kg) into the right and left atrium, and by their fiber conduction velocities (Coleridge and Coleridge, 1977). Conduction velocities were measured by stimulating the cervical vagus nerve through two pairs of electrodes, 2 cm apart, fixed in a shielded assembly; conduction velocity was derived from the conduction time between the stimulating electrodes. The approximate location of the receptors was determined by probing the lung: receptors not located in the lung were discarded. Impulses were counted in 1 sec bins by ratemeters whose window discriminators were set to accept potentials of a particular amplitude. Nerve filaments from which recordings were made contained a single active fiber, or two fibers whose action potentials were of sufficiently different amplitude and configuration to be clearly distinguishable.
Vagal cooling. The left vagus nerve was placed in a groove on the platform (width, 2 cm) of a silver cooling device through which alcohol of different temperatures was
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Fig. 1. Progressive vagal cooling attenuates conduction in an RAR fiber. RAR stimulated at intervals of 1-2 min by briefly hyperinflating the lungs (3 VT), as indicated by the increases in tracheal pressure (PT). Note virtual abolition of recorded activity at 7 ° C and restoration of activity as nerve rewarmed. Other abbreviations: Pco2, tidal Pco2; ABP, arterial blood pressure; temp (needle), temperature of vagus nerve recorded by needle thermistor; temp (platform), temperature of cooling platform; IF, impulse frequency recorded by ratemeter. Between 28 °C and 6 ° C, the vagus nerve was cooled at the rate of 1.8 ° C/min. Before vagal cooling, the RAR was stimulated by injecting histamine (20 #g/kg) into the right atrium; when the vagus nerve had been cooled to 7-6 ° C, injection of histamine (hist; note decrease in ABP and increase in PT) had no effect on RAR activity recorded rostral to the cooling platform.
circulated. The sides and bottom of the platform were insulated with a layer of silicone elastomer. To reduce thermal gradients the nerve was covered with a warm (40°C) solution of 4 ~ agar in saline, which, upon cooling, gelled to form a semisolid layer approximately 0.5 cm thick. The temperature of the platform was measured by thermistor (Yellow Springs Instrument 729), and the temperature of the nerve by a needle thermistor (Yellow Springs Instrument 524) inserted obliquely so that its tip was between the sheath and the nerve. When measured simultaneously (fig. 1), the two temperatures were within 1 °C; vagal temperature was taken as the average of the two. Care was taken not to exert tension on the nerve when placing it upon the cooling platform because tension is known to elevate the blocking temperature of nerve fibers (Paintal, 1965a).
Coolingprotocol. We examined the effect of cooling the lower cervical vagus nerve on pulmonary afferent activity recorded rostral to the cooling platform, both during control ventilation and when the lungs were briefly distended to FRC + 3 VT (peak tracheal
COLD-BLOCKADE OF PULMONARY AFFERENT INPUT
23
pressure, 20-25 cm H 2 0 ) (fig. 1). We also examined the effect ofvagal cooling on C fiber activity evoked by injecting c apsaicin (10 #g/kg) into the right atrium through a c atheter inserted via an external jugular vein. In most experiments the vagus nerve was cooled in steps, temperature being maintained at each step for 3 min before measurement of impulse activity; in others the nerve was cooled gradually from 37 °C to 0 °C at the rate of 1-2°C per min. Results obtained using the two methods of cooling were identical, and have been combined. Impulse activity was measured at vagal temperatures of 37, 15, 12, 9, 7, 5, 3, 1 and 0 ° C. In a few experiments we continued cooling to - 1 or - 2 ° C in order to determine the blocking temperature of a C fiber whose conduction was not blocked at 0°C. Usually, however, we did not cool the nerve below 0°C, because we examined several fibers in each dog and wished to avoid damaging the nerve by repeated cooling to very low temperatures.
Analysis of data. During control ventilation the impulse activity of all pulmonary afferents was averaged over five ventilatory cycles at each vagal temperature and expressed as impulses/sec. The activity evoked by lung distension was also measured at each temperature. Activity in PSRs was counted from the peak of inflation at FRC + 2 VT to the peak of inflation at FRC + 3 VT (as determined from the tracheal pressure record), and expressed as impulses/sec. Because RARs and pulmonary C fibers were sometimes silent during the early stages of distension and fired a relatively short burst of impulses during the third inflation, their response was counted over the 2 sec at which firing was at a maximum and was expressed as impulses/sec. C fiber activity evoked by capsaicin was also measured at each temperature and was averaged over 2 sec at the peak of the response and expressed as impulses/sec. Impulse activities at each temperature were expressed as means + SE. For each of the three types of pulmonary afferent, we plotted the impulse frequency transmitted across the cooling platform at each temperature as a function of the impulse frequency transmitted at 37 ° C, which was taken as a measure of the discharge frequency of the receptor itself. To obtain a wide range of frequencies in individual fibers, each scatter plot included baseline activity during control ventilation and increased activity evoked by lung distension and, in the case of C fibers, by injection of capsaicin. The relationship between the impulse frequency at 37°C and the transmitted impulse frequency at each temperature was determined by linear regression ( C R U N C H statistical software). Regression equations were of the form Fx = aT + bT(F37oc), where Far is the impulse frequency transmitted across the cooling platform at temperature T, and a T and bx are respectively the y intercept and the slope of the regression equation. The plots for PSRs and RARs showed an inflection point suggesting an impulse frequency below which the data lay on the line of identity (Fa- = F37oc) and above which there was attenuation. Therefore the regression equations were constructed using only data above this inflection point. The inflection point thus represented the
24
A. JONZON et al.
maximum frequency transmitted without attenuation at that temperature. The position of the inflection point was determined by iterative calculation of the regression line, omitting points below the putative inflection point until the regression equation became constant. Regression lines for C fibers were constructed using all data points. At each temperature, the slopes of the regression lines for each type of afferent were compared using the standard errors of the slopes to calculate the 95 % confidence intervals for a statistically discernible difference between each pair of slopes.
Results General We recorded the impulse activity of 42 slowly adapting pulmonary stretch receptors (PSRs), 43 rapidly adapting receptors (RARs) and 42 C fibers (38 pulmonary and 4 bronchial). Fiber conduction velocities of 27 PSRs ranged from 17 to 66 m/sec (mean, 32.0 + 2.0), of 29 RARs from 11 to 52 m/sec (mean, 31.9 + 1.7), and of 30 C fibers from 0.5 to 2.5 m/sec (mean, 1.3 + 0.1). During control ventilation, PSRs discharged in a regular, phasic fashion and their average frequency was relatively high (24.8 + 2.5 impulses/sec). By contrast, RARs discharged irregularly and average frequency was low (2.7 + 0.5 impulses/sec); however, the activity of individual RARs varied widely and 16~o of RARs were inactive during control ventilation. If five unusually active receptors with control discharges of 6.1-15.7 impulses/sec are excluded, RAR frequencies during control ventilation averaged 1.7 + 0.3 impulses/sec. C fibers also discharged irregularly, and their activity
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Fig. 2. Progressive cold blockade of activity in myelinated pulmonary fibers. Data are means + SE. Observations were made on: (A), 42 PSRs and (B), 43 RARs; note differentcalibration scales in A and B. Open bars, effect of cooling on activity during control ventilation; hatched bars, effect of cooling on activity evoked by hyperinflatingthe lungs (3 VT).
COI.D-BLOCKADE OF PULMONARY AFFERENT INPUT
25
was very low (0.5 + 0.1 impulses/see), 3 6 ~ of C fibers being inactive. In all but the four bronchial C fibers, activity increased significantly when the lungs were hyperinflated, reaching 71.2 + 5.2 impulses/see in PSRs, 16.7 + 1.7 impulses/see in RARs and 10.1 + 0.9 impulses/see in pulmonary C fibers. Activity in both pulmonary and bronchial C fibers increased after right atrial injection of capsaicin, to 31.0 + 2.2 impulses/see.
Effects of cooling on myelinatedfibers.
Impulse frequency recorded rostral to the cooling platform was attenuated in a generally similar fashion in PSR and RAR fibers as the vagus nerves were cooled (fig. 2A,B). Although input in some PSRs and RARs A
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Fig. 3. Transmitted impulse frequencies in myelinated pulmonary fibers at different vagal temperatures related to impulse frequencies at 37 ° C. Dashed line is line of identity. On left, scatter plots for individual PSRs (A) and RARs (C) show impulse frequencies conducted across the cooled region of nerve at 15 ° C. Note that low frequencies (up to 13-14 impulses/see in PSRs and 4-5 impulses/see in RARs) were transmitted without attenuation at 15 ° C, but that higher frequencies were reduced to a relatively constant fraction of those at 37 ° C. Solid lines are drawn from regression equations (table 1) for these data points. On right, solid lines are drawn from regression equations (table 1) for PSRs (B) and RARs (D) at each vagal temperature, calculations from a minimum of 60 data points. Note that the transmitted fraction of PSR and RAR impulse frequencies (slope of regression line) was similar at 15 ° C, but was significantly greater for RARs at temperatures of 12°C and below. Note different calibration scales for PSRs and RARs.
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A. J O N Z O N et al.
decreased during control ventilation when vagal temperature was reduced to 15 ° C, it was not attenuated significantly until the vagus was cooled to 12°C. However, when activity was increased by hyperinflating the lungs, the input frequency of both PSRs and RARs was attenuated significantly at 15°C (fig. 2). The proportionate decrease in activity across the cooled region of nerve was greater for PSRs than for RARs. At 9°C, PSR input frequency during hyperinflation was only a third of that during control ventilation at 37 °C (fig. 2A), whereas RAR input frequency during hyperinflation did not fall below the control level until the vagus was cooled to 7°C (fig. 2B). Cooling to 7 °C reduced the transmitted input in PSR and RAR fibers to a similar low level. In some myelinated fibers, transmission across the cooling platform persisted at low temperatures, and was more likely to do so during hyperinflation than at normal tidal volume. At 5 ° C, 28 Yo of PSR fibers and 46 ~o of RAR fibers continued to conduct 1-3 of the impulses evoked by hyperinflation. Even at 1 ° C, isolated impulses from one PSR and three RARs were still conducted across the cooled region of nerve; these were blocked at 0°C. The impulse frequency arriving at a cooled region of nerve influences conduction across it (Paintal, 1965b; Franz and Iggo, 1968). We therefore examined the transmitted impulse frequency in individual fibers at each temperature as a function of the receptor discharge frequency (measured as the transmitted impulse frequency at 37°C). To obtain a wide range of frequencies, we included data obtained during control ventilation and hyperinflation. Receptor discharge frequencies ranged from 1 to 108 impulses/sec
TABLE 1 Frequency-dependent effects of vagal cooling on pulmonary afferent input PSRs
RARs
Pulm Cs
Vagal temp ( ° C)
MF (imp/sec)
b r +_ SE
MF (imp/sec)
b w _+ SE
MF bT _+ SE (imp/sec)
15 12 9 7 5 3
14.7 11.0 5.8 1.9
0.57 0.22 0.02 0.00
3.4 2.7 1.2 0.3 0.0
0.61 0.36 0.19 0.10 0.04
-0.7 1.5 1.7 0.8 1.0 0.8
+ + + +
0.05 0.04 0.02 0.01
+ 0.04 + 0.04 + 0.03 + 0.02 _+ 0.01
0.84 0.63 0.43 0.38 0.22 0.13
+ 0.04 + 0.06 + 0.06 + 0.05 _+ 0.04 _+ 0.04
Values calculated at each vagal temperature from regression equation: F T = aT + bx(F37oc), where F x was the impulse frequency transmitted across the cooling platform at temperature T, and a T and b T were respectively the y intercept and the slope of the regression equation; MF, maximal frequency transmitted without attenuation at each vagal temperature (i.e., point at which regression line intersects line of identity, see Methods). Data obtained during control ventilation and during lung hyperinflation, with a minimum of 60 data points each for PSRs and RARs and 43 for C fibers. Range of impulse frequencies at 37°C: PSRs (n = 42), 1-108 impulses/sec; RARs (n = 43), 1-44 impulses/sec; pulmonary C fibers (n = 38), 1-22 impulses/sec.
27
COLD-BLOCKADE OF PULMONARY AFFERENT INPUT
for PSRs and from 1 to 44 impulses/sec for RARs. The scatter plots obtained for both PSRs and RARs (fig. 3A,C) revealed that low frequencies were transmitted across the cooled region without attenuation. Above a critical frequency (MF, table 1), the transmitted input at each temperature was best described as a linear function of the receptor discharge frequency, the slope of the regression line indicating the fraction of incoming impulses transmitted (fig. 3B,D; table 1). Cooling to 15°C had little effect on PSR fibers fLring at less than 14 impulses/sec, or on RAR fibers firing at less than 3.5 impulses/sec (fig. 3A,C; table 1). Above these frequencies, the attenuation of impulse transmission, as indicated by the slopes of the regression lines, did not differ significantly between PSRs (0.57) and RARs (0.61). At temperatures below 12 ° C, the fraction of activity passing the cooled region of nerve was greater for RARs than for PSRs, the slope of the regression line for RARs being greater than that for PSRs (P < 0.05). This was partly, but not entirely, due to the higher maximum frequencies of the PSR discharge. When the regression equations were recalculated using only data points for PSRs whose discharge frequencies were within the range of the RAR frequencies (1--44 impulses/sec), the difference in slopes was statistically discernible only at 7°C. Below 9°C, the cooled region of nerve acted as a 'low pass filter' to PSR activity, transmission being limited on average to 5-6 impulses/sec at 9°C and to 2 impulses/sec at 7 ° C, whatever the incoming frequency (fig. 3B; table 1). Consequently hyperinflation no longer increased PSR input central to the cold block. This was not true of the RARs,
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Fig. 4. Attenuation by progressive vagal cooling of conduction in 2 pulmonary C fibers; (A) and (B) in different dogs. (A) C fiber activity evoked by hyperinflating the lungs (3 VT); the records depict the second and third inflations above FRC; final record (37°C) when vagus nerve rewarmed. AP, action potentials; PT, tracheal pressure. (B) C fiber activity evoked by injecting capsaicin (10 #g/kg) into the right atrium; interval of 10 min between injections; final record (37°C) when vagus nerve rewarmed; IF, impulse frequency recorded by ratemeter.
28
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Fig. 5. Effect of vagal cooling on conduction in lung C fibers. Data are means + SE. Open bars, effect of cooling on 38 C fibers stimulated by hyperinflating the lungs (3 VT); hatched bars, effect of cooling on 20 C fibers stimulated by injecting capsaicin (10 #g/kg) into the right atrium. Note that at a vagal temperature of 7 ° C, C fiber activity evoked by the two methods of stimulation was 44 ~o and 39 ~o, respectively, of the corresponding activity at 37°C. IF, impulse frequency.
whose regres sion lines still had a positive slope at 9 o C and 7 ° C (fig. 3D; table 1). Below 7°C, attenuation was virtually complete in both types of myelinated fiber (table 1).
Effects of cooling on Cfibers.
The low-frequency discharge in C fibers during control ventilation was not attenuated until vagal temperature was reduced to 7 o C. Below 7 ° C, complete block of increasing numbers of fibers was more important than reduction in the impulse frequency in individual fibers in decreasing the mean transmitted discharge of C fibers. Ten C fibers continued to transmit occasional impulses during control ventilation at 0 ° C. We did not attempt to determine the absolute blocking temperature of these fibers. By contrast, C fiber input evoked by hyperinflating the lungs or by injecting capsaicin was significantly attenuated at 15 ° C, and decreased in an approximately linear fashion as temperature was reduced further (figs. 4,5). Attenuation was less than that in myelinated fibers, so that at 9 °C the hyperinflation-induced input in pulmonary C fibers exceeded that in RAR fibers, and at 7 ° C it exceeded the combined input of RARs and P SRs (fig. 6). At 3 ° C, 61 ~o of pulmonary C fibers still transmitted some of the activity evoked by hyperinflation, the average discharge passing the cold block amounting to 20~o of that at 37°C. Even at 0°C, a third of the C fibers still transmitted isolated impulses evoked by hyperinflation or capsaicin; in three of these fibers, transmission was blocked at - 1 ° C, and in one at - 2 ° C . We did not attempt to determine the absolute blocking temperature of the remainder.
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Fig. 6. Comparison of effects ofvagal cooling on the recorded impulse frequency (IF) of the 3 types of lung afferent stimulated by hyperinflating the lungs (3 VT). Data are means _+ SE; hatched bars, 42 PSRs; stippled bars, 43 RARs; open bars, 38 lung C fibers. (A) Results obtained over complete range of vagal temperatures; calibration for PSRs on left, that for RARs and C fibers on right. (B) Results obtained over lower range of vagal temperatures, plotted on a common expanded calibration scale.
Quantitative differences in the effects of cooling on C fibers and myelinated fibers were demonstrated even more clearly when the transmitted frequencies in individual fibers at each temperature were expressed as a function of the frequencies at 37 ° C. The attenuating effects of cooling on C fibers and R A R s were c o m p a r e d over a similar range of firing frequencies by including in the scatter plots the response of C fibers to capsaicin (fig. 7A). The frequencies transmitted in C fibers, like those in myelinated fibers, were reduced by cooling to a relatively constant fraction of the frequency at 37 °C (fig. 7A), but the transmitted fraction in C fibers (fig. 7B) was significantly greater than that in myelinated fibers at all temperatures. This difference ( P < 0.01) held whether the frequencies were restricted to those evoked by lung distension (table 1) or whether the range was extended by including the higher frequencies evoked by capsaicin (fig. 7). The data for C fiber activity recorded above the cooling platform did not show such a clear point of inflection as did that for R A R s and P S R s (table 1). However, at 3 ° C, when the slope of the regression line was not significantly different from zero, low frequencies (average, 1.5 impulses/sec) continued to pass the cooled region of nerve.
30
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Fig. 7. Transmitted impulse frequencies in individual C fibers at different vagal temperatures related to impulse frequencies at 37°C. Data points (n = 62) include those obtained by injection of capsaicin. (A) Scatter plots for individual C fibers show impulse frequencies conducted across the cooling platform at 15°C. Dashed line is line of identity; solid line is drawn from regression equation for these data points (n = 62). (B) Lines drawn from regression equations at various temperatures.
Discussion Our primary purpose was not to determine the absolute blocking temperature of the various pulmonary afferents, but rather to examine the progressive attenuation of their input to the respiratory centers by cooling the vagus nerve to temperatures commonly used to examine pulmonary reflexes. The input evoked by hyperinflating the lungs was attenuated by cooling in all three types of afferent, attenuation being least marked in C fibers. At a vagal temperature of 37°C, the activity evoked by hyperinflation was much less in pulmonary C fibers than in PSRs and RARs, but when temperature was reduced to 7°C, C fiber activity began to dominate the transmitted input to the respiratory centers, the input in myelinated fibers being only a small fraction of that at 37°C. Even so, in some myelinated fibers impulse transmission persisted at low temperatures; in a few, occasional impulses evoked by hyperinflation were transmitted at temperatures as low as 1-3 o C. Average blocking temperatures reported for myelinated fibers range from 6.5 °C to 8.0°C (Paintal, 1965a, 1967; Franz and Iggo, 1968; Linden etal., 1981); however, examples of myelinated fibers with blocking temperatures between 1.8 ° C and 3 ° C are cited in three of these studies (Paintal, 1965a, 1967; Franz and Iggo, 1968). Average blocking temperatures reported for C fibers range from 2.7 °C to 4.3 °C (Paintal, 1967; Franz and Iggo, 1968; Linden etal., 1981), but Franz and Iggo found that 23~/o of C
COLD-BLOCKADE OF PULMONARY A F F E R E N T INPUT
31
fibers were blocked between 0.3 °C and 0.8 °C and speculated that blocking temperatures for 'undisturbed non-myelinated axons' were likely to be close to 0 ° C. In a third of our C fibers activity evoked by hyperinflation was not completely blocked at 0 ° C, an observation consistent with the finding that C fiber compound action potentials can still be evoked in the rabbit vagus during prolonged cooling at 0 ° C (Patberg and Veringa, 1985). Thus all investigators agree that myelinated fibers block at significantly higher temperatures than non-myelinated ones, but the absolute blocking temperatures vary in estimates from different laboratories, as do the temperatures at which vagal reflexes are abolished. Because differences in cooling method and pattern of discharge influence blocking temperature (Paintal, 1965a; Franz and Iggo, 1968), it is desirable to use similar cooling methods and stimuli when comparing the effects of nerve blockade on afferent impulse traffic and reflexes. Effects of vagal cooling on impulse conduction are frequency dependent (Paintal, 1965b; Franz and Iggo, 1968). We examined the influence of the impulse frequency arriving at the cooled region of nerve on the transmission of impulses across it, expressing the frequency transmitted at each temperature as a function of the receptor discharge (i.e., the frequency transmitted at 37 ° C). The frequencies and vagal temperatures of most interest to us (because they are most applicable to reflex studies), were at the lower end of the ranges of frequency and temperature examined by investigators who stimulated nerves electrically (Paintal, 1965b; Franz and Iggo, 1968; Linden et al., 1981). At intermediate temperatures low frequencies were transmitted without attenuation, whereas higher frequencies were attenuated to a relatively constant fraction of the receptor discharge frequency. The transmitted fraction of activity at each temperature was considerably higher in C fibers than in either PSR or RAR fibers. This was not due to a lower firing frequency in C fibers, because when the activity evoked by capsaicin was included to extend the upper range of frequencies in C fibers to match those in RAR fibers the difference remained significant at all temperatures. It seems likely that the mode of conduction is a crucial factor, the saltatory conduction in myelinated fibers largely accounting for their greater susceptibility to cooling (Paintal, 1967). Although complete blockade of conduction in all myelinated fibers occurs over the same temperature range (Palintal, 1965a; Franz and Iggo, 1968), two differences in the effects of cooling on PSR and RAR fibers emerged in our experiments. First, the maximum discharge frequency passing the cold block without attenuation was higher in PSR fibers than in RAR fibers at all temperatures (fig. 3). Second, the fraction of impulses blocked by cooling was greater in PSR fibers than in RAR fibers at temperatures of 12°C and below (fig. 3; table 1). The higher discharge frequencies of PSRs were responsible in part for the second observation, but differences in fn'ing pattern probably played a role in both. Whereas impulses from PSRs are spaced relatively evenly, the irregular discharge of RARs results in a wide range of interspike intervals. Cooling attenuates impulse transmission by increasing the refractory period of axons (Paintal, 1965b; Franz and Iggo, 1968). As the refractory period is progressively lengthened by cooling, attenuation will begin with the shortest interspike intervals, while the longest
32
A. J O N Z O N et al.
interspike intervals will remain unimpaired until complete blockade. RAR discharges, having the wider scatter of interspike intervals, will be attenuated over a wider temperature range than PSR discharges of the same average frequency. Differences in fiber diameter are unlikely, to play a part in these differential effects of cooling. In dogs and guinea pigs, the only species in which conduction velocities have been measured in a sufficiently large number of fibers to allow inferences as to mean diameter, conduction velocities of PSR and RAR fibers overlap extensively (Sampson, 1977; Bergren and Sampson, 1982; present results). In evaluating the effects of cooling on reflexes mediated by myelinated pulmonary afferents, it is important to distinguish the blocking temperature of the afferent activity present at normal tidal volumes from the blocking temperature of the hyperinflationevoked activity producing a reflex response. Our results suggest that a reflex evoked by hyperinflation and triggered by PSRs will be abolished at a temperature at least 4°C higher than that required to abolish all PSR input at normal tidal volumes. Owing to the high discharge frequency of PSRs at normal tidal volumes, the capacity of the cooled region of axon to transmit hyperinflation-evoked activity is less in these fibers than in RAR fibers at the same temperature. Thus when the vagus nerve was cooled to 9°C, hyperinflation did not increase PSR input to the medullary centers because the impulse frequency transmitted during control ventilation was already the maximum that could pass the cold block (fig. 2A). By contrast, the sparser control discharge of RARs still allowed a fourfold increase in firing to be transmitted at 9°C when the lungs were distended (fig. 2B). These results are in general agreement with the observation (Widdicombe, 1954) that vagal cooling distinguishes two components of the reflex phrenic response to a large, abrupt lung inflation: an inhibitory Hering-Breuer component attributed to input from PSRs and blocked at 9-15°C, and an excitatory component attributed to input from RARs and blocked at 5-11 °C. Cooling the vagus nerves to 7 °C is often used to distinguish the reflex effects of myelinated and non-myelinated afferents. Our results indicate that hyperinflationevoked activity in both A and C fiber afferents is attenuated at this temperature, attenuation being much greater in the former. At 7 ° C, virtually none of the increase (A) in PSR activity evoked by hyperinflation and only 10~o of the increase in RAR activity passes the cold block - indeed the hyperinflation-evoked activity transmitted across the cooled nerve in RAR fibers is now less than the receptor discharge during control ventilation. In contrast, almost 40 Y/oof the hyperinflation-evoked increase (A) in C fiber activity reaches the medullary centers - an input almost ten times that during control ventilation at 37 ° C. If one assumes that reflex effects are attenuated in proportion to the attenuation of afferent input, it seems reasonable to conclude from the results depicted in fig. 6B that a hyperinflation-evoked reflex whose amplitude is attenuated by less than 60~o when the vagus nerves are cooled from 9.5-6°C to 5.5-2°C (Roberts et al., 1988) is triggered by C fibers. The effects of cooling on pulmonary C fiber input evoked by right atrial injection of capsaicin (fig. 4B) are consistent with the observation that the pulmonary chemoreflex survives vagal cooling below 7 °C (for references see Coleridge and Coleridge, 1984).
COLD-BLOCKADE OF PULMONARY AFFERENT INPUT
33
In conclusion, vagal cooling can be used to distinguish the reflex effects of the various pulmonary afferents stimulated by hyperinflation if the results are interpreted with an awareness of the degree of attenuation of both the tonic and inflation-evoked discharge of each fiber type.
Acknowledgements.We thank Albert Dangel and Ronald Brown for technical assistance and Rolinda Wang for typing the manuscript. This investigation was supported by Grant HL-07192 and Project Grants HL-24136 and HL-25847 from the National Heart, Lung and Blood Institute. A.J. was Julius H. Comroe Jr. Research Fellow of the Cardiovascular Research Institute; he was also supported by grants from the Swedish Medical Research Council, the Sven Jerring Foundation, and the Sigurd and Elsa Golje Foundation.
References Bergen, D.R. and S.R. Sampson (1982). Characterization of intrapulmonary rapidly adapting receptors of guinea pigs. Respir. Physiol. 47: 83-95. Coleridge, H.M. and J.C.G. Coleridge (1977). Impulse activity in afferent vagal C-fibres with endings in the intrapulmonary airways of dogs. Respir. Physiol. 29: 125-142. Coleridge, H.M. and J.C.G. Coleridge (1986). Reflexes from the tracheobronchial tree and lungs. In: Handbook of Physiology. Section 3. The Respiratory System. Vol. 2. Control of Breathing. Part 1, edited by N. S. Cherniack and J.G. Widdicombe. Washington, D.C. American Physiological Society, pp. 395--429. Coleridge, J. C. G. and H.M. Coleridge (1984). Afferent vagai C fibre innervation of the lungs and airways and its functional significance. Rev. Physiol. Biochem. Pharmacol. 99:1-110. Franz, D.N. and A. Iggo (1968). Conduction failure in myelinated and non-myelinated axons at low temperatures. J. Physiol. (London) 199: 319-345. Head, H. (1889). On the regulation of respiration. Part. I. Experimental. J. Physiol. (London) 10: 1-70. Linden, R.J., D.A.S.G. Mary and D. Weatherill (1981). The effect of cooling on transmission of impulses in vagal nerve fibres attached to atrial receptors in the dog. Q. J. Exp. Physiol. 66: 321-332. Paintal, A. S. (1965a). Block of conduction in mammalian myelinated nerve fibres by low temperatures. J. Physiol. (London) 180: 1-19. Paintal, A. S. (1965b). Effects of temperature on conduction in single vagal and saphenous myelinated nerve fibres of the cat. J. Physiol. (London) 180: 20--49. Paintal, A. S. (1967). A comparison of the nerve impulses of mammalian non-medullated nerve fibres with those of the smallest diameter medullated fibres. J. Physiol. (London) 193: 523-533. Patberg, W.R. and F. Veringa (1985). Graded blocking of non-myelinated vagal fibres in the rabbit. J. PhysioL (London) 366: 31P. Pisarri, T. E., J. Yu, H. M. Coleridge and J. C. G. Coleridge (1986). Background activity in pulmonary vagal C-fibers and its effects on breathing. Respir. Physiol. 64: 29--43. Roberts, A. M., H. M. Coleridge and J. C. G. Coleridge (1988). Reciprocal action of pulmonary vagal afferents on tracheal smooth muscle tension in dogs. Respir. Physiol. 72: 35-46. Sampson, S.R. (1977). Sensory neurophysiology of airways. Am. Rev. Respir. D/s. 115: 107-115. Widdicombe, J.G. (1954). Respiratory reflexes excited by inflation of the lungs. J. Physiol. (London) 123: 105-115.
19
RSP 01389
Attenuation of pulmonary afferent input by vagal cooling in dogs A. Jonzon, T.E. Pisarri, A . M . Roberts, J. C. G. Coleridge and H . M . Coleridge Cardiovascular Research Institute and Department of Physiology, University of California San Francisco, San Francisco, CA 94143-0130, U.S.A. (Accepted for publication 16 November 1987) Abstract. In open chest, artificially ventilated, anesthetized dogs, we examined the effect of vagal cooling
on the pulmonary afferent input evoked by hyperinflating the lungs to 3 VT, recording the activity of slowly adapting pulmonary stretch receptors (PSRs), rapidly adapting receptors (RARs) and pulmonary C fibers rostral to the cooling platform. At 15 ° C and below, input in all three types of fiber was significantly reduced, attenuation being least marked in C fibers. Between 12°C and 7°C, attenuation of RAR input was significantly less than that of PSRs. At 7°C, virtually none of the hyperinflation-evoked increase in PSR activity and only 10~o of that in RARs passed the cooling platform - indeed RAR input was less than during normal ventilation at 37 ° C; by contrast, 40 ~o of the hyperinflation-evoked increase in C fiber activity was still transmitted. Cooling had similar effects on C fiber input evoked by capsaicin. If reflexes are attenuated in proportion to the attenuation of afferent input, our results suggest that a hyperinflation-evoked reflex that survives vagal cooling below 6°C is almost certainly triggered by C fibers.
Cold block of lung afferents; Conduction velocities of lung afferents; Lung C fibers; Pulmonary C fibers and capsaicin; Pulmonary stretch receptors; Rapidly adapting receptors
Cooling the vagus nerves is often used to investigate reflexes of lower airway origin. However, attempts to identify the afferents responsible for a given reflex by comparing the blocking temperature of the various pulmonary afferents with the blocking temperature of the reflex itself continue to arouse controversy (Coleridge and Coleridge, 1984, 1986). Much of what is known about the attenuating effects of cooling on conduction in single myelinated (A) and non-myelinated (C) fibers has been derived from experiments in which nerve trunks were stimulated electrically at constant frequency, beginning with trains of pulses delivered at the maximal frequency the individual axon could follow and continuing with progressively lower frequencies as the nerve was cooled (Paintal, 1965b; Franz and Iggo, 1968; Linden et al., 1981). Experiments of this type have established that C fibers continue to conduct impulses at temperatures that Correspondence address: Dr. H.M. Coleridge, Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA 94143-0130, U.S.A. 0034-5687/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
20
A. J O N Z O N et al.
block conduction in A fibers. They have also shown that, regardless of fiber type, the effects of cooling are frequency dependent, the higher the firing frequency the greater the attenuation produced at a given vagal temperature. In most reflex studies, however, pulmonary afferent input to the medullary centers is evoked not by stimulating nerve fibers electrically at constant frequency but by applying a mechanical or chemical stimulus to the lower respiratory tract to excite the afferent terminals themselves. The various types of pulmonary ending respond in different ways to a given stimulus, the pattern and frequency of the evoked discharge varying with the threshold, sensitivity and rate of adaptation of the ending. Consequently, when attempts are made to correlate the effect ofvagal cooling on pulmonary afferent input with that on a particular reflex response, it is essential to stimulate the endings themselves in both cases, and to use the same stimulus. However, there is little detailed information about the attenuating effects of progressive vagal cooling on naturally evoked activity in the various types of pulmonary afferent. In a previous study in this laboratory, Pisarri et al. (1986) described the effect of vagal cooling on the baseline activity in pulmonary afferent fibers when the lungs were ventilated at normal tidal volume. We have extended these observations, examining the attenuating effects of cooling on input from a large number of slowly adapting pulmonary stretch receptors, rapidly adapting receptors and pulmonary C fibers in dogs, both during artificial ventilation at normal tidal volumes and during lung distension (3 VT). Distension of the lungs is known to stimulate all three types of pulmonary afferent, and was shown by Head (1889) in rabbits to have reflex effects on breathing that could be reversed as the vagus nerves were cooled (Head's 'paradoxical' reflex). In a reflex study in dogs, described in a companion paper (Roberts et al., 1988), vagal cooling was also found to reverse the effects of lung distension on airway smooth muscle tone, distension evoking tracheal relaxation when the vagus nerves were at body temperature but contraction when the nerves were cooled below 8-7 ° C. The present experiments shed light on the afferents responsible for this reversal of the tracheal response. In addition, because capsaicin stimulates lower airway C fibers, and is often used to evoke lower airway reflexes, we examined the effects of vagal cooling on capsaicin-evoked input in pulmonary and bronchial C fibers.
Methods General. Eighteen dogs (14-27 kg) were given promazine HC1 (Sparine, Wyeth, 50 mg i.m.); 30 min later they were anesthetized with 0.25 ml/kg i.v. of a 1 : 1 mixture of solutions of Dial Compound (allobarbital 100 mg/ml, urethane 400 mg/ml, Ciba) and sodium pentobarbital (50 mg/ml). Supplemental doses were given as required to maintain surgical anesthesia. The trachea was cannulated low in the neck and the chest was opened in the midsternal line. The lungs were ventilated with 50~o 02 in air by a Harvard respirator (model 613) whose expiratory outlet was placed under 3-5 cm water. Tidal CO 2 was
COLD-BLOCKADE OF PULMONARY A F F E R E N T INPUT
21
monitored by a Beckman LB-1 gas analyzer. Tidal volume was set at approximately 15 ml/kg, and ventilator frequency (10-16 cycles/rain) was adjusted to maintain endtidal Pco2 at approximately 35 mm Hg. Arterial Po2, Pco~, pH and base excess were measured at regular intervals with an automatic analyzer (Coming 175 blood gas/pH analyzer). Any metabolic acidosis was corrected by administration o f N a H C O 3 solution i.v.
Tracheal pressure was recorded from a sidearm of the tracheal cannula, and systemic arterial blood pressure was recorded from a femoral artery. Pressures were recorded with Statham P23Gb strain gauges. An electrocardiogram (lead II) was recorded. Afferent vagal impulses were recorded and were counted by ratemeters (see below). The signals representing tidal CO2, pressures, ratemeter counts of impulse frequency, and vagal temperature (see below) were recorded by a Grass polygraph. Vagal action potentials, ratemeter counts and other selected variables were also recorded by an ultraviolet light recorder (SE Laboratories) or by a Gould (ES 1000) electrostatic recorder.
Dissection of neck. The anterior muscles of the neck were dissected on the left side of the trachea and retracted to form two separate pools. In the rostral pool, 3-4 cm of the left cervical vagus nerve was freed from the carotid sheath and placed upon a small platform (1 cm wide). After the sheath had been removed from the vagus nerve on the platform, the rostral pool was filled with mineral oil, and fine vagal strands were dissected for the recording of afferent impulses (see below). In the caudal pool, 7 cm of the left vagus nerve was freed from the carotid sheath, and the nerve was placed upon a cooling platform (see below). Rostral to the cooling platform, the nerve was placed upon stimulating electrodes for measurement of fiber conduction velocities.
Pulmonary afferent activity. Using conventional techniques, we recorded impulses from afferent vagal fibers arising from the left lung. Slowly adapting pulmonary stretch receptors (PSRs), rapidly adapting receptors (RARs) and pulmonary and bronchial C fibers were identified by their characteristic patterns of discharge, by their responses to lung distension (3 VT) and to injection of capsaicin (10 #g/kg) into the right and left atrium, and by their fiber conduction velocities (Coleridge and Coleridge, 1977). Conduction velocities were measured by stimulating the cervical vagus nerve through two pairs of electrodes, 2 cm apart, fixed in a shielded assembly; conduction velocity was derived from the conduction time between the stimulating electrodes. The approximate location of the receptors was determined by probing the lung: receptors not located in the lung were discarded. Impulses were counted in 1 sec bins by ratemeters whose window discriminators were set to accept potentials of a particular amplitude. Nerve filaments from which recordings were made contained a single active fiber, or two fibers whose action potentials were of sufficiently different amplitude and configuration to be clearly distinguishable.
Vagal cooling. The left vagus nerve was placed in a groove on the platform (width, 2 cm) of a silver cooling device through which alcohol of different temperatures was
22
A. JONZON et al.
35 Pc°2 mm Hgol ABP 40
temp
~~.~.~needle)
°c 5~ eo
~ ~ IF
L
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~
.
.
.
.
.
. I
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hist
I
I
I
I
I
I
I
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1
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I
Fig. 1. Progressive vagal cooling attenuates conduction in an RAR fiber. RAR stimulated at intervals of 1-2 min by briefly hyperinflating the lungs (3 VT), as indicated by the increases in tracheal pressure (PT). Note virtual abolition of recorded activity at 7 ° C and restoration of activity as nerve rewarmed. Other abbreviations: Pco2, tidal Pco2; ABP, arterial blood pressure; temp (needle), temperature of vagus nerve recorded by needle thermistor; temp (platform), temperature of cooling platform; IF, impulse frequency recorded by ratemeter. Between 28 °C and 6 ° C, the vagus nerve was cooled at the rate of 1.8 ° C/min. Before vagal cooling, the RAR was stimulated by injecting histamine (20 #g/kg) into the right atrium; when the vagus nerve had been cooled to 7-6 ° C, injection of histamine (hist; note decrease in ABP and increase in PT) had no effect on RAR activity recorded rostral to the cooling platform.
circulated. The sides and bottom of the platform were insulated with a layer of silicone elastomer. To reduce thermal gradients the nerve was covered with a warm (40°C) solution of 4 ~ agar in saline, which, upon cooling, gelled to form a semisolid layer approximately 0.5 cm thick. The temperature of the platform was measured by thermistor (Yellow Springs Instrument 729), and the temperature of the nerve by a needle thermistor (Yellow Springs Instrument 524) inserted obliquely so that its tip was between the sheath and the nerve. When measured simultaneously (fig. 1), the two temperatures were within 1 °C; vagal temperature was taken as the average of the two. Care was taken not to exert tension on the nerve when placing it upon the cooling platform because tension is known to elevate the blocking temperature of nerve fibers (Paintal, 1965a).
Coolingprotocol. We examined the effect of cooling the lower cervical vagus nerve on pulmonary afferent activity recorded rostral to the cooling platform, both during control ventilation and when the lungs were briefly distended to FRC + 3 VT (peak tracheal
COLD-BLOCKADE OF PULMONARY AFFERENT INPUT
23
pressure, 20-25 cm H 2 0 ) (fig. 1). We also examined the effect ofvagal cooling on C fiber activity evoked by injecting c apsaicin (10 #g/kg) into the right atrium through a c atheter inserted via an external jugular vein. In most experiments the vagus nerve was cooled in steps, temperature being maintained at each step for 3 min before measurement of impulse activity; in others the nerve was cooled gradually from 37 °C to 0 °C at the rate of 1-2°C per min. Results obtained using the two methods of cooling were identical, and have been combined. Impulse activity was measured at vagal temperatures of 37, 15, 12, 9, 7, 5, 3, 1 and 0 ° C. In a few experiments we continued cooling to - 1 or - 2 ° C in order to determine the blocking temperature of a C fiber whose conduction was not blocked at 0°C. Usually, however, we did not cool the nerve below 0°C, because we examined several fibers in each dog and wished to avoid damaging the nerve by repeated cooling to very low temperatures.
Analysis of data. During control ventilation the impulse activity of all pulmonary afferents was averaged over five ventilatory cycles at each vagal temperature and expressed as impulses/sec. The activity evoked by lung distension was also measured at each temperature. Activity in PSRs was counted from the peak of inflation at FRC + 2 VT to the peak of inflation at FRC + 3 VT (as determined from the tracheal pressure record), and expressed as impulses/sec. Because RARs and pulmonary C fibers were sometimes silent during the early stages of distension and fired a relatively short burst of impulses during the third inflation, their response was counted over the 2 sec at which firing was at a maximum and was expressed as impulses/sec. C fiber activity evoked by capsaicin was also measured at each temperature and was averaged over 2 sec at the peak of the response and expressed as impulses/sec. Impulse activities at each temperature were expressed as means + SE. For each of the three types of pulmonary afferent, we plotted the impulse frequency transmitted across the cooling platform at each temperature as a function of the impulse frequency transmitted at 37 ° C, which was taken as a measure of the discharge frequency of the receptor itself. To obtain a wide range of frequencies in individual fibers, each scatter plot included baseline activity during control ventilation and increased activity evoked by lung distension and, in the case of C fibers, by injection of capsaicin. The relationship between the impulse frequency at 37°C and the transmitted impulse frequency at each temperature was determined by linear regression ( C R U N C H statistical software). Regression equations were of the form Fx = aT + bT(F37oc), where Far is the impulse frequency transmitted across the cooling platform at temperature T, and a T and bx are respectively the y intercept and the slope of the regression equation. The plots for PSRs and RARs showed an inflection point suggesting an impulse frequency below which the data lay on the line of identity (Fa- = F37oc) and above which there was attenuation. Therefore the regression equations were constructed using only data above this inflection point. The inflection point thus represented the
24
A. JONZON et al.
maximum frequency transmitted without attenuation at that temperature. The position of the inflection point was determined by iterative calculation of the regression line, omitting points below the putative inflection point until the regression equation became constant. Regression lines for C fibers were constructed using all data points. At each temperature, the slopes of the regression lines for each type of afferent were compared using the standard errors of the slopes to calculate the 95 % confidence intervals for a statistically discernible difference between each pair of slopes.
Results General We recorded the impulse activity of 42 slowly adapting pulmonary stretch receptors (PSRs), 43 rapidly adapting receptors (RARs) and 42 C fibers (38 pulmonary and 4 bronchial). Fiber conduction velocities of 27 PSRs ranged from 17 to 66 m/sec (mean, 32.0 + 2.0), of 29 RARs from 11 to 52 m/sec (mean, 31.9 + 1.7), and of 30 C fibers from 0.5 to 2.5 m/sec (mean, 1.3 + 0.1). During control ventilation, PSRs discharged in a regular, phasic fashion and their average frequency was relatively high (24.8 + 2.5 impulses/sec). By contrast, RARs discharged irregularly and average frequency was low (2.7 + 0.5 impulses/sec); however, the activity of individual RARs varied widely and 16~o of RARs were inactive during control ventilation. If five unusually active receptors with control discharges of 6.1-15.7 impulses/sec are excluded, RAR frequencies during control ventilation averaged 1.7 + 0.3 impulses/sec. C fibers also discharged irregularly, and their activity
imp/sec
imp/sec
A
80
20 -
60
1I iF
40
:?.~:
i!8
20
37
15
12
9
7
5
3
vagal temperature
37
15
12
9
?
S
3
°C
Fig. 2. Progressive cold blockade of activity in myelinated pulmonary fibers. Data are means + SE. Observations were made on: (A), 42 PSRs and (B), 43 RARs; note differentcalibration scales in A and B. Open bars, effect of cooling on activity during control ventilation; hatched bars, effect of cooling on activity evoked by hyperinflatingthe lungs (3 VT).
COI.D-BLOCKADE OF PULMONARY AFFERENT INPUT
25
was very low (0.5 + 0.1 impulses/see), 3 6 ~ of C fibers being inactive. In all but the four bronchial C fibers, activity increased significantly when the lungs were hyperinflated, reaching 71.2 + 5.2 impulses/see in PSRs, 16.7 + 1.7 impulses/see in RARs and 10.1 + 0.9 impulses/see in pulmonary C fibers. Activity in both pulmonary and bronchial C fibers increased after right atrial injection of capsaicin, to 31.0 + 2.2 impulses/see.
Effects of cooling on myelinatedfibers.
Impulse frequency recorded rostral to the cooling platform was attenuated in a generally similar fashion in PSR and RAR fibers as the vagus nerves were cooled (fig. 2A,B). Although input in some PSRs and RARs A
I00
B
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/
/
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~
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FREQUENCY (imp/sec),
37 *'C
410
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Fig. 3. Transmitted impulse frequencies in myelinated pulmonary fibers at different vagal temperatures related to impulse frequencies at 37 ° C. Dashed line is line of identity. On left, scatter plots for individual PSRs (A) and RARs (C) show impulse frequencies conducted across the cooled region of nerve at 15 ° C. Note that low frequencies (up to 13-14 impulses/see in PSRs and 4-5 impulses/see in RARs) were transmitted without attenuation at 15 ° C, but that higher frequencies were reduced to a relatively constant fraction of those at 37 ° C. Solid lines are drawn from regression equations (table 1) for these data points. On right, solid lines are drawn from regression equations (table 1) for PSRs (B) and RARs (D) at each vagal temperature, calculations from a minimum of 60 data points. Note that the transmitted fraction of PSR and RAR impulse frequencies (slope of regression line) was similar at 15 ° C, but was significantly greater for RARs at temperatures of 12°C and below. Note different calibration scales for PSRs and RARs.
26
A. J O N Z O N et al.
decreased during control ventilation when vagal temperature was reduced to 15 ° C, it was not attenuated significantly until the vagus was cooled to 12°C. However, when activity was increased by hyperinflating the lungs, the input frequency of both PSRs and RARs was attenuated significantly at 15°C (fig. 2). The proportionate decrease in activity across the cooled region of nerve was greater for PSRs than for RARs. At 9°C, PSR input frequency during hyperinflation was only a third of that during control ventilation at 37 °C (fig. 2A), whereas RAR input frequency during hyperinflation did not fall below the control level until the vagus was cooled to 7°C (fig. 2B). Cooling to 7 °C reduced the transmitted input in PSR and RAR fibers to a similar low level. In some myelinated fibers, transmission across the cooling platform persisted at low temperatures, and was more likely to do so during hyperinflation than at normal tidal volume. At 5 ° C, 28 Yo of PSR fibers and 46 ~o of RAR fibers continued to conduct 1-3 of the impulses evoked by hyperinflation. Even at 1 ° C, isolated impulses from one PSR and three RARs were still conducted across the cooled region of nerve; these were blocked at 0°C. The impulse frequency arriving at a cooled region of nerve influences conduction across it (Paintal, 1965b; Franz and Iggo, 1968). We therefore examined the transmitted impulse frequency in individual fibers at each temperature as a function of the receptor discharge frequency (measured as the transmitted impulse frequency at 37°C). To obtain a wide range of frequencies, we included data obtained during control ventilation and hyperinflation. Receptor discharge frequencies ranged from 1 to 108 impulses/sec
TABLE 1 Frequency-dependent effects of vagal cooling on pulmonary afferent input PSRs
RARs
Pulm Cs
Vagal temp ( ° C)
MF (imp/sec)
b r +_ SE
MF (imp/sec)
b w _+ SE
MF bT _+ SE (imp/sec)
15 12 9 7 5 3
14.7 11.0 5.8 1.9
0.57 0.22 0.02 0.00
3.4 2.7 1.2 0.3 0.0
0.61 0.36 0.19 0.10 0.04
-0.7 1.5 1.7 0.8 1.0 0.8
+ + + +
0.05 0.04 0.02 0.01
+ 0.04 + 0.04 + 0.03 + 0.02 _+ 0.01
0.84 0.63 0.43 0.38 0.22 0.13
+ 0.04 + 0.06 + 0.06 + 0.05 _+ 0.04 _+ 0.04
Values calculated at each vagal temperature from regression equation: F T = aT + bx(F37oc), where F x was the impulse frequency transmitted across the cooling platform at temperature T, and a T and b T were respectively the y intercept and the slope of the regression equation; MF, maximal frequency transmitted without attenuation at each vagal temperature (i.e., point at which regression line intersects line of identity, see Methods). Data obtained during control ventilation and during lung hyperinflation, with a minimum of 60 data points each for PSRs and RARs and 43 for C fibers. Range of impulse frequencies at 37°C: PSRs (n = 42), 1-108 impulses/sec; RARs (n = 43), 1-44 impulses/sec; pulmonary C fibers (n = 38), 1-22 impulses/sec.
27
COLD-BLOCKADE OF PULMONARY AFFERENT INPUT
for PSRs and from 1 to 44 impulses/sec for RARs. The scatter plots obtained for both PSRs and RARs (fig. 3A,C) revealed that low frequencies were transmitted across the cooled region without attenuation. Above a critical frequency (MF, table 1), the transmitted input at each temperature was best described as a linear function of the receptor discharge frequency, the slope of the regression line indicating the fraction of incoming impulses transmitted (fig. 3B,D; table 1). Cooling to 15°C had little effect on PSR fibers fLring at less than 14 impulses/sec, or on RAR fibers firing at less than 3.5 impulses/sec (fig. 3A,C; table 1). Above these frequencies, the attenuation of impulse transmission, as indicated by the slopes of the regression lines, did not differ significantly between PSRs (0.57) and RARs (0.61). At temperatures below 12 ° C, the fraction of activity passing the cooled region of nerve was greater for RARs than for PSRs, the slope of the regression line for RARs being greater than that for PSRs (P < 0.05). This was partly, but not entirely, due to the higher maximum frequencies of the PSR discharge. When the regression equations were recalculated using only data points for PSRs whose discharge frequencies were within the range of the RAR frequencies (1--44 impulses/sec), the difference in slopes was statistically discernible only at 7°C. Below 9°C, the cooled region of nerve acted as a 'low pass filter' to PSR activity, transmission being limited on average to 5-6 impulses/sec at 9°C and to 2 impulses/sec at 7 ° C, whatever the incoming frequency (fig. 3B; table 1). Consequently hyperinflation no longer increased PSR input central to the cold block. This was not true of the RARs,
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Fig. 4. Attenuation by progressive vagal cooling of conduction in 2 pulmonary C fibers; (A) and (B) in different dogs. (A) C fiber activity evoked by hyperinflating the lungs (3 VT); the records depict the second and third inflations above FRC; final record (37°C) when vagus nerve rewarmed. AP, action potentials; PT, tracheal pressure. (B) C fiber activity evoked by injecting capsaicin (10 #g/kg) into the right atrium; interval of 10 min between injections; final record (37°C) when vagus nerve rewarmed; IF, impulse frequency recorded by ratemeter.
28
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Fig. 5. Effect of vagal cooling on conduction in lung C fibers. Data are means + SE. Open bars, effect of cooling on 38 C fibers stimulated by hyperinflating the lungs (3 VT); hatched bars, effect of cooling on 20 C fibers stimulated by injecting capsaicin (10 #g/kg) into the right atrium. Note that at a vagal temperature of 7 ° C, C fiber activity evoked by the two methods of stimulation was 44 ~o and 39 ~o, respectively, of the corresponding activity at 37°C. IF, impulse frequency.
whose regres sion lines still had a positive slope at 9 o C and 7 ° C (fig. 3D; table 1). Below 7°C, attenuation was virtually complete in both types of myelinated fiber (table 1).
Effects of cooling on Cfibers.
The low-frequency discharge in C fibers during control ventilation was not attenuated until vagal temperature was reduced to 7 o C. Below 7 ° C, complete block of increasing numbers of fibers was more important than reduction in the impulse frequency in individual fibers in decreasing the mean transmitted discharge of C fibers. Ten C fibers continued to transmit occasional impulses during control ventilation at 0 ° C. We did not attempt to determine the absolute blocking temperature of these fibers. By contrast, C fiber input evoked by hyperinflating the lungs or by injecting capsaicin was significantly attenuated at 15 ° C, and decreased in an approximately linear fashion as temperature was reduced further (figs. 4,5). Attenuation was less than that in myelinated fibers, so that at 9 °C the hyperinflation-induced input in pulmonary C fibers exceeded that in RAR fibers, and at 7 ° C it exceeded the combined input of RARs and P SRs (fig. 6). At 3 ° C, 61 ~o of pulmonary C fibers still transmitted some of the activity evoked by hyperinflation, the average discharge passing the cold block amounting to 20~o of that at 37°C. Even at 0°C, a third of the C fibers still transmitted isolated impulses evoked by hyperinflation or capsaicin; in three of these fibers, transmission was blocked at - 1 ° C, and in one at - 2 ° C . We did not attempt to determine the absolute blocking temperature of the remainder.
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Fig. 6. Comparison of effects ofvagal cooling on the recorded impulse frequency (IF) of the 3 types of lung afferent stimulated by hyperinflating the lungs (3 VT). Data are means _+ SE; hatched bars, 42 PSRs; stippled bars, 43 RARs; open bars, 38 lung C fibers. (A) Results obtained over complete range of vagal temperatures; calibration for PSRs on left, that for RARs and C fibers on right. (B) Results obtained over lower range of vagal temperatures, plotted on a common expanded calibration scale.
Quantitative differences in the effects of cooling on C fibers and myelinated fibers were demonstrated even more clearly when the transmitted frequencies in individual fibers at each temperature were expressed as a function of the frequencies at 37 ° C. The attenuating effects of cooling on C fibers and R A R s were c o m p a r e d over a similar range of firing frequencies by including in the scatter plots the response of C fibers to capsaicin (fig. 7A). The frequencies transmitted in C fibers, like those in myelinated fibers, were reduced by cooling to a relatively constant fraction of the frequency at 37 °C (fig. 7A), but the transmitted fraction in C fibers (fig. 7B) was significantly greater than that in myelinated fibers at all temperatures. This difference ( P < 0.01) held whether the frequencies were restricted to those evoked by lung distension (table 1) or whether the range was extended by including the higher frequencies evoked by capsaicin (fig. 7). The data for C fiber activity recorded above the cooling platform did not show such a clear point of inflection as did that for R A R s and P S R s (table 1). However, at 3 ° C, when the slope of the regression line was not significantly different from zero, low frequencies (average, 1.5 impulses/sec) continued to pass the cooled region of nerve.
30
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Fig. 7. Transmitted impulse frequencies in individual C fibers at different vagal temperatures related to impulse frequencies at 37°C. Data points (n = 62) include those obtained by injection of capsaicin. (A) Scatter plots for individual C fibers show impulse frequencies conducted across the cooling platform at 15°C. Dashed line is line of identity; solid line is drawn from regression equation for these data points (n = 62). (B) Lines drawn from regression equations at various temperatures.
Discussion Our primary purpose was not to determine the absolute blocking temperature of the various pulmonary afferents, but rather to examine the progressive attenuation of their input to the respiratory centers by cooling the vagus nerve to temperatures commonly used to examine pulmonary reflexes. The input evoked by hyperinflating the lungs was attenuated by cooling in all three types of afferent, attenuation being least marked in C fibers. At a vagal temperature of 37°C, the activity evoked by hyperinflation was much less in pulmonary C fibers than in PSRs and RARs, but when temperature was reduced to 7°C, C fiber activity began to dominate the transmitted input to the respiratory centers, the input in myelinated fibers being only a small fraction of that at 37°C. Even so, in some myelinated fibers impulse transmission persisted at low temperatures; in a few, occasional impulses evoked by hyperinflation were transmitted at temperatures as low as 1-3 o C. Average blocking temperatures reported for myelinated fibers range from 6.5 °C to 8.0°C (Paintal, 1965a, 1967; Franz and Iggo, 1968; Linden etal., 1981); however, examples of myelinated fibers with blocking temperatures between 1.8 ° C and 3 ° C are cited in three of these studies (Paintal, 1965a, 1967; Franz and Iggo, 1968). Average blocking temperatures reported for C fibers range from 2.7 °C to 4.3 °C (Paintal, 1967; Franz and Iggo, 1968; Linden etal., 1981), but Franz and Iggo found that 23~/o of C
COLD-BLOCKADE OF PULMONARY A F F E R E N T INPUT
31
fibers were blocked between 0.3 °C and 0.8 °C and speculated that blocking temperatures for 'undisturbed non-myelinated axons' were likely to be close to 0 ° C. In a third of our C fibers activity evoked by hyperinflation was not completely blocked at 0 ° C, an observation consistent with the finding that C fiber compound action potentials can still be evoked in the rabbit vagus during prolonged cooling at 0 ° C (Patberg and Veringa, 1985). Thus all investigators agree that myelinated fibers block at significantly higher temperatures than non-myelinated ones, but the absolute blocking temperatures vary in estimates from different laboratories, as do the temperatures at which vagal reflexes are abolished. Because differences in cooling method and pattern of discharge influence blocking temperature (Paintal, 1965a; Franz and Iggo, 1968), it is desirable to use similar cooling methods and stimuli when comparing the effects of nerve blockade on afferent impulse traffic and reflexes. Effects of vagal cooling on impulse conduction are frequency dependent (Paintal, 1965b; Franz and Iggo, 1968). We examined the influence of the impulse frequency arriving at the cooled region of nerve on the transmission of impulses across it, expressing the frequency transmitted at each temperature as a function of the receptor discharge (i.e., the frequency transmitted at 37 ° C). The frequencies and vagal temperatures of most interest to us (because they are most applicable to reflex studies), were at the lower end of the ranges of frequency and temperature examined by investigators who stimulated nerves electrically (Paintal, 1965b; Franz and Iggo, 1968; Linden et al., 1981). At intermediate temperatures low frequencies were transmitted without attenuation, whereas higher frequencies were attenuated to a relatively constant fraction of the receptor discharge frequency. The transmitted fraction of activity at each temperature was considerably higher in C fibers than in either PSR or RAR fibers. This was not due to a lower firing frequency in C fibers, because when the activity evoked by capsaicin was included to extend the upper range of frequencies in C fibers to match those in RAR fibers the difference remained significant at all temperatures. It seems likely that the mode of conduction is a crucial factor, the saltatory conduction in myelinated fibers largely accounting for their greater susceptibility to cooling (Paintal, 1967). Although complete blockade of conduction in all myelinated fibers occurs over the same temperature range (Palintal, 1965a; Franz and Iggo, 1968), two differences in the effects of cooling on PSR and RAR fibers emerged in our experiments. First, the maximum discharge frequency passing the cold block without attenuation was higher in PSR fibers than in RAR fibers at all temperatures (fig. 3). Second, the fraction of impulses blocked by cooling was greater in PSR fibers than in RAR fibers at temperatures of 12°C and below (fig. 3; table 1). The higher discharge frequencies of PSRs were responsible in part for the second observation, but differences in fn'ing pattern probably played a role in both. Whereas impulses from PSRs are spaced relatively evenly, the irregular discharge of RARs results in a wide range of interspike intervals. Cooling attenuates impulse transmission by increasing the refractory period of axons (Paintal, 1965b; Franz and Iggo, 1968). As the refractory period is progressively lengthened by cooling, attenuation will begin with the shortest interspike intervals, while the longest
32
A. J O N Z O N et al.
interspike intervals will remain unimpaired until complete blockade. RAR discharges, having the wider scatter of interspike intervals, will be attenuated over a wider temperature range than PSR discharges of the same average frequency. Differences in fiber diameter are unlikely, to play a part in these differential effects of cooling. In dogs and guinea pigs, the only species in which conduction velocities have been measured in a sufficiently large number of fibers to allow inferences as to mean diameter, conduction velocities of PSR and RAR fibers overlap extensively (Sampson, 1977; Bergren and Sampson, 1982; present results). In evaluating the effects of cooling on reflexes mediated by myelinated pulmonary afferents, it is important to distinguish the blocking temperature of the afferent activity present at normal tidal volumes from the blocking temperature of the hyperinflationevoked activity producing a reflex response. Our results suggest that a reflex evoked by hyperinflation and triggered by PSRs will be abolished at a temperature at least 4°C higher than that required to abolish all PSR input at normal tidal volumes. Owing to the high discharge frequency of PSRs at normal tidal volumes, the capacity of the cooled region of axon to transmit hyperinflation-evoked activity is less in these fibers than in RAR fibers at the same temperature. Thus when the vagus nerve was cooled to 9°C, hyperinflation did not increase PSR input to the medullary centers because the impulse frequency transmitted during control ventilation was already the maximum that could pass the cold block (fig. 2A). By contrast, the sparser control discharge of RARs still allowed a fourfold increase in firing to be transmitted at 9°C when the lungs were distended (fig. 2B). These results are in general agreement with the observation (Widdicombe, 1954) that vagal cooling distinguishes two components of the reflex phrenic response to a large, abrupt lung inflation: an inhibitory Hering-Breuer component attributed to input from PSRs and blocked at 9-15°C, and an excitatory component attributed to input from RARs and blocked at 5-11 °C. Cooling the vagus nerves to 7 °C is often used to distinguish the reflex effects of myelinated and non-myelinated afferents. Our results indicate that hyperinflationevoked activity in both A and C fiber afferents is attenuated at this temperature, attenuation being much greater in the former. At 7 ° C, virtually none of the increase (A) in PSR activity evoked by hyperinflation and only 10~o of the increase in RAR activity passes the cold block - indeed the hyperinflation-evoked activity transmitted across the cooled nerve in RAR fibers is now less than the receptor discharge during control ventilation. In contrast, almost 40 Y/oof the hyperinflation-evoked increase (A) in C fiber activity reaches the medullary centers - an input almost ten times that during control ventilation at 37 ° C. If one assumes that reflex effects are attenuated in proportion to the attenuation of afferent input, it seems reasonable to conclude from the results depicted in fig. 6B that a hyperinflation-evoked reflex whose amplitude is attenuated by less than 60~o when the vagus nerves are cooled from 9.5-6°C to 5.5-2°C (Roberts et al., 1988) is triggered by C fibers. The effects of cooling on pulmonary C fiber input evoked by right atrial injection of capsaicin (fig. 4B) are consistent with the observation that the pulmonary chemoreflex survives vagal cooling below 7 °C (for references see Coleridge and Coleridge, 1984).
COLD-BLOCKADE OF PULMONARY AFFERENT INPUT
33
In conclusion, vagal cooling can be used to distinguish the reflex effects of the various pulmonary afferents stimulated by hyperinflation if the results are interpreted with an awareness of the degree of attenuation of both the tonic and inflation-evoked discharge of each fiber type.
Acknowledgements.We thank Albert Dangel and Ronald Brown for technical assistance and Rolinda Wang for typing the manuscript. This investigation was supported by Grant HL-07192 and Project Grants HL-24136 and HL-25847 from the National Heart, Lung and Blood Institute. A.J. was Julius H. Comroe Jr. Research Fellow of the Cardiovascular Research Institute; he was also supported by grants from the Swedish Medical Research Council, the Sven Jerring Foundation, and the Sigurd and Elsa Golje Foundation.
References Bergen, D.R. and S.R. Sampson (1982). Characterization of intrapulmonary rapidly adapting receptors of guinea pigs. Respir. Physiol. 47: 83-95. Coleridge, H.M. and J.C.G. Coleridge (1977). Impulse activity in afferent vagal C-fibres with endings in the intrapulmonary airways of dogs. Respir. Physiol. 29: 125-142. Coleridge, H.M. and J.C.G. Coleridge (1986). Reflexes from the tracheobronchial tree and lungs. In: Handbook of Physiology. Section 3. The Respiratory System. Vol. 2. Control of Breathing. Part 1, edited by N. S. Cherniack and J.G. Widdicombe. Washington, D.C. American Physiological Society, pp. 395--429. Coleridge, J. C. G. and H.M. Coleridge (1984). Afferent vagai C fibre innervation of the lungs and airways and its functional significance. Rev. Physiol. Biochem. Pharmacol. 99:1-110. Franz, D.N. and A. Iggo (1968). Conduction failure in myelinated and non-myelinated axons at low temperatures. J. Physiol. (London) 199: 319-345. Head, H. (1889). On the regulation of respiration. Part. I. Experimental. J. Physiol. (London) 10: 1-70. Linden, R.J., D.A.S.G. Mary and D. Weatherill (1981). The effect of cooling on transmission of impulses in vagal nerve fibres attached to atrial receptors in the dog. Q. J. Exp. Physiol. 66: 321-332. Paintal, A. S. (1965a). Block of conduction in mammalian myelinated nerve fibres by low temperatures. J. Physiol. (London) 180: 1-19. Paintal, A. S. (1965b). Effects of temperature on conduction in single vagal and saphenous myelinated nerve fibres of the cat. J. Physiol. (London) 180: 20--49. Paintal, A. S. (1967). A comparison of the nerve impulses of mammalian non-medullated nerve fibres with those of the smallest diameter medullated fibres. J. Physiol. (London) 193: 523-533. Patberg, W.R. and F. Veringa (1985). Graded blocking of non-myelinated vagal fibres in the rabbit. J. PhysioL (London) 366: 31P. Pisarri, T. E., J. Yu, H. M. Coleridge and J. C. G. Coleridge (1986). Background activity in pulmonary vagal C-fibers and its effects on breathing. Respir. Physiol. 64: 29--43. Roberts, A. M., H. M. Coleridge and J. C. G. Coleridge (1988). Reciprocal action of pulmonary vagal afferents on tracheal smooth muscle tension in dogs. Respir. Physiol. 72: 35-46. Sampson, S.R. (1977). Sensory neurophysiology of airways. Am. Rev. Respir. D/s. 115: 107-115. Widdicombe, J.G. (1954). Respiratory reflexes excited by inflation of the lungs. J. Physiol. (London) 123: 105-115.