- Email: [email protected]
Effects of vagotomy on ventilatory responses to CO2 in alligators
Respiration Physiology, 87 (1992)63-76 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0034-5687/92/$03.50
63
RESP 01845
Effects of vagotomy on ventilatory responses to C02 in alligators M.A. Douse* and G.S. Mitchell Department of Comparative Biosciences, University of Wisconsin, Madison, Wisconsin, U.S.A. (Accepted 26 August 1991) Abstract. Reptiles increase ventilation during hypercapnia at a constant temperature. In this study, the contributions ofvagal vs non-vagal receptors to CO2 ventilatory responses were investigated in 16 sedated Alligator missbsippiensb (25 mg/kg pentobarbital; 3 days prior to data collection). Four animals served as controls to assess the effects of time and/or anesthetic drift on ventilation and blood gases; significant ventilatory drift was not detected during the observation period. The effects of bilateral vagotomy on CO2 ventilatory responses were determined during spontaneous breathing (n = 6) and unidirectional ventilation (UDV; n = 6) at two body temperatures (Tb = 30 and 20 °C). Resting Paco~, minute ventilation (Q0, tidal volume (VT) and breathing frequency if) were elevated at 30 °C relative to 20 °C in spontaneously breathing alligators. Increasing inspired CO2 to 5 ~ increased Paco2, f, V3"and ~/l at both levels of Tb. Ventilatory sensitivity to CO2 (S = A~'l/APaco~) was higher at 30 °C with a temperature coeffecient (Q~o) of 2.3. Vagotomy increased Paco2 and VT, decreased f and had no effect on VI at either Tb. After vagotomy, hypercapnia had no effects on ventilation. When CO2 feedback loops were opened by UDV at a high flow rate (> 2 L/min), Tb had no effects on ventilatory efforts at constant Pea2, but hypercapnia significantly increased f, VT and Vt. S was variable with a Qio of 2.1. After vagotomy, a significant CO2-ventilatory response remained during UDV, but S was unaffected by Tb (Qto = 0.8). The results indicate that non-vagal chemoreceptors contribute to CO2 ventilatory responses in alligators, although their contribution following vagotomy is evident only during unidirectional ventilation. Although tentative, the data also suggest that COa-sensitive vagal receptors may be necessary for the temperature dependency of S.
Control of breathing, CO2 sensitivity, reptile; Lung receptors, CO2, reptile; Reptile, Alligatormissbsippiensis; Vagus nerve, CO2 sensitivity, reptile
Many reptiles have vigorous, temperature dependent ventilatory responses to hypercapnia (cf Shelton et al., 1986). However, the relative roles of central ¢s peripheral CO2-sensitive receptors in the hypercapnic ventilatory response of reptiles are unclear. Respiratory chemoreflexes have been elicited from chemosensitive areas in the central nervous system (Hitzig, 1982), peripheral arterial chemoreceptors (Benchetrit and
Correspondence to: G.S. Mitchell, Department of Comparative Biosciences, University of Wisconsin, 2015 Linden Drive West, Madison, WI 53706, U.S.A. * Present address: Department of Physiology, University of Toronto, Toronto, Ontario, Canada.
64
M.A. DOUSE AND G.S. MITCHELL
Dejours, 1980) and CO2-sensitive intrapulmonary receptors (Gatz et al., 1975; Milsom and Jones, 1979). Removal of peripheral arterial chemoreceptor and pulmonary receptor input by vagotomy nearly completely abolishes the ventilatory response to inspired CO2 in snakes (Gratz, 1984), but only diminishes the ventilatory response of Chelonia (Benchetrit and Dejours, 1980; Milsom and Jones, 1980). The reasons for this difference are not clear, but may involve species differences in central CO2 sensitivity or constraints to ventilation imposed by large tidal volumes following vagotomy in some species. In the present study, our objectives were to determine the roles ofvagal vs non-vagal (presumably central) CO2-sensitive receptors in the hypercapnic ventilatory response of a crocodilian species, Alligator mississippiensis. Thus, we determined the effects of bilateral cervical vagotomy on hypercapnic ventilatory responses during both spontaneous breathing and unidirectional ventilation. Since resting Paco, and the CO2 ventilatory response are increased at elevated body temperature in many reptiles (Shelton et al., 1986), including alligators (Davies etal., 1982), we investigated the effects of vagotomy on CO2 responsiveness at two temperatures.
Methods
Animals Experiments were performed on 16 juvenile American alligators (,4. mississippiensis) ranging from 1.4 to 4.2 kg body mass (mean = 2.3 ± 1.0 kg ( ± SD)) obtained from the Rockefeller Wildlife Reserve in Grand Chenier, LA. Prior to experimentation, the animals were fed a diet of mice, with free access to water, and housed at 30 °C. Animals were fasted for > 1 week before experiments to avoid complications due to alkaline tide (Coulson and Hernandez, 1983). Three series of experiments were conducted.
Series !: Effects of anesthesia and time. General anesthesia was induced in four alligators with pentobarbital (approx. 25 mg/kg; i.p.) 3 days prior to data collection. This procedure resulted in a surgical level of anesthesia on the preparation day, and a light level of anesthesia on the two subsequent days of data collection. Further anesthetic (0.2-0.5 mi at 6.5 mg/ml pentobarbitai) was administered as required to minimize the withdrawal response to toe pinch. The animals were studied in ventral recumbancy while body temperature (Tb) was monitored with a cloacal thermistor and regulated with a temperature controlled table. The femoral artery and vein were cannulated (PE-60) and the animals were intubated with a cuffed French endotracheai tube (no. 3.0-4.0) to ensure that upper airway CO2-sensitive receptors (Ballam, 1985) did not contribute to ventilatory responses. Ventilatory parameters were measured in sedated alligators breathing room air on 2 days. Measurements were made three times each day (09: 00, 14: 00 and 18: 00 hrs) for 1 h to assess possible effects of time and/or anesthetic drift. During each measurement period, an arterial blood sample was withdrawn randomly with respect to the
VENTILATORY RESPONSES IN ALLIGATORS
65
phase of the ventilator), cycle over 1-2 min. Body temperature was maintained constant throughout the experiment at either 30 °C (n = 2) or 20 °C (n = 2).
Series H: Spontaneous ventilation. Six alligators were anesthetized and prepared as described for Series I. In addition, the vagus nerves were isolated in the mid-cervical region for subsequent denervation. On the first experimental day, ventilatory responses to an increased inspired CO2 fraction (Fico2) were determined (0.0, 0.05 and return to 0.0 CO2 in 20-30% 02; balance N2). At least 30 min were allowed to reach steady-state at each Fit:o: level. Measurements of ventilation were recorded over the next 30 rain, and an arterial blood sample was obtained. Three alligators were studied initially at 30 °C and three at 20 ° C. Body temperature was decreased to 20 °C or increased to 30 °C over I h, and the protocol was repeated. On the second experimental day, the vagi were sectioned bilaterally under local anesthetic (2% lidocaine), and the entire protocol was repeated. Vagal denervation and removal of peripheral arterial chemoreceptor input was confirmed by the absence of response to intravenous injection of NaCN (50-100 #g/kg), a dose that elicits a brisk ventilatory response in intact alligators. An example of the ventilatory response to NaCN before and after bilateral cervical vagotomy in one alligator is presented in Fig. 1. There was no response to NaCN in any of the 12 alligators tested after bilateral vagotomy (Series II and Series Ill).
Ail 4. 8 0 0 r -
vt -tO0 !
NoON
tso potko)
BII
v,
÷800I
t
Na ON
(6o,uO#kp)
t
¢100pOtko)
Fig. 1. VentUatory responses to intravenous NaCN in a unidirectionally ventilated alligator before (A) and after (B) bilateral cervical vagotomy, in A, 50 #g/kg NaCN elicits a brisk ventilatory response within 30 s. In B, neither 50 nor 100 #g/kg NaCN elicited a ventilatory response, although the alligator did take a breath > 2 rain after injection of NaCN. This animal could still respond to increased Paco2.
66
M.A. DOUSE AND G.S. MITCHELL
Series III: Unidirectional ventilation. Ventilatory responses to CO2 were also studied
during open loop conditions created by unidirectional ventilation (UDV) with a high flow rate. Six alligators were anesthetized and prepared as described for Series II. Both lungs were exposed by midline incision. Three heat-flared PE-240 cannulae (i.d. 2.0 mm) were inserted at the base and sides of each lung and secured with purse-string sutures. The lungs were repositioned into the body cavity, the incision edges were covered with 2% lidocaine jelly (Astra), and the body wall and skin were closed. Incisions were infiltrated with local anesthetic (2% lidocaine) throughout the experiment. UDV began by passing a humidified gas stream (4% COa; 30-40% 02 in N2; > 2 L/rain) through the cannulae, into each lung and out into the atmosphere via the endotracheal tube. Water temperature in the humidifierwas maintained at the alligator's Tb, ensuring that lung temperature approximated Tb. Respiratory responses to CO2 were determined by changing Fxco2 levels in the UDV gas stream. At least three different CO2 levels were delivered to the lungs. The first and last levels were always FI¢o., = 0.04. The sequence of the remaining levels was random and consisted of two or more of the following: FIco 2 = 0.02, 0.06, 0.08 and/or 0.09. High CO2 levels were occasionally necessary since some alligators did not have respiratory movements at lower levels. At least 30 min were allowed to achieve steady-state at each FIco., level. Ventilation measurements were then made over 30 rain and an arterial blood sample was obtained. Three alligators were studied initially at 30 °C and three at 20 °C. Body temperature was changed to 20 or 30 °C, respectively, over I h, and the protocol was repeated. On the second experimental day, the vagi were sectioned bilaterally under local anesthesia, and the entire protocol was repeated. Efficacy of vagal (and peripheral arterial chemoreceptor) denervation was confirmed by the lack of a ventilatory response to NaCN. Measurements
Femoral arterial blood pressure and airway pressure were monitored with pressure transducers (Statham, P23-id). Levels of CO 2 and 02 in the inspiratory gas stream were measured with an infrared CO2 analyzer (Anarad, model pro-20) and an oxygen analyzer (Teledyne, model 331BX). Ventilation was monitored with a pneumotachograph attached to the endotracheal tube (Fleisch, type 00). The pressure drop across the pneumotaehograph was measured with a differential pressure transducer (Validyne, MP45), a carrier/demodulator (Validyne, C15) and integrated to give tidal volume (Gould). The continuous airflow during UDV in Series Ill was electronically offset so that ventilatory efforts about this bias flow could be observed. The pneumotachograph was calibrated during apneic periods. The response of this system was confirmed to be linear over the ventilation range encountered during these experiments. Arterial blood was sampled by withdrawing 0.2-0.5 ml into a syringe to remove dead space, and then withdrawing 0.3 ml anaerobically into a heparinized 0.5 ml glass syringe. At the beginning and end of each experimental day, an additional aliquot of blood
VENTILATORY RESPONSES IN ALLIGATORS
67
was withdrawn to determine hematocrit and lactate concentration. The blood-saline mixture was returned to the animal and the cannula was flushed with fresh heparinized saline. Analysis of arterial Pco_, and Po2 began within 1 rain of sampling using a micro gas analyzer (Radiometer, model BMS 3, MK II; PHM 73) maintained at the animal's body temperature. Electrodes were calibrated before and after each measurement; the Po_~electrode was calibrated with air-saturated water; the P¢o, electrode was calibrated with saturated gases provided by a gas-mixing device (5.61% and 11.22% CO:,; Radiometer GMA2) and a precision-calibrated gas tank (2.53 ~o CO2). Accuracy of the calibration gases was confirmed with a WOsthoffgas mixing pump. Lactate concentrations were determined with a YSI lactate analyzer (model 23L).
Analysis Respiratory flow traces were analyzed in terms of inspiratory (T0, expiratory (TE) and non-ventilatory periods (Tnvp). The distinction between TE (within a breath cluster) and a non-ventilatory period separating breath clusters is somewhat arbitrary; any period ofapnea longer than the mean breath length was designated as Tnvp (see Milsom and Jones, 1980). Both tidal volume (VT) and inspiratory minute ventilation (Qi) were corrected to BTPS conditions and normalized to alligator body mass. Ventilatory sensitivity to Pa¢o~ (S = AVi/APa¢o_~) was assessed by standard regression techniques. Tests of significance were performed using t-tests or paired t-tests (one-tailed), with the Bonferroni correction for multiple comparisons where appropriate. An overall probability of error less than 0.05 was considered significant.
Results
Series I: Eff:cts of anesthesia and time. There were no significant changes in any of the measured variables over the 2 days of experimentation. Therefore, measurements were averaged for each alligator and the resulting values were used to calculate a mean value at each Tb (Table 1). There were no significant changes in hematocrit or lactate concentration in Series I. The overall averages were: Hct = 18.7 + 1.8% and lactate concentration = 0.5 + 0.1 mM/L. Series 11: Spontaneous ventilation. In intact alligators breathing room air, mean VI and VT were significantly higher at 30 °C than at 20 °C (P < 0.01; Table 1), but there were no significant effects on frequency, TI, TE or Tnvp. Despite increased ventilation, mean Paco2 at 30 °C was significantlyhigher than at 20 °C (P < 0.01; Table 1), indicating that metabolic CO2 production increased more than effective ventilation. Arterial Po2 was also higher at 30°C than at 20°C ( P < 0.05, Table 1). Increasing Flco2 to 0.05 at 30 °C increased Paco2, V[, VT and f(all P < 0.05; Figs. 2 and 3). Increased f was due to a significant decrease in Tnvp (P < 0.05), without effect on Tl or TE. At 20 °C, 5% CO2 increased Paco2 and VT (P < 0.01; Fig. 3), but neither
68
M.A. D O U S E A N D G.S. M I T C H E L L TABLE I
Respiratory variables of alligators breathing room air (Series I and ll) or 4~o CO~J30-40~ 02 (Series Ill) before and after bilateral cervical vagotomy. Numbers in parentheses are + SE. Series I
Series II
Intact
Intact
(n = 2) 30 °C ~'l (L'0.5 h - D.kg-J) VT
Series I I I Vagotomy
Intact
Vagotomy (n = 6)
(n = 6)
0.524 (0.131) 0.055 a
(0.074) 0.040 a
0.602 ~ (0.0"/6) 0.116 b'~
0.180 (0.116) 0.022
0.118 c (0.082) 0.019 ~
(L. kg- ~)
(0.011)
(0.008)
(0.007)
(0.008)
(0.012)
f
9.5 (0.4) 47. ! ~ (0.6) 97 a (4)
12.2 (!.4) 45.5 ~ (I.5) 89 u'c (5)
5.3 b (0.8) 62.9 h.~ (4.8) 59 b'c (8)
6.7 (4.7) 41.5 (!.6) ! 51 c (10)
2.0 (I.4) 45.9 c (3.8) 138 c (10)
0.169 ~ (0.026) 0,024 '~
0.434 c (0.108)
VT
0. I 71 (0.066) 0-027'~
0.207 (0.149) 0.019
0.066 c (0.051) 0.020 c
(L. kg - ')
(0.001)
(0,006)
(0.007)
(0.007)
(0.013)
(breaths. 0.5 h - ') Paco2 (mmHg)
PaD, (mmHg)
0.444 ~
20°C ~/I
(L,O.5 h- I,kg- ')
f (breaths, 0.5 h ~ I) Paco2 (mmHg)
Pao~ (mmHg)
6.4
8,2
O. 108 b'c
3.8 h
3.5
1.0
(2.7) 36,8"
(2,2) 32,6"'~
(I. I) 40. I b
(3.3) 37.2~
(0.1) 84 '~
(I,0) 70" ,'~
(2.7) 79 b
(2,5) 120 c
(!.7) 127 C
(6)
(5)
(13)
(10)
(12)
(0.7) 36.7
" P < 0,05 comparing 30 vs 20 °C within a series. b p < 0,05 comparing intact vs vagotomy within a series. c p < 0,05 comparing Series !1 vs Series II!.
f nor its components were altered. Although all animals appeared to increase VI with increased Pace , at 20 oC, the increase was highly variable and did not attain significance (0.I > P > 0.05; Fig, 2). Mean ventilatory sensitivity to CO 2 in intact alligators (S = &VI/APaco~) was significantly greater than zero at 30 °C (P < 0.025), but not at 20 °C (P > 0, I; Table 2). S was significantly higher at 30 °C than at 20 °C (P < 0.05; Table 2) with a temperature coeffecient (Qio) of S = 2.3. Bilateral cervical vagotomy had no significant effect on VI, but increased Pace ~ and VT and decreased f at both Tb (P < 0,05; Table l). VT increased 200~o at 30 °C and 400~ at 20 °C after vagotomy, thus attaining Tb-independent values. Decreased f resulted from increases in TI, TE and Tnvp (all P < 0.05). Vagotomy decreased PaD2 at 30 ° C, but not at 20 °C (Table I). Increasing Fico 2 had no effect on VI, VT or f at
69
VENTILATORY RESPONSES IN ALLIGATORS 4.0
4.0
A.
B.
T
I.S
_~
,.5
M'D
.~ ~
l.O
~.o
O.
°3;
4;
50 60 PoC0a (,~i 9)
70"
80
O'.~O
40 '
50, 6; PoC0a (ram9)
70'
80
Fig. 2. Effects of Paco2 on minute ventilation (VI) in spontaneously breathing alligators at (A) 30 °C (n = 6) and (B) 20 °C (n = 5) before and after bilateral cervical vagotomy (Series II). Filled symbols arc means ( _+SE) pre-vagotomy; open symbols are means ( + SE) post-vagotomy. Squares = 30 °C; circles = 20 °C.
either temperature after vagotomy (P> 0.2; Figs. 2 and 3). Since S values postvagotomy were indistinguishable from zero ( P > 0.5; Table 2), a Qio could not be calculated. Restoring 0% CO2 after 5% CO2 returned both Paco2 and ventilation to initial levels prior to, but not following vagotomy (Figs. 2 and 3). There were no significant changes in hematocrit during Series II experiments (18.4 + 0.9%). .15
.15
A.
,10
.I0
.08
.08
0 40
I
O,
i
I
j
3O
..~
i
a0
e.
!
i
I
I
,0
50
6'0
,;
3O 40
10
30
40
50
60
PoC0~ (m.X9)
7
80
o;
80
PoC0a (,~19)
Fig. 3. Tidal volume (VT) and breathing frequency (f) as a function of Pace2 in spontaneously breathing alligators at 30 °C (A,C) and 20 °C (B,D) before and after bilateral cervical vagotomy (Series II). Filled symbols are means (+ SE) pre-vagotomy; open symbols are means (+ SE) post-vagotomy. Squares = 30 °C; circles = 20 °C.
70
M,A. D O U S E AND G.S. M I T C H E L L TABLE 2
Ventilatory sensitivity to Paco2 (S = A~'t/APaco2) in Series 11 and Series 111 before and after vagotomy at Tb = 3 0 ° C and 20°C. Series 11
Intact
Vagotomy
Series I!I
30 °C
20 °C
30 °C
20 °C
0.146~''b
0.063~
0.079b
0.038h
+ 0.039 (r 2 = 0.89)
+ 0.041 (r 2 = 0.78)
+ 0.031 (r 2 = 0.90)
+ 0.010 (r 2 = 0.86)
0.003
- 0.001
0.026b
0.034 b
+ 0.005
_+0,005
+_0.005
_+0.009
(r 2 ~- 0.70)
(r 2 = 0.34)
(r" = 0.84)
(r 2 = 0.76)
"Indicates significantly different 30 v s 20 °C within a series (P < 0.05; one-tailed test); h Indicates significantly different from zero (P < 0.05; one-tailed test).
During UDV, initial conditions were established at both 30 and 20 °C by ventilating the alligators with 4% CO 2. This CO2 level in the UDV gas stream established a mean Pace_, that was indistinguishable from spontaneously breathing values at 30 °C (Table 1), but higher than during spontaneous breathing at 20 °C (P < 0.05; Table 1). Levels of ~/1, VT and f were not significantly different during UDV vs spontaneous breathing at either Tb (Table 1), nor were there significant differences between variables measured during UDV with 4% CO2 at 30 vs 20 ° C (Table I). Increasing CO2 in the unidirectional gas stream significantly increased Pace2, '~l, VT and f at both temperatures (P < 0.05; Figs. 4 and 5). Increased f was not matched by significant changes in T[, Te or Tnvp. Mean values of S were greater than zero at both Series I l l : Unidirectional ventilation.
2,0 A,
2.0 ill.
"'
Ijt /
1.5
.~ ~ 1.0
~
1.0 0.S
,o
6;
Po(:0t (0~49)
,;
t
ao %
,o
so
6o
PoCOa(md~j)
,o
eo
Fig,4. Mean values ( _+SE)of~t vs Paco: in unidirectionally ventilated alligators at (A) 30°C (n = 6) and (B) 20°C (n = 6) before and after bilateral vagotomy (Series ill). FilLed symbols are means (+_SE) prevagotomy; open symbols are means ( + SE) post-vagotomy. Squares = 30 °C; circles = 20 °C.
VENTILATORY RESPONSES IN ALLIGATORS
.15 [A.
.15 B.
^m ,101
',-
.lO
.05
.05
0
0
46 C.
40 D.
30
30
20
20
IOn
10n O0
71
41)
51) 61) PoC0z (md49)
, ?0
60
0i 30
40
50 60 PoC02 (mH9)
70
60
Fig. 5. Effects of Pace2 on VT and fin unidirectionally ventilated alligators at 30 *C (A,C) and 20 *C (B,D) before and after bilateral vagotomy (Series !!1). Filled symbols are means (+ SE) pre-vagotomy; open symbols are means ( + SE) post-vagotomy. Squares ffi 30 °C; circles = 20 °C.
Tb (Table 2), although the apparent difference between them did not quite attain statistical significance (0.05 < P < 0.1; Table 2). During Series !II, the apparent Qio for S was 2.1. In contrast to spontaneously breathing animals, bilateral cervical vagotomy had no effect on any variable during UDV at either Tb. Also in contrast to vagotomized, spontaneously breathing animals, hypercapnia significantly increased VI, V'r and f at both Tb in vagotomized animals during UDV (P < 0.05; Figs. 4 and 5). There were no significant effects on Tl, Tv or Tnvp. Values of S were significantly different from zero at both Tb post-vagotomy (P < 0.01), but were not different from each other (P ,> 0.05; Table 2). The Qlo of S was 0.8. The level of 02 in the UDV gas stream was held constant at 30-40% in every condition, resulting in a higher Pao: than during spontaneous breathing (P < 0.05; Table 1). There was a significant decrease in hematocrit on the first experimental day in Series III (18.8 + 2.0% to 16.9 +_ 2.6%; P < 0.05), and a nonsignificant decrease on day 2 (15.0 + 2.5?/0 to 13.4 + 2.4%; P > 0.05). There were no significant changes in lactate concentration over the duration of an experiment (overall mean = 5.8 + 2.4 raM/L). However, lactate levels were significantly higher in Series III than in Series I (P < 0.05).
72
M.A. DOUSE AND G.S. MITCHELL
Discussion
The results of this study show that non-vagai, presumably central chemosensitive areas respond to hypercapnia and stimulate ventilation in alligators. However, increased ventilation during hypercapnia was not seen in spontaneously breathing alligators after vagotomy (Series II) and was evident only during the open loop conditions of unidirectional ventilation (Series Ill). The effects of temperature on ventilatory sensitivity to Paco: were highly variable, but suggest that the temperature-dependent CO2 response may require intact vagi. Critique of methods. The American alligator has a preferred body temperature of 32 °C (Johnson et aL, 1978). The temperatures used in this study are within the range experienced by juvenile alligators in their natural habitat (McNease and Joanen, 1974), and they adapt rapidly to temperature changes in terms of ventilatory control and acid-base status (Douse and Mitchell, 1991). Results from Series I experiments indicate that there were no significant changes in respiratory variables due to time or anesthetic drift. However, all of the animals were hypoventilating, as indicated by the relatively high Paco, in Series ! and II compared to unanesthetized, unrestrained alligators (cf Douse and Mitchell, 1991). It may be that ventilatory sensitivity to CO2 was underestimated in this study due to anesthesia. Nonetheless, all alligators had the same anesthetic protocol, allowing comparisons between conditions, in two Series Ill experiments, relative hypoxemia combined with decreased hematocrit and a slightly increased lactate concentration suggested inadequate oxygen delivery in some vascular beds. These factors may have affected the hypvrcapnic ventUatory response, possibly contributing to the large variability of S in Series !!!.
Opening C02-feedback loops by unidirectional ventilation at high flow rates had no significant effects on ventilatory efforts relative to spontaneously breathing animals. However, there were some differences in blood gas values (Table 1). Resting values of VT in Series I! and ill were similar to those in previous reports for alligators (Davies et al. 1982), although breathing frequencies were lower. In addition, temperature had no effect on VT in their studies (Davies et ai., 1982) in contrast with our results. These differences may be due to residual anesthetic effects on the alligators in our study and/or the use of restrained, disturbed alligators in the study of Davies and colleagues. Low Paco: values and an apparent metabolic acidosis (cf Douse and Mitchell, 1991) suggest that restraint caused stress-related hyperventilation in the study of Davies et al. (1982). Our calculations of S are also lower than those estimated for alligators from the data of Davies and co-workers (1982) over a similar Paco, range. Again, part of this difference may be related to anesthetic effects or stress in the respective studies. In our study, CO2 sensitivity of vagally intact alligators during UDV was smaller than during spontaneous breathing. In birds there is also a decreased response to CO2 in open vs closed feedback loop conditions (Jones and Barnford, 1976), possibly due to oscillatory vs constant CO2 levels affecting chemoreceptor reflexes, or differences in proprioceptor feedback during a respiratory cycle. Vagally t, tact animals.
VENTILATORY RESPONSES IN ALLIGATORS
73
Vagotomized animals. In spontaneously breathing alligators, increased VT was offset by decreased frequency following vagotomy, resulting in constant VI. Similar effects of vagotomy have been observed in other reptilian species (Benchetrit and Dejours, 1980; Gratz, 1984; Milsom and Jones, 1980). Pace: was signific&ntlyhigher post-va.gotomy at both temperatures, indicating a relative hypoventilation despite constant VI. The hypercapnia may have resulted from increased metabolic rate, increased dead space ventilation, ventilation-perfusion mismatch or cardiopulmonary shunt. An increased shunt or venous admixture would increase Pace2 if the animal could not respond to the Paco~ stimulus by increasing ventilation (cf. West, 1985). Indeed, since further Pace2 increases had no effect on spontaneous ventilation after vagotomy, the alligators failed to respond to this apparent stimulus. In contrast, bilateral vagotomy during unidirectional ventilation had no effect on VT, and a CO2 response could still be elicited. Since there were increases in both VT and f during hypercapnia in vagotomized, unidirectionaUy ventilated alligators, non-vagal (presumably central) chemosensitive areas alone can respond to changes in Paco~ and increase ventilation. A similar disparity between open and closed loop experimental conditions has been observed in chickens where bilateral vagotomy nearly abolishes the CO2 response in spontaneously breathing (Mitchell and Osborne, 1979), but not unidirectionally ventilated animals (Peterson and Fedde, 1971). The reasons underlying differences in post-vagotomy responses to hypercapnia in open vs closed loop conditions are not clear, but may involve one or more of the following: (l)the ventilatory response to Pace2 during spontaneous breathing may already be at maximum levels post-vagotomy due to. ~he high Pace, when breathing room air; a lower Paco~ is obtained with UDV and m~, be within the operative Pace, response range; (2) oscillatory (Series ll) vs constant (Series Ill) COa delivery to the lungs may alter the ventUatory response to Pace:; (3)the large VT (or elevated lung volume) in spontaneously breathing alligators may reach a mechanical limit or inhibit further increases in ventilation via inhibitory chest wall proprioceptors; or (4) alterations in central nervous system integration may result from loss of vagal inputs. Since increases in ventilation could still be obtained at high levels of Pa¢o~ during UDV, and CO2 oscillations are minimized in this condition, the first two mechanisms are unlikely. Inhibition of breathing by chest wall afferents and/or physical mechanical limits are more likely to cause reduced CO2 responsiveness after vagotomy during spontaneous breathing. However, based on data from Nile crocodiles (Perry, 1988), increased VT after vagotomy is unlikely to reach maximal inflation volumes unless end expiratory lung volume has also increased. The alligators did exhibit a bloated appearance similar to that of vagotomized snakes (Gratz, 1984), an observation that may have been due to increased expiratory lung volume. Stimulation of chest wall afferents by large lung inflations inhibits breathing in mammals (Shannon, 1986). While this may explain why tidal volume does not increase after vagotomy, the lack of frequency increase during hypercapnia in spontaneously breathing alligators suggests at least some involvement of central neural mechanisms in the diminished CO2 response. In spontaneously breathing tortoises, snakes, turtles and chickens, bilateral vagotomy
74
M.A. DOUSE AND G.S. MITCHELL
increases VT and decreases f(Benchetrit and Dejours, 1980; Gratz, 1984; Milsom and Jones, 1980; Mitchell and Osborne, 1979). Increasing CO2 post-vagotomy does not further increase VT or VI in snakes and chickens (Gratz, 1984; Mitchell and Osborne, 1979), but does increase VT and "¢, in Chelonia (Benchetrit and Dejours, 1980; Milsom and Jones, 1980). Thus, it is possible that post-vagotomy tidal volumes in snakes, chickens and alligators are large enough to restrict further increases in ventilation, wherea~ Chelonia may not reach this limit. During UDV, vagotomy did not increase VT in alligators. This may reduce the mechanical or reflex inhibition of the ventilatory response, allowing expression of CO2 sensitivity during UDV in alligators (this study) or chickens (Peterson and Fedde, 1971). Temperature. Increased temperature significantly increased spontaneous ventilation and Pace2, but had no effects on ventilatory efforts during UDV at constant CO2 (Table 1). Pace2 is relatively high at 20 °C during UDV with 4~o CO2 compared to spontaneous ventilation at the same temperature, and is expected to have increased ventilation according to the 20 °C CO2 response curve. Without the relative hyper= capnia, ventilation is expected to have been lower at 20 ° C. Indeed, it has been proposed that appropriate regulation ofventilation and Pace2 in normal, spontaneously breathing animals depends on changes in ventilatory sensitivity to CO2 and metabolic CO2 production at different temperatures (Funk and Milsom, 1987). As shown previously in alligators (Davies etal., 1982), there was an increased hypercapnic ventilatory response at elevated Tb. Although the apparent Qm of S in this study (2.3) was slightly higher than an estimated value (1.5) from the study of Davies et al. (1982), it compares favorably with the Qm of S for turtles (2.0; Funk and Milsom, 1987) and tegu lizards (1.9; Crawford et al., 1977) over the same temperature range. However, all reptiles do not show an effect of Tb on S between 20 and 30 °C (snakes: Nolan and Frankel, 1982; turtles: Jackson et ai., 1974), although an effect can often be discerned in these studies at lower Tb. Results from Series 111 suggest that the temperature-dependent CO2 response in alligators relies, at least in part, on intact vagal receptors. This hypothesis must remain tentative since the temperature effects on S were highly variable during UDV, resulting in an apparently higher value at 30 °C that was not quite significantly different from the value at 20 °C (Qm = 2.1; 0.05 < P < 0.10; Table 2). However, temperature clearly had no effect on S during UDV after vagotomy, although the alligators could still respond to CO2 (apparent Qm ~ 1.0). Thus, there were no temperature effects on central CO,-sensitive areas and/or central integrative structures involved in the CO2 response after removal of vagal afferent feedback. This hypothesis is supported by previous studies showing temperature-independent central CO2/pH responses in turtles (Hitzig, 1982), but large temperature effects on vagal receptors in many reptiles (cf. Douse et ai., 1989). Temperature has a large effect on CO2-sensitive intrapulmonary chemoreceptor discharge in alligators (Q~o--3.2) and a smaller effect on the CO2 sensitivity of pulmonary stretch receptor discharge (Qto -- 2.2; Douse et ai., 1989), but the temperature effect on peripheral arterial chemoreceptors in reptiles is unknown. The CO2/pH
VENTILATORY RESPONSES IN ALLIGATORS
75
response of mammalian carotid bodies has a Qlo of 5.9 (Gallego et al., 1979), and it is likely that reptilian peripheral arterial chemoreceptors have a relatively high Qto as well. Thus, it may be argued that central CO 2 sensitivity and/or brainstem integrative areas with a low Q~o (i.e. < 1.0) overcome the large Tb dependency of vagal CO2_ -sensitive receptors (i.e. Qto "" 3.0), thereby producing the overall system response (i.e. Qto " 2.0). Species-specific regulation of resting Paco 2 at a temperature-dependent set point may therefore result from temperature effects on intrapulmonary and peripheral arterial chemoreceptor CO 2 sensitivity, their integration in the central nervous system and changes in metabolic CO2 production.
Acknowledgements.We are grateful to "fed Joanen and Larry McNease of the Rockefeller Wildlife Refuge for their help in procurement of alligators. G.S.M. was supported by an NIH Research Career Development Award (HL01494).
References Bailam, G.O. (1985). Breathing response of the tegu lizard to I-4% CO2 in the mouth and nose or inspired into the lungs. Respir. Physiol. 62: 375-386. Benehetrit, G. and P. Dejours (1980). Ventilatory CO2 drive in the tortoise, Testudo horsfieidi. J. Exp. Biol. 87: 229-236. Coulson, R.A. and T. Hernandez (1983). Alligator metabolism: Studies on chemical reactions in vivo. Oxford: Pergamon Press, pp. 1-182. Crawford, E.C., R.N. Gatz and J. Piiper (! 977). Ventilatory response of the tegu lizard to inspired CO2 at different body temperatures. Physiologist 20: 19. Davies, D.G., J.L. Thomas and E.N. Smith (1982). Effect of body temperature on ventilatory control in the alligator, J. Appl. Physiol. 52: 114-118. Douse, M. A,, F.L. Powell, W.K. Milsom and G.S. Mitchell (1989). Temperature effects on pulmonary receptor responses to airway pressure and CO2 in Alligator mississipplensls. Respir. Physiol. 78:33 !-344. Douse, M.A. and G.S. Mitchell (1991). Time course of temperature effects on arterial acid-base status in Alligator misslssipplensis. Respir, Physiol. 83: 87-102. Funk, G. D. and W.K. Milsom (1987). Changes in ventilation and breathing pattern produ©edby changing body temperature and inspired CO2 concentration in turtles. Respir. Physiol. 67: 37-51. Gallego, R., C. Eyzaguirre and L. Monti-Bloch (1979). Thermal and osmotic responses of arterial receptors. J. Neurophysiol. 42: 665-680. Gatz, R.N., M.R. Fedde and E.C. Crawford (1975). Lizard lungs: CO2-sensitive receptors in Tupinambis nigropunctatus. £xperientia 31: 455-456. Gratz, R.K. (1984). Effect of bilateral vagotomy on the ventilatory responses of the water snake, Nerodia sipedon. Am. J. Physiol. 246: R221-R227. Hitzig, B.M. (1982). Temperature-induced changes in turtle CSF pH and central control of ventilation. Respir. Physiol. 49: 205-222. Jackson, D.C., S.E. Palmer and W.L. Meadow (1974). The effects of temperature and carbon dioxide breathing on ventilation and acid-base status of turtles. Respir. Physiol. 20: 131-146. Johnson, C.R., W.G. Voigt and E.N. Smith (1978). Thermoregulation in crocodilians - Ill. Thermal preferenda, voluntary maxima, attd heating and cooling rates in the American alligator, Alligator mississippiensis. Zool. J. Linn. Soc. 62: 179-188. Jones, D. R. and O. S. Bamford (1976). Open-loop respiratory chemosensitivity in chickens and ducks. Am. J. Physiol. 230: 861-867.
76
M.A. DOUSE AND G.S. MITCHELL
McNease, L. and T. Joanen (1974). A study ofimmature alligators on Rockefeller Refuge, Louisiana. Proc. Southeastern Assoc. Game and Fish Commissioners Conf. 28: 482-500. Milsom, W.K. and D.R. Jones (1979). Pulmonary receptor chemosensitivity and the ventilatory response to inhaled CO2 in the tunic. Respir. Physiol. 37: 101-107. Milsom, W.K. and D.R. Jones (1980). The role of vagal afferent information and hypercapnia in control of the breathing pattern in Chelonia. J. Exp. Biol. 87: 53-63. Mitchell, G. S. and ,l. L. Osborne (! 979). Ventilatory responses to carbon dioxide inhalation after vagotomy in chickens. Respir. Physiol. 36: 81-88. Nolan, W. F. and H. M. Frankel (1982). Ventilatory responses to CO2 at different temperatures in the snake, Coulber constricter. Experientia 38: 943-945. Perry, S.F. (1988). Functional morphology of the lungs of the nile crocodile, Crocodylus niloticus: nonrespiratory parameters. J. Exp. BioL 134:99-117. Peterson, D.F. and M.R. Fedde (1971). Avian intrapulmonary CO2-sensitive receptors: A comparative study. Comp. Biochem. Physiol. 40A: 425-430. Shannon, R. (1986). Reflexes from respiratory muscles and costovertebral joints. In: Handbook of Physiology, Section 3: The Respiratory System, Vol. 2; edited by A.P. Fishman, N.S. Cherniack, .I.G. Widdicombe and S.R. Geiger. Washington, DC: American Physiological Society, pp. 431-447. Shelton, G., D.R. Jones and W.K. Milsom (1986). Control of breathing in ¢ctothermic vertebrates. In: Handbook of Physiology, Section 3: The Respiratory System, Voi, 2; edited by A.P. Fishman, N.S. Cherniack, J.G. Widdicombe and S.R. Geiger. Washington, DC: American Physiological Society, pp. 857-909. West, ,l. B. (1985). Respiratory Physiology. The Essentials. Third Edition. Baltimore: Williams and Wilkins, 183 pp.
63
RESP 01845
Effects of vagotomy on ventilatory responses to C02 in alligators M.A. Douse* and G.S. Mitchell Department of Comparative Biosciences, University of Wisconsin, Madison, Wisconsin, U.S.A. (Accepted 26 August 1991) Abstract. Reptiles increase ventilation during hypercapnia at a constant temperature. In this study, the contributions ofvagal vs non-vagal receptors to CO2 ventilatory responses were investigated in 16 sedated Alligator missbsippiensb (25 mg/kg pentobarbital; 3 days prior to data collection). Four animals served as controls to assess the effects of time and/or anesthetic drift on ventilation and blood gases; significant ventilatory drift was not detected during the observation period. The effects of bilateral vagotomy on CO2 ventilatory responses were determined during spontaneous breathing (n = 6) and unidirectional ventilation (UDV; n = 6) at two body temperatures (Tb = 30 and 20 °C). Resting Paco~, minute ventilation (Q0, tidal volume (VT) and breathing frequency if) were elevated at 30 °C relative to 20 °C in spontaneously breathing alligators. Increasing inspired CO2 to 5 ~ increased Paco2, f, V3"and ~/l at both levels of Tb. Ventilatory sensitivity to CO2 (S = A~'l/APaco~) was higher at 30 °C with a temperature coeffecient (Q~o) of 2.3. Vagotomy increased Paco2 and VT, decreased f and had no effect on VI at either Tb. After vagotomy, hypercapnia had no effects on ventilation. When CO2 feedback loops were opened by UDV at a high flow rate (> 2 L/min), Tb had no effects on ventilatory efforts at constant Pea2, but hypercapnia significantly increased f, VT and Vt. S was variable with a Qio of 2.1. After vagotomy, a significant CO2-ventilatory response remained during UDV, but S was unaffected by Tb (Qto = 0.8). The results indicate that non-vagal chemoreceptors contribute to CO2 ventilatory responses in alligators, although their contribution following vagotomy is evident only during unidirectional ventilation. Although tentative, the data also suggest that COa-sensitive vagal receptors may be necessary for the temperature dependency of S.
Control of breathing, CO2 sensitivity, reptile; Lung receptors, CO2, reptile; Reptile, Alligatormissbsippiensis; Vagus nerve, CO2 sensitivity, reptile
Many reptiles have vigorous, temperature dependent ventilatory responses to hypercapnia (cf Shelton et al., 1986). However, the relative roles of central ¢s peripheral CO2-sensitive receptors in the hypercapnic ventilatory response of reptiles are unclear. Respiratory chemoreflexes have been elicited from chemosensitive areas in the central nervous system (Hitzig, 1982), peripheral arterial chemoreceptors (Benchetrit and
Correspondence to: G.S. Mitchell, Department of Comparative Biosciences, University of Wisconsin, 2015 Linden Drive West, Madison, WI 53706, U.S.A. * Present address: Department of Physiology, University of Toronto, Toronto, Ontario, Canada.
64
M.A. DOUSE AND G.S. MITCHELL
Dejours, 1980) and CO2-sensitive intrapulmonary receptors (Gatz et al., 1975; Milsom and Jones, 1979). Removal of peripheral arterial chemoreceptor and pulmonary receptor input by vagotomy nearly completely abolishes the ventilatory response to inspired CO2 in snakes (Gratz, 1984), but only diminishes the ventilatory response of Chelonia (Benchetrit and Dejours, 1980; Milsom and Jones, 1980). The reasons for this difference are not clear, but may involve species differences in central CO2 sensitivity or constraints to ventilation imposed by large tidal volumes following vagotomy in some species. In the present study, our objectives were to determine the roles ofvagal vs non-vagal (presumably central) CO2-sensitive receptors in the hypercapnic ventilatory response of a crocodilian species, Alligator mississippiensis. Thus, we determined the effects of bilateral cervical vagotomy on hypercapnic ventilatory responses during both spontaneous breathing and unidirectional ventilation. Since resting Paco, and the CO2 ventilatory response are increased at elevated body temperature in many reptiles (Shelton et al., 1986), including alligators (Davies etal., 1982), we investigated the effects of vagotomy on CO2 responsiveness at two temperatures.
Methods
Animals Experiments were performed on 16 juvenile American alligators (,4. mississippiensis) ranging from 1.4 to 4.2 kg body mass (mean = 2.3 ± 1.0 kg ( ± SD)) obtained from the Rockefeller Wildlife Reserve in Grand Chenier, LA. Prior to experimentation, the animals were fed a diet of mice, with free access to water, and housed at 30 °C. Animals were fasted for > 1 week before experiments to avoid complications due to alkaline tide (Coulson and Hernandez, 1983). Three series of experiments were conducted.
Series !: Effects of anesthesia and time. General anesthesia was induced in four alligators with pentobarbital (approx. 25 mg/kg; i.p.) 3 days prior to data collection. This procedure resulted in a surgical level of anesthesia on the preparation day, and a light level of anesthesia on the two subsequent days of data collection. Further anesthetic (0.2-0.5 mi at 6.5 mg/ml pentobarbitai) was administered as required to minimize the withdrawal response to toe pinch. The animals were studied in ventral recumbancy while body temperature (Tb) was monitored with a cloacal thermistor and regulated with a temperature controlled table. The femoral artery and vein were cannulated (PE-60) and the animals were intubated with a cuffed French endotracheai tube (no. 3.0-4.0) to ensure that upper airway CO2-sensitive receptors (Ballam, 1985) did not contribute to ventilatory responses. Ventilatory parameters were measured in sedated alligators breathing room air on 2 days. Measurements were made three times each day (09: 00, 14: 00 and 18: 00 hrs) for 1 h to assess possible effects of time and/or anesthetic drift. During each measurement period, an arterial blood sample was withdrawn randomly with respect to the
VENTILATORY RESPONSES IN ALLIGATORS
65
phase of the ventilator), cycle over 1-2 min. Body temperature was maintained constant throughout the experiment at either 30 °C (n = 2) or 20 °C (n = 2).
Series H: Spontaneous ventilation. Six alligators were anesthetized and prepared as described for Series I. In addition, the vagus nerves were isolated in the mid-cervical region for subsequent denervation. On the first experimental day, ventilatory responses to an increased inspired CO2 fraction (Fico2) were determined (0.0, 0.05 and return to 0.0 CO2 in 20-30% 02; balance N2). At least 30 min were allowed to reach steady-state at each Fit:o: level. Measurements of ventilation were recorded over the next 30 rain, and an arterial blood sample was obtained. Three alligators were studied initially at 30 °C and three at 20 ° C. Body temperature was decreased to 20 °C or increased to 30 °C over I h, and the protocol was repeated. On the second experimental day, the vagi were sectioned bilaterally under local anesthetic (2% lidocaine), and the entire protocol was repeated. Vagal denervation and removal of peripheral arterial chemoreceptor input was confirmed by the absence of response to intravenous injection of NaCN (50-100 #g/kg), a dose that elicits a brisk ventilatory response in intact alligators. An example of the ventilatory response to NaCN before and after bilateral cervical vagotomy in one alligator is presented in Fig. 1. There was no response to NaCN in any of the 12 alligators tested after bilateral vagotomy (Series II and Series Ill).
Ail 4. 8 0 0 r -
vt -tO0 !
NoON
tso potko)
BII
v,
÷800I
t
Na ON
(6o,uO#kp)
t
¢100pOtko)
Fig. 1. VentUatory responses to intravenous NaCN in a unidirectionally ventilated alligator before (A) and after (B) bilateral cervical vagotomy, in A, 50 #g/kg NaCN elicits a brisk ventilatory response within 30 s. In B, neither 50 nor 100 #g/kg NaCN elicited a ventilatory response, although the alligator did take a breath > 2 rain after injection of NaCN. This animal could still respond to increased Paco2.
66
M.A. DOUSE AND G.S. MITCHELL
Series III: Unidirectional ventilation. Ventilatory responses to CO2 were also studied
during open loop conditions created by unidirectional ventilation (UDV) with a high flow rate. Six alligators were anesthetized and prepared as described for Series II. Both lungs were exposed by midline incision. Three heat-flared PE-240 cannulae (i.d. 2.0 mm) were inserted at the base and sides of each lung and secured with purse-string sutures. The lungs were repositioned into the body cavity, the incision edges were covered with 2% lidocaine jelly (Astra), and the body wall and skin were closed. Incisions were infiltrated with local anesthetic (2% lidocaine) throughout the experiment. UDV began by passing a humidified gas stream (4% COa; 30-40% 02 in N2; > 2 L/rain) through the cannulae, into each lung and out into the atmosphere via the endotracheal tube. Water temperature in the humidifierwas maintained at the alligator's Tb, ensuring that lung temperature approximated Tb. Respiratory responses to CO2 were determined by changing Fxco2 levels in the UDV gas stream. At least three different CO2 levels were delivered to the lungs. The first and last levels were always FI¢o., = 0.04. The sequence of the remaining levels was random and consisted of two or more of the following: FIco 2 = 0.02, 0.06, 0.08 and/or 0.09. High CO2 levels were occasionally necessary since some alligators did not have respiratory movements at lower levels. At least 30 min were allowed to achieve steady-state at each FIco., level. Ventilation measurements were then made over 30 rain and an arterial blood sample was obtained. Three alligators were studied initially at 30 °C and three at 20 °C. Body temperature was changed to 20 or 30 °C, respectively, over I h, and the protocol was repeated. On the second experimental day, the vagi were sectioned bilaterally under local anesthesia, and the entire protocol was repeated. Efficacy of vagal (and peripheral arterial chemoreceptor) denervation was confirmed by the lack of a ventilatory response to NaCN. Measurements
Femoral arterial blood pressure and airway pressure were monitored with pressure transducers (Statham, P23-id). Levels of CO 2 and 02 in the inspiratory gas stream were measured with an infrared CO2 analyzer (Anarad, model pro-20) and an oxygen analyzer (Teledyne, model 331BX). Ventilation was monitored with a pneumotachograph attached to the endotracheal tube (Fleisch, type 00). The pressure drop across the pneumotaehograph was measured with a differential pressure transducer (Validyne, MP45), a carrier/demodulator (Validyne, C15) and integrated to give tidal volume (Gould). The continuous airflow during UDV in Series Ill was electronically offset so that ventilatory efforts about this bias flow could be observed. The pneumotachograph was calibrated during apneic periods. The response of this system was confirmed to be linear over the ventilation range encountered during these experiments. Arterial blood was sampled by withdrawing 0.2-0.5 ml into a syringe to remove dead space, and then withdrawing 0.3 ml anaerobically into a heparinized 0.5 ml glass syringe. At the beginning and end of each experimental day, an additional aliquot of blood
VENTILATORY RESPONSES IN ALLIGATORS
67
was withdrawn to determine hematocrit and lactate concentration. The blood-saline mixture was returned to the animal and the cannula was flushed with fresh heparinized saline. Analysis of arterial Pco_, and Po2 began within 1 rain of sampling using a micro gas analyzer (Radiometer, model BMS 3, MK II; PHM 73) maintained at the animal's body temperature. Electrodes were calibrated before and after each measurement; the Po_~electrode was calibrated with air-saturated water; the P¢o, electrode was calibrated with saturated gases provided by a gas-mixing device (5.61% and 11.22% CO:,; Radiometer GMA2) and a precision-calibrated gas tank (2.53 ~o CO2). Accuracy of the calibration gases was confirmed with a WOsthoffgas mixing pump. Lactate concentrations were determined with a YSI lactate analyzer (model 23L).
Analysis Respiratory flow traces were analyzed in terms of inspiratory (T0, expiratory (TE) and non-ventilatory periods (Tnvp). The distinction between TE (within a breath cluster) and a non-ventilatory period separating breath clusters is somewhat arbitrary; any period ofapnea longer than the mean breath length was designated as Tnvp (see Milsom and Jones, 1980). Both tidal volume (VT) and inspiratory minute ventilation (Qi) were corrected to BTPS conditions and normalized to alligator body mass. Ventilatory sensitivity to Pa¢o~ (S = AVi/APa¢o_~) was assessed by standard regression techniques. Tests of significance were performed using t-tests or paired t-tests (one-tailed), with the Bonferroni correction for multiple comparisons where appropriate. An overall probability of error less than 0.05 was considered significant.
Results
Series I: Eff:cts of anesthesia and time. There were no significant changes in any of the measured variables over the 2 days of experimentation. Therefore, measurements were averaged for each alligator and the resulting values were used to calculate a mean value at each Tb (Table 1). There were no significant changes in hematocrit or lactate concentration in Series I. The overall averages were: Hct = 18.7 + 1.8% and lactate concentration = 0.5 + 0.1 mM/L. Series 11: Spontaneous ventilation. In intact alligators breathing room air, mean VI and VT were significantly higher at 30 °C than at 20 °C (P < 0.01; Table 1), but there were no significant effects on frequency, TI, TE or Tnvp. Despite increased ventilation, mean Paco2 at 30 °C was significantlyhigher than at 20 °C (P < 0.01; Table 1), indicating that metabolic CO2 production increased more than effective ventilation. Arterial Po2 was also higher at 30°C than at 20°C ( P < 0.05, Table 1). Increasing Flco2 to 0.05 at 30 °C increased Paco2, V[, VT and f(all P < 0.05; Figs. 2 and 3). Increased f was due to a significant decrease in Tnvp (P < 0.05), without effect on Tl or TE. At 20 °C, 5% CO2 increased Paco2 and VT (P < 0.01; Fig. 3), but neither
68
M.A. D O U S E A N D G.S. M I T C H E L L TABLE I
Respiratory variables of alligators breathing room air (Series I and ll) or 4~o CO~J30-40~ 02 (Series Ill) before and after bilateral cervical vagotomy. Numbers in parentheses are + SE. Series I
Series II
Intact
Intact
(n = 2) 30 °C ~'l (L'0.5 h - D.kg-J) VT
Series I I I Vagotomy
Intact
Vagotomy (n = 6)
(n = 6)
0.524 (0.131) 0.055 a
(0.074) 0.040 a
0.602 ~ (0.0"/6) 0.116 b'~
0.180 (0.116) 0.022
0.118 c (0.082) 0.019 ~
(L. kg- ~)
(0.011)
(0.008)
(0.007)
(0.008)
(0.012)
f
9.5 (0.4) 47. ! ~ (0.6) 97 a (4)
12.2 (!.4) 45.5 ~ (I.5) 89 u'c (5)
5.3 b (0.8) 62.9 h.~ (4.8) 59 b'c (8)
6.7 (4.7) 41.5 (!.6) ! 51 c (10)
2.0 (I.4) 45.9 c (3.8) 138 c (10)
0.169 ~ (0.026) 0,024 '~
0.434 c (0.108)
VT
0. I 71 (0.066) 0-027'~
0.207 (0.149) 0.019
0.066 c (0.051) 0.020 c
(L. kg - ')
(0.001)
(0,006)
(0.007)
(0.007)
(0.013)
(breaths. 0.5 h - ') Paco2 (mmHg)
PaD, (mmHg)
0.444 ~
20°C ~/I
(L,O.5 h- I,kg- ')
f (breaths, 0.5 h ~ I) Paco2 (mmHg)
Pao~ (mmHg)
6.4
8,2
O. 108 b'c
3.8 h
3.5
1.0
(2.7) 36,8"
(2,2) 32,6"'~
(I. I) 40. I b
(3.3) 37.2~
(0.1) 84 '~
(I,0) 70" ,'~
(2.7) 79 b
(2,5) 120 c
(!.7) 127 C
(6)
(5)
(13)
(10)
(12)
(0.7) 36.7
" P < 0,05 comparing 30 vs 20 °C within a series. b p < 0,05 comparing intact vs vagotomy within a series. c p < 0,05 comparing Series !1 vs Series II!.
f nor its components were altered. Although all animals appeared to increase VI with increased Pace , at 20 oC, the increase was highly variable and did not attain significance (0.I > P > 0.05; Fig, 2). Mean ventilatory sensitivity to CO 2 in intact alligators (S = &VI/APaco~) was significantly greater than zero at 30 °C (P < 0.025), but not at 20 °C (P > 0, I; Table 2). S was significantly higher at 30 °C than at 20 °C (P < 0.05; Table 2) with a temperature coeffecient (Qio) of S = 2.3. Bilateral cervical vagotomy had no significant effect on VI, but increased Pace ~ and VT and decreased f at both Tb (P < 0,05; Table l). VT increased 200~o at 30 °C and 400~ at 20 °C after vagotomy, thus attaining Tb-independent values. Decreased f resulted from increases in TI, TE and Tnvp (all P < 0.05). Vagotomy decreased PaD2 at 30 ° C, but not at 20 °C (Table I). Increasing Fico 2 had no effect on VI, VT or f at
69
VENTILATORY RESPONSES IN ALLIGATORS 4.0
4.0
A.
B.
T
I.S
_~
,.5
M'D
.~ ~
l.O
~.o
O.
°3;
4;
50 60 PoC0a (,~i 9)
70"
80
O'.~O
40 '
50, 6; PoC0a (ram9)
70'
80
Fig. 2. Effects of Paco2 on minute ventilation (VI) in spontaneously breathing alligators at (A) 30 °C (n = 6) and (B) 20 °C (n = 5) before and after bilateral cervical vagotomy (Series II). Filled symbols arc means ( _+SE) pre-vagotomy; open symbols are means ( + SE) post-vagotomy. Squares = 30 °C; circles = 20 °C.
either temperature after vagotomy (P> 0.2; Figs. 2 and 3). Since S values postvagotomy were indistinguishable from zero ( P > 0.5; Table 2), a Qio could not be calculated. Restoring 0% CO2 after 5% CO2 returned both Paco2 and ventilation to initial levels prior to, but not following vagotomy (Figs. 2 and 3). There were no significant changes in hematocrit during Series II experiments (18.4 + 0.9%). .15
.15
A.
,10
.I0
.08
.08
0 40
I
O,
i
I
j
3O
..~
i
a0
e.
!
i
I
I
,0
50
6'0
,;
3O 40
10
30
40
50
60
PoC0~ (m.X9)
7
80
o;
80
PoC0a (,~19)
Fig. 3. Tidal volume (VT) and breathing frequency (f) as a function of Pace2 in spontaneously breathing alligators at 30 °C (A,C) and 20 °C (B,D) before and after bilateral cervical vagotomy (Series II). Filled symbols are means (+ SE) pre-vagotomy; open symbols are means (+ SE) post-vagotomy. Squares = 30 °C; circles = 20 °C.
70
M,A. D O U S E AND G.S. M I T C H E L L TABLE 2
Ventilatory sensitivity to Paco2 (S = A~'t/APaco2) in Series 11 and Series 111 before and after vagotomy at Tb = 3 0 ° C and 20°C. Series 11
Intact
Vagotomy
Series I!I
30 °C
20 °C
30 °C
20 °C
0.146~''b
0.063~
0.079b
0.038h
+ 0.039 (r 2 = 0.89)
+ 0.041 (r 2 = 0.78)
+ 0.031 (r 2 = 0.90)
+ 0.010 (r 2 = 0.86)
0.003
- 0.001
0.026b
0.034 b
+ 0.005
_+0,005
+_0.005
_+0.009
(r 2 ~- 0.70)
(r 2 = 0.34)
(r" = 0.84)
(r 2 = 0.76)
"Indicates significantly different 30 v s 20 °C within a series (P < 0.05; one-tailed test); h Indicates significantly different from zero (P < 0.05; one-tailed test).
During UDV, initial conditions were established at both 30 and 20 °C by ventilating the alligators with 4% CO 2. This CO2 level in the UDV gas stream established a mean Pace_, that was indistinguishable from spontaneously breathing values at 30 °C (Table 1), but higher than during spontaneous breathing at 20 °C (P < 0.05; Table 1). Levels of ~/1, VT and f were not significantly different during UDV vs spontaneous breathing at either Tb (Table 1), nor were there significant differences between variables measured during UDV with 4% CO2 at 30 vs 20 ° C (Table I). Increasing CO2 in the unidirectional gas stream significantly increased Pace2, '~l, VT and f at both temperatures (P < 0.05; Figs. 4 and 5). Increased f was not matched by significant changes in T[, Te or Tnvp. Mean values of S were greater than zero at both Series I l l : Unidirectional ventilation.
2,0 A,
2.0 ill.
"'
Ijt /
1.5
.~ ~ 1.0
~
1.0 0.S
,o
6;
Po(:0t (0~49)
,;
t
ao %
,o
so
6o
PoCOa(md~j)
,o
eo
Fig,4. Mean values ( _+SE)of~t vs Paco: in unidirectionally ventilated alligators at (A) 30°C (n = 6) and (B) 20°C (n = 6) before and after bilateral vagotomy (Series ill). FilLed symbols are means (+_SE) prevagotomy; open symbols are means ( + SE) post-vagotomy. Squares = 30 °C; circles = 20 °C.
VENTILATORY RESPONSES IN ALLIGATORS
.15 [A.
.15 B.
^m ,101
',-
.lO
.05
.05
0
0
46 C.
40 D.
30
30
20
20
IOn
10n O0
71
41)
51) 61) PoC0z (md49)
, ?0
60
0i 30
40
50 60 PoC02 (mH9)
70
60
Fig. 5. Effects of Pace2 on VT and fin unidirectionally ventilated alligators at 30 *C (A,C) and 20 *C (B,D) before and after bilateral vagotomy (Series !!1). Filled symbols are means (+ SE) pre-vagotomy; open symbols are means ( + SE) post-vagotomy. Squares ffi 30 °C; circles = 20 °C.
Tb (Table 2), although the apparent difference between them did not quite attain statistical significance (0.05 < P < 0.1; Table 2). During Series !II, the apparent Qio for S was 2.1. In contrast to spontaneously breathing animals, bilateral cervical vagotomy had no effect on any variable during UDV at either Tb. Also in contrast to vagotomized, spontaneously breathing animals, hypercapnia significantly increased VI, V'r and f at both Tb in vagotomized animals during UDV (P < 0.05; Figs. 4 and 5). There were no significant effects on Tl, Tv or Tnvp. Values of S were significantly different from zero at both Tb post-vagotomy (P < 0.01), but were not different from each other (P ,> 0.05; Table 2). The Qlo of S was 0.8. The level of 02 in the UDV gas stream was held constant at 30-40% in every condition, resulting in a higher Pao: than during spontaneous breathing (P < 0.05; Table 1). There was a significant decrease in hematocrit on the first experimental day in Series III (18.8 + 2.0% to 16.9 +_ 2.6%; P < 0.05), and a nonsignificant decrease on day 2 (15.0 + 2.5?/0 to 13.4 + 2.4%; P > 0.05). There were no significant changes in lactate concentration over the duration of an experiment (overall mean = 5.8 + 2.4 raM/L). However, lactate levels were significantly higher in Series III than in Series I (P < 0.05).
72
M.A. DOUSE AND G.S. MITCHELL
Discussion
The results of this study show that non-vagai, presumably central chemosensitive areas respond to hypercapnia and stimulate ventilation in alligators. However, increased ventilation during hypercapnia was not seen in spontaneously breathing alligators after vagotomy (Series II) and was evident only during the open loop conditions of unidirectional ventilation (Series Ill). The effects of temperature on ventilatory sensitivity to Paco: were highly variable, but suggest that the temperature-dependent CO2 response may require intact vagi. Critique of methods. The American alligator has a preferred body temperature of 32 °C (Johnson et aL, 1978). The temperatures used in this study are within the range experienced by juvenile alligators in their natural habitat (McNease and Joanen, 1974), and they adapt rapidly to temperature changes in terms of ventilatory control and acid-base status (Douse and Mitchell, 1991). Results from Series I experiments indicate that there were no significant changes in respiratory variables due to time or anesthetic drift. However, all of the animals were hypoventilating, as indicated by the relatively high Paco, in Series ! and II compared to unanesthetized, unrestrained alligators (cf Douse and Mitchell, 1991). It may be that ventilatory sensitivity to CO2 was underestimated in this study due to anesthesia. Nonetheless, all alligators had the same anesthetic protocol, allowing comparisons between conditions, in two Series Ill experiments, relative hypoxemia combined with decreased hematocrit and a slightly increased lactate concentration suggested inadequate oxygen delivery in some vascular beds. These factors may have affected the hypvrcapnic ventUatory response, possibly contributing to the large variability of S in Series !!!.
Opening C02-feedback loops by unidirectional ventilation at high flow rates had no significant effects on ventilatory efforts relative to spontaneously breathing animals. However, there were some differences in blood gas values (Table 1). Resting values of VT in Series I! and ill were similar to those in previous reports for alligators (Davies et al. 1982), although breathing frequencies were lower. In addition, temperature had no effect on VT in their studies (Davies et ai., 1982) in contrast with our results. These differences may be due to residual anesthetic effects on the alligators in our study and/or the use of restrained, disturbed alligators in the study of Davies and colleagues. Low Paco: values and an apparent metabolic acidosis (cf Douse and Mitchell, 1991) suggest that restraint caused stress-related hyperventilation in the study of Davies et al. (1982). Our calculations of S are also lower than those estimated for alligators from the data of Davies and co-workers (1982) over a similar Paco, range. Again, part of this difference may be related to anesthetic effects or stress in the respective studies. In our study, CO2 sensitivity of vagally intact alligators during UDV was smaller than during spontaneous breathing. In birds there is also a decreased response to CO2 in open vs closed feedback loop conditions (Jones and Barnford, 1976), possibly due to oscillatory vs constant CO2 levels affecting chemoreceptor reflexes, or differences in proprioceptor feedback during a respiratory cycle. Vagally t, tact animals.
VENTILATORY RESPONSES IN ALLIGATORS
73
Vagotomized animals. In spontaneously breathing alligators, increased VT was offset by decreased frequency following vagotomy, resulting in constant VI. Similar effects of vagotomy have been observed in other reptilian species (Benchetrit and Dejours, 1980; Gratz, 1984; Milsom and Jones, 1980). Pace: was signific&ntlyhigher post-va.gotomy at both temperatures, indicating a relative hypoventilation despite constant VI. The hypercapnia may have resulted from increased metabolic rate, increased dead space ventilation, ventilation-perfusion mismatch or cardiopulmonary shunt. An increased shunt or venous admixture would increase Pace2 if the animal could not respond to the Paco~ stimulus by increasing ventilation (cf. West, 1985). Indeed, since further Pace2 increases had no effect on spontaneous ventilation after vagotomy, the alligators failed to respond to this apparent stimulus. In contrast, bilateral vagotomy during unidirectional ventilation had no effect on VT, and a CO2 response could still be elicited. Since there were increases in both VT and f during hypercapnia in vagotomized, unidirectionaUy ventilated alligators, non-vagal (presumably central) chemosensitive areas alone can respond to changes in Paco~ and increase ventilation. A similar disparity between open and closed loop experimental conditions has been observed in chickens where bilateral vagotomy nearly abolishes the CO2 response in spontaneously breathing (Mitchell and Osborne, 1979), but not unidirectionally ventilated animals (Peterson and Fedde, 1971). The reasons underlying differences in post-vagotomy responses to hypercapnia in open vs closed loop conditions are not clear, but may involve one or more of the following: (l)the ventilatory response to Pace2 during spontaneous breathing may already be at maximum levels post-vagotomy due to. ~he high Pace, when breathing room air; a lower Paco~ is obtained with UDV and m~, be within the operative Pace, response range; (2) oscillatory (Series ll) vs constant (Series Ill) COa delivery to the lungs may alter the ventUatory response to Pace:; (3)the large VT (or elevated lung volume) in spontaneously breathing alligators may reach a mechanical limit or inhibit further increases in ventilation via inhibitory chest wall proprioceptors; or (4) alterations in central nervous system integration may result from loss of vagal inputs. Since increases in ventilation could still be obtained at high levels of Pa¢o~ during UDV, and CO2 oscillations are minimized in this condition, the first two mechanisms are unlikely. Inhibition of breathing by chest wall afferents and/or physical mechanical limits are more likely to cause reduced CO2 responsiveness after vagotomy during spontaneous breathing. However, based on data from Nile crocodiles (Perry, 1988), increased VT after vagotomy is unlikely to reach maximal inflation volumes unless end expiratory lung volume has also increased. The alligators did exhibit a bloated appearance similar to that of vagotomized snakes (Gratz, 1984), an observation that may have been due to increased expiratory lung volume. Stimulation of chest wall afferents by large lung inflations inhibits breathing in mammals (Shannon, 1986). While this may explain why tidal volume does not increase after vagotomy, the lack of frequency increase during hypercapnia in spontaneously breathing alligators suggests at least some involvement of central neural mechanisms in the diminished CO2 response. In spontaneously breathing tortoises, snakes, turtles and chickens, bilateral vagotomy
74
M.A. DOUSE AND G.S. MITCHELL
increases VT and decreases f(Benchetrit and Dejours, 1980; Gratz, 1984; Milsom and Jones, 1980; Mitchell and Osborne, 1979). Increasing CO2 post-vagotomy does not further increase VT or VI in snakes and chickens (Gratz, 1984; Mitchell and Osborne, 1979), but does increase VT and "¢, in Chelonia (Benchetrit and Dejours, 1980; Milsom and Jones, 1980). Thus, it is possible that post-vagotomy tidal volumes in snakes, chickens and alligators are large enough to restrict further increases in ventilation, wherea~ Chelonia may not reach this limit. During UDV, vagotomy did not increase VT in alligators. This may reduce the mechanical or reflex inhibition of the ventilatory response, allowing expression of CO2 sensitivity during UDV in alligators (this study) or chickens (Peterson and Fedde, 1971). Temperature. Increased temperature significantly increased spontaneous ventilation and Pace2, but had no effects on ventilatory efforts during UDV at constant CO2 (Table 1). Pace2 is relatively high at 20 °C during UDV with 4~o CO2 compared to spontaneous ventilation at the same temperature, and is expected to have increased ventilation according to the 20 °C CO2 response curve. Without the relative hyper= capnia, ventilation is expected to have been lower at 20 ° C. Indeed, it has been proposed that appropriate regulation ofventilation and Pace2 in normal, spontaneously breathing animals depends on changes in ventilatory sensitivity to CO2 and metabolic CO2 production at different temperatures (Funk and Milsom, 1987). As shown previously in alligators (Davies etal., 1982), there was an increased hypercapnic ventilatory response at elevated Tb. Although the apparent Qm of S in this study (2.3) was slightly higher than an estimated value (1.5) from the study of Davies et al. (1982), it compares favorably with the Qm of S for turtles (2.0; Funk and Milsom, 1987) and tegu lizards (1.9; Crawford et al., 1977) over the same temperature range. However, all reptiles do not show an effect of Tb on S between 20 and 30 °C (snakes: Nolan and Frankel, 1982; turtles: Jackson et ai., 1974), although an effect can often be discerned in these studies at lower Tb. Results from Series 111 suggest that the temperature-dependent CO2 response in alligators relies, at least in part, on intact vagal receptors. This hypothesis must remain tentative since the temperature effects on S were highly variable during UDV, resulting in an apparently higher value at 30 °C that was not quite significantly different from the value at 20 °C (Qm = 2.1; 0.05 < P < 0.10; Table 2). However, temperature clearly had no effect on S during UDV after vagotomy, although the alligators could still respond to CO2 (apparent Qm ~ 1.0). Thus, there were no temperature effects on central CO,-sensitive areas and/or central integrative structures involved in the CO2 response after removal of vagal afferent feedback. This hypothesis is supported by previous studies showing temperature-independent central CO2/pH responses in turtles (Hitzig, 1982), but large temperature effects on vagal receptors in many reptiles (cf. Douse et ai., 1989). Temperature has a large effect on CO2-sensitive intrapulmonary chemoreceptor discharge in alligators (Q~o--3.2) and a smaller effect on the CO2 sensitivity of pulmonary stretch receptor discharge (Qto -- 2.2; Douse et ai., 1989), but the temperature effect on peripheral arterial chemoreceptors in reptiles is unknown. The CO2/pH
VENTILATORY RESPONSES IN ALLIGATORS
75
response of mammalian carotid bodies has a Qlo of 5.9 (Gallego et al., 1979), and it is likely that reptilian peripheral arterial chemoreceptors have a relatively high Qto as well. Thus, it may be argued that central CO 2 sensitivity and/or brainstem integrative areas with a low Q~o (i.e. < 1.0) overcome the large Tb dependency of vagal CO2_ -sensitive receptors (i.e. Qto "" 3.0), thereby producing the overall system response (i.e. Qto " 2.0). Species-specific regulation of resting Paco 2 at a temperature-dependent set point may therefore result from temperature effects on intrapulmonary and peripheral arterial chemoreceptor CO 2 sensitivity, their integration in the central nervous system and changes in metabolic CO2 production.
Acknowledgements.We are grateful to "fed Joanen and Larry McNease of the Rockefeller Wildlife Refuge for their help in procurement of alligators. G.S.M. was supported by an NIH Research Career Development Award (HL01494).
References Bailam, G.O. (1985). Breathing response of the tegu lizard to I-4% CO2 in the mouth and nose or inspired into the lungs. Respir. Physiol. 62: 375-386. Benehetrit, G. and P. Dejours (1980). Ventilatory CO2 drive in the tortoise, Testudo horsfieidi. J. Exp. Biol. 87: 229-236. Coulson, R.A. and T. Hernandez (1983). Alligator metabolism: Studies on chemical reactions in vivo. Oxford: Pergamon Press, pp. 1-182. Crawford, E.C., R.N. Gatz and J. Piiper (! 977). Ventilatory response of the tegu lizard to inspired CO2 at different body temperatures. Physiologist 20: 19. Davies, D.G., J.L. Thomas and E.N. Smith (1982). Effect of body temperature on ventilatory control in the alligator, J. Appl. Physiol. 52: 114-118. Douse, M. A,, F.L. Powell, W.K. Milsom and G.S. Mitchell (1989). Temperature effects on pulmonary receptor responses to airway pressure and CO2 in Alligator mississipplensls. Respir. Physiol. 78:33 !-344. Douse, M.A. and G.S. Mitchell (1991). Time course of temperature effects on arterial acid-base status in Alligator misslssipplensis. Respir, Physiol. 83: 87-102. Funk, G. D. and W.K. Milsom (1987). Changes in ventilation and breathing pattern produ©edby changing body temperature and inspired CO2 concentration in turtles. Respir. Physiol. 67: 37-51. Gallego, R., C. Eyzaguirre and L. Monti-Bloch (1979). Thermal and osmotic responses of arterial receptors. J. Neurophysiol. 42: 665-680. Gatz, R.N., M.R. Fedde and E.C. Crawford (1975). Lizard lungs: CO2-sensitive receptors in Tupinambis nigropunctatus. £xperientia 31: 455-456. Gratz, R.K. (1984). Effect of bilateral vagotomy on the ventilatory responses of the water snake, Nerodia sipedon. Am. J. Physiol. 246: R221-R227. Hitzig, B.M. (1982). Temperature-induced changes in turtle CSF pH and central control of ventilation. Respir. Physiol. 49: 205-222. Jackson, D.C., S.E. Palmer and W.L. Meadow (1974). The effects of temperature and carbon dioxide breathing on ventilation and acid-base status of turtles. Respir. Physiol. 20: 131-146. Johnson, C.R., W.G. Voigt and E.N. Smith (1978). Thermoregulation in crocodilians - Ill. Thermal preferenda, voluntary maxima, attd heating and cooling rates in the American alligator, Alligator mississippiensis. Zool. J. Linn. Soc. 62: 179-188. Jones, D. R. and O. S. Bamford (1976). Open-loop respiratory chemosensitivity in chickens and ducks. Am. J. Physiol. 230: 861-867.
76
M.A. DOUSE AND G.S. MITCHELL
McNease, L. and T. Joanen (1974). A study ofimmature alligators on Rockefeller Refuge, Louisiana. Proc. Southeastern Assoc. Game and Fish Commissioners Conf. 28: 482-500. Milsom, W.K. and D.R. Jones (1979). Pulmonary receptor chemosensitivity and the ventilatory response to inhaled CO2 in the tunic. Respir. Physiol. 37: 101-107. Milsom, W.K. and D.R. Jones (1980). The role of vagal afferent information and hypercapnia in control of the breathing pattern in Chelonia. J. Exp. Biol. 87: 53-63. Mitchell, G. S. and ,l. L. Osborne (! 979). Ventilatory responses to carbon dioxide inhalation after vagotomy in chickens. Respir. Physiol. 36: 81-88. Nolan, W. F. and H. M. Frankel (1982). Ventilatory responses to CO2 at different temperatures in the snake, Coulber constricter. Experientia 38: 943-945. Perry, S.F. (1988). Functional morphology of the lungs of the nile crocodile, Crocodylus niloticus: nonrespiratory parameters. J. Exp. BioL 134:99-117. Peterson, D.F. and M.R. Fedde (1971). Avian intrapulmonary CO2-sensitive receptors: A comparative study. Comp. Biochem. Physiol. 40A: 425-430. Shannon, R. (1986). Reflexes from respiratory muscles and costovertebral joints. In: Handbook of Physiology, Section 3: The Respiratory System, Vol. 2; edited by A.P. Fishman, N.S. Cherniack, .I.G. Widdicombe and S.R. Geiger. Washington, DC: American Physiological Society, pp. 431-447. Shelton, G., D.R. Jones and W.K. Milsom (1986). Control of breathing in ¢ctothermic vertebrates. In: Handbook of Physiology, Section 3: The Respiratory System, Voi, 2; edited by A.P. Fishman, N.S. Cherniack, J.G. Widdicombe and S.R. Geiger. Washington, DC: American Physiological Society, pp. 857-909. West, ,l. B. (1985). Respiratory Physiology. The Essentials. Third Edition. Baltimore: Williams and Wilkins, 183 pp.