- Email: [email protected]
Ventilatory response to hypoxia and hypercapnia in the torpid bat, Eptesicus fuscus
Respiration Physiologr, 88 (1992) 217-232 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0034-5687/92/$05.00
217
RESP 01887
Ventilatory response to hypoxia and hypercapnia in the torpid bat, Eptesicusfuscus Joseph M. Szewczak and Donald C. Jackson Division of Biology and Medicine. Brown University. Providence, Rhode Island. U.S.A.
(Accepted 6 January 1992) Abstract. Ventilatory pattern and ventilatory responses to hypercapnia and hypoxia were investigated in torpid big brown bats at body temperatures of 5, 10, 20. 30 and 37 °C. The pattern of breathing at temperatures below 30 °C was intermittent, consisting of rhythmic breathing bouts separated by apneic periods with occasional sporadic, non-rhythmic breathing episodes. Overall ventilation (~te) was matched consistently to overall oxygen consumption (1~!o.,)over the entire range of temperatures with a mean air convection requirement (f,/e/l~lo:) of 1.28 L/mmol. However, calculating the air convection requirement using only oxygen uptake acquired during ventilation yielded an ectotherm-like temperature relationship. Ventilation was stimulated at all temperatures by either increased inspired CO., or decreased inspired 02. At 20 ° C, graded hypercapnic stimulation increased the duration of the rhythmic bouts and decreased the duration of apneas until at high CO., (> 3°~,)breathing was continuous. Hypoxic stimulation below about 7°, 02 increased ventilation by selectively increasing the non-rhythmic ventilations and decreasing rhythmic bouts.
Control of breathing, hypoxia, hyp~rcapnia, torpidity (bat); Hypercapnia, eentilation, torpidity (bat); Hypoxia, ventilation, torpidity (bat); Mammal, bat (Eptesicusfuscus); Pattern of breathing, intermittent breathing, torpidity (bat)
During heterothermic torpor below a body temperature (Tb) of 30 oC the big brown bat, Eptesk'us./i,scus, breathes intermittently leading to significant oscillations in blood gases and pH. Samples collected at the end of ventilatory bouts reveal a heterotherm-like ApHa/ATb slope of -0.011 U/°C, whereas samples not correlated with ventilatory state yield a ApHa/ATb slope of -0.0054 U/°C, which is typical of heterothermic mammals (Szewczak and Jackson, 1992; Heisler, 1986). This animal's acid-base and ventilatory regulation strategies are difficult to interpret from these data. The objective of this study was to further understand this bat's heterothermic control strategy by investigating its ventilatory response to temperature and inspired gases. The ratio of ventilatory volume (Ve) to metabolic rate (lVlo:), termed the air con-
Correspondence to: Permanoat address: J.M. Szewczak, Deep Springs College, HC 72, Box 45001, via Dyer, Nevada 89010, U.S.A.
218
J.M. SZEWCZAK AND D.C. JACKSON
vection requirement (ACR) (Dejours et al., 1970), can provide insight into an air breathing animal's respiratory strategy with changing temperature (Jackson, 1971). At constant temperature, pulmonary ventilation generally varies directly with 02 consumption. This is manifested by a constant ACR and Paco_, throughout the range of euthermic metabolic scope (Dejours, 1981). As temperature changes, however, so do the various physicochemical relationships on which the blood acid-base state depends (e.g., blood gas solubilities, buffering capacities, and equilibrium constants). Therefore the Paco. set point would be expected to change with temperature. in general, ectothermic vertebrates employ an acid-base control strategy that defends the net charge state of proteins as temperature varies (Reeves, 1969). This strategy requires increasing convective ventilation (ACR) with decreasing Tb. In contrast, observations of heterothermic mammals reveal a nearly constant ACR as Tb varies (Heisler, 1986). Determining the ACR of torpid Eptesicusfuscus was complicated by non-ventilatory 02 uptake during apnea (J.M. Szewczak and D.C. Jackson, submitted). This study considered both the overall ACR and the separate ACR calculated within ventilation episodes.
Melhods
Animals. Big brown bats, Eptesicus./hscus, were managed as described in our acid-base study of Eptesicus./it~'cus (Szewczak and Jackson, 1992). Body temperature. Body temperature was measured as described in our acid-base study of l~'ptesicusJiescus (Szewczak and Jackson, 1992). A skin contact temperature probe was used for convenience and bvcausc rectal probes agitate the bats and prevent entry into torpor. EaT~erime,ud proced, re. The chamber used was previously described in detail (Szcwczak and Jackson, 1992). Bats were placed in the chamber at selected ambient temperatures to reach target Tbs of 5, 10, 20, or 3() ° C, while monitoring Tb, ventilation, metabolic rate, and ECG. At least 2 h of steady-state conditions were observed before acquiring data. During experiments, gas flowed through the chamber from pressurized tanks: at other times, room air flowed through the chamber to maintain the bat during Tb equilibration, setting of gas mixtures, and analyzer calibration (Fig. 1). Ventilatio. recordi, g. Ventilatory movements were acquired by whole body plethysmography (Drorbaugh and Fcnn, 1955; Malan, 1973). Respiratory movements were detected with a Grass PT5 differential pressure transducer. Incoming and outgoing chamber air passed through pneumotachs constructed by fitting 2.5 cm pieces of PE 10 tubing in 3 mm i.d. polyvinyl tubing (Tygon, # 3603), (Cameron, 1986). These pneumotachs permitted continuous airflow through the chamber while facilitating the detection of transient ventilatory pressure variations, i.e. they effectively functioned as low-pass
VENTILATORY RESPONSE, HYPOXIA, CO,,, TORPID BAT
219
bypass
bgpass .circuit
needle valves
T~
H20 ECG
room air
C02 absorbent
ebsorbanL
pressure ~transducer
needle valve
bypass carbon dioxide enolgzer ~ /
"--" differential oxggen onolgzer
Fig. !. Schematic diagram of physiological chamber arrangement for ventilation experiments.
filters. The ventilation trace was recorded on a Grass polygraph and simultaneously sent to a computerized data acquisition system which monitored ventilatory rate and pattern (IBM XT with Data Translation DT2800 A/D board, ASYST data acquisition and data management software)using software we developed. i
O.wgen c,msmnption. A continuous record of 02 consumption was acquired using an Applied Electrochemistry (Sunnyvale, CA)differential 02 analyzer (model S-3A)in an
220
J.M. SZEWCZAK AND D.C. JACKSON
open circuit arrangement. Both inspired and expired 0 2 concentrations were acquired by the data acquisition system and used to calculate an instantaneous 02 consumption (Depocas and Hart, 1957; Hill, 1972). This data was displayed during the experiment, and stored for later analysis. Flow rate through the chamber was 50 mi per min for most experiments. The internal air volume of the chamber was approximately 83 ml. The bat's perch and sensors displaced about 10 ml, and with a 20-25 g bat the air volume in the chamber was on the order of 50 ml. When smaller bats (15-20 g)were placed in the chamber an insert was used that displaced about 5 ml, so the air volume remained about 50 ml. Tubing (60 c m x 0.3-cm lumen) and fittings connecting the chamber to the O 2 analyzer added about 5 ml. With this arrangement, the response lag of the 02 analyzer was on the order of 0.5 min, and provided a sensitivity sufficient to reveal 02 uptake from single breaths. Gas mixtures were adjusted with a needle vaive/flowmeter mixer. Following the mixer the flow split into two circuits, a reference circuit and the chamber circuit. The calibration procedure consisted of setting the differential 02 analyzer to a zero difference using the identical gases and flows for the experiment. The chamber itself was bypassed during calibration to avoid removing and disturbing the torpid bat, which was provided with an auxiliary supply of room air during this procedure. The bypass circuit was adjusted to an equivalent flow resistance as the chamber. This calibration procedure yielded a zero metabolic rate when tested with a ,/~mx bat in the chamber. When gas mi×tures werc changed, there was a time lag as the gas resident in the circuit was flushed. Th~ progression of gas mixtures to equilibrium was monitored by graphic display on the computer. When th~ desired mixtur~ was achieved, the flow rate was fine tuned by switching flow to the flowmctcr and adjusting th~ appropriate needle valve. Flow was th~n diverted from the flowm~t~r and the differential 0,, analyzer set to zero upon ~quilibrium, The experimental gas mixture was th~n switched to flow through the chamber, with the progression to equilibrium again monitored on the computer display as room air in the chamber was flushed out. Data records would be initiated only after a steady equilibriumwas reached. The flowmeter was not used continuously in the circuit because its volume attenuated the response to changes in O, uptake. Furthermore, the small pressure pulsations caused by movement of the float added substantial baseline noise to the ventilation trace. To compensate, the time of flow adjustment was recorded, and the final flow and time was checked, ifthere was any variation, a linear drift was assumed and a correction made to the record. As flow rates with the system were quite constant, this was necessary less than 10",, ofthe time" and corrections were at most 5 °~ ofthe total. There /O was no measured difference in O: uptake with the flowmeter in the circuit compared with it out of the circuit. When hypcrcapnic mixtures wcrc used, the afferent and efferent CO: absorbers were bypassed during mixing and the flow went to a CO~ analyzer. When the desired CO~ concentration was achieved, the efferent CO, absorber was reconnected, flow rate adjusted, and calibration proceeded as above. Metabolic rate cafibration.
VENTILATORY RESPONSE, HYPOXIA, CO_., TORPID BAT
221
Trial runs of the set-up revealed that the differential 02 zero drifted linearly over the time course of a typical experiment. As a correction for this drift, the time of the zero calibration was noted, and a final check of the zero calibration was made at the end of the experiment, and that time recorded. A correction was then applied to the record to compensate for this drift. Data from experiments were not used if the end zero drift exceeded 10"o of the mean 02 differential.
Duration of acquisition. With most experiments, the duration of each experimental data acquisition was adjusted to obtain at least a dozen cycles of ventilatory bouts in each record. This eliminated complications from the cyclic nature of the intermittent ventilation. With normocapnic, normoxic conditions this required about 1 h at Tb = 20 °C. However, with normocapnic, normoxic conditions at Tb = 10 °C, this was not always possible within 4 h, the maximum file duration that could be managed by the data acquisition system. In such a case, the data was evaluated in phase with the ventilation/apnea cycle. With continuous ventilation, data was typically acquired for 20-30 rain. Calcu&tion of vemilatoO' and metabolic rates. Mean respiratory frequency (fR) was calculated from the entire count for an experimental record divided by that record's time. The respiratory frequency within a ventilatory about (fR*) was calculated from the breath count within a bout divided by that bout's duration. Instantaneous metabolic rate (IVlo:) was acquired every millisecond, from which 5 s means were calculated and stored. Mean metabolic rates were calculated by integrating with respect to time. Steady-state apneic sections from the continuous O., consumption recordings were separately integrated with respect to time to determine rates of apneic O2 uptake. From the ventilation recordings, the time fraction apneic was calculated, and the apneic 02 uptake rates were then applied to determine the total contribution of apneic O2 uptake. Use of resp/ratory rate halieu o./'mhnae vo&me. The plethysmograph was not accurately calibrated for the determination of volume changes on the scale elicited from this animars respiratory movements. However, within the ranges used in this study (except for severe hypoxia and hypercapnia at Tb = 5 °C) average tidal volumes appeared to remain constant, with rates adjusted by changes in inter-ventilation periods. It has been reported that reptiles and hibernating ground squirrels adjust ventilation primarily by changing the total non-ventilatory period upon exposure to hypercapnia and hypoxia (Milsom, 1988). Hibernating ground squirrels have been shown to increase tidal volume (VT) in response to hypercapnia (Milsom et al., 1986; Milsom, 1988), but a VT change was not apparent in Eptesicusfuscus. Further supporting evidence for this is a constant metabolic cost per breath over the range of respiratory frequencies used in this study (unpublished observation). Thus, it seems reasonable to use fR as an indicator of overall volume of ventilation (Ve) in Eptesicusfuscus. For purposes of comparison, an assumed VT was used to estimated ~'e. A physiological scaling equation for mammalian VT yields a value of 0.1125 mi for 15-g
222
J.M. SZEWCZAK AND D.C.*JACKSON
bats (Mead, 1960). (Although bats gain 5-10 g of body weight in captivity, it is from gaining fat content; there is no reason to expect such a gain affected respiratory morphometry; 15 g is the normal body weight of freshly captured bats), Allometric scaling of total lung volume in bats indicates bat lung volumes to be 1.54 times greater than terrestrial mammal lung volumes for 15-g animals (Maina and King, 1984). Applying this correction factor to VT yields 0.173 nil. As a check, we can use the alveolar gas equation:
' (0.7)'~] PAco, = RT'\(1(4o, ~:--~ where 0.7 is the respiratory quotient, R is the gas constant, T is absolute temperature, and PAt, o: is the alveolar CO2 partial pressure. If the arterial CO2 partial pressure (Pa¢,o,) is assumed to be equivalent to the alveolar value, then there are sufficient values to solve for VT (Szewczak and Jackson, 1992). For Tb = 30 °C, at which ventilation was nearly continuous, VT solved to be 0.170 ml. Taking the arithmetic mean of ventilatory and apneic Paco,S at Tb - 20 °C, and solving for VT yields 0.165 ml. So, a value of 0.17 ml was a used as an estimate for VT in calculating ~'e.
Results Re.~7~h'ato~v response to temperature. Overall ventilation remained well coupled to ovm'all met:tboli¢ rate from Tb -~ 5 to 37 °C with a mean ACR of 1.21 L/mmol (Table 1). This is herein referred to as 'standard ACR'. 'Ventilatory ACR' is calculated using O, uptake acquired exclusively front ventilation. From T b - 5 to 20 °C, ventilatory ACR differed fi'om standard ACR. However, for Tb > 30 °C they were equivalent,
TABLE I Metabolic and ventilatory r~tes, and ACRs in the torpid bat, Eptesicusfuscus, at various body temperatures Tb °C
Respiration rate (breaths/rain)
Minute volume (ml/g, h)
Total metabolic rate #reel 0,4(8' h)
Standard ACR (L/retool)
Ventilatory ACR (L/retool)
5 10 20 30 37
3.63 + 0,89 (3) 3,30 + 0,73 (5) 12,2 +0,9 (25) 61,5 + 5,52 (5) 132 (I)
1.87 ± 0.47 1.37 +0.21 5.67 + 0.61 24.8 + 3.9 50.2
1.21 + 0.29 (4) 1.51 +0.11 (4) 4.76 + 0.32 (24) 18.7 + 2.0 (4) 423 (I)
1.53 ± 0.29 (3) 0.99+0.26(4) 0.99 + 0.11 (9) 1.36 ± 0.26 (4) !.18 (I)
2.36 ± 0.12 (3) 2,27 ± 0.32 (5)
1.37 ± 0.19 (9)
Minute volume is calculated from an estimated Vr (see Methods). Standard ACR is calculated from total oxygen uptake, includhtg non-ventilatory (see text). Ventilatory ACR is calculate ] using only oxygen uptake from ventilation. Data are from 8 bats; _+SE (el).
VENTILATORY RESPONSE, HYPOXIA, CO_~, TORPID BAT
293
3.0
- - o2.5
~...
.... B-- Vent. ACR
........ ""--o
2.0
.---l-- Turtle ACR lelmloQlll °
§
ggZ
¢0
~
]~
Std. ACR
T'"'°'-..
i
i
i
i
u
a
o
e
u
l
l
T
e
i
n
-1:
e
e
me~
qb
0.5 0.0
. . . .
0
I
5
-
-
-
'
l
l
10
.
.
.
.
l
.
15
*
l
.
l
.
20
.
l
l
25
l
l
,
l
,
.
.
.
30
1
1
35
ii
I
40
Body Temperature (°C) Fig. 2. Ventilatory ACR and standard ACR ofthe torpid bat, Eptesicusfuscus, at Tb = 5, 10, 20, and 30 °C and the ACR of the turtle Pseudemysscripta elegans for comparison (Jackson, 197 I). The linear regression ofventilatory ACR vs Tb is more consistent than the standard ACR, r 2 = 0.87 and r 2 - 0.016, respectively. Minute ventilation is calculated from an estimated VT (see Methods). Data are from 8 bats.
because there is no significant apneic 02 uptake, i.e. when 02 uptake is unimodal, standard ACR and ventilatory ACR are equivalent. The slope of the ventilatory ACR v~"Tb is -0.0391 L/(mmol. °C), r 2 = 0.87, which is somewhat less than the slope of a representative ectotherm, the turtle Pseudeno's scripta elegans, -0.0589 L/(mmol. °C), r 2 = 0.96 (Jackson, 1971)(Fig. 2). However, the bat's ventilatory ACR vs Tb slope is greater than, and more consistent than, that of its standard ACR' - 0.0022 L/(mmoi. oC), r-" - 0.016. Although the turtle is also an intermittent breather, it is submerged between ventilatory bouts with little potential for non-ventilatory 02 uptake (although it may have some cutaneous CO2 exchange). Thus, the standard ACR for the turtle may be considered equal to its ventilator), ACR by the above definition.
Normal breathing pattern. Above Tb --- 30 • C Eptesicusfuscus breathed continuously in a pattern typical of many euthermic mammals. Short apneas first became noticeable at about Tb - 30 °C which increased in duration as Tb decreased down to about 10 oC. When breathing intermittently Eptesicus fuscus exhibited two qualitatively distinct patterns of ventilation. The first is a rhythmic pattern characteristic of intermittent ventilatory bouts. This pattern had a rather consistent VT and evenly spaced breaths. The quality of the second pattern was sporadic, less rhythmic, and exhibited more variation in VT. At Tb = 20 °C a typical normoxic, normocapnic respiratory cycle included both patterns: a rhythmic bout, followed by a period of apnea with perhaps one to three sporadic breath events prior to resuming another rhythmic bout (Figs. 3 and 4, top trace). Sporadic breaths were less common below T b - 20°C. These patterns were consistent among the 8 bats observed in this study. In general, relatively
224
£M. SZEWCZAK AND D.C. JACKSON
.~ 1 minute
I ~.. _._ ,._
..
~_ { ~F r ' . . . . . .
~ IF
;1. . . . . . . . . _. . . . . . . . . . . . . l~In'~ . . . . - I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
P1co2 : 0.0 torr
~iI~ . I~n . . . . . . . .
t lr
. "
~L ,, - . . . . . . ,,,,~..,,,,~ I r'- '" ' l i l ~ ....... ~'""~....
13 breaths/ m i n u t e
Ptco= = 15.3 torr
55 breaths/ minute
PICa2
:
120 breaths/ m i n u t e
Ptco~
: 32.0 torr
22.9 torr
236 breaths/
minute
Fig. 3. Ventilatory response to hypercapnia in Eptesicusfuscus at Tb = 20 °C.
1 minute ..... .
.
.
.
.
.
.
.
ILl ......... lF ~ .
.
.
.
It TF
....................
Plea = 162 torr
___LLifL~,_JI~]._ L Ill [i ~ d iI1~_ _ 1'11
~'.-
q'"-
"-,~"-vq
~l-~l
tl p c , - . -
PloI " 75,4 torr
L[,
.... __ _ _ IL . . . .
tL
....
13 b r e a t h s / m i n u t e
..,
_~_,!ll,l .... •r !
.. I~I_ 'T
L_]L i ! -ll'-IW'
-
l lI.Jl._ll I I~ 7w"]ll-
II ~l I' I~1
ill ~||
Ii ~r
dlill r ~
9 breaths/minute
AI.I.uI~JaJ k_a al._|.k ~jl It ~1J di• ~l t tdiL_ll J. | J i I d it l.~_t _a IJoL | i, j| .tJI I .|. l_ ~t | . i l . r'"'~""'v"
"~"'w'-r"w'-r'F"'m
I" "
T ' " ~ ' P'.r ', ~p',r ~e It' n I ~ " v ' . - W ' v ' " ~
~ , ~ ' I~ m 1 " P
Plos -" S6,0 torr
19 breaths/ m i n u t e
Pto~ = 30.3 torr
30 breathe/ minute
IV' w - ~ ' - w " - ~ p
w' . '
"~ - ~ v ~
t~.,,~
n'-Vv" ~ ' "
Fig, 4, Ventilatory response to hypoxia in Eptesicusfuscus at Tb = 20 QC,
longer apncas wcrc rollowcd by relatively longer ventilatory bouts. Ventilatory bouts averaged 1.0 + 0,1, 6.3 + 1.2, and 0.90 + 0.16 rain for Tb = 20, 10, and 5 °C, respectively. The intervals between ventilatory bouts (not counting occasional sporadic breaths) were 6.1 +0.35, maximum: 13.7; 56.7 + 15.3, maximum 147; and 6,5 + 0,8: maximum: 40.9 rain for Tb = 20, 10, and 5 °C, respectively.
VENTILATORY RESPONSE, HYPOXIA, CO2, TORPID BAT
225
Respirato:y response to graded hypercapnia. The overall duration of the rhythmic venti!atory bouts, and the frequency of breaths within them (fR*), increased in response to hypercapnia (Fig. 3). Additionally, as hypercapnia progressed, the apneic periods shortened, and sporadic breathing events were gradually eliminated from the remaining TABLE 2 Ventilatory response to hypercapnia in the torpid bat, Eptesicusfuscus, at Tb = 5, 10, 20, and 30 °C Tb
Inspired ?0CO,
Respiration rate (breaths/min)
Instantaneous respiration rate (breaths/rain)
n
5 5
0.00 + 0.00 5.80 + 0.80
3.63 + 0.89 51.8 + 0.2
34.4 + 2. ! 51.8 + 0.2
3 2
10 10
0.00 + 0.00 5.04 + 0.04
3.30 + 0.73 87.1 + 3.4
33.3 + 3.7 87.1 + 3.4
5 5
20 20 20 20 20
0.00 + 0.00 2.40 + 0.14 3.28 + 0.18 4.13 + 0.09 5.1q+0.07
12.2 + 0.9 52.5 + 5.5 82.5 + 13.2 136 + 43 172 +22
61,7 89.4 87.2 143 172
+ 1.8 + 7.8 + 5.6 + 38 +22
17 7 4 4 7
30 30
0.00 + 0.00 5.10± 0. I0
61.5 + 5.5 141 + 23
72.3 _+ 2.5 141 + 23
4 2
Instantaneous respiration rate is the rate within a ventilatory bout. Data are from 8 bats, + SE.
Eptewlcua fuacue 30 25 20
[]
30 °
0
20 °
¢
10 °
gg °
5°
15 • • ••
-
8e Q
human
10
5
tO, ; ? ? , • o, ,. : ' "
.
•-
0 ° m •
.:'::~. "-,;.......,.........,......'.',..'::: ........ "6
0 0
I
2
3
4
5
6
7
Inspired %C02 Fig. 5. Respiratory response to hypercapnia in the torpid bat, Eptesicusfuscus, at Tb = 5, 10, 20, and 30 °C. Change in respiratory rate is calculated in reference to the rate without CO2 for individual bats. Human data is from Davies et al. (1925). The regression line is calculated from the Tb = 20 °C data, Data are from 8 bats.
226
J.M. SZEWCZAK AND D.C. JACKSON
apnea. Eventually, breathing became continuous, and additional increases in hypercapnia continued to increase .fR. The quantitative ventilatory response to hypercapnia at Tb = 5, 10, 20, and 30 °C is presented in Table 2. The change in fR in response to hypercapnia is calculated with respect to each bat's mean fR in the absence of CO2, and is depicted in Fig. 5 with a typical euthermic (human) response included for reference (Davies etal., 1925). Similarly, change in the respiratory frequency within a bout, fR*, is normalized to each bat's mean ./R* without CO2, and is shown in Fig. 6 against the same euthermic
Epteelcue fuecus .i
ill
ii. |
i.i
i.
i
•
.
l * * l i i J ~ , l ,
0
I
.
~
2
.
J
.
.
.
.
.
J
l
l
i
*
.
.
a
3
.
i
.
,
.
*
.
l
.
l
.
i
4
Inspired
.
.
.
*
.
.
.
.
.
.
~
l
l
l
l
l
5
""
n
30 °
0
20 °
O
10 °
A
5° •
human
l
6
7
%COs
Fit~.6. Cha,se in i,statlttuleous respiratory rate (./k within a bout) in response to hypereapnia in the torpid bat, L:pt¢.vi¢,s/itst.,s, at Tb ~ 5, 10, 20, and 30 '~C. Cha,se in instantaneous respiratory rate is calculated in ret~rence to the rate without COz for individual bats. Human data is from Davies et al. (1925). The ret;r~ssio, line is calculated from the Tb ~ 20 ~C data. Data are from 8 bats. TABLE 3 Ventilatory response to hypoxia in the torpid bat, Eptesicusfuscus, at Tb = 20 °C Inspired % O2
R~spiration frequency (breaths,'min)
Change in respiration frequ~,:ncy
21,0 + 0,0 10,0 + 0,0 7,30 + 0,10 6.33 + 0.12 5,10 + 0,10 4,05 _+0,05 3.00
12,7 + 0,98 10,5 + 1,75 12,6 + 3,37 17.9 + 4.92 27,4 + 4,75 62,4 + 1,40 87.7
1,0(', + 0.00 0,08 0,9~i + 0.04 1.39 + 0.28 2,28 :!: 0.19 3,88 t 0.39 5.83 (),8(,~ +
21 6 2 3
4 2 1
Change in respiration frequencyis in reference to the t~equencyofth~,"same animal breathing 21% 02. Data arc from 5 bats, + SE,
VENTILATORY RESPONSE, HYPOXIA, CO,, TORPID BAT
227
Eptesi©us fuscus 7 6
o
Tb=20°C
s
m
O. sO
Q or
2 1
.
O
.
0
. .
.
.
.
. I
.
, ........ .
.
5
.
a
10
i
i
,
•
. I
•
15
•
- ~'0-- . . . . ,
•
a
20
,
.
~
|
25
Inspired %0= Fig. 7. Respiratory respo.lse to hypoxia in the torpid bat, £ptesicusf , scus, at Tb = 20 °C. Change in respiratory rate is calculated in reference to the rate with normoxia for individual bats. The respiration rate at 10°o inspired O, is significantly less than normoxia; P < 0.05. Data are from 5 bats.
response. Note that the change in fR* is more consistent to the euthermic response than is the change in ./k.
Respiratoo' response to graded hypoxia,
in response to hypoxia, the duration of the rhythmic bouts decreased and were superseded by an increase in non-rhythmic ventilations. At lower O., concentrations, even, rhythmic breathing was entirely supplanted by the uneven pattern of breathing, and the elimination of apneas (Fig. 4). At Tb -- 20 ° C, hypoxia did not elicit a significant ventilatory increase until inspired O., dropped to about 6"~, (48 Torr). The respiratory response to hypoxia at Tb - 20 °C is normaUzed to individual mean normoxic fR and presented in Table 3, and displayed in Fig. 7. The respiratory rate at 10"o~ inspired O2 was significantly less than the rate at 21 ",, inspired O.,, P < 0.05. There was no noticeable change in respiratory rate and pattern between pure 02 and 2 1 ° O2.
Discussion
Respiratory response to temperature.
Eptesicus fuscus main:ained a close match of
overall ventilation with overall metabolic rate through[~¢~ its range of physiologic Tbs (Table 1). This suggests a ventilatory strategy consistent with observations of other mammalian heterotherms (Reeves, 1969; Musacchia and Volkert, 1971; Malan et al., 1973; Heisler, 1986). However, below T b - 30 °C, ventilation is not steady-state in Eptesicusfi¢scus, and its control strategy cannot necessarily be assessed from the average effect reflected by this 'standard ACR'. Certainly, a steady-state ACR could not generate the observed oscillations in acid-base state and blood gases.
228
J.M. SZEWCZAK AND D.C. JACKSON
By definition, the ACR is calculated from the total volume of ventilation divided by the total 02 uptake (Dejours et ai., 1970). Ordinarily, this relates ventilatory activity to the animal's 02 requirement. However, Eptesicus ~lscus acquires a substantial portion of its total 02 uptake (but not CO2 elimination) by non-ventilatory airway diffusion during apnea (J. M. Szcwczak and D.C. Jackson, submitted). Thus, this bat becomes a bimodal breather in torpor. A calculation of the ACR including the apneic 02 uptake does not accurately characterize the ventilatory controller relative to metabolism, because it is incorrectly attributing 02 uptake to vcntilatory action. Instead, a calculation of the ACR using only gas exchanged during ventilation, i.e. the 'ventilatory ACR', reflects the operation of the vcntilatory controller and the parameters to which it is responding. The temperature dependency of ventilatory ACR in Eptesicus fuscus is more characteristic of an cctothcrm (Fig. 2). This is consistent with the end-ventilatory acid-base data which also exhibit an cctothermic-like relationship (Szewczak and Jackson, 1992). These data suggest that this animal overventilates with respect to this O.~ requirement during ventilatory bouts; i.e. although its 02 needs are satisfied, it continues ventilation until a satisfactory Paco, is achieved. That the ventilatory bouts are CO.,-depcndent rather than O,-dependent is further supported by the rhythmic pattern of ventilation, which is characteristic of hypercapnic stimulation (see below). Unfortunately, we were not able to measure I¢1co: with the equipment available for this study. Following the respiratory gas quotient through a ventilatory bout would be infimuative. Calculating the ACR using IVl<,o~instead of 1¢1o:would obviate the complication of passive apneic gas exchange, A study of central neural control of respiration in hypothermic cats concluded that the respiratory responses to altered Tb could be entirely explained by temperature effects of neurons, rather than by any o,'ganismal effort to maintain a constant protein charge state (Kiley et al., 1984). The heterothermic response to hypercapnia of Eptesicusfuscus contradicts that conclusion, because its hypercapnic response was uniformly temperature-indepe,~dent from Tb = 10-30 °C (and also perhaps at 5 °C, see below). The temperature-independence of Eptesicus,/iescus' CO2 response suggests a feedback loop linked to protein charge state, distinct from neuronal temperature effects. The contradictory response observed in hypothermic cats may arise from their lack of adaptation to heterothermy. Because hypothermia is not a natural state for that species, its respiratory controller may be incapable of defending homeostasis under such an abnormal condition. Pam, r, ¢~.l'breaehi,g. The pattern ofintermittent breathing in Eptesic~,.sfuscus is different in some respects from other torpid mammals. Golden hamsters, Mesocricetus auratus (Kristofferson and Soivio, 1966), hedgehogs, Erinaceus europaeus (Pembrey and Pitts, 1899; Kristofferson and Soivio, 1964, 1967; Tllhti, 1975), dormice, Myoxus avellanarius (Pembrey and Pitts, 1899); Elion[vs quercinus L. (Pajunen, 1970, 1974), and marmots, Marmota marmota (Pembrey and Pitts, 1899), have all been shown to displ,~y a pattern of intermittent breathing characterized by a bout of rhythmic breathing followed by
VENTILATORY RESPONSE, HYPOXIA, CO2, TORPID BAT
229
uninterrupted apnea until the next bout of rhythmic breathing. Eptesicus fuscus differs from these animals by the presence of sporadic, non-rhythmic breaths interspersed during apnea (Figs. 3 and 4, top trace). Some similar sporadic breathing appears during torpor in the ground squirrel, Spermophilus lateralis (Milsom et al., 1986; Riedesel etal., 1986), but the overall pattern is not as well defined and repeating as in Eptesicusfuscus. At Tb below 6 °C, this ground squirrel changes from intermittent breathing to single breaths (Miisom et ai., 1986); with a similar change induced from mild anesthesia at higher temperatures (Milsom, 1991). Eptesicus fuscus maintained its intermittent breathing pattern down to Tb = 3 °C; however, mild anesthesia also induced a single breath pattern as high as Tb = 25 °C.
Respirator3, response to graded hypercapnia. Hibernators have previously been characterized as insensitive to hypercapnia or of possessing a deficiency of respiratory regulation (Rasmussen, 1916; Kayser, 1961). Subsequent work has found this to be an inaccurate generalization. Hibernating hamsters (Mesocricetus auratus) have been shown to respond to hypercapnia similarly to humans (Lyman, 1951). However, hibernating ground squirrels and marmots do not respond to hypercapnia as precisely or predictably as other hibernators, possibly owing to their fossorial adaptation and a blunted CO., response (Endres and Taylor, 1930; Lyman, 1951; Milsom et aL, 1986; Riedesel etaL, 1986). Ground squirrels decrease the apneic period, and increase the duration of the ventilatory period and ./R* (Riedesel etal., 1986; Milsom etaL, 1986). In hibernating hedgehogs, Bi6rck et al. (1956) found variations in their hypercapnic response. However, T'ahti (1975) found that 5% CO2 was sufficient to abolish intermittent breathing in hibernating hedgehogs, and that apneic periods began to shorten with as little as 1% CO.,, but the ventilatory period remained constant. The overall frequency response to hypercapnia in Eptesicus fuscus depended on vcntilatory stat0. Continuously-ventilating bats at Tb = 30 ° C were similar to the typical euth0rmic hypercapnic response, whereas intermittently-ventilating bats responded with a much greater increase in fR (Fig. 5). Instantaneous respiratory rates, however, all fall near the normal euthermic response, except for Tb = 5 ~C (Fig. 6). At this temperature, the bats failed to increase fR from 5 % to 6.6% inspired CO2, and instead appeared to increase VT to augment Ve. Additionally, the respiratory movements (as registered on the polygraph trace) wore noticeably sluggish. Thus, the actual ~/e at Tb = 5 °C may fall along the relationship with the others if Ve could have been directly measured. The ventilatory control of intermittent breathing may be presumed to switch between 'on' and 'off' states based upon thresholds of the integrated status of blood gases and pH. By this reasoning, an animal's 1Vie:and setpoints for those thresholds will influence the duration of apneas, and consequently the fR. We believe this complication may be alleviated by using fR* as a basis for comparing ventilatory responses, particularly between continuous and intermittent-breathing. For the intermittent breather, the fR* might be considered a direct indication of the urgency to breathe when the system is switched to the 'on' state. Viewed by this rationale, the hypercapnic response in torpid,
230
J.M. SZEWCZAK AND D.C. JACKSON
intermittently-breathing Eptesicus fuscus is not inherently different from euthermic, continuously-breathing mammals (Fig. 6). Respiratory response to graded hypoxia. Inconsistencies exist in the literature concerning hypoxic responsiveness among torpid mammals. Riedesel et al. (1986) exposed hibernating ground squirrels (Spermophilus lateralis) to 100~o N2, and observed no significant respiratory response. However, Milsom et ai. (1986)found a graded hypoxic response in two species of ground squirrels (Spermophilus lateralis and Spermophilus cohmlbitmus) that correlated with the temperature dependent shift of the O 2 dissociation curve. BiOrck et al. (1956)exposed hibernating hedgehogs to 100~o N2 and reported no significant respiratory response during exposures of 50-120 min duration. In contrast, T',lhti (1975) found a graded hypoxic response in hibernating hedgehogs, with respiration becoming continuous with 3)o inspired 02, a similar response to that observed in Eptesicusfuscus by this study. These discrepancies may result from varying levels or depths of torpor, similar to the documented differences in respiratory control during the various levels of sleep in euthermic mammals (Walker and Berger, 1988). Epu,sicusfu.~cus exhibited a sensitive response to hypoxia during torpor at Tb = 20 ° C (Table 3). Although the response to inspired Po., < :50 Torr was brisk, little response occurred until inspired O2 dropped to about 75 Torr. This may be explained by considering the expected left shift of the O2 dissociation curve at Tb = 20 °C compared to euthermi:t. There was actually a slight decrease in ventilation with moderate hypoxia which was associated with an increase in non=rhythmic breaths, and a decrease in the duration of apnea (Fig. 7). The shorter, more frequent ventilation pattern of mild hypoxia probably supports lower tissue Pco~ levels compared with the longer apneas of normoxia, which require extended ventilation episodes to release this tissue store (refer to ACR discussion above). Thus, a more even exchange of O2 and COa is maintained, it should be noted there was no measurable reduction of l~'lo, during hypoxia that could account for the reduction in ~'e with moderate hypoxia.
Com'h~shm, The temperature dependence of the airway convection requirement in Eptesicus/it~'cusis similarto that of ectotherms ifit is calculated using oxygen uptake exclusivelyfrom activeventilation,Hypercapnia and hypoxia both stimulateventilation, but are manifested by qualitativelydistinctpatterns.Overventilationwith respectto 02, and the patternof ventilationduring ventilatorybouts,indicatethat Pace _,isthe primary feedback variable controlling these bouts, The quality of the ventilatory patterns suggests that a hypercapnic response utilizesthe central rhythm generator more elegantly than does a hypoxic response. This may result from the integration of dissimilar chemoreceptor sites and their actions, or because hypoxia-stimulated b,'eathing is relegated to a secondary 'back up' role which does not warrant the organismal investment to support a smooth feedback/control loop.
VENTILATORY RESPONSE, HYPOXIA, CO.,, TORPID BAT
231
Acknowledgements. We thank Dr. Andr~ Malan for his comments and encouragement of this investigation. This work was supported by Grant DCB8802045 from the National Science Foundation (DO).
References Bi6rck, G,, B.W. Johansson and H. Schmid (1956). Reactions of hedgehogs, hibernating and non-hibernating, to the inhalation of oxygen, carbon dioxide, and nitrogen. Acta Physiol. Scand. 37: 71-83. Cameron, J.N. (1986). Principles of Physiological Measurement. Orlando, FL: Academic Press, pp. 169-170. Davies, H.W., G. R. Brow and C. A.L. Binger (1925). The respiratory response to carbon dioxide. J. Exp. Med. 41: 37-52. Dejours, P., W. F. Garey and H. Rahn (I 970). Comparisons ofventilatory and circulatory flow rates between animals in various physiological conditions. Respir. Physiol. 9: 108-177. Dejours, P. (198 i). Principles of Comparative Respiratory Physiology. New York: Elsevier/North Holland Biomedical Press, 265 p. Depocas, F. and J.S. Hart (1957). Use of the Pauling oxygen analyzer for measurement of oxygen consumption of animals in a short-lag, closed-circuit apparatus. J. Appl. Physiol. 10: 388-392. Drorbaugh, J. E. and W.O. Fenn (1955). A barometric method for measuring ventilation in newborn infants. Pediatrics 16: 81-87. Endres, G. and H. Taylor (1930). Observations on certain physiological processes of the marmot. Ii. The respiration. Prec. R. Soc. (London) 107: 231-240. Heisler, N. (198o). Comparative aspects of acid-base regulation. In: Acid-Base Regulation in Animals, edited by N. Heisler. New York: Elsevier, pp. 397-450. Hill, R. W, (1972). Determination of oxygen consumption by use of the paramagnetic oxygen analyzer. J. Appl. Physiol. 33: 261-263. Jackson, D.C. ( 1971). The effect of temperature on the turtle, Pseudemys scripta elegans. Respir. PhysioL 12: 131-141. Kayser, Ch. (1961). The Physiology of Natural Hibernation. New York: Pergamon Press, 325 p. Kiley, P.K., F.L. Eldridge and D.E. Millhorn (1984). The effect of hypothermia on central neural control of respiration. Respir. Ph.vsiol. 58: 295-312, Kristoflbrson, R. and A. Soivio (1964). Hibernation in the hedgehog (£rinaceus europaeus L.). Ann. Acad. $ci. Fenn. 82: 1-17. Kristofferson, R. and A. Soivio (1966). Duration of hyperthermia periods and type of respiration in the hibernating golden hamster, Mesocrtcetus auratus Waterh. Ann. Zool. Fenn. 3: 66-67. Kristofferson, R. and A. Soivio (1967). Studies i~1the periodicity of hibernation the hedgehog (Erinacaeus europaeus L.) !I. Changes in respiratory rhythm, heart rate and body temperature at the onset of spontaneous arousals. Area. Zool. Fema. 4: 595-597. Lyman, C.P. (1951). Effect of increased CO, on respiration and heart rate of hibernating hamsters and ground squirrels. Am. J. Physiol. 167: 638-643. Maina, J.N. and A.S. King (1984). Correlations between structure and function in the design of the bat lung: a morphometric study. J. Exp. Biol. I I 1: 43-61. Malan, A. (1973). Ventilation measured by whole body plethysmography in hibernating animals and poikilotherms. Respir. Physiol. 17: 32-44. Malan, A., H. Arens and A. Waechter (1973). Pulmonary respiration and acid-base state in hibernating marmots and hamsters. Respir. Physiol. 17: 45-61. Mead, J. (1960). Control of respiratory frequency. J. Appl. Physiol. 15: 325-336. Milsom, W. K., M. D. McArthur and C. L. Webb (1986). Control ofbreathing in hibernating ground squirrels. in: Living in the Cold, edited by H.C. Heller, X.J. Musacchia and L. C. H. Wang. New York: Elsevier, pp. 469-475. Milsom, W.K. (1988). Control of arrhythmic breathing in aerial breathers. Can. J. Zool. 66: 99-108.
232
J.M. SZEWCZAK AND D.C. JACKSON
Milsom, W.K. (1991). Intermittent breathing in vertebrates. Annu. Rev. Physiol. 53: 87-105. Musacchia, X.J. and W.A. Volkert (1971). Blood gases in hibernating and active ground squirrels: HbO2 affinity at 6 and 38 °C. Am. J.Physiol. 221: 128-130. Pajunen, !. (1970). Body temperature, heart rate, breathing pattern, weight loss and periodicity of hibernation in the Finnish garden dormouse, Eliomys quercinus L. Ann. Zool. Fenn. 7: 251-266. Pajunen, I. (1974). Body temperature, heart rate, breathing pattern, weight loss and periodicity of hibernation in the French garden dormouse, Eiiomys quercinus L. at 4.2 + 0.5 °C. Ann. Zooi. Fenn. 1I: 107-119. Pembrey, M. S. and A. G. Pitts (1899). The relationship between the internal temperature and the respiratory movements of hibernating animals. J. Physiol. 24:305-316. Rasmussen. A.T. (1916). A further study of the blood gases during hibernation in the woodchuck (Marmota marmcml ). The respiratory capacity of the blood. Am. J. Physiol 41: |62-172. Reeves. R. B. (1969). Role ofbody temperature in determining the acid-base state in vertebrates. Fed. Proc. 28:1204-1208. Riedesel, M.L., J.E. Oriego and D.C. Espinosa (1986). Respiratory patterns in hibernating Spermophilus t~#e)alis. In: Living in the Cold, edited by H.C. Heller, X.J. Musacchia and L.C.H. Wang. New York: Elsevier, pp. 461-468. Szewczak, J.M. and D.C. Jackson (1992). Acid-base state and intermittent breathing in the torpid bat, Eptesicus fuscus. Respir. PhysioL 88: 205-215. T',lhti, H. (1975). Effects of changes in CO, and 02 concentrations in the inspired gas on respiration in the hibernating hedgehog (Erinacaeus europaeus L.) Ann. Zool. Fenn. 12: 183-187. Walker, J.W. and R.J. Berger (1988). Sleep as an adaptation for energy conservation functionally related to hibernation and torpor. Frog. Brain Res. 53: 255-278.
217
RESP 01887
Ventilatory response to hypoxia and hypercapnia in the torpid bat, Eptesicusfuscus Joseph M. Szewczak and Donald C. Jackson Division of Biology and Medicine. Brown University. Providence, Rhode Island. U.S.A.
(Accepted 6 January 1992) Abstract. Ventilatory pattern and ventilatory responses to hypercapnia and hypoxia were investigated in torpid big brown bats at body temperatures of 5, 10, 20. 30 and 37 °C. The pattern of breathing at temperatures below 30 °C was intermittent, consisting of rhythmic breathing bouts separated by apneic periods with occasional sporadic, non-rhythmic breathing episodes. Overall ventilation (~te) was matched consistently to overall oxygen consumption (1~!o.,)over the entire range of temperatures with a mean air convection requirement (f,/e/l~lo:) of 1.28 L/mmol. However, calculating the air convection requirement using only oxygen uptake acquired during ventilation yielded an ectotherm-like temperature relationship. Ventilation was stimulated at all temperatures by either increased inspired CO., or decreased inspired 02. At 20 ° C, graded hypercapnic stimulation increased the duration of the rhythmic bouts and decreased the duration of apneas until at high CO., (> 3°~,)breathing was continuous. Hypoxic stimulation below about 7°, 02 increased ventilation by selectively increasing the non-rhythmic ventilations and decreasing rhythmic bouts.
Control of breathing, hypoxia, hyp~rcapnia, torpidity (bat); Hypercapnia, eentilation, torpidity (bat); Hypoxia, ventilation, torpidity (bat); Mammal, bat (Eptesicusfuscus); Pattern of breathing, intermittent breathing, torpidity (bat)
During heterothermic torpor below a body temperature (Tb) of 30 oC the big brown bat, Eptesk'us./i,scus, breathes intermittently leading to significant oscillations in blood gases and pH. Samples collected at the end of ventilatory bouts reveal a heterotherm-like ApHa/ATb slope of -0.011 U/°C, whereas samples not correlated with ventilatory state yield a ApHa/ATb slope of -0.0054 U/°C, which is typical of heterothermic mammals (Szewczak and Jackson, 1992; Heisler, 1986). This animal's acid-base and ventilatory regulation strategies are difficult to interpret from these data. The objective of this study was to further understand this bat's heterothermic control strategy by investigating its ventilatory response to temperature and inspired gases. The ratio of ventilatory volume (Ve) to metabolic rate (lVlo:), termed the air con-
Correspondence to: Permanoat address: J.M. Szewczak, Deep Springs College, HC 72, Box 45001, via Dyer, Nevada 89010, U.S.A.
218
J.M. SZEWCZAK AND D.C. JACKSON
vection requirement (ACR) (Dejours et al., 1970), can provide insight into an air breathing animal's respiratory strategy with changing temperature (Jackson, 1971). At constant temperature, pulmonary ventilation generally varies directly with 02 consumption. This is manifested by a constant ACR and Paco_, throughout the range of euthermic metabolic scope (Dejours, 1981). As temperature changes, however, so do the various physicochemical relationships on which the blood acid-base state depends (e.g., blood gas solubilities, buffering capacities, and equilibrium constants). Therefore the Paco. set point would be expected to change with temperature. in general, ectothermic vertebrates employ an acid-base control strategy that defends the net charge state of proteins as temperature varies (Reeves, 1969). This strategy requires increasing convective ventilation (ACR) with decreasing Tb. In contrast, observations of heterothermic mammals reveal a nearly constant ACR as Tb varies (Heisler, 1986). Determining the ACR of torpid Eptesicusfuscus was complicated by non-ventilatory 02 uptake during apnea (J.M. Szewczak and D.C. Jackson, submitted). This study considered both the overall ACR and the separate ACR calculated within ventilation episodes.
Melhods
Animals. Big brown bats, Eptesicus./hscus, were managed as described in our acid-base study of Eptesicus./it~'cus (Szewczak and Jackson, 1992). Body temperature. Body temperature was measured as described in our acid-base study of l~'ptesicusJiescus (Szewczak and Jackson, 1992). A skin contact temperature probe was used for convenience and bvcausc rectal probes agitate the bats and prevent entry into torpor. EaT~erime,ud proced, re. The chamber used was previously described in detail (Szcwczak and Jackson, 1992). Bats were placed in the chamber at selected ambient temperatures to reach target Tbs of 5, 10, 20, or 3() ° C, while monitoring Tb, ventilation, metabolic rate, and ECG. At least 2 h of steady-state conditions were observed before acquiring data. During experiments, gas flowed through the chamber from pressurized tanks: at other times, room air flowed through the chamber to maintain the bat during Tb equilibration, setting of gas mixtures, and analyzer calibration (Fig. 1). Ventilatio. recordi, g. Ventilatory movements were acquired by whole body plethysmography (Drorbaugh and Fcnn, 1955; Malan, 1973). Respiratory movements were detected with a Grass PT5 differential pressure transducer. Incoming and outgoing chamber air passed through pneumotachs constructed by fitting 2.5 cm pieces of PE 10 tubing in 3 mm i.d. polyvinyl tubing (Tygon, # 3603), (Cameron, 1986). These pneumotachs permitted continuous airflow through the chamber while facilitating the detection of transient ventilatory pressure variations, i.e. they effectively functioned as low-pass
VENTILATORY RESPONSE, HYPOXIA, CO,,, TORPID BAT
219
bypass
bgpass .circuit
needle valves
T~
H20 ECG
room air
C02 absorbent
ebsorbanL
pressure ~transducer
needle valve
bypass carbon dioxide enolgzer ~ /
"--" differential oxggen onolgzer
Fig. !. Schematic diagram of physiological chamber arrangement for ventilation experiments.
filters. The ventilation trace was recorded on a Grass polygraph and simultaneously sent to a computerized data acquisition system which monitored ventilatory rate and pattern (IBM XT with Data Translation DT2800 A/D board, ASYST data acquisition and data management software)using software we developed. i
O.wgen c,msmnption. A continuous record of 02 consumption was acquired using an Applied Electrochemistry (Sunnyvale, CA)differential 02 analyzer (model S-3A)in an
220
J.M. SZEWCZAK AND D.C. JACKSON
open circuit arrangement. Both inspired and expired 0 2 concentrations were acquired by the data acquisition system and used to calculate an instantaneous 02 consumption (Depocas and Hart, 1957; Hill, 1972). This data was displayed during the experiment, and stored for later analysis. Flow rate through the chamber was 50 mi per min for most experiments. The internal air volume of the chamber was approximately 83 ml. The bat's perch and sensors displaced about 10 ml, and with a 20-25 g bat the air volume in the chamber was on the order of 50 ml. When smaller bats (15-20 g)were placed in the chamber an insert was used that displaced about 5 ml, so the air volume remained about 50 ml. Tubing (60 c m x 0.3-cm lumen) and fittings connecting the chamber to the O 2 analyzer added about 5 ml. With this arrangement, the response lag of the 02 analyzer was on the order of 0.5 min, and provided a sensitivity sufficient to reveal 02 uptake from single breaths. Gas mixtures were adjusted with a needle vaive/flowmeter mixer. Following the mixer the flow split into two circuits, a reference circuit and the chamber circuit. The calibration procedure consisted of setting the differential 02 analyzer to a zero difference using the identical gases and flows for the experiment. The chamber itself was bypassed during calibration to avoid removing and disturbing the torpid bat, which was provided with an auxiliary supply of room air during this procedure. The bypass circuit was adjusted to an equivalent flow resistance as the chamber. This calibration procedure yielded a zero metabolic rate when tested with a ,/~mx bat in the chamber. When gas mi×tures werc changed, there was a time lag as the gas resident in the circuit was flushed. Th~ progression of gas mixtures to equilibrium was monitored by graphic display on the computer. When th~ desired mixtur~ was achieved, the flow rate was fine tuned by switching flow to the flowmctcr and adjusting th~ appropriate needle valve. Flow was th~n diverted from the flowm~t~r and the differential 0,, analyzer set to zero upon ~quilibrium, The experimental gas mixture was th~n switched to flow through the chamber, with the progression to equilibrium again monitored on the computer display as room air in the chamber was flushed out. Data records would be initiated only after a steady equilibriumwas reached. The flowmeter was not used continuously in the circuit because its volume attenuated the response to changes in O, uptake. Furthermore, the small pressure pulsations caused by movement of the float added substantial baseline noise to the ventilation trace. To compensate, the time of flow adjustment was recorded, and the final flow and time was checked, ifthere was any variation, a linear drift was assumed and a correction made to the record. As flow rates with the system were quite constant, this was necessary less than 10",, ofthe time" and corrections were at most 5 °~ ofthe total. There /O was no measured difference in O: uptake with the flowmeter in the circuit compared with it out of the circuit. When hypcrcapnic mixtures wcrc used, the afferent and efferent CO: absorbers were bypassed during mixing and the flow went to a CO~ analyzer. When the desired CO~ concentration was achieved, the efferent CO, absorber was reconnected, flow rate adjusted, and calibration proceeded as above. Metabolic rate cafibration.
VENTILATORY RESPONSE, HYPOXIA, CO_., TORPID BAT
221
Trial runs of the set-up revealed that the differential 02 zero drifted linearly over the time course of a typical experiment. As a correction for this drift, the time of the zero calibration was noted, and a final check of the zero calibration was made at the end of the experiment, and that time recorded. A correction was then applied to the record to compensate for this drift. Data from experiments were not used if the end zero drift exceeded 10"o of the mean 02 differential.
Duration of acquisition. With most experiments, the duration of each experimental data acquisition was adjusted to obtain at least a dozen cycles of ventilatory bouts in each record. This eliminated complications from the cyclic nature of the intermittent ventilation. With normocapnic, normoxic conditions this required about 1 h at Tb = 20 °C. However, with normocapnic, normoxic conditions at Tb = 10 °C, this was not always possible within 4 h, the maximum file duration that could be managed by the data acquisition system. In such a case, the data was evaluated in phase with the ventilation/apnea cycle. With continuous ventilation, data was typically acquired for 20-30 rain. Calcu&tion of vemilatoO' and metabolic rates. Mean respiratory frequency (fR) was calculated from the entire count for an experimental record divided by that record's time. The respiratory frequency within a ventilatory about (fR*) was calculated from the breath count within a bout divided by that bout's duration. Instantaneous metabolic rate (IVlo:) was acquired every millisecond, from which 5 s means were calculated and stored. Mean metabolic rates were calculated by integrating with respect to time. Steady-state apneic sections from the continuous O., consumption recordings were separately integrated with respect to time to determine rates of apneic O2 uptake. From the ventilation recordings, the time fraction apneic was calculated, and the apneic 02 uptake rates were then applied to determine the total contribution of apneic O2 uptake. Use of resp/ratory rate halieu o./'mhnae vo&me. The plethysmograph was not accurately calibrated for the determination of volume changes on the scale elicited from this animars respiratory movements. However, within the ranges used in this study (except for severe hypoxia and hypercapnia at Tb = 5 °C) average tidal volumes appeared to remain constant, with rates adjusted by changes in inter-ventilation periods. It has been reported that reptiles and hibernating ground squirrels adjust ventilation primarily by changing the total non-ventilatory period upon exposure to hypercapnia and hypoxia (Milsom, 1988). Hibernating ground squirrels have been shown to increase tidal volume (VT) in response to hypercapnia (Milsom et al., 1986; Milsom, 1988), but a VT change was not apparent in Eptesicusfuscus. Further supporting evidence for this is a constant metabolic cost per breath over the range of respiratory frequencies used in this study (unpublished observation). Thus, it seems reasonable to use fR as an indicator of overall volume of ventilation (Ve) in Eptesicusfuscus. For purposes of comparison, an assumed VT was used to estimated ~'e. A physiological scaling equation for mammalian VT yields a value of 0.1125 mi for 15-g
222
J.M. SZEWCZAK AND D.C.*JACKSON
bats (Mead, 1960). (Although bats gain 5-10 g of body weight in captivity, it is from gaining fat content; there is no reason to expect such a gain affected respiratory morphometry; 15 g is the normal body weight of freshly captured bats), Allometric scaling of total lung volume in bats indicates bat lung volumes to be 1.54 times greater than terrestrial mammal lung volumes for 15-g animals (Maina and King, 1984). Applying this correction factor to VT yields 0.173 nil. As a check, we can use the alveolar gas equation:
' (0.7)'~] PAco, = RT'\(1(4o, ~:--~ where 0.7 is the respiratory quotient, R is the gas constant, T is absolute temperature, and PAt, o: is the alveolar CO2 partial pressure. If the arterial CO2 partial pressure (Pa¢,o,) is assumed to be equivalent to the alveolar value, then there are sufficient values to solve for VT (Szewczak and Jackson, 1992). For Tb = 30 °C, at which ventilation was nearly continuous, VT solved to be 0.170 ml. Taking the arithmetic mean of ventilatory and apneic Paco,S at Tb - 20 °C, and solving for VT yields 0.165 ml. So, a value of 0.17 ml was a used as an estimate for VT in calculating ~'e.
Results Re.~7~h'ato~v response to temperature. Overall ventilation remained well coupled to ovm'all met:tboli¢ rate from Tb -~ 5 to 37 °C with a mean ACR of 1.21 L/mmol (Table 1). This is herein referred to as 'standard ACR'. 'Ventilatory ACR' is calculated using O, uptake acquired exclusively front ventilation. From T b - 5 to 20 °C, ventilatory ACR differed fi'om standard ACR. However, for Tb > 30 °C they were equivalent,
TABLE I Metabolic and ventilatory r~tes, and ACRs in the torpid bat, Eptesicusfuscus, at various body temperatures Tb °C
Respiration rate (breaths/rain)
Minute volume (ml/g, h)
Total metabolic rate #reel 0,4(8' h)
Standard ACR (L/retool)
Ventilatory ACR (L/retool)
5 10 20 30 37
3.63 + 0,89 (3) 3,30 + 0,73 (5) 12,2 +0,9 (25) 61,5 + 5,52 (5) 132 (I)
1.87 ± 0.47 1.37 +0.21 5.67 + 0.61 24.8 + 3.9 50.2
1.21 + 0.29 (4) 1.51 +0.11 (4) 4.76 + 0.32 (24) 18.7 + 2.0 (4) 423 (I)
1.53 ± 0.29 (3) 0.99+0.26(4) 0.99 + 0.11 (9) 1.36 ± 0.26 (4) !.18 (I)
2.36 ± 0.12 (3) 2,27 ± 0.32 (5)
1.37 ± 0.19 (9)
Minute volume is calculated from an estimated Vr (see Methods). Standard ACR is calculated from total oxygen uptake, includhtg non-ventilatory (see text). Ventilatory ACR is calculate ] using only oxygen uptake from ventilation. Data are from 8 bats; _+SE (el).
VENTILATORY RESPONSE, HYPOXIA, CO_~, TORPID BAT
293
3.0
- - o2.5
~...
.... B-- Vent. ACR
........ ""--o
2.0
.---l-- Turtle ACR lelmloQlll °
§
ggZ
¢0
~
]~
Std. ACR
T'"'°'-..
i
i
i
i
u
a
o
e
u
l
l
T
e
i
n
-1:
e
e
me~
qb
0.5 0.0
. . . .
0
I
5
-
-
-
'
l
l
10
.
.
.
.
l
.
15
*
l
.
l
.
20
.
l
l
25
l
l
,
l
,
.
.
.
30
1
1
35
ii
I
40
Body Temperature (°C) Fig. 2. Ventilatory ACR and standard ACR ofthe torpid bat, Eptesicusfuscus, at Tb = 5, 10, 20, and 30 °C and the ACR of the turtle Pseudemysscripta elegans for comparison (Jackson, 197 I). The linear regression ofventilatory ACR vs Tb is more consistent than the standard ACR, r 2 = 0.87 and r 2 - 0.016, respectively. Minute ventilation is calculated from an estimated VT (see Methods). Data are from 8 bats.
because there is no significant apneic 02 uptake, i.e. when 02 uptake is unimodal, standard ACR and ventilatory ACR are equivalent. The slope of the ventilatory ACR v~"Tb is -0.0391 L/(mmol. °C), r 2 = 0.87, which is somewhat less than the slope of a representative ectotherm, the turtle Pseudeno's scripta elegans, -0.0589 L/(mmol. °C), r 2 = 0.96 (Jackson, 1971)(Fig. 2). However, the bat's ventilatory ACR vs Tb slope is greater than, and more consistent than, that of its standard ACR' - 0.0022 L/(mmoi. oC), r-" - 0.016. Although the turtle is also an intermittent breather, it is submerged between ventilatory bouts with little potential for non-ventilatory 02 uptake (although it may have some cutaneous CO2 exchange). Thus, the standard ACR for the turtle may be considered equal to its ventilator), ACR by the above definition.
Normal breathing pattern. Above Tb --- 30 • C Eptesicusfuscus breathed continuously in a pattern typical of many euthermic mammals. Short apneas first became noticeable at about Tb - 30 °C which increased in duration as Tb decreased down to about 10 oC. When breathing intermittently Eptesicus fuscus exhibited two qualitatively distinct patterns of ventilation. The first is a rhythmic pattern characteristic of intermittent ventilatory bouts. This pattern had a rather consistent VT and evenly spaced breaths. The quality of the second pattern was sporadic, less rhythmic, and exhibited more variation in VT. At Tb = 20 °C a typical normoxic, normocapnic respiratory cycle included both patterns: a rhythmic bout, followed by a period of apnea with perhaps one to three sporadic breath events prior to resuming another rhythmic bout (Figs. 3 and 4, top trace). Sporadic breaths were less common below T b - 20°C. These patterns were consistent among the 8 bats observed in this study. In general, relatively
224
£M. SZEWCZAK AND D.C. JACKSON
.~ 1 minute
I ~.. _._ ,._
..
~_ { ~F r ' . . . . . .
~ IF
;1. . . . . . . . . _. . . . . . . . . . . . . l~In'~ . . . . - I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
P1co2 : 0.0 torr
~iI~ . I~n . . . . . . . .
t lr
. "
~L ,, - . . . . . . ,,,,~..,,,,~ I r'- '" ' l i l ~ ....... ~'""~....
13 breaths/ m i n u t e
Ptco= = 15.3 torr
55 breaths/ minute
PICa2
:
120 breaths/ m i n u t e
Ptco~
: 32.0 torr
22.9 torr
236 breaths/
minute
Fig. 3. Ventilatory response to hypercapnia in Eptesicusfuscus at Tb = 20 °C.
1 minute ..... .
.
.
.
.
.
.
.
ILl ......... lF ~ .
.
.
.
It TF
....................
Plea = 162 torr
___LLifL~,_JI~]._ L Ill [i ~ d iI1~_ _ 1'11
~'.-
q'"-
"-,~"-vq
~l-~l
tl p c , - . -
PloI " 75,4 torr
L[,
.... __ _ _ IL . . . .
tL
....
13 b r e a t h s / m i n u t e
..,
_~_,!ll,l .... •r !
.. I~I_ 'T
L_]L i ! -ll'-IW'
-
l lI.Jl._ll I I~ 7w"]ll-
II ~l I' I~1
ill ~||
Ii ~r
dlill r ~
9 breaths/minute
AI.I.uI~JaJ k_a al._|.k ~jl It ~1J di• ~l t tdiL_ll J. | J i I d it l.~_t _a IJoL | i, j| .tJI I .|. l_ ~t | . i l . r'"'~""'v"
"~"'w'-r"w'-r'F"'m
I" "
T ' " ~ ' P'.r ', ~p',r ~e It' n I ~ " v ' . - W ' v ' " ~
~ , ~ ' I~ m 1 " P
Plos -" S6,0 torr
19 breaths/ m i n u t e
Pto~ = 30.3 torr
30 breathe/ minute
IV' w - ~ ' - w " - ~ p
w' . '
"~ - ~ v ~
t~.,,~
n'-Vv" ~ ' "
Fig, 4, Ventilatory response to hypoxia in Eptesicusfuscus at Tb = 20 QC,
longer apncas wcrc rollowcd by relatively longer ventilatory bouts. Ventilatory bouts averaged 1.0 + 0,1, 6.3 + 1.2, and 0.90 + 0.16 rain for Tb = 20, 10, and 5 °C, respectively. The intervals between ventilatory bouts (not counting occasional sporadic breaths) were 6.1 +0.35, maximum: 13.7; 56.7 + 15.3, maximum 147; and 6,5 + 0,8: maximum: 40.9 rain for Tb = 20, 10, and 5 °C, respectively.
VENTILATORY RESPONSE, HYPOXIA, CO2, TORPID BAT
225
Respirato:y response to graded hypercapnia. The overall duration of the rhythmic venti!atory bouts, and the frequency of breaths within them (fR*), increased in response to hypercapnia (Fig. 3). Additionally, as hypercapnia progressed, the apneic periods shortened, and sporadic breathing events were gradually eliminated from the remaining TABLE 2 Ventilatory response to hypercapnia in the torpid bat, Eptesicusfuscus, at Tb = 5, 10, 20, and 30 °C Tb
Inspired ?0CO,
Respiration rate (breaths/min)
Instantaneous respiration rate (breaths/rain)
n
5 5
0.00 + 0.00 5.80 + 0.80
3.63 + 0.89 51.8 + 0.2
34.4 + 2. ! 51.8 + 0.2
3 2
10 10
0.00 + 0.00 5.04 + 0.04
3.30 + 0.73 87.1 + 3.4
33.3 + 3.7 87.1 + 3.4
5 5
20 20 20 20 20
0.00 + 0.00 2.40 + 0.14 3.28 + 0.18 4.13 + 0.09 5.1q+0.07
12.2 + 0.9 52.5 + 5.5 82.5 + 13.2 136 + 43 172 +22
61,7 89.4 87.2 143 172
+ 1.8 + 7.8 + 5.6 + 38 +22
17 7 4 4 7
30 30
0.00 + 0.00 5.10± 0. I0
61.5 + 5.5 141 + 23
72.3 _+ 2.5 141 + 23
4 2
Instantaneous respiration rate is the rate within a ventilatory bout. Data are from 8 bats, + SE.
Eptewlcua fuacue 30 25 20
[]
30 °
0
20 °
¢
10 °
gg °
5°
15 • • ••
-
8e Q
human
10
5
tO, ; ? ? , • o, ,. : ' "
.
•-
0 ° m •
.:'::~. "-,;.......,.........,......'.',..'::: ........ "6
0 0
I
2
3
4
5
6
7
Inspired %C02 Fig. 5. Respiratory response to hypercapnia in the torpid bat, Eptesicusfuscus, at Tb = 5, 10, 20, and 30 °C. Change in respiratory rate is calculated in reference to the rate without CO2 for individual bats. Human data is from Davies et al. (1925). The regression line is calculated from the Tb = 20 °C data, Data are from 8 bats.
226
J.M. SZEWCZAK AND D.C. JACKSON
apnea. Eventually, breathing became continuous, and additional increases in hypercapnia continued to increase .fR. The quantitative ventilatory response to hypercapnia at Tb = 5, 10, 20, and 30 °C is presented in Table 2. The change in fR in response to hypercapnia is calculated with respect to each bat's mean fR in the absence of CO2, and is depicted in Fig. 5 with a typical euthermic (human) response included for reference (Davies etal., 1925). Similarly, change in the respiratory frequency within a bout, fR*, is normalized to each bat's mean ./R* without CO2, and is shown in Fig. 6 against the same euthermic
Epteelcue fuecus .i
ill
ii. |
i.i
i.
i
•
.
l * * l i i J ~ , l ,
0
I
.
~
2
.
J
.
.
.
.
.
J
l
l
i
*
.
.
a
3
.
i
.
,
.
*
.
l
.
l
.
i
4
Inspired
.
.
.
*
.
.
.
.
.
.
~
l
l
l
l
l
5
""
n
30 °
0
20 °
O
10 °
A
5° •
human
l
6
7
%COs
Fit~.6. Cha,se in i,statlttuleous respiratory rate (./k within a bout) in response to hypereapnia in the torpid bat, L:pt¢.vi¢,s/itst.,s, at Tb ~ 5, 10, 20, and 30 '~C. Cha,se in instantaneous respiratory rate is calculated in ret~rence to the rate without COz for individual bats. Human data is from Davies et al. (1925). The ret;r~ssio, line is calculated from the Tb ~ 20 ~C data. Data are from 8 bats. TABLE 3 Ventilatory response to hypoxia in the torpid bat, Eptesicusfuscus, at Tb = 20 °C Inspired % O2
R~spiration frequency (breaths,'min)
Change in respiration frequ~,:ncy
21,0 + 0,0 10,0 + 0,0 7,30 + 0,10 6.33 + 0.12 5,10 + 0,10 4,05 _+0,05 3.00
12,7 + 0,98 10,5 + 1,75 12,6 + 3,37 17.9 + 4.92 27,4 + 4,75 62,4 + 1,40 87.7
1,0(', + 0.00 0,08 0,9~i + 0.04 1.39 + 0.28 2,28 :!: 0.19 3,88 t 0.39 5.83 (),8(,~ +
21 6 2 3
4 2 1
Change in respiration frequencyis in reference to the t~equencyofth~,"same animal breathing 21% 02. Data arc from 5 bats, + SE,
VENTILATORY RESPONSE, HYPOXIA, CO,, TORPID BAT
227
Eptesi©us fuscus 7 6
o
Tb=20°C
s
m
O. sO
Q or
2 1
.
O
.
0
. .
.
.
.
. I
.
, ........ .
.
5
.
a
10
i
i
,
•
. I
•
15
•
- ~'0-- . . . . ,
•
a
20
,
.
~
|
25
Inspired %0= Fig. 7. Respiratory respo.lse to hypoxia in the torpid bat, £ptesicusf , scus, at Tb = 20 °C. Change in respiratory rate is calculated in reference to the rate with normoxia for individual bats. The respiration rate at 10°o inspired O, is significantly less than normoxia; P < 0.05. Data are from 5 bats.
response. Note that the change in fR* is more consistent to the euthermic response than is the change in ./k.
Respiratoo' response to graded hypoxia,
in response to hypoxia, the duration of the rhythmic bouts decreased and were superseded by an increase in non-rhythmic ventilations. At lower O., concentrations, even, rhythmic breathing was entirely supplanted by the uneven pattern of breathing, and the elimination of apneas (Fig. 4). At Tb -- 20 ° C, hypoxia did not elicit a significant ventilatory increase until inspired O., dropped to about 6"~, (48 Torr). The respiratory response to hypoxia at Tb - 20 °C is normaUzed to individual mean normoxic fR and presented in Table 3, and displayed in Fig. 7. The respiratory rate at 10"o~ inspired O2 was significantly less than the rate at 21 ",, inspired O.,, P < 0.05. There was no noticeable change in respiratory rate and pattern between pure 02 and 2 1 ° O2.
Discussion
Respiratory response to temperature.
Eptesicus fuscus main:ained a close match of
overall ventilation with overall metabolic rate through[~¢~ its range of physiologic Tbs (Table 1). This suggests a ventilatory strategy consistent with observations of other mammalian heterotherms (Reeves, 1969; Musacchia and Volkert, 1971; Malan et al., 1973; Heisler, 1986). However, below T b - 30 °C, ventilation is not steady-state in Eptesicusfi¢scus, and its control strategy cannot necessarily be assessed from the average effect reflected by this 'standard ACR'. Certainly, a steady-state ACR could not generate the observed oscillations in acid-base state and blood gases.
228
J.M. SZEWCZAK AND D.C. JACKSON
By definition, the ACR is calculated from the total volume of ventilation divided by the total 02 uptake (Dejours et ai., 1970). Ordinarily, this relates ventilatory activity to the animal's 02 requirement. However, Eptesicus ~lscus acquires a substantial portion of its total 02 uptake (but not CO2 elimination) by non-ventilatory airway diffusion during apnea (J. M. Szcwczak and D.C. Jackson, submitted). Thus, this bat becomes a bimodal breather in torpor. A calculation of the ACR including the apneic 02 uptake does not accurately characterize the ventilatory controller relative to metabolism, because it is incorrectly attributing 02 uptake to vcntilatory action. Instead, a calculation of the ACR using only gas exchanged during ventilation, i.e. the 'ventilatory ACR', reflects the operation of the vcntilatory controller and the parameters to which it is responding. The temperature dependency of ventilatory ACR in Eptesicus fuscus is more characteristic of an cctothcrm (Fig. 2). This is consistent with the end-ventilatory acid-base data which also exhibit an cctothermic-like relationship (Szewczak and Jackson, 1992). These data suggest that this animal overventilates with respect to this O.~ requirement during ventilatory bouts; i.e. although its 02 needs are satisfied, it continues ventilation until a satisfactory Paco, is achieved. That the ventilatory bouts are CO.,-depcndent rather than O,-dependent is further supported by the rhythmic pattern of ventilation, which is characteristic of hypercapnic stimulation (see below). Unfortunately, we were not able to measure I¢1co: with the equipment available for this study. Following the respiratory gas quotient through a ventilatory bout would be infimuative. Calculating the ACR using IVl<,o~instead of 1¢1o:would obviate the complication of passive apneic gas exchange, A study of central neural control of respiration in hypothermic cats concluded that the respiratory responses to altered Tb could be entirely explained by temperature effects of neurons, rather than by any o,'ganismal effort to maintain a constant protein charge state (Kiley et al., 1984). The heterothermic response to hypercapnia of Eptesicusfuscus contradicts that conclusion, because its hypercapnic response was uniformly temperature-indepe,~dent from Tb = 10-30 °C (and also perhaps at 5 °C, see below). The temperature-independence of Eptesicus,/iescus' CO2 response suggests a feedback loop linked to protein charge state, distinct from neuronal temperature effects. The contradictory response observed in hypothermic cats may arise from their lack of adaptation to heterothermy. Because hypothermia is not a natural state for that species, its respiratory controller may be incapable of defending homeostasis under such an abnormal condition. Pam, r, ¢~.l'breaehi,g. The pattern ofintermittent breathing in Eptesic~,.sfuscus is different in some respects from other torpid mammals. Golden hamsters, Mesocricetus auratus (Kristofferson and Soivio, 1966), hedgehogs, Erinaceus europaeus (Pembrey and Pitts, 1899; Kristofferson and Soivio, 1964, 1967; Tllhti, 1975), dormice, Myoxus avellanarius (Pembrey and Pitts, 1899); Elion[vs quercinus L. (Pajunen, 1970, 1974), and marmots, Marmota marmota (Pembrey and Pitts, 1899), have all been shown to displ,~y a pattern of intermittent breathing characterized by a bout of rhythmic breathing followed by
VENTILATORY RESPONSE, HYPOXIA, CO2, TORPID BAT
229
uninterrupted apnea until the next bout of rhythmic breathing. Eptesicus fuscus differs from these animals by the presence of sporadic, non-rhythmic breaths interspersed during apnea (Figs. 3 and 4, top trace). Some similar sporadic breathing appears during torpor in the ground squirrel, Spermophilus lateralis (Milsom et al., 1986; Riedesel etal., 1986), but the overall pattern is not as well defined and repeating as in Eptesicusfuscus. At Tb below 6 °C, this ground squirrel changes from intermittent breathing to single breaths (Miisom et ai., 1986); with a similar change induced from mild anesthesia at higher temperatures (Milsom, 1991). Eptesicus fuscus maintained its intermittent breathing pattern down to Tb = 3 °C; however, mild anesthesia also induced a single breath pattern as high as Tb = 25 °C.
Respirator3, response to graded hypercapnia. Hibernators have previously been characterized as insensitive to hypercapnia or of possessing a deficiency of respiratory regulation (Rasmussen, 1916; Kayser, 1961). Subsequent work has found this to be an inaccurate generalization. Hibernating hamsters (Mesocricetus auratus) have been shown to respond to hypercapnia similarly to humans (Lyman, 1951). However, hibernating ground squirrels and marmots do not respond to hypercapnia as precisely or predictably as other hibernators, possibly owing to their fossorial adaptation and a blunted CO., response (Endres and Taylor, 1930; Lyman, 1951; Milsom et aL, 1986; Riedesel etaL, 1986). Ground squirrels decrease the apneic period, and increase the duration of the ventilatory period and ./R* (Riedesel etal., 1986; Milsom etaL, 1986). In hibernating hedgehogs, Bi6rck et al. (1956) found variations in their hypercapnic response. However, T'ahti (1975) found that 5% CO2 was sufficient to abolish intermittent breathing in hibernating hedgehogs, and that apneic periods began to shorten with as little as 1% CO.,, but the ventilatory period remained constant. The overall frequency response to hypercapnia in Eptesicus fuscus depended on vcntilatory stat0. Continuously-ventilating bats at Tb = 30 ° C were similar to the typical euth0rmic hypercapnic response, whereas intermittently-ventilating bats responded with a much greater increase in fR (Fig. 5). Instantaneous respiratory rates, however, all fall near the normal euthermic response, except for Tb = 5 ~C (Fig. 6). At this temperature, the bats failed to increase fR from 5 % to 6.6% inspired CO2, and instead appeared to increase VT to augment Ve. Additionally, the respiratory movements (as registered on the polygraph trace) wore noticeably sluggish. Thus, the actual ~/e at Tb = 5 °C may fall along the relationship with the others if Ve could have been directly measured. The ventilatory control of intermittent breathing may be presumed to switch between 'on' and 'off' states based upon thresholds of the integrated status of blood gases and pH. By this reasoning, an animal's 1Vie:and setpoints for those thresholds will influence the duration of apneas, and consequently the fR. We believe this complication may be alleviated by using fR* as a basis for comparing ventilatory responses, particularly between continuous and intermittent-breathing. For the intermittent breather, the fR* might be considered a direct indication of the urgency to breathe when the system is switched to the 'on' state. Viewed by this rationale, the hypercapnic response in torpid,
230
J.M. SZEWCZAK AND D.C. JACKSON
intermittently-breathing Eptesicus fuscus is not inherently different from euthermic, continuously-breathing mammals (Fig. 6). Respiratory response to graded hypoxia. Inconsistencies exist in the literature concerning hypoxic responsiveness among torpid mammals. Riedesel et al. (1986) exposed hibernating ground squirrels (Spermophilus lateralis) to 100~o N2, and observed no significant respiratory response. However, Milsom et ai. (1986)found a graded hypoxic response in two species of ground squirrels (Spermophilus lateralis and Spermophilus cohmlbitmus) that correlated with the temperature dependent shift of the O 2 dissociation curve. BiOrck et al. (1956)exposed hibernating hedgehogs to 100~o N2 and reported no significant respiratory response during exposures of 50-120 min duration. In contrast, T',lhti (1975) found a graded hypoxic response in hibernating hedgehogs, with respiration becoming continuous with 3)o inspired 02, a similar response to that observed in Eptesicusfuscus by this study. These discrepancies may result from varying levels or depths of torpor, similar to the documented differences in respiratory control during the various levels of sleep in euthermic mammals (Walker and Berger, 1988). Epu,sicusfu.~cus exhibited a sensitive response to hypoxia during torpor at Tb = 20 ° C (Table 3). Although the response to inspired Po., < :50 Torr was brisk, little response occurred until inspired O2 dropped to about 75 Torr. This may be explained by considering the expected left shift of the O2 dissociation curve at Tb = 20 °C compared to euthermi:t. There was actually a slight decrease in ventilation with moderate hypoxia which was associated with an increase in non=rhythmic breaths, and a decrease in the duration of apnea (Fig. 7). The shorter, more frequent ventilation pattern of mild hypoxia probably supports lower tissue Pco~ levels compared with the longer apneas of normoxia, which require extended ventilation episodes to release this tissue store (refer to ACR discussion above). Thus, a more even exchange of O2 and COa is maintained, it should be noted there was no measurable reduction of l~'lo, during hypoxia that could account for the reduction in ~'e with moderate hypoxia.
Com'h~shm, The temperature dependence of the airway convection requirement in Eptesicus/it~'cusis similarto that of ectotherms ifit is calculated using oxygen uptake exclusivelyfrom activeventilation,Hypercapnia and hypoxia both stimulateventilation, but are manifested by qualitativelydistinctpatterns.Overventilationwith respectto 02, and the patternof ventilationduring ventilatorybouts,indicatethat Pace _,isthe primary feedback variable controlling these bouts, The quality of the ventilatory patterns suggests that a hypercapnic response utilizesthe central rhythm generator more elegantly than does a hypoxic response. This may result from the integration of dissimilar chemoreceptor sites and their actions, or because hypoxia-stimulated b,'eathing is relegated to a secondary 'back up' role which does not warrant the organismal investment to support a smooth feedback/control loop.
VENTILATORY RESPONSE, HYPOXIA, CO.,, TORPID BAT
231
Acknowledgements. We thank Dr. Andr~ Malan for his comments and encouragement of this investigation. This work was supported by Grant DCB8802045 from the National Science Foundation (DO).
References Bi6rck, G,, B.W. Johansson and H. Schmid (1956). Reactions of hedgehogs, hibernating and non-hibernating, to the inhalation of oxygen, carbon dioxide, and nitrogen. Acta Physiol. Scand. 37: 71-83. Cameron, J.N. (1986). Principles of Physiological Measurement. Orlando, FL: Academic Press, pp. 169-170. Davies, H.W., G. R. Brow and C. A.L. Binger (1925). The respiratory response to carbon dioxide. J. Exp. Med. 41: 37-52. Dejours, P., W. F. Garey and H. Rahn (I 970). Comparisons ofventilatory and circulatory flow rates between animals in various physiological conditions. Respir. Physiol. 9: 108-177. Dejours, P. (198 i). Principles of Comparative Respiratory Physiology. New York: Elsevier/North Holland Biomedical Press, 265 p. Depocas, F. and J.S. Hart (1957). Use of the Pauling oxygen analyzer for measurement of oxygen consumption of animals in a short-lag, closed-circuit apparatus. J. Appl. Physiol. 10: 388-392. Drorbaugh, J. E. and W.O. Fenn (1955). A barometric method for measuring ventilation in newborn infants. Pediatrics 16: 81-87. Endres, G. and H. Taylor (1930). Observations on certain physiological processes of the marmot. Ii. The respiration. Prec. R. Soc. (London) 107: 231-240. Heisler, N. (198o). Comparative aspects of acid-base regulation. In: Acid-Base Regulation in Animals, edited by N. Heisler. New York: Elsevier, pp. 397-450. Hill, R. W, (1972). Determination of oxygen consumption by use of the paramagnetic oxygen analyzer. J. Appl. Physiol. 33: 261-263. Jackson, D.C. ( 1971). The effect of temperature on the turtle, Pseudemys scripta elegans. Respir. PhysioL 12: 131-141. Kayser, Ch. (1961). The Physiology of Natural Hibernation. New York: Pergamon Press, 325 p. Kiley, P.K., F.L. Eldridge and D.E. Millhorn (1984). The effect of hypothermia on central neural control of respiration. Respir. Ph.vsiol. 58: 295-312, Kristoflbrson, R. and A. Soivio (1964). Hibernation in the hedgehog (£rinaceus europaeus L.). Ann. Acad. $ci. Fenn. 82: 1-17. Kristofferson, R. and A. Soivio (1966). Duration of hyperthermia periods and type of respiration in the hibernating golden hamster, Mesocrtcetus auratus Waterh. Ann. Zool. Fenn. 3: 66-67. Kristofferson, R. and A. Soivio (1967). Studies i~1the periodicity of hibernation the hedgehog (Erinacaeus europaeus L.) !I. Changes in respiratory rhythm, heart rate and body temperature at the onset of spontaneous arousals. Area. Zool. Fema. 4: 595-597. Lyman, C.P. (1951). Effect of increased CO, on respiration and heart rate of hibernating hamsters and ground squirrels. Am. J. Physiol. 167: 638-643. Maina, J.N. and A.S. King (1984). Correlations between structure and function in the design of the bat lung: a morphometric study. J. Exp. Biol. I I 1: 43-61. Malan, A. (1973). Ventilation measured by whole body plethysmography in hibernating animals and poikilotherms. Respir. Physiol. 17: 32-44. Malan, A., H. Arens and A. Waechter (1973). Pulmonary respiration and acid-base state in hibernating marmots and hamsters. Respir. Physiol. 17: 45-61. Mead, J. (1960). Control of respiratory frequency. J. Appl. Physiol. 15: 325-336. Milsom, W. K., M. D. McArthur and C. L. Webb (1986). Control ofbreathing in hibernating ground squirrels. in: Living in the Cold, edited by H.C. Heller, X.J. Musacchia and L. C. H. Wang. New York: Elsevier, pp. 469-475. Milsom, W.K. (1988). Control of arrhythmic breathing in aerial breathers. Can. J. Zool. 66: 99-108.
232
J.M. SZEWCZAK AND D.C. JACKSON
Milsom, W.K. (1991). Intermittent breathing in vertebrates. Annu. Rev. Physiol. 53: 87-105. Musacchia, X.J. and W.A. Volkert (1971). Blood gases in hibernating and active ground squirrels: HbO2 affinity at 6 and 38 °C. Am. J.Physiol. 221: 128-130. Pajunen, !. (1970). Body temperature, heart rate, breathing pattern, weight loss and periodicity of hibernation in the Finnish garden dormouse, Eliomys quercinus L. Ann. Zool. Fenn. 7: 251-266. Pajunen, I. (1974). Body temperature, heart rate, breathing pattern, weight loss and periodicity of hibernation in the French garden dormouse, Eiiomys quercinus L. at 4.2 + 0.5 °C. Ann. Zooi. Fenn. 1I: 107-119. Pembrey, M. S. and A. G. Pitts (1899). The relationship between the internal temperature and the respiratory movements of hibernating animals. J. Physiol. 24:305-316. Rasmussen. A.T. (1916). A further study of the blood gases during hibernation in the woodchuck (Marmota marmcml ). The respiratory capacity of the blood. Am. J. Physiol 41: |62-172. Reeves. R. B. (1969). Role ofbody temperature in determining the acid-base state in vertebrates. Fed. Proc. 28:1204-1208. Riedesel, M.L., J.E. Oriego and D.C. Espinosa (1986). Respiratory patterns in hibernating Spermophilus t~#e)alis. In: Living in the Cold, edited by H.C. Heller, X.J. Musacchia and L.C.H. Wang. New York: Elsevier, pp. 461-468. Szewczak, J.M. and D.C. Jackson (1992). Acid-base state and intermittent breathing in the torpid bat, Eptesicus fuscus. Respir. PhysioL 88: 205-215. T',lhti, H. (1975). Effects of changes in CO, and 02 concentrations in the inspired gas on respiration in the hibernating hedgehog (Erinacaeus europaeus L.) Ann. Zool. Fenn. 12: 183-187. Walker, J.W. and R.J. Berger (1988). Sleep as an adaptation for energy conservation functionally related to hibernation and torpor. Frog. Brain Res. 53: 255-278.