A method for discriminating between chemically induced changes in locomotor activity and whole-body oxygen consumption in mice

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

31, 390-402 (1975)

A Method for Discriminating between Chemically Induced Changes in Locomotor Activity and Whole-Body Oxygen Consumption in Mice1 ROBERTKRUSZYNAAND

ROGER P. S~~ITH

Department of Pharmacology and Toxieolo~~y, Dwft7701~fl7 Hanover, New Hampshire 03755

Medical

Scl7ool.

Received June 29, 1974: accepted Artgglnf 13, 1974 A Method for Discriminating between Chemically Jnduced Changes in Locomotor Activity and Whole-Body Oxygen Consumption in Mice. KRLJSZYNA, R. AND SMITH, R. P. (1975). Toxicol. Appl. Phamacol. 31, 390-402. Concurrent measurements of O2 consumption and locomotor activity were made in adult female mice at 20 and 34 C. As anticipated. activity and 0, consumption were positively correlated with a high degree of statistical significance. A 30-fold increase in spontaneous motor activity, however, was accompanied by only a 30”: increase in O2 consumption. Extrapolation of the regression relationship to zero activity provided a novel means of estimating the basal metabolic rate (BMR) of mice. That value agreed with traditional estimates based on resting or sleeping animals. Under our conditions the respiratory quotient for mice was 0.83 at 20’ C and 1.0 at 34°C. Oxygen consumption averaged over all levels of spontaneous activity was 4.79 ml g-’ hr-’ at 20-C and 2.21 ml g-’ hr’ at 34 C. These estimates are actually lower than those of others who employed physical restraint to control activity. Because the BMR of mice constitutes about 70% of the OL consumption at the highest levels of spontaneous motor activity, drug-induced changes in motor activity may not significantly affect OZ consumption. Alternatively. it may be obvious that the two changes are not correlated. Forexample, in our hands pentobarbital decreased motor activity without affecting 0, consumption, and amphetamine increased motor activity but decreased OZ consumption. When the changes are in the same direction, the problem of distinguishing between a metabolic or respiratory effect of the drug and a change related to increased or decreased motor activity becomes more difficult. Analysis of covariance oKers one possible approach to this problem. One of the most fundamental and commonly measured parameters in biology is O2 consumption. Perhaps because it is such a common measurement, the list of factors known to influence the O2 consumption of intact animals is imposing: activity, temperature, nutrition, body size, stageof life cycle, season, time of day, previous 0, experience, genetic background (Prosserand Brown, 1961). and probably others not yet recognized. Most of these variables, however, are common in biologic research and are corrected

for in any well-designed and adequately controlled experiment. ’ This investigation was supported by USPHS Grant HL 14127 from the National Heart and Lung Institute. 390 Copyright Q 1975 by Academic press, Inc. All rights of reproduction Printed in Great Britain

in any form reserved.

ACTIVITY

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The factor which is the most difficult to control, and one that is generally believed to

have a large influence on 0, consumption,

is muscular activity. Approaches to the control of this variable employed in the past include attempts to minimize muscular activity by restraint or the use of curare or anesthesia, reducing ambient noise levels, controlling the degree of illumination, and habituating the animal to the apparatus. An alternative approach has been to make measurements at fixed levels of forced activity such as swimming or running (Prosser and Brown, 1961). Some of the above approaches are time consuming or cumbersome, and others place the test animal under highly artificial conditions. We are not aware of previous attempts to quantitate spontaneous locomotor activity in small animals in order to correlate it with 0, consumption. In our experience the latter technique offers some advantage over the more classical procedures. Potentially, the most important of these is that it might provide a rational basis for assessing a general metabolic or respiratory effect of a chemical in the face of a concomitant effect on locomotor activity. We believe this report establishes the feasibility of one approach while recording a number of technical pitfalls that can be avoided.

METHODS Apparatus The apparatus used in these experiments and the essential features of its operation are indicated in Figs. l-3. Briefly, the Aloe Scientific Spirometer was modified to permit concurrent measurement of 0, consumption, locomotor activity, and the ambient temperature inside the sealed system. When the apparatus is in actual operation with a mouse and a container of soda lime to absorb exhaled COz, the volume of the sealed system steadily decreases while atmospheric pressure is maintained. Because the system is closed, the ratio of 02/Nz decreases during an experimental trial, but the actual value of the PO, can be computed for any point in time, This complication can be avoided by flushing the system with 100 “/! 0, at the start; however, that delays the start of observations and places the animal under an unusually high O2 tension. The apparatus also records faithfully any increase in volume at atmospheric pressure such as would occur with an increase in internal temperature. In the absence of soda lime, but with a mouse present, the pen records the net effects of O2 consumption, CO, evolution, and changes in internal temperature. Because it was anticipated that the mouse would also be a heat source, an attempt was made to measure the temperature changes independently of the net changes in volume of the respiratory gases. A sensitive air thermometer consisting of a hollow annular ring made of thinwalled brass was introduced into the system. It occupied about 10 y0 of the total volume of 3 liters. As shown in Fig. 4 a predictable and linear calibration of air thermometer readings vs spirometer pen travel was obtained with a static heat source, but a linear calibration could not be achieved when a mouse was also introduced into the system. Thus, merely correcting for the role of the mouse as a heat source did not obviate or even identify other variables concomitantly introduced with the animal. Nevertheless, the air thermometer continued to be helpful in indicating when unusually large temperature changes occurred.

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Fro. 1. The Aloe Scientific Model 160 Minute Oxygen Uptake Spirometer. The top has been removed to show the internal components. Number I is a servocontrolled motor that drives a piston into a calibrated cylindrical reservoir of air (No. 2). Rubber hose connections to the animal chamber (Fig. 2) and the spirometer (No. 3) are seen at the end of No. 2. The small plastic spirometer (No. 3) responds to differences between atmospheric pressure and the pressure in the closed system connected with the animal chamber. The spirometer has a volume discrimination of 0.25 ml and its cap is counterbalanced between electrical contacts such that, when contact is made, No. I is activated to restore the closed system to atmospheric pressure. The spirometer is so sensitive that there is almost zero pressure loading of the system. The position of the piston is indicated by an ink pen (No. 4) mounted on one arm of the piston drive shaft.

4i

5

FIG. 2. Modified animal chamber with the cover detached. The total air space is about 3 liters. The water bath (No. 1) serves as a heat sink and seal. Number 2 indicates one of the two connections to the air thermometer, which is not visible under the grid floor of the animal chamber. The central cylinder directs animal movement primarily in a circular path, and it also serves to protect the container of soda lime directly under it from animal excreta. Number 3 shows one of the four photocells which are positioned at 90” angles around the outside of the chamber. A Lucite reflecting cone (No. 4) is placed at the bottom of a well in the cover. The light source (cf. Fig. 3) is located above this well, and the cone provides reflection to activate all four photocells. A temporary interruption of the light beam to any cnc of the four photocells registers one count of motor activity on the display (No. 5).

ACTIVITY

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CONSUMPTION

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FIG. 3. Fully assembled apparatus with ancillary components. Number 1 is a microscope illuminator used as a light source to activate the photocells. Number 2 indicates a rubber tubing connection between the air thermometer and the pressure (50.04 psi) transducer (No. 5). The meter (No. 6) provides readout in arbitrary units from No. 5. The short lengths of rubber tubing opposite Nos. 2 and 3 are clamped shut during an experiment. Number 3 is the tubing connection to the spirometer. Number 7 indicates the ink pen on a strip of chart which marks the position of the piston in the air reservoir (cf. Fig. 1). Number 8 shows a wheel with a notched rim which is rotated by a synchronous-speed motor. The notch results in I-min time interval marks on the line traced by the pen. Number 4 indicates the grid floor in the animal chamber directly under which is located the doughnut-shaped air thermometer with its nesting container of soda lime. Under experimental conditions, 1 mm of pen travel represents a 0.673-ml change in volume.

Attempts also were made to control the ambient temperature inside the sealedsystem with various arrangements of copper coils and constant temperature water-circulating devices. In terms of temperature response the spirometer was more sensitive (few hundredths of a degree)than common thermostatted water circulators (few tenths of a degree). Although a thermal steady state could ultimately be achieved over a range of preselectedtemperatures, periods of 1 hr or more were required. Such a waiting period wastoo long for our purposes.No doubt more sensitivewater circulators are available, but it wasnot clear to us that they would necessarilydecreasethe time required to reach the thermal steady state. Although the water bath is needed as a seal, it probably also servesas a useful heat sink. Empirical

Correction

Factors

Attempts to control the internal temperature of the apparatus involved delays that were incompatible with our purposes, and mice introduced variables other than heat (Fig. 4). For these reasons,we attempted to derive an empirical set of corrections for application to the raw O2 consumption data. Since some drug effects could be highly

394

KRUSZYNA

L

-40

I

-30

mm

AND

I

-IO

-20

PEN

SMITH

n 0

/

IO

20

I

30

TRAVEL/INTERVAL

FIG. 4. Attempts to calibrate the air thermometer (strain gauge readings) against the spirometer (mm pen travel). The symbol n shows thecalibration with a static heat source in the form of a 6-V lamp. The symbols 3 and 0 show two extreme results when a mouse was also present. Increases in volume within the closed system are plotted here as negative values. No soda lime was present.

this approach offered the advantage that observations could begin within a few minutes after the animal was placed in the chamber. The empirical corrections were obtained by placing saline-injected (10 ml/kg, ip) mice in the apparatus without soda lime and recording the pen travel in each 5-min interval for up to 70 min. The selection of 5-min intervals was arbitrary, but these appeared to reveal the essentialfeatures of the changeswith time (Fig. 5). The points in Fig. 5 represent the net effects of O2 consumption, CO2 evolution, and changesin temperature on pen travel. A net increase in the volume of the system has been plotted as a negative

evanescent,

value. As in Fig. 4 a net increase in volume

is the predominant

effect at the beginning

of

an experiment, causingthe recording pen to be driven backwards. If the changesin Fig. 5 were due only to temperature, we estimate that at the peak of the effect the internal temperature could have increased by no more than 0.5‘C. As shown in Fig. 5, the system slowly drifts toward a steady state which is reached in about 45 min. The pen travel then settlesto a relatively constant positive value for each time interval. We believe that this steady state value is a reflection of the respiratory quotient, and we calculate it to be 0.83. Although we found no comparable estimates

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395

for mice in the literature, our value is similar to the findings of others for rats (Spector, 1956). If the steady state value of about +3 mm pen travel/5 min interval is due to the respiratory quotient, then empirical corrections for other effects of the mouse can be separatedout by subtracting that value from the total observed pen travel in each 5-min interval. The mean corrections for each time interval are listed in the legend to Fig. 5. In correcting experimental data, the appropriate values are added to the observed pen travel since an increase in the volume of the system tends to mask the actual 0, consumption. These data were obtained at various times, and each new set of control animals was compared with the existing data baseby t tests(Snedecor and Cochran, 1967). A failure to detect significant differences on any of these occasions suggeststhat although the corrections are empirical they are reproducibile.

TIME

INTERVAL

(5 mid

FIG. 5. The mm of pen travel per 5-min time interval when mice were placed in the apparatus without soda lime. Each point is the mean value for 28 saline-injected control animals. Pen travel under these circumstances reflects the net effects of O2 consumption, CO2 evolution, and changes in the internal temperature of the closed system. Increases in volume within the closed system are plotted here as negative values. These data have been used to derive empirical correction factors for raw oxygen consumption data as indicated in the text. The mean + SD values for 28 mice in 10 consecutive 5-min intervals were as follows: (1) 6 i: 2.6; (2) 9 + 3.5; (3) 7 + 3.5; (4) 5 + 3.0; (5) 4 + 2.4; (6) 3 + 1.6; (7) 2 f 1.9; (8) 1 + 2.2; (9) 1 t 1.9; (10) I f 1.7.

An examination of the above correction factors, however, suggestedthat an anomaly may exist in the first 5-min interval. When applied to actual 0, consumption data (always obtained in the presenceof soda lime), the correction for the first interval appeared to be too small by a factor of about 2. For individual animals this anomaly occasionally appearedin the second5-min interval aswell as the first. For all results presented hereafter (except Fig. 6) resultsobtained for the first 5 min have beenomitted; sometimes the second 5-min interval was omitted as well. It seemslikely, however, that this phenomenon and that in Fig. 4 are related and may be due to a delay in the establishment of a steady state between CO, production by the mouse and its absorption by soda lime. Carbon dioxide begins to accumulate as soon as a mouseis introduced into the system, but CO, absorption by soda lime must necessarilylag behind production because of mixing problems in the chamber. Eventually, a standing gradient of CO, is established so that a steady state then exists with respect to production and absorption. If this hypothesis is valid, the steady state concentration of CO2 in the chamber should be an estimate of the amount of CO2 that wasgeneratedinitially in establishingthe standing gradient. Thus, the difference between the value for the apparent 0, consumption in the

356

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first time interval and the corresponding values for later time intervals (all of which are about equivalent as shown in Fig. 6) should equal the steady state CO, concentration. As calculated, that difference could be accounted for by generation of a CO, concentration of about 0.6 %. Gas samples taken from a central area of the chamber at 30 and at 70 min contained 0.64 and 0.62% CO,, respectively, on analysis in a Scholander apparatus. The above resul? may also provide some insight into the failure to obtain a linear calibration between the air thermometer and the spirometer (Fig. 4). This lack of agreement may be due to a changing respiratory quotient in the initial time intervals of a trial. Since the mouse is usually placed in the chamber immediately after an ip injection, it is probably hyperventilating. Thus, its apparent respiratory quotient would be abnormally high in the initial time intervals although a stable respiratory quotient is eventually achieved (Fig. 5).

TIME

(min)

FIG. 6. Changes in 0, consumption with time for saline-injected control animals and mice giben 2 mmol/kg of 3-CPT 24 hr previously. The initial apparent increase in 0, consumption is an artifact due to thermal and respiratory quotient changes of the animals. Ambient temperature was 20’ C.

The statement above suggeststhat the thermal contribution ofthe mouseis a relatively unimportant source of error in this system, but the question was further examined with drug-treated animals. Drugs or chemicals which affect O1 consumption and/or motor activity are also apt to have effects on body temperature. 3-Chloro-p-toluidine (3-CPT) under the conditions used here reduces the rectal temperature of mice an average of 6’C below that of control animals (Felsenstein et al., 1974). Yet, empirical corrections obtained with 3-CPT-treated animals (N = 4) in the absenceof soda lime did not differ significantly from those of saline-treated mice (legend to Fig. 5). Similarly. the dose of amphetamine used here would be expected to provoke a significant hyperpyrexia (Askew, 1961; Dolfini et al., 1569). We did not make actual measurementsof body temperature, but again a set of empirical corrections obtained with 6 amphetaminetreated mice did not differ significantly from those of Fig. 5. Finally. empirical corrections for saline-treated mice obtained at an ambient temperature of 34°C (N = 7) also

ACTIVITY

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397

were not different from those obtained at an ambient temperature of 20°C (Fig. 5). Even so, the 34°C corrections were actually used for evaluating drug effects at that temperature. Thus, if new conditions are found which affect the empirical corrections, the solution is simply to derive a new set of corrections for the new conditions. The steady state readings obtained after about 45 min for the empirical corrections at an ambient temperature of 34°C (data not shown) were zero instead of the +3 mm/5 min obtained at 20°C (Fig. 5). Otherwise, the shapesof the two curves were similar. Since we believe (as above) that the steady state readings are proportional to the respiratory quotient, this result yields a respiratory quotient of 1.Ofor this thermoneutral temperature for mice (e.g., Cassin, 1963). Animals

and Data Processing

The mice used in these experiments were Charles River CD1 25- to 30-gfemales.No control was exercised over the stage of estrus or the light-dark cycle of the animals’ environment except that imposed by the regular working hours of the animal caretakers. Observations on drug-treated animals were made at the sametime of day as the respective control observations. All drugs were given ip. At selectedtimes after saline and drug treatments mice were placed in the apparatus. Observations were made at the end of each 5-min interval over 60-70 min so that each trial involved 12-14 separate measurementsof O2 consumption and locomotor activity. The O2 consumption data were corrected for body weight, and then the empirical corrections of Fig. 5 were added. Anomalous observations during the first 5 or 10 min were discarded. Since each animal had previously been given a control trial with a saline injection, the O2 consumption and motor activity data before and after drug treatment could be paired for each 5-min interval. For most statistical purposes,however, the mean 0, consumption and the mean motor activity over the entire trial for each animal were paired with respectto before-after drug treatment. As anticipated. both 0, consumption and motor activity tended to fall off with time during a trial. In determining whether 0, consumption and motor activity were correlated, regressionanalysis wasuseful, and in evaluating drug effects on both these parameters analysis of covariance was sometimeshelpful (Snedecor and Cochran, 1967). As a check on the accuracy and appropriateness of the locomotor counts of activity, an arbitrary subjective rating scaleof animal behavior was devised (Table 1). Thesesubjective ratings were assignedat each 5-min interval independently of the motor activity counts. TABLE 1 SUBJECTIVE

Code H V A L I S

ACTIVITY

RATING

SCALE

Description Highly active, running about frantically Very active, moving about rapidly Active, regular movements,curiousexploring Lessactive, occasionalmovement,grooming Inactive, sedentary,staying mostly in one place Sleeping

398

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RESULTS

When the mean values for O2 consumption/5 min interval and the mean values for motor activity/5 min interval obtained in observation periods of about 1 hr on 24 control mice were subjected to regression analysis, the correlation coefficient was 0.62 (probability (F) = 0.001). The regression relationship was L = 0.045C + 15.9, where L = pen travel per 5 min interval and C = counts of motor activity per 5 min interval. If one setsC equal to zero in this expressionand corrects to the appropriate units, the result should be a novel estimate of the basal metabolic rate (BMR) of mice, namely 4.24 ml g-l hr-’ at 20°C. This estimate of the BMR agreeswith the result obtained in sleeping or resting mice (seebelow). Perhaps a more revealing treatment of the data is summarized in Table 2. The data base has been expanded to 31 control mice, but each 5-min time interval has been grouped in accord with the subjective classification schemeof Table 1. The subjective activity ratings show a good agreement with actual mean counts of motor activity. A good correlation between actual counts of motor activity and O2 consumption was found using only the four points of grouped data. The most interesting aspect is the steepnessof the regressionrelationship. A 30-fold increasein spontaneous locomotor activity is accompaniedby only a 30 % increasein 0, consumption. Obviously, the BMR of mice is a very large fraction (70:;;) of the O2 consumption at the highest levels of spontaneous activity observed. It does not appear that motor activity in theseanimals is asimportant a determinant of O2 consumption asis widely believed. This result would also lead one to anticipate a low “signal-to-noise” ratio when looking at the effects of drug treatments. TABLE RELATIONSHIP

Activity ratingb __--____~ A L 1 S

BETWEEN ACTIVITY IN CONTROL

2 AND OXYGEN MICE”

Percentof total population”

Activity (counts/interval)

14 26 25 35

124 i- 21 68 f- 20 21 + 16 4+ 10

CONSUMPTION

OLconsumption (ml g-’ hr-‘) 5.3 4.8 4.5 4.1

_+ 0.8 i 0.8 i 0.8 i 0.8

(i Data derived from observations on 31 saline-injected mice, but the total population of observations is the sum ofall 5-min intervals for all animals. Values are means k SD. b Rating according to the subjective activity scale of Table 1.

The effects of various drug and chemical treatments on O2 consumption and locomotor activity in mice are summarized in Table 3. As indicated by paired-sample t tests, thyroxin significantly increasedboth motor activity and O2 consumption. The sametrend is apparent with dinitrophenol although the results did not achieve statistical significance with this small sample size. At an ambient temperature of 2O”C, 3-CPT significantly depressedmotor activity. The mean 0, consumption was also decreased, but the unusually large SD precluded that result from reaching statistical significance. The most dramatic effects on OZ consumption were observed in control

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TABLE 3 EFFECT OF VARIOUS TREATMENTS ON THE LOCOMOTOR ACTIVITY AND OXYGEN CONSUMPTION OF MICE’

Activity, counts/interval Treatment Thyroxin, 0.4 mg/kg/day tested at 7 days Dinitrophenol, 10 mg/kg/day testedat 24 hr As above, tested at 48 hr 3-CPT, 2 mmol/kg testedat 24 hr As above* Amphetamine, 10 mg/kg Pentobarbital, 45 mg/kg Depressedphase Excited phase

O2Consumption, ml g-r hr-’

Control

Treated

Control

Treated

26k 21

52 * 14b

4.3 * 0.4

5.0 + 0.2’

36& 15

82 f 58

4.8 Ifr 0.3

5.4 + 0.5

42* 19 27 + 27 55 f44

43 + 32 4 + 4b 3+3 129*47b

4.8 t 0.2 3.0 10.6 4.7 f 0.5

5.0 & 0.6 3.4 * 1.2 2.1 + 0.26 4.3 + 0.4b

5.6 & 1.0 4.7 f 0.6

5.5 + 0.6 4.8 + 0.6

64 + 20 31 k23

22 * 146 83+ 60

RValuesaremean& SD for 4-6 animals,wherethe parameters for the individualanimalsrepresent their meansover a 1-hrobservationperiod.Statisticalanalyses wereby paired-sample (before-after treatment)t tests. bp < 0.05. cp < 0.01. d Ambienttemperatureof 34°C;all othersat ambienttemperatureof 20°C. animals when the ambient temperature was raised to 34°C. Despite this large changein control mice, 3-CPT was still effective in further decreasingboth activity and O2 consumption. Amphetamine increasedmotor activity more than two-fold but significantly decreased 0, consumption. The effects of pentobarbital over the I-hr observation period were clearly divided into two phasesof about equal duration. In the initial (depressed)phase, mice actually lost their righting reflex. On recovery (excited phase) the mice appeared to be somewhat hyperactive with respect to the controls, but the 0, consumption was not changed in either phase.Although only 5 drugs were tested, each in small groups of animals, a number of statistically significant changeswere observed. No doubt additional degreesof freedom would have allowed other trends apparent in the data to have reached comparable levels of statistical significance. Clearly, there is no correlation between drug-induced changesin activity and in 0, consumption with respect to some of the agents tested in Table 3. In other cases, however, this approach still does not allow one to distinguish between an effect of a drug on O2 consumption and an effect on motor activity which secondarily influences Oz consumption. The most troublesome casesare those in which both parameters are changed by the drug in the samedirection : e.g., thyroxin and 3-CPT. A simpleregression analysisdoesnot provide sufficient information to make this distinction. A more powerful statistical test is an analysis of covariance (Snedecor and Cochran, 1967). When appropriately applied, this analysisallows one to determine whether two distinct regression relationships exist instead of a single one and, if two are present, to determine whether or not their slopesare different within experimental error.

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The results of an analysis of covariance on the effects of thyroxin are summarized in Table 4. Unlike the analyses in Table 3, the results of Table 4 are based on individually paired 5-min observation periods obtained with 4 mice. We recognize that this procedure introduces an inappropriately large number of degrees of freedom, but we are interested only in establishing the feasibility of this approach. An analysis of covariance properly performed on only four points of mean data pairs showed the same trends as those of Table 4, but no comparison achieved statistical significance. The results of Table 4 suggest that two distinct regression relationships exist (comparison between adjusted means) although their slopes are the same within experimental error (comparison of slopes). Thus, the effects of thyroxin would best be described as a set, proportional increase in 0, consumption at all levels of motor activity. TABLE EFFECTS

4

OF THYROXIN ON LOCOMOTOR ACTIVITY AND O2 CONSUMPTION IN MICE: SUMMARY OF THE ANALYSIS OF COVARIANCE

Deviations from regression Regression Degreesof coefficient freedom Saline Thyroxin Difference betweenslopes Commonslope Adjusted means Total

0.0262 0.0241

46 43

0.0248

90

0.0307

91

I I

Sumof squares Mean square 407.121 551.126

0.154 958.4

78.671 1037. I

8.85 12.817

0.154” 10.649

78.671’ Il.396

aComparison of slopes,F= 0.0143,p = 0.9. bComparison betweenadjustedmeans,F= 7.388,p = 0.008. An analysisof covariance was also performed with the 3-CPT data obtained at 20’.C. The results(not shown) were similar to those ofTable 4 except in the opposite direction; i.e., a set, proportional decreasein 0, consumption appeared to accompany all levels of motor activity. We are lesscertain about the validity of the result here, however, becausefor all practical purposes treated animals had zero motor activity (Table 3). A more valid comparison might have been made between control sleepingmice (Table 2) and 3-CPT mice, in which case0, consumption is clearly decreasedin the latter. Therefore, even an analysis of covariance might not be the best statistical comparison in all cases.Before deciding on a statistical procedure it is helpful to examine a plot of the data. DISCUSSION Some of the drug effects listed in Table 3 deserve special comment. The results obtained with thyroxin and dinitrophenol are at least consistent in direction with their known metabolic effects. Recent evidence suggeststhat 3-CPT does not affect aerobic tissue metabolism, but it does appear to be a directly acting central respiratory depressant (Borison, Snow, Longnecker and Smith, 1975). The two phases of

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motor activity observed with pentobarbital may be somewhat artifactual. During the second phase, the motor activity of control animals is considerably decreased from that of the first phase because of the phenomenon of habituation (Table 3). The effect of pentobarbital could then be viewed as a postponement of the characteristic initial phase of high motor activity associated with exploration, etc. The effects of amphetamine were remarkable. We cannot explain these results, but it may be relevant that Borbely et al. (1974) have shown that the hyperthermic effects of amphetamine in rats are not associatedwith the concomitant increase in motor activity. As indicated above, spontaneousmotor activity during a trial tends to decreasewith time becauseof habituation. As shown in Fig. 6,0, consumption also tends to decrease gradually with time. It is possiblethat these two phenomenarepresent causeand effect, but our finding that the 0, consumption is relatively insensitive to spontaneousmotor activity in mice (Table 2) led us to seek some other explanation. Among the factors that influence the 0, consumption of mice is the ambient partial pressureof O2 itself. Cassin(1963) observed that at the thermoneutral temperature for mice (34°C) 0, consumption remainsfairly constant when the P,,, is decreasedover the range from 150 to 80 mm Hg. Thereafter, 0, consumption falls gradually with the decreasing PO,. At 2O”C, however, the O2 consumption drops much more abruptly with the decreasingPo2. In our system the 0, consumption of control mice is such that it reducesthe PO2by 15-20 ‘!“, or to about 120mm Hg over the course of an average trial. According to Cassin (1963) even this modest reduction in O2 tension at 20°C might be sufficient to influence 0, consumption in and of itself. Thus. there is a reasonablepossibility that the decrease in 0, consumption with time in Fig. 6 is related more closely to the gradually decreasing PO, than to the spontaneousdecreasein motor activity. We can offer no direct evidence either way on this point, but the question arisesas to the influence of this effect if it is manifested independently of a drug-induced change. For example, when thyroxin is given, the PO2will obviously fall more rapidly with time than in the caseof saline controls. Thus the metabolic effect of thyroxin to increase0, consumption will be partially masked becausethe more rapid decreasein the PO, will have the opposing effect to slow O2 consumption. On the other hand, as in the caseof 3-CPT, the PO will fall more slowly in the caseof treated animals than in the case of controls. In th:s situation, the PO2effect is more pronounced in controls and the more rapid decreasein PO,for control mice will partially mask the effect of 3-CPT to decrease0, consumption. This hypothesis is supported by our data. As tested at 2O”C, 3-CPT reduced O2 consumption by about 30 O/b (Table 3), but at 34C, where the 0, consumption is independent of the partial pressureof O2 in the range encompassedby our conditions. the reduction in O1 consumption induced by 3-CPT was 40>;,. Therefore, irrespective of the direction of the drug effect on O2 consumption, the partial pressureeffect in our experimental designacts in the. opposite direction. Drug effects tend to be underestimated, but at least thosereported here cannot be ascribed to an artifact involving changes in the partial pressure of 0,. Despite our failure to correct for the O2 tension effect, our values for the O2consumption of control mice are comparable to those obtained by other investigators (Table 5). Our values in Table 5 represent an average 0, consumption over all levels of spontaneous locomotor activity. Although we did not correct for motor activity, our values

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are lower than those of Cassin (1963), who made observations on mice physically restrained by wrapping them in wire cloth (Cassin and Vogh, 1962). Our results, however, are a little higher than those of Pennycuik (1967), who used techniques similar to ours. The agreement with Pennycuik (1967) at the thermoneutral temperature is better than the agreement with Cassin (1963). Thus, although our results suggest that motor activity in mice is not as important a determinant of 0, consumption as is generally believed, either correcting for it by methods described here, or ignoring it, would appear to be superior to restraint. TABLE

5

OXYGEN CONSUMPTION OF CONTROL MICE

Temperature 20°C

Investigators

34°C

Or uptake, ml g-’ hr-’

Cassin(1963) Pennycuik (I 967) This report’

2.8

6.03 4.06 4.79

2.31

2.12

a Values are means that include all levels of spontaneous activity. ACKNOWLEDGMENTS

Dr. C. DennisThron provided help with the statistical analyses.Helpful suggestions were made by Dr. Donald Bartlett, Jr.. Dr. John Remmers,and Dr. George Stibitz. REFERENCES ASKEW, B. M. (1961). Amphetamine

toxicity

in aggregated

mice. J. Pharttt. Phamacol.

13,

701-703.

BORBELY, A. A., BAUMANN, 1. R. AND WASER, P. G. (1974). Amphetamine and thermoregulation: Studies in the unrestrained and curarized rat. Nattttytt-Sdmiedebergs Arch. Phavmakol. Exp. Pathoi. 281, 327-340. CASSIN, S. AND VOGH, B. (1962). An automatic small-animal volumeter. J. Appl. Physiol. 17, 150-151. CASSIN, S. (1963). 205,325-330.

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