Camp. Biochem. Phvsiol. Vol. Printed in Great B&in

78C, No. 2. pp. 373-376, 1984 0306-449284 $3.00 + 0.00 ‘( 1984 Pergamon Press Ltd

DECREASED THERMAL TOLERANCE IN

MUS MUSCULUS WITH MELATONIN AND

CHLORPROMAZINE

VICTOR H. HUTCHISON and NINETTE HART

Department of Zoology, University of Oklahoma, Norman, OK 73019, USA. Telephone: (405) 325-4821

(Received 1 Nol?ember 1983)

Abstract-l. Adult Mus musculus, previously acclimatized for two weeks to 25-C and an LD 12: 12 photoperiod, were injected (i.p.) with daily doses of melatonin (4.0 mg) or chlorpromazine (20.0 mg kg ‘) for 2 days.

2. Both drugs significantly reduced the critical thermal maximum (CTM) and the elevated defended temperature (EDT).

3. Melatonin significantly increased the length of EDT but chlorpromazine had no effect on EDT. 4. Both drugs reduce thermal tolerance in ectotherms, but this is apparently the first demonstration

that melatonin and chlorpromazine increase sensitivity to high temperature in an endotherm.

INTRODUCTION

The pineal organ and a major secretory product,

melatonin (5-methyl-N-acetyltryptamine), have been attributed several unique physiological functions in vertebrates with a role in adaptation to the environ- ment (Quay, 1969). Melatonin is implicated as a major element in behavioural and physiological thermoregulation in vertebrates (Ralph et al., 1979). Melatonin appears to be involved in the normal functioning of an endogenous clock controlling the cyclic nature of activity and body temperature. Administration of melatonin and chlorpromazine significantly decreased mean selected temperature (MST) of the three-toed box turtle, Terrupene curo- lina triunguis, compared with control animals over 24-hr periods (Erskine and Hutchison, 1981). In the mudpuppy, Necturus maculosus, melatonin and chlorpromazine injections significantly decreased temperatures selected by salamanders in a linear thermal gradient and eliminated the normal die1 cycle in behavioural thermoregulation shown by control animals (Hutchison et al., 1979). Chronic melatonin administration produces an increase in daily torpor in the white-footed mouse, Peromyscus leucopus (Lynch et al., 1978).

The pineal organ and melatonin also play a role in vertebrate thermal tolerance. The critical thermal maximum (CTM) has been a popular tool for mea- suring temperature sensitivity in terrestrial and aquatic ectotherms (Hutchison, 1961; Hutchison and Kosh, 1965; Hutchison et a/., 1966; Cox, 1974; Ma- ness and Hutchison, 1979). Parietalectomy of the lizard, Anolis carolinensis, resulted in significantly lowered CTM (Kosh and Hutchison, 1972). Melatonin and chlorpromazine administration significantly reduced the CTM below that of control animals in the mudpuppy, Necturus masculosus (Erskine and Hutchison, 1982a). The CTM has also

been demonstrated as a useful tool for examining thermal tolerance in endotherms (Wright et a/., 1977; Erskine and Hutchison, 1982b,c). This study was undertaken to determine the influence of exogenous and endoqenous melatonin on the thermal tolerance of white mice, Mus musculus.

MATERIALS AND METHODS

Mixed-strain male and nonpregnant female white mice, Mus musculus, were randomly selected from an inbred laboratory stock. Mice were housed 4-6 per cage in Sherer environmental chambers at 25 k I C and photoperiod of LD 12: I2 with photophase centred at noon CST. Purina Chow pellets and water were available ad libitum. All animals were acclimatized for at least 14 days prior to CTM determinations.

Animals were randomly placed into control and experi- mental groups. One group received intraperitoneal (i.p.) injections of melatonin (4.0mg). one group received i.p. injections of chlorpromazine (20.0 mg kg ‘), and one group received equal volume injections of saline. Injections were administered 25 hr and 1 hr (0900 CST) prior to testing. All CTM determinations began at approximately IO00 CST in the period February-April.

Animals were removed from acclimation chambers and weighed to the nearest 0.1 g. A 36-gauge copper/constantan thermocouple sheathed in polyethylene tubing was inserted through the rectum into the colon and taped to the tail. The animals were maintained for a l&20 min period to allow body temperature to stabilize.

A Temp-Air Convector System (Scientific Instruments. Skokie, Illinois, USA) provided dry heated air (relative humidity, 15%) to the testing apparatus. The heated air was blown into a centralized separator chamber and then chan- neled to each of four test chambers (13 cm wide x 18 cm long x 1Ocm high) through tygon tubing fitted with Hoffman pinch clamps to control air flow. The entire apparatus was insulated with styrofoam.

Test chamber temperatures of 42 + I C were monitored with a Yellow Springs Model 47 Telethermometer equipped

373

314 VICTOR H. HUXHIKIN and NINETTE HART

r

I I I I I I I I

TYPE I

’ . 8. 1

i . 1 TYPE II TYPE ID

91 A’

ELEVATED DEFENDED TEMPERATURE

0 50 100 I50

TIME (hIIN)

Fig. 1. Examples of Type I, II and III heating patterns (Ohara et al., 1975; Wright et al., 1977) plotted for colonic temperature in Mus musculus. Best-fit lines for each segment were computed by the method of least squares. The final point on each curve is the critical thermal maximum (CTM) for each individual. From Erskine and Hutchison (1982~).

with thermister probes. Animal temperatures were moni- tored continuously with a Bailey BAT-8 Digital Ther- mometer and recorded every 5min.

The onset of spasms, characterized by uncoordinated twitching of the limbs, was used as the endpoint for the CTM determination. Following exposure to the CTM, the animal was immediately removed from the test chamber and cooled as rapidly as possible. The thermocouple was removed and the animal was reweighed.

Heating curves were determined for each animal from plots of colonic temperature (T,) recorded every 5 min. Each curve was inspected to determine if an individual exhibited a Type I, II or III heating pattern (Fig. I). Mean body temperature around which T, fluctuated during segment 2 for Type III animals was computed. This increased level in body temperature calculated for segment 2 was termed elevated defended temperature (EDT) (Erskine and Hutch- ison, 1982b, 1982~). The total time (length) of the EDT was measured.

All data were tested for normality and homogeneity and were normally distributed. Single classification analysis of

variance and Student’s l-test were performed with the Statistical Analysis System (Barr et al., 1979).

RESULTS

Intraperitoneal injections of melatonin and chlor- promazine significantly decreased the initial tem- perature, EDT and CTM when compared to control animals. Melatonin also significantly increased the time to thermoregulatory breakdown. Chlor- promazine showed no effects on length of elevated defended temperature (Fig. 2).

Chlorpromazine, which may block the breakdown of endogenous melatonin by inhibiting catabolic hepatic enzymes (Ozaki et al., 1976), showed less significant effects in all cases. The results of a multiple comparison test are summarized in Table 1.

Type III heating pattern predominated in all groups, with three Type I in chlorpromazine group, and one Type I and one Type II in melatonin group.

There were no sex-related differences in CTM, EDT, EDT-length or weight loss. The mortality rate 24 hr after the CTM determination was 100(x in controls and animals treated with melatonin and 807: in animals injected with chlorpromazine.

DISCUSSION

The pineal complex and its secretions, specifically melatonin, play a critical role in behavioural and physiological temperature regulation of vertebrates (Ralph et al., 1979; Hutchison and Maness, 1979). Both melatonin and chlorpromazine reduce behaviourally-selected temperatures in ectotherms and lower body temperatures in endotherms (Hutch- ison et al., 1979). Chlorpromazine administered sys- temically to rabbits and rats produces hypothermia, possibly by direct action of brain monamines; chlor- promazine has a complex mechanism of action and interferes with several brain neuronal systems (Lin, 1979) and thus may have had direct or indirect action on the lowering of initial body temperature, CTM and EDT. However, the observed outcomes may result from the action of chlorpromazine as a block- ing agent of the hepatic enzymes which catabolize melatonin (Ozaki et al., 1976). The effect of chlor- promazine on the CTM of pinealectomized mice should be determined.

Our results must be considered as preliminary, since they were obtained with pharmacological rather

Table 1. The effects of melatonin (MEL) and chlorpromazine (CPZ) on critical thermal maximum (CTM), elevated defended temperature (EDT), elevated defended temperature length, initial colonic temperature (T,), and per cent weight loss in Mus musculus

CTM EDT EDT Initial T, Weight

Group (‘C) P ("C) P (min) P (‘C) P BUSS (%I P

MEL 41.5 i 0.31 38.4 f 0.20 395.0 f 38.5 33.6 k 0.41 10.7 k 1.46

(4 mg) (IO) (8) (8) (10) (IO) 0.0041 0.0006 0.0260 0.000 I NS

Control 42.7 f 0.21 39.6 k 0.21 249.0 f 43.3 37.8 i 0.36 9.8 + 1.18

(IO) (IO) (10) (IO) (10) 0.0260 0.0143 NS 0.0001 0.0266

CPZ 41.8 k 0.33 38.5 i 0.41 211.4f62.5 34.3 k 0.38 6.0 f I .08

(20 mg/kg) (10) (7) (7) (IO) (10)

Means and i 1 SE are shown. Sample sizes shown in parentheses. P, probability from Student’? f-test of preceding adjacent groups. NS, no significant dilTerence.

t , 1 i , t I 1 t I I I f t 8 I I , 5 8 I 1 , I , I r

LENGTH OF ELEVATED DEFENDED TEMPERATURE

CONTROL

-ii*, 4OmgMel

-b31

20.0 mll CPZ

IIItIIIIt! ,,,,,,,,,,,,,,,,, I ~ 100 200 300 400 500 600

MINUTES

Fig. 2. The effect of melatonin (MEL) and chlorpromazine (CPZ) on initial colonic temperature, elevated defended temperature, critical thermal maximum, and length of elevated defended temperature in MUS muscuius. The horizontal line is the range, the vertical line is the mean of each treatment, and the shaded rectangles represent two SEMs. Sample sizes are shown in parentheses. Dosages shown were given 25 hr

and 1 hr prior to exposure to 4YC test temperature. Controls were given saline.

Mouse thermal tolerance

CONTROL

- (101

4 0mg Me,

-(to)

20.0mg CPZ

-(IO)

I I 1 1

ELEVATED OEFENDEO TEMPERATURE

I 1 I 1 1 I 1 I ,

CRITICAL THERMAL MAXIMUM

CONTROL

*(IO,

4.Omg Mel

ski,

20 Omg CPZ

--J=---w

I‘,,,i,ilil,,f,,,,,,,,,,,,,

31 32 33 34 35 36 37 38 39 40 41 42 43 44

BODY TEMPERATURE “C

375

than physiological doses. Melatonin content of the pineal gland of small rodents is usually less than 2000 pg and is often less than 100 pg gland -I, but depends upon the portion of the 24 hr cycle from which samples are taken (Reiter, 1982). The half-life of melatonin elimination from blood is less than 1 hr following injection (i.p.) (Gibbs and Vriend, 1981). Thus, our rather high doses may be necessary to maintain elevated levels of circulatng melatonin over the 8-9 hr required for the animals to reach the CTM.

The low initial body temperatures in the groups treated with melatonin and chlorpromazine (Table 1; Fig. 1) are apparently not important influences on the

results obtained for CTM, since the times spent at elevated temperatures (EDT) during heating experi- ments are not correlated with the CTM (Erskine and Hutchison, 1982b). Although the lengths of the EDT were significantly different in the two treated groups, the initial temperatures were not (Table 1).

The role of the pineai complex in thermal tolerance has not been studied extensively. Exogenous and endogenous melatonin reduce thermal tolerance in ectotherms (Erskine and Hutchison, 1982a). The results presented here are apparently the first to demonstrate that melatonin also increases sensitivity to higher temperatures in mammals. The mechanisms

316 VICTOR H. HUTCHISON and NINETTE HART

whereby the pineal complex and melatonin exert their Melatonin and chlorpromazine: thermal selection in the

effects on thermal tolerance are unknown. mudpuppy, Necturus maculosus. L$r Sci. 25, 527-530. Hutchison V. H., Vinegar A. and Kosh R. J. (1966) Criticai

thermal maxima in turtles. ~erpet~l~~ica 22, 32-41. Kosh R. J. and Hutchison V. H. (1972) Thermal tolerance

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