Blockade of arousal from hibernation by inhibition of norepinephrine synthesis in the golden hamster

Life Sciences Vol. 9, Part I, pp. 1117-1125, 1970. Printed in Great Britain

Pergamon Press

BLOCKADE OF AROUSAL FROM HIBERNATION BY INHIBITION OF NOREPINEPHRINE SYNTHESIS IN THE GOLDEN RAMS TER Dale D . Peist 1 Department of Zoology, University of California, Berkeley, California 94720

(Received in final form 7 August 1970) Physiological and pharmacological studies of the mechanisms of thermoregulation (1,2,3,4), sleep, and arousal (5) support the involvement of the sympathetic nervous system in the phenomenon of hibernation . The activity of the peripheral sympathetic nervous system seems essential for normal arousal from deep hibernation (3) . A causal relationship between the activity of central adrenergic neurons and the onset of entrance into hibernation has been suggested (4) . Central adrenergie neurons may also be involved in the triggering of arousal from hibernation . In view of the evidence for the importance of the sympathetic nervous system in the process of arousal from hibernation, it seemed reasonable to postulate the following . The initiation and/or maintenance of sympathetic activity necessary for complete arousal (or arousal thermogenesis) might depend upon newly synthesized norepinephrine (NE) at some critical site or sites (e .g . hypothalamus, reticular activating system, brown fat) . If this is true, then normal arousal might be blocked, either completely or in part, by giving an animal in deep hibernation an inhibitor of NE synthesis . The experiments presented here were designed to begin testing the effect of inhibition of NE synthesis on the triggering and maintenance of arousal in the hibernating golden hamster (Mesocricetus auratus ) .

1 Present address : Department of Physiology I, Karolinska Institute, Stockholm 60, Sweden

111 8

AROUBAL FROM HIBERNATION

Vol . 9, No. 19

Materials and Methods Adult golden hamsters

(Mesocricetus auratus ) of both sexes were

housed individually in metal oéges with wood chip bedding in either a wars room at 22t 1 0 0 or in a cold room at 411 0 C . They were kept under artificial illumination from 8 AM to 4 PM (PST) each day and given Purina rat chow and water ad libitum . The animals were subdivided as follows : 1) normal controls maintained at 22* 1 0 C ; 2) cold acclimated animals kept at 4 t 1 0 C, having hibernated, but not in hibernation at the time of use ; 3) hibernating animals kept at 4 t 1 0 0 with a rectal or cheek pouch temperature of 5-7 00 ; 4) newly aroused animal s which attained a rectal temperature of 36-3800 at an ambient temperature of 411 00 after artificial stimulation to arouse from hibernation by taking the rectal or cheek pouch temperature . Temperatures were monitored with a small animal thermistor probe attached to a Yellow Springs Instrument Co . telethermomster . In all of the experiments, he- tore were exposed to cold for 3-4 months . They experienced between 5 to 10 weeks of hibernation period before autopsy at an age of 7-8 months . Experiments were performed between mid January and early October . In the first series of experiments, deeply hibernating hamsters were given various dose levels (40-200 mg/ 1008 body weight, i .p . injection) of the tyrosine hydroxylase inhibitor D,Iralpha-methyl-p-tyrosine methyl ester HC1 (ac-Mpfe, Regis) . The inhi bitor was dissolved or suspended in equal volumes of saline and 0 .1 M PC 4 buffer, pH 7 .0 to give a final pH of 6 and a final concentration of 40 mg/ml . Diluent adjusted to pH 6 with HC1 was given to control animals . At the time of autopsy, certain tissues were excised from some of the animals and frozen on dry ice for eventual determination of A&, tyrosine, and ac-methyl-tyrosine (89-AT) levels . In the second set of experiments, hamsters were given a dose of K -MpTme (120mg/100g) at different times after artificial initiation of

AROUSAL FROM HIBERNATION

Vol. 9, No. 19

111 9

arousal . This was done in order to determine whether arousal would go to successful completion regardless of inhibition of HE synthesis after some critical phase in the arousal process . Horepinephrine, tyrosine, andoc-methyl-tyrosine were extracted and isolated from tissues essentially by the Al 2 0 3 procedure of Anton and Sayre (6) . Borepinephrine, tyrosine, and oc-methyl-tyrosine were assayed fluorometrioally (7,8) . L~norepinephrine bitartrate (B&K Labs), L-tyrosine (A grade Cal Biochem), and D,L- oc-methyl-p-tyrosine (Regis) were used to prepare standard solutions . Values for ATE were corrected for loss during extraction and isolation . Calculation of all values given in the results was based on the free amine or amino acid . Results Normal body temperature changes during arousal Cheek pouch and rectal temperatures and arousal time data were combined from 52 male and female hamsters to construct a generalized body temperature curve for control hibernating animals arousing from 5 .5 .t .20 C to 36 .5 ; .3 0C at an ambient temperature of 4 t 1 0 0 after artifioial stimulation to arouse . As shown in Figure 1, the cheek pouch temperature rises very gradually during the first two hours of arousal . A more rapid rise in body temperature occurs during the second half of arousal . The mean arousal time for these hamsters was 253110 minutes . Effect of eC -HpTme on arousal time and body temperature Table 1 sters

presents the results of experiments in which hibernating ham-

were given increasing dose levels of "-MpTme to season the effect

on arousal . Doses of 40 mg/100g body weight and 60 mg/100g did not block arousal or significantly alter the arousal time . This lack of effect appeared to be due to a low level of inhibitor in the tissues . For example, the @I-MT concentration (hg/g tissue) in intersoapular brown fat ;N=5) than previously found in these hamsters was markedly lower (44f 2 in brown fat of normal controls (225 313tH=4) and cold acclimated

112 0

AROUSAL FROM HIBERNATION

40

Vol. 9, No. 19

embimr tomp .e 4f1 0C Ineun arouml timos 253t10 minuth (Mm 221

36 .5

30

body temp . oC

20

10

a 1

0 initiotion of oroulol

30

1

1

1

60

120

150

1

1!0

210

1

240

270

time (minute

FIG . 1 Changes in Body Temperature of Hamsters during Arousal from Hibernation after Artificial Stimulation to Arouse . Each point and vertical line represents the mean t S .E . of determinations on at least 4 animals . animals (229148 ;H=4) at four hours after injection of the same dose . Doses of 80 mg/100g or higher blocked completion of arousal in most hamsters tested (Table 1) . Seventy two percent (18/25) of the animals failed to arouse from hibernation after a dose of 80 mg/100g or higher . All but a few of these showed definite evidence (breathing activity) of being alive at four hours or later . In most cases these hamsters showed a slight increase in cheek pouch temperature (1_2 0C) after four hours (above that recorded at the onset of arousal) . The response of hamsters given a dose of 80 mg/100g or higher usually fell into one of several categories . 1) Some hamsters showed no affect of the inhibitor on arousal time or body temperature within the normal arousal period . However, usually within several hours after normal arousal these animals began to show the affect of the inhibitor by a gradual decline in body temperature . 2) Some animals showed a partial arousal as evidenced by a partially elevated body temperature by the end of normal arousal time . 3) Post

Vol. 9, No . 19

AROUSAL FROM RIBERNA77ON

112 1

TABLE 1 The Effect of Different Doses ofec-methyl-p-tyrosine methyl eater HC1 on Arousal from Hibernation Does 1 (mg/100g body wt .)

Arousal Time 3

(aroused/total)

(minutes)

22/22

253 t10

40

5/5

229 :L19

60

3/3

279(mean)

80

1/3

242

100

3/6

273 (mean)

120

2/132

231 9 348

200

0/3

----

0

1=

Humber of Animals

dose given as a single i . p . injection ; controls received diluent alone

2 = included one partial arousal to 31 .5 0 C 3 = arousal time given for hamsters which aroused in each dose group as single value, mean, or meant 3 .B . hamsters failed to arouse in the time and showed only slightly elevated body temperatures . All hamsters given these high doses of inhibitor while in deep hibernation usually died within 24 hours at an ambient temperature . of 4 oC . Effect of !f-MpTme on body temperature of normal, cold acclimated animals Four hours after injection of 120mg/100g b .wt . of N -MpTme, the body temperature of normal hamsters at 22 0C remained normal (36 .5 to 37 0 C in 4 hamsters) while that of cold acclimated animals at 4 o C declined by several degrees (28 to 36 0 C ; mean = 32 .9 0C for 4 hamsters) . Tissue HE and inhibitor levels after a(-MpTme Tissue NE and K-MT levels were compared in interecapular brown fat of normal controls, cold acclimated, and hibernating hamsters at 4 hours

AROUSAL FROM HIBERNATION

1122

Vol. 9, No. 19

after a dose of 120 mg/100g . As shown in Table 2, the inhibitor levels (dug/g ties .) were of comparable magnitude in brown fat of all three groups but lowest in the hibernating group which failed to arouse . TABLE 2 Levels of the Tyrosine Hydroxylase Inhibitor cc-methyl-tyrosine (ac-1!T) in Interecapular Brown Fat of Hamsters at 4 Hours after i .p . injection of 120 mg « -YpTme/1009 b .wt . Experimental group

Se-11T (139/9 tissue)

normal control

593148(4)

Cdld acclimated

729 :t 39(4)

Hibernating

4581128(3)

Values represent mean t S .E .(number of animals) . IIaximal inhibition of tyrosine hydroxylase has been assumed when the inhibitor level exceeds twice the endogenous tyrosine (9) . Endogenous tyrosine in brown fat of hamsters has been found to range from 18 to 25 xig/g tissue (10) . As shown in Figure 2, after a high dose of rc_äpTme, the AE level was reduced in brown fat of normal and cold acclimated animals . However, the HE content in the hibernating group was not different from the level in normal deep hibernators . This is consistent with a low rate of NE turnover in brown fat of hibernating hamsters which received the inhibitor. Effect of ac -ï -Tme at times after the start of arousal Experiments were performed in which separate groups of hamsters were given a single dose of ac -YpTme (120 mg/100g b .wt .) at 30 minute time intervals between 0 and 210 minutes after initiation of arousal . Of the 16 hamsters receiving the inhibitor between 0-60 minutes, 87 .5% failed to arouse by the mean control arousal time . Of the two hems tern which aroused, one received an injection at 0 time and one at 60 minutes . The body temperatures of animals beyond 60 minutes (but before

Vol . 9, No. 19

AROUSAL FROM HIBERNATION

nerval control 22 0C

cold ecclüe . 40C

Albare. ,0C

1123

newly aroused 40C

]PIG . 2 The Effect of Inhibition of HE Synthesis on the Level of YE in Interecapular Brown !at from Hamsters Yen a Single i .p . Injection of «-Ypfe (120 mg/100g b .wt .f . The number in each column = number of animals . injection) were quite variable .

Seventy one percent of these 14 animals

failed to arouse normally . However, beyond 60 minutes of arousal, animals receiving the inhibitor at the same body temperature regardless of the time after the start of arousal showed considerable variation in their response to the drug . The responses of hamsters given the inhibitor at the same time or body temperature ranged from apparent death before 4 hours to complete arousal in the normal time . Discussion The results indicate that a dose of tyrosine hydroxylase inhibitor which does not affect the body temperature of the normal hamster at 22 00 after 4 hours and which reduces the body temperature of the cold accli mated animal by several degrees after 4 hours, has a profound affect on the arousal of hamsters in deep hibernation . In most deeply hibernating animals, a high does of o -IIpTae precluded an increase of more than 1-2 00

1124

AROUSAL FROM HIBERNATION

Vol. 9, No. 19

in body temperature during the normal period of arousal . The inhibitor accumulation at the anterior end of the body was probably rapid as gauged by the brown fat d-NT content in animals failing to arouse after 4 hours . Previous studies of the effect of cc-methyl-tyrosine on tissue NE levels in rate have shown a profound and rapid decrease of brain NE (9) . The brain catecholamine levels were decreased to a greater extent than peripheral levels (11) . The most probable explanation for those animals which aroused °normally" from hibernation after u-11IpTme would be the failure of distribution of the inhibitor to critical sites of NE synthesis . Since most of the hamsters given at-llpTme in hibernation, while failing to arouse, still maintain a body temperature above ambient temperature, the inhibition of NE synthesis would seem to involve the central triggering or sustaining of arousal or arousal thermogenesis rather than the loss of integrity of peripheral mechanisms for heat production and conservation . NE has been suggested as an important mediator of central thersoregulatory processes (12) . The hypothalamus plays an essential role in temperature regulation (1) . The hypothalamus contains the highest concentration of NE compared to other brain regions (13) . The effect of ,c-NpTas in blocking arousal could be exerted upon NE containing neurons in the hypothalamus . However, the question of the localisation of the effect remains to be answered . It has been shown previously that the sympathetic nervous system is necessary for normal arousal from hibernation (3) . Theoretically arousal the aogenesis and thermoconservation could be initiated and maintained by the release of previously synthesised and stored NE at central and peripheral noradrenergic nerve endings . The present results suggest that stored NS is not sufficient to allow for arousal and that newly synthesized BE may be necessary for successful initiation and maintenance of sympathetic stimulation of arousal .

Vol . 9, No . 19

1125

AROUSAL FROM HIBERNATION Acknowledg_e nent

I would like to thank Dr . Wilbur B . Quay for helpful support and advice during the experiments and preparation of the manuscript . This work was aided by IISPHS, HIH grants 1-F1-GM-32,590 and HB-06296 . References 1 . J . Bligh, Biol . Rev . 41, 317 (1966) . 2 . H . T . Hammel, Ins Mammalian Hibernation III, ad . by K.C . Fisher, A .R . Dawe, C .P . Lyman, E . Sc Snbaum, F .E . South,Jr ., Elsevier, N .Y ., p . 86 (1967) . 3 . C .P . Lyman and R .C . O'Brien, J . Physiol . 168, 477

(1963) .

4 . P .R . Draskoos3y and C .P . Lyman, J . Phmrmacol . Exp . Thsr . 1J, 101

(1967) .

5 . W .C . Dement, Ins M-iian Hibernation III ,ed . by K .C . Fisher, A .R . Dowe, C .P . Lyman, E . Schdnbaum, F .E . South,Jr ., Elsevier, N .Y ., p . 175 (1967) . 6 . A .H . Anton and D.F . Sayre, J . Pharmacol . Exp . Ths r. .!M, 360 (1962) 7 . R . Laverty and K .Y. Taylor, Analyt . Biochem. 22, 269 (1968) . 8 . T .P . Waalkes and S . IIdenfriend, J . Lab . Clin . Yed . 50, 733 (1957) . 9 . S . Spector, A . Sjosrdsma, and S . IIdenfriend, J . Phmrmacol . Exp . Thsr . 141, 65 (1965) . 10 . D .D . Feiet, Comp . Gen . Pharmacol ., in press (1970) . 11 . C .C . Porter, J .A . Totmro, A . Burcin, E .R . Yynosky, Biochem . Pharmacol . 15, 583 (1966) . 12 . W . Feldberg and R .D . Meyers, J . Phyeiol .(Lond . ) 173, 226 (1964) . 13 . J . Glowinski and J . Baldessarini, Pharm . Revs . 18, 1201

(1966) .