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Distribution of epinephrine and norepinephrine in the brain of Citellus lateralis during the hibernating cycle
Comp. g~a. Pharma¢., I97o, x, 47-53
47
D I S T K I B U T I O N OF EPINEPHRINE AND N O R E P I N E P H R I N E IN THE BRAIN OF CITELLUS LATERALIS D U R I N G THE HIBEKNATING CYCLE* JOHN W. TWENTE, W I L L I A M H. CLINE, jun., Atop JANET A. TWENTE Space Sciences Research Center, Department of Zoology, and Department of Pharmacology, University of Missouri, Columbia, U.S.A. (Pacdwd 27 oTune, x969) ABSTRACT i. The regional concentration of epinephrine and norepinephrine in the brain of golden-mantled ground squirrels (Citdlus lateralis) was measured at different intervals of the hibernating cycle. 2. The concentratiom of epinephrine and norepinephrine in the hypothalamus and cerebellum were comtant during induction into hibernation, during three segments of the hibernating period, and during the period of activity between hibernation periods. 3- The concentration of epinephrine in the medulla oblongata (with pore) showed no variation related to sampling time; however, the concentration of norepinephrine in this tissue was significantly reduced during the period of activity. 4. The data indicate that progressive irritability during the hibernating period is probably not accounted for by a genexalized accumulation of epinephrine in the hypothalamus as was previously proposed.
Hmcm~x~No periods of undisturbed, goldenmantled ground squirrels (Citdlus lateralis) proceed until spontaneous arousals characterize their tc.iaination. These arousals have been considered to be the result of a centrally initiated stimulus (Twente and Twente, x968a). The duration of the hibernating period (Fig. I) is a linear function of body temperature over a range of 2-25 ° C. (Twente and Twente, I965a ). Because the Arrhenius plot derived from these data was also linear (Twente and Twente, I965a), it was considered that a single, temperature-dependent metabolic process may control spontaneous arousal from hibernating periods and therefore regulate the duration of the hibernating
period. As each hibexnating period progresses, an animal becomes increasingly sensitive to sound or tactile stimuli (Twcnte and Twente, *This work was supported in part by U.S. Public Health Service Grant 5 Rot AMIt32o.
t965b , x968a). Premature arousals are not evoked by i.p. injectiom of 0. 5 nil. of isotonic saline if administered before 5° per cent of an animal's predicted hibernating period has elapsed. After 5° per cent of the predicted hibernating period has elapsed, the frequency of premature arousals in response to saline increases as the normal termination of the hibernating period is approached. This increasing semitivity has been designated as progressive irritability (Twente and Twente, I968a ). Epinephrine administered i.p. evoked arousal early in the hibernating period in a linear dose--response relationship (Twente and Twente, I968b ). A per animal dosage of 5opg. L-epinephrine hydrochloride in o. 5 ml. saline evoked premature arousal at the beginning of the hibernating period (o per cent), whereas 2.5 pg. per animal did not evoke premature arousal until approximately 33 per cent of the predicted hibernating period had elapsed. Intermediate
48
vE rE
dosages fell on a line between these points and caused arousal at appropriate percentages of the predicted hibernating periods. It was implied that the arousing effect was probably the result of circulating epinephrine reaching the central nervous system. Comparable injections of T-norepinephrine did not evoke premature arousal or otherwise affect the pattern of progressive irritability. 1.8 " I
*
1.4 1.2
1.o
norepinephrine from whole brains of Citellus trideeemlineatus showed no differences between active and hibernating animals. They did not, however, determine whether the ratio of epinephrine to norepinephrine changed during the hibernating period. Twente and Twente (z968b) postulated that the duration of the hibernating period may be limited by the development of
.
.
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HIBERNATING CYCLE
/
25 HIBERNATING PERIOD 5 !
0
!0 HOURS
0
!
75 lOO ELAPSED HIBERNATION EXPRESSEDAS PERCENTAGE OF EXPECTEDTOTAL
%
Fxo. x.kThe hypothalamlc concentrations of epinephrine and norepinephrine (lag. per g.) and the ratio of epinephrine to norepinephrine arc plotted with respect to the interval of the hibernating cycle at which determinations were made. The average concentrations for each group of data are indicated by a horizontal line ( - ). The bottom segment represents the core temperature during the hibernating cycle plotted against the expected duration of the hibernating period expressed as the percentage of the mean duration (individuals ranged from 128 to 2~9 hours). The duration of arousal, the period of activity, and induction are plotted in actual hours since there is litde variation between animah for these segments of the hibernating cycle. Uusp'~i (t963) reported concentrations of norepinephrine in whole brains from active and hibernating hedgehogs (Erinaceus europaeus). A significant reduction in the concentration of norepinephrine was found during hibernation when compared to aroused animals Drasktczy and Lyman (I967) suggested that the turnover of catecholamines ceased before induction into hibernation. Their measurements of epinephrine and
progressive irritability which culminates in spontaneous arousal. They further postulated that progressive irritability and the consequential spontaneous arousal may result from an accumulation of epinephrine at the level of the hypothalamus. Pohorecky, Zigmond, Karten, and Wurtman (i969) have demonstrated the presence and unequal distribution of phenylethanolaminc-N-methyl transferase, an enzyme which alIows for the conversion
I97o, I
BRAIN CATECHOLAMINES ]~N H I B E R N A T I O N
of norepinephrine to epinephrine, in the brain o f rats, chickens, a n d one turtle. This study was initiated to determine whether changes occurred in the concentrations o f epinephrine, norepinephrine, or their ratio as the hibernating period progressed. I f significant differences could be d e m o n strated, it was t h o u g h t t h a t mechanisms indicative o f selective rates of production a n d a c c u m u l a t i o n as opposed to degradation a n d secretion could possibly be related to the temperature d e p e n d e n c y o f the duration o f the hibernating period. This investigation involved the determination o f regional levels o f epinephrine a n d norepinephrine in the brain. T h e p r i m a r y region o f interest was the hypothalamus. T h e region o f secondary interest, because o f its a u t o n o m i c centres, was the m e d u l l a oblongata (with pons). T h e cerebellum was included as a control region where no change was expected in the relatively low content o f the catecholamines. METHODS Adult Citellus lateralis of both sexes were individually housed in stainless-steel cages with cedar shavings. Cut oranges and sunflower seeds were provided for food. The hibernating ground squirrels were maintained in cold rooms at 4 and 5° C. with maximum environmental temperature fluctuations of q-o.25 ° C. Recordings of temperature from iron-constantan thermocouples, either implanted on the rib (Twente and Twente, 1965b) or placed beneath the animal in its nest, were continuously monitored and printed at 6-minute intervals on time-printed chart paper by Leeds and Northrup Speedomax G 16-point recording potentiometers. These records yielded data which accurately determined the duration, in hours, of each hibernating period and the period of activity between hibernating periods. It was first established that each ground squirrel was exhibiting the winter pattern of hibernating behaviour (Twente and Twente, 1967). Criteria for the determination of the winter pattern included: (a) hibernating periods of relatively constant duration; (b) periods of activity between hibernating periods of relatively short (less than I x hours) and constant duration; and (c) hibernating body temperatures within 0.5° C. of the temperature of the micro-environment. A single hibernating period of an undisturbed ground squirrel exhibiting the winter pattern of hibernating behaviour seldom varies more than :klO per cent from the average duration for hat animal. The variation in the duration of
9
hibernating periods for each animal used for this study was less than + 6 per cent. The average durations of hibernating periods of different animals at the same body temperature may be highly variable. Ground squirrels with different average durations of hibernating periods (those used in this study ranged from Ia8 to 229 hours) have been compared by means of a normalizing process which equates each animal's average duration as constituting xoo per cent. Fractions thereof, expressed as percentages, have been considered to be physiologically comparable. This technique has proved valid in respect to the dose-response relationship derived from injections of epinephrine (Twente and Twente, 1968b) Animals were sacrificed by decapitation within x minute from the time the cage was removed from the cold room. The regions of the brain were dissected on ice. The anatomical guidelines described by Glowinski and Iversen (I966) were followed; the tissues were weighed within 5 - i i minutes of decapitation. Intervals of the hibernating cycle that were sampled included: (a) from the termination of arousal to I t hours after arousal (the end of the period of activiW and beginning of induction); (b) during the induction into hibernation at core temperatures from x5.6 to 8.o° C.; (c) at the beginning of the hibernating period (o-xo per cent); (d) at the middle of the hibernating period (48-55 per cent) ; and (e) at the end of the hibernating period (95-too per cent). Because of the limited number of samples that could be analysed at any one time and because it was considered that storage could influence the results, not all regions from every animal were analysed. The concentrations of epinephrine and norepinephrine were determined for each region of the brain individually. Each tissue was homogenized in 15 ml. I o per cent trichloro-acetic acid with a Polytron homogenizer (PT io generator) according to the method of yon Euler and Lishajko (1959). Catecholamines were concentrated by adsorption onto o'5g. Alumina (British Drug House) and eluted with 2 ml. of cold o'2 ~/" acetic acid followed by 2 ml. of cold deionized water. Differential determination of epinephrine and norepinephrine was accomplished in these small samples by combining the automated trihydroxyindole fluorometric methods of Robinson and Watts (I965) and Fiorica (i965). Both methods employ the Technicon AutoAnalyzer system. A Technicon (Model I) fluorometer was used. The manifold of the Fiorica system was modified (a) • by adding a debubbler to the sodium hydroxide/ ascorbic acid stream and (b) by adding a single mixing coil to the final stream. The reagents of Robinson and Watts (I965) were employed for the determination of norepinephrine. Their method was modified by measuring epinephrine at pH 3"5- This allowed for the determination of a higher epinephrine to norepinephrine ratio than
5o
TWZm~ ET AL.
could be accomplished at 10H 6. The sensitivity was increased by eliminating the dialysis step. This procedure required that the blank for each sample be determined. Since epinephrine represented less than Io per cent of the total in the samples measured, the combination of methods provided a better differentiation of the two catccholamines than either method alone. Aliquots were used for duplicate determinations of both epinephrine and norepinephrine. Single catecholamine standards (25 rig. per ml. epinephrine and 5ong. per nil. norepinephrine),
averages and standard deviations o f the weights of the hypothalamus, medulla oblonRata (with pons), and cerebellum are shown in Table L These data are similar in regard to both absolute weight and degree of variation to those reported for similar dissections of the brain of the rat by Glowinski and Iversen (1966). The data regarding the concentrations of epinephrine and norepinephrine in the hypothalamus and the ratio of these two catecholamines are shown in relation to the cycle of hibernation in Fig. I. Regression analyses of the data during the hibernating period ( o - l o o per cent) indicate that no significant change occurs either in the concentration of epinephrine and norepinephrine or the ratio of the two. Regression analyses of thc data obtaincd for the medulla oblongata (with pons) and cercbellurn showed that no significant change occurrcd in the concentrations of the two catccholamincs or their ratio in either tissue as the hibernating period progressed.
Table L--WEIoH'r8 OF THE RROION'S OP TIIE BRAIN Or G R O U N D SQUIRRELS ANALYgED FOR EPINEPHRINE AND NOREPINEPHRINE
Tmstm
No.
MEAN WRIGHT
3I
29
o.x ~o (4-0.020) 0.263 (4-0"o94)
15
0"353 (4-0.o66)
Hypothalamus Medulla oblongata (with pore) Cerebellum
Coral0° gen. Pluzrma¢.
(g.4-S.D.)
~"able II.~MI~AN ~ONCT,NTRATION OF NOREFINEPHRINE IN THE BRAIN OF GROUND SQUIRRELS DURING THE HIBERNATING CYCLE
TIME or Sxm, LmO
MEDULLA OBLONOATA (wrrHPDN~
HYPo~s
DURING THE HIBERNATING
CYCLB
No.
Pefiodofacfivity Induction ~-xo per cent 48-55 per cent 95-moper cent Allperiods
6 6
7 4 8
3I
NE (~g. per g,±S.D.) I'I6 (4-0"33) 1.34 (4-O'OI) I'~9 (4-0"24) I'I7 (4-0"30 1'30 (+Oq6) X'24 (4-0"24)
No. 5* 6 6 4 8 24
CEREBELLUM
NE (gg. per g.:I:S.D.) No. 0"42 (4-0"05) 0"67 (4-oq4) o.66 (4-o'x6) o'6x (4-0'06) 0"65 (4-0"07)
0"65 (4-o',,)
NE (gg. per g.4-S.D.)
5 2 4
0.20 (+0"05) o.x8 (+0.02) 0.22 (+0.06)
X
3
o.x 9 0.26 (4-0.02)
I5
o'I9 (4-0"05)
*Because of statistical significance (P
The average concentrations of norepinephrine in the three regions of the brain at the different times of sampling are shown in Table II. Student's totest was used to compare the mean concentrations. No significant differences existed between any two sampling periods or any single sampling period and groupings of other sampling periods for concentrations of norepinephrine in the hypothalamus or cerebellum. A comparison
5t
BRAIN CATECHOLAMINES IN HIBERNATION
'970, x
each sample determination. T h e averages of these data for the different regions of the brain at the different times of sampling are shown in Table IV. Statistical evaluations of these data (analyses of regression, variance, and t-tests) indicated that the relative concentrations of the two catecholamines for each region of the brain was independent of the time of sampling. T h e total concentration of epinephrine and norepinephrine for each tissue for all
of the mean values for the medulla oblongata (with pons), however, showed that the average concentration of norepinephrine during the period of activity was significantly lower (P
Table III.--ME.~ CONCZSTI~OS OF EPINEPHRINEIN THE Bma~mo~ GROUNDSQUIRRELSDURINGTHE
I-ImERNATmGCYCLE oF SAMPLING HYPOTHALAMUS DURING THE HmERNAT~O E CYCLE No. (gg. per g.±S.D.)
MEDULLA OBLONOATA (WITH PONS)
T~
Period of activity [nduction >-,o per cent ~-55 per cent p5-xoo per cent All periods
6 6 7 4 8 31
No.
o . , o (±0"05) o . , , (±0"04) o.,o (4-0.03) o ' , 4 (±0"04) o.,x (4-0"04) o . , o (4-0"04)
CEREBELLUM
E
(gg. per g.±S.D.)
4
0"041 ('4-0"016)
6 6 4 8 =8
0"0'5 (±0"009) o'o30 (±o'oI6) 0.008 (±o'oo4)
E
No.
(gg. per g.±S.D.)
5
0.oo8 (±o-o, i) o'oo9 (±o"oo9) o.ol i (±o'oo9) o.oo6 o.oo7 (+0-007) o.oo9 (±0.008)
0
4 I
0.003 (±o'o14)
3
0.006 (4-o'o14)
'5
Table IV.--RATm O~ CONCZm~.ATmmO~ EPINEPHRINETO NOREPINEPHRINEIN THE BRAINOF GROUND
SQummzLs nuRiNo THE HmERNATINOCYCLE Tn~z oF SAMPLING
MEDULLAOBLONOATA (wrrH PONS)
HYPOTHALAMUS
DURING THE
CEREBELLUM
Hm~.RNATINO CYCLE
Period of activity [nducfion )--to per cent i~-55 per cent }5-1oo per cent MI periods
No.
N-~E(±S.D.)
6 6 7
o.ioo (±0-058) 0.080 (±o.o0o) o.mo (±0"005)
4
O'10I ( ± 0 " 0 1 8 )
8 3x
0"087 (±0"028) o'o96 (-I-o"o34)
No.
~(±S.D.)
4 6 6 4
0"078 (4-0"053) 0.008 (±o'o2o) o'o53 (±o'o35) 0"049 (±o'o16)
8
0"040 (z~O'OI8)
08
0-048 (±0"033)
measured are shown in Table I I L Statistical evaluations of these data (the same as those indicated for norepinephrine) showed no significant variation with regard to time of sampling during the hibernating cycle. T h e ratio of the concentration of epinephrine to norepinephrine was calculated for
No.
~(±S.D.)
5 " 4 x 3 I5
0"047 (-4-0-o58) 0"035 (±0"007) 0"057 (-4-o'o5x) 0"030 0"044 (±0"040) 0"047 (±0"043)
sampling times (excepting norepinephrine in the medulla oblongata (with ports) during the period of activity) showed average values as follows: hypothalamns, t.36 ttg. per g. (8.8 per cent epinephrine); medulla oblongata (with pons), o.68 ~tg. per g. (3.6 per cent epinephrine); and cerebellum, o.2o ttg. per g. (4"3 per cent epinephrine).
5~
vwz~rm ST AL.
DISCUSSION The relatively constant levels of epinephrine in the hypothalamns and medulla oblongata (with pons) found throughout the hibernating period do not support the original hypothesis of an accumulation of this monoamine. The possibility that epinephr/ne is involved with progressive irritability and with the ' t r i g g e r ' for arousal from the hibernating period is not negated, however. Progressive irritability may result from the lowering of thresholds of particular central neurons which are involved in the process which initiates arousal from the hibernating period. I f this were the case, then no change in the level of epinephrine or the rate of release would be necessary to elicit arousal, but rather an increased response to a low concentration of the transmitter would
suffice. There is also a possibilitythat cpincphrinc may reach the central nervous system from the peripheral circulation. The arousing effectof epincphrinc injectedi.p.would seem to enhance this argument (Twcntc and Twcntc, I968b ). Wcil-Malherbc, Whitby, and Axclrod (x9 6 i) demonstratcd that small amounts of epinephrine and norepincphrine pass the blood-brain barrier in the region of the hypothalamus ofthc cat,but arc cxcludcd from thc remainder of the brain. The statistically significant lower level of norepinephrine during the period of activity should be investigated more thoroughly in order to interpret the biological significance. The unequal distribution ofnorepinephrine in the brain of the golden-mantled ground squirrel is in accordance with the findings for the brain of the cat (Vogt, i954) , of the rat (Glowinski and Iversen, 1966; Manshardt and Wurtman, x968), and of man (Sano, Gamo, Kakimoto, Taniguchi, Takesada, and Nishinuma, I959). The hypothalamus was shown to contain the highest regional concentration of norepinephrinc in all of these studies. The regional distributionof epincphrinc in the brain has bccn neglected, generally, until this study, primarily because of the difficulty of measuring the low conccntra°
Comp. gen. Pl~rmac.
tions. The unequal distribution of epinephrine that parallels that of norepinephrine in these three regions of the brain does not seem to have been reported previously for any species. The unequal regional distribution of epinephrine is of interest in regard to this monoamine as a central neurotransmitter. Several of the criteria for a neurotransmitter are met when the regional distribution is considered in conjunction with the finding of the presence and unequal distribution of phenylethanolamine-N-methyl transferase (Pohorecky and others, I969). ACKNOW/.~DOEMENT$
We are indebted to Horst-Dieter Dellmann for his assistance in teaching one of us (J. W.T.) the techniques of dissection of the regions of the brain. REFERENCES DRASK6CZY, P. R., and LYMAN, C. P. (x967),
'Turnover of catecholamines in active and hibernating ground squirrels',07. P/~rmac. exp. 7~f., I 5 ~ IOI--I I I.
yon EULZR,U. S., and LmRAJKO,F. (x959), ' The estimation of catecholamlnes in urine ', Acta physiol, scand., 45t 122-x 32. FIosJcA, V. (1965), 'An improved semi-automated procedure for fluorometric determination of plasma eatecholamines ', Clinica chim. Acta, x ~ z9I-I97. GLOWn~SKI,J., and IVERSEN,L. (I966), ' Regional studies of catecholamlnes in rat brain, I. The disposition of (SH) norepinephrine and (SH) dopa in various regions of the brain ', 07. aVeurochem.,z$, 655-669. M~SHAngT, J., and WURTm~, R. J. (I968), 'Daily rhythm in the noradrenaline content of rat hypothalamus'; Nature, Lond., ax7, 574-575. POHORECKY, L. A., ZIGMOND, M. J., K~TEN, H.J., and WUgT~'% R.J. (i 969), ' Enzymatic conversion of norepinephrine to epinephrine by the brain ', 07. Pharmac. exp. Ther., x65, x9o-195. ROBINSON,R. L., and WATTS, D. T. (I965), 'An
automatcd trihydroxyindoleprocedure for the differentialanalysis of catccholamines', Clin. Chem., xx, 986--997. SnNO, I., G,~o, T., KnraMowo, Y., T,~mGUCHX,K., T,~,Y.anVA, M., and NmHm-UMA, L. (x959), ' Distribution of catechol compounds in human brain ', Biochim. biophys. Acta, 32, 586-587. TW~NTE, J. W., and TWENTY, J. A. (x965a), ' Regulation of hibernating periods by temperature ', Proc. natn. Acad. Sci. U.S.A., 54, Io58xo6x.
197o, I
BRAIN CATECHOLAMINES IN HIBERNATION
TwE~rE, J. W., and Twe~rrE, J. A. 0965b), ' Effects of core temperature upon duration of hibernation of Citellva lateralis ', 07. appl. Physiol., 2 0 , 4x x-4x6. . . . . . (I967) , 'Seasonal variation in the hibernating behaviour of Citellus iateralis ', in Mammalian Hibernation (ed. FmrmR, K. C., DAWE, A. W., LYMAN, C. P., SCH(JNBAUM,E., and SOUTH, F. E.), vol. I I I , pp. 47-63. Edinburgh: Oliver and Boyd. (I968a), 'Progressive irritability of hibernating Citdlus iateralis ', Comp. Biochem. Physiol., ~5, 467-474. (I968b), ' Effects of epinephrine upon progressive irritability of hibernating Citellus lateralis ', Ibid., 25, 475-483. UUSv~A, V. T. (x963) , ' The catecholamine content of the brain and heart of the hedgehog (Erinaceus europaeus) during hibernation and in
53
an active state ', Anals Med. exp. Biol. Finn., 4 x,
340-348.
VOOT, M. (1954) , ' The concentration of sympathin in different parts of the central nervous system under normal conditions and after the administration of drugs ', .7. Physiol., Lond., x~3,
451-481.
WV_aL-MAL~RB~, H., WHrreY, L. G., and AXELROD,J. (196i), ' The blood-braln barrier for catecholamincs in different regions of the brain ', in Regional vVeurodmnistr) (ed. KXTY, S., and ELKES, J.), pp. 284-992. Oxford: Pergamon . . . . . . . . . . K~ Word Index: Brain, catecholamlncs, cerebellum, Citdlus lateralis, duration of the hibernating period, regional distribution of epinephrine, ground squirrel, hibernation, hypothalamus, medulla oblongata, regional distribution of norepinephrine.
47
D I S T K I B U T I O N OF EPINEPHRINE AND N O R E P I N E P H R I N E IN THE BRAIN OF CITELLUS LATERALIS D U R I N G THE HIBEKNATING CYCLE* JOHN W. TWENTE, W I L L I A M H. CLINE, jun., Atop JANET A. TWENTE Space Sciences Research Center, Department of Zoology, and Department of Pharmacology, University of Missouri, Columbia, U.S.A. (Pacdwd 27 oTune, x969) ABSTRACT i. The regional concentration of epinephrine and norepinephrine in the brain of golden-mantled ground squirrels (Citdlus lateralis) was measured at different intervals of the hibernating cycle. 2. The concentratiom of epinephrine and norepinephrine in the hypothalamus and cerebellum were comtant during induction into hibernation, during three segments of the hibernating period, and during the period of activity between hibernation periods. 3- The concentration of epinephrine in the medulla oblongata (with pore) showed no variation related to sampling time; however, the concentration of norepinephrine in this tissue was significantly reduced during the period of activity. 4. The data indicate that progressive irritability during the hibernating period is probably not accounted for by a genexalized accumulation of epinephrine in the hypothalamus as was previously proposed.
Hmcm~x~No periods of undisturbed, goldenmantled ground squirrels (Citdlus lateralis) proceed until spontaneous arousals characterize their tc.iaination. These arousals have been considered to be the result of a centrally initiated stimulus (Twente and Twente, x968a). The duration of the hibernating period (Fig. I) is a linear function of body temperature over a range of 2-25 ° C. (Twente and Twente, I965a ). Because the Arrhenius plot derived from these data was also linear (Twente and Twente, I965a), it was considered that a single, temperature-dependent metabolic process may control spontaneous arousal from hibernating periods and therefore regulate the duration of the hibernating
period. As each hibexnating period progresses, an animal becomes increasingly sensitive to sound or tactile stimuli (Twcnte and Twente, *This work was supported in part by U.S. Public Health Service Grant 5 Rot AMIt32o.
t965b , x968a). Premature arousals are not evoked by i.p. injectiom of 0. 5 nil. of isotonic saline if administered before 5° per cent of an animal's predicted hibernating period has elapsed. After 5° per cent of the predicted hibernating period has elapsed, the frequency of premature arousals in response to saline increases as the normal termination of the hibernating period is approached. This increasing semitivity has been designated as progressive irritability (Twente and Twente, I968a ). Epinephrine administered i.p. evoked arousal early in the hibernating period in a linear dose--response relationship (Twente and Twente, I968b ). A per animal dosage of 5opg. L-epinephrine hydrochloride in o. 5 ml. saline evoked premature arousal at the beginning of the hibernating period (o per cent), whereas 2.5 pg. per animal did not evoke premature arousal until approximately 33 per cent of the predicted hibernating period had elapsed. Intermediate
48
vE rE
dosages fell on a line between these points and caused arousal at appropriate percentages of the predicted hibernating periods. It was implied that the arousing effect was probably the result of circulating epinephrine reaching the central nervous system. Comparable injections of T-norepinephrine did not evoke premature arousal or otherwise affect the pattern of progressive irritability. 1.8 " I
*
1.4 1.2
1.o
norepinephrine from whole brains of Citellus trideeemlineatus showed no differences between active and hibernating animals. They did not, however, determine whether the ratio of epinephrine to norepinephrine changed during the hibernating period. Twente and Twente (z968b) postulated that the duration of the hibernating period may be limited by the development of
.
.
".
Corftp. gO?LP ~ l c .
ET AL.
NE
." a " . • .. •
ee
-
0.8
Y
,° 1 .
0.0
..,
E
lie
:
0.0
•
"
HIBERNATING CYCLE
/
25 HIBERNATING PERIOD 5 !
0
!0 HOURS
0
!
75 lOO ELAPSED HIBERNATION EXPRESSEDAS PERCENTAGE OF EXPECTEDTOTAL
%
Fxo. x.kThe hypothalamlc concentrations of epinephrine and norepinephrine (lag. per g.) and the ratio of epinephrine to norepinephrine arc plotted with respect to the interval of the hibernating cycle at which determinations were made. The average concentrations for each group of data are indicated by a horizontal line ( - ). The bottom segment represents the core temperature during the hibernating cycle plotted against the expected duration of the hibernating period expressed as the percentage of the mean duration (individuals ranged from 128 to 2~9 hours). The duration of arousal, the period of activity, and induction are plotted in actual hours since there is litde variation between animah for these segments of the hibernating cycle. Uusp'~i (t963) reported concentrations of norepinephrine in whole brains from active and hibernating hedgehogs (Erinaceus europaeus). A significant reduction in the concentration of norepinephrine was found during hibernation when compared to aroused animals Drasktczy and Lyman (I967) suggested that the turnover of catecholamines ceased before induction into hibernation. Their measurements of epinephrine and
progressive irritability which culminates in spontaneous arousal. They further postulated that progressive irritability and the consequential spontaneous arousal may result from an accumulation of epinephrine at the level of the hypothalamus. Pohorecky, Zigmond, Karten, and Wurtman (i969) have demonstrated the presence and unequal distribution of phenylethanolaminc-N-methyl transferase, an enzyme which alIows for the conversion
I97o, I
BRAIN CATECHOLAMINES ]~N H I B E R N A T I O N
of norepinephrine to epinephrine, in the brain o f rats, chickens, a n d one turtle. This study was initiated to determine whether changes occurred in the concentrations o f epinephrine, norepinephrine, or their ratio as the hibernating period progressed. I f significant differences could be d e m o n strated, it was t h o u g h t t h a t mechanisms indicative o f selective rates of production a n d a c c u m u l a t i o n as opposed to degradation a n d secretion could possibly be related to the temperature d e p e n d e n c y o f the duration o f the hibernating period. This investigation involved the determination o f regional levels o f epinephrine a n d norepinephrine in the brain. T h e p r i m a r y region o f interest was the hypothalamus. T h e region o f secondary interest, because o f its a u t o n o m i c centres, was the m e d u l l a oblongata (with pons). T h e cerebellum was included as a control region where no change was expected in the relatively low content o f the catecholamines. METHODS Adult Citellus lateralis of both sexes were individually housed in stainless-steel cages with cedar shavings. Cut oranges and sunflower seeds were provided for food. The hibernating ground squirrels were maintained in cold rooms at 4 and 5° C. with maximum environmental temperature fluctuations of q-o.25 ° C. Recordings of temperature from iron-constantan thermocouples, either implanted on the rib (Twente and Twente, 1965b) or placed beneath the animal in its nest, were continuously monitored and printed at 6-minute intervals on time-printed chart paper by Leeds and Northrup Speedomax G 16-point recording potentiometers. These records yielded data which accurately determined the duration, in hours, of each hibernating period and the period of activity between hibernating periods. It was first established that each ground squirrel was exhibiting the winter pattern of hibernating behaviour (Twente and Twente, 1967). Criteria for the determination of the winter pattern included: (a) hibernating periods of relatively constant duration; (b) periods of activity between hibernating periods of relatively short (less than I x hours) and constant duration; and (c) hibernating body temperatures within 0.5° C. of the temperature of the micro-environment. A single hibernating period of an undisturbed ground squirrel exhibiting the winter pattern of hibernating behaviour seldom varies more than :klO per cent from the average duration for hat animal. The variation in the duration of
9
hibernating periods for each animal used for this study was less than + 6 per cent. The average durations of hibernating periods of different animals at the same body temperature may be highly variable. Ground squirrels with different average durations of hibernating periods (those used in this study ranged from Ia8 to 229 hours) have been compared by means of a normalizing process which equates each animal's average duration as constituting xoo per cent. Fractions thereof, expressed as percentages, have been considered to be physiologically comparable. This technique has proved valid in respect to the dose-response relationship derived from injections of epinephrine (Twente and Twente, 1968b) Animals were sacrificed by decapitation within x minute from the time the cage was removed from the cold room. The regions of the brain were dissected on ice. The anatomical guidelines described by Glowinski and Iversen (I966) were followed; the tissues were weighed within 5 - i i minutes of decapitation. Intervals of the hibernating cycle that were sampled included: (a) from the termination of arousal to I t hours after arousal (the end of the period of activiW and beginning of induction); (b) during the induction into hibernation at core temperatures from x5.6 to 8.o° C.; (c) at the beginning of the hibernating period (o-xo per cent); (d) at the middle of the hibernating period (48-55 per cent) ; and (e) at the end of the hibernating period (95-too per cent). Because of the limited number of samples that could be analysed at any one time and because it was considered that storage could influence the results, not all regions from every animal were analysed. The concentrations of epinephrine and norepinephrine were determined for each region of the brain individually. Each tissue was homogenized in 15 ml. I o per cent trichloro-acetic acid with a Polytron homogenizer (PT io generator) according to the method of yon Euler and Lishajko (1959). Catecholamines were concentrated by adsorption onto o'5g. Alumina (British Drug House) and eluted with 2 ml. of cold o'2 ~/" acetic acid followed by 2 ml. of cold deionized water. Differential determination of epinephrine and norepinephrine was accomplished in these small samples by combining the automated trihydroxyindole fluorometric methods of Robinson and Watts (I965) and Fiorica (i965). Both methods employ the Technicon AutoAnalyzer system. A Technicon (Model I) fluorometer was used. The manifold of the Fiorica system was modified (a) • by adding a debubbler to the sodium hydroxide/ ascorbic acid stream and (b) by adding a single mixing coil to the final stream. The reagents of Robinson and Watts (I965) were employed for the determination of norepinephrine. Their method was modified by measuring epinephrine at pH 3"5- This allowed for the determination of a higher epinephrine to norepinephrine ratio than
5o
TWZm~ ET AL.
could be accomplished at 10H 6. The sensitivity was increased by eliminating the dialysis step. This procedure required that the blank for each sample be determined. Since epinephrine represented less than Io per cent of the total in the samples measured, the combination of methods provided a better differentiation of the two catccholamines than either method alone. Aliquots were used for duplicate determinations of both epinephrine and norepinephrine. Single catecholamine standards (25 rig. per ml. epinephrine and 5ong. per nil. norepinephrine),
averages and standard deviations o f the weights of the hypothalamus, medulla oblonRata (with pons), and cerebellum are shown in Table L These data are similar in regard to both absolute weight and degree of variation to those reported for similar dissections of the brain of the rat by Glowinski and Iversen (1966). The data regarding the concentrations of epinephrine and norepinephrine in the hypothalamus and the ratio of these two catecholamines are shown in relation to the cycle of hibernation in Fig. I. Regression analyses of the data during the hibernating period ( o - l o o per cent) indicate that no significant change occurs either in the concentration of epinephrine and norepinephrine or the ratio of the two. Regression analyses of thc data obtaincd for the medulla oblongata (with pons) and cercbellurn showed that no significant change occurrcd in the concentrations of the two catccholamincs or their ratio in either tissue as the hibernating period progressed.
Table L--WEIoH'r8 OF THE RROION'S OP TIIE BRAIN Or G R O U N D SQUIRRELS ANALYgED FOR EPINEPHRINE AND NOREPINEPHRINE
Tmstm
No.
MEAN WRIGHT
3I
29
o.x ~o (4-0.020) 0.263 (4-0"o94)
15
0"353 (4-0.o66)
Hypothalamus Medulla oblongata (with pore) Cerebellum
Coral0° gen. Pluzrma¢.
(g.4-S.D.)
~"able II.~MI~AN ~ONCT,NTRATION OF NOREFINEPHRINE IN THE BRAIN OF GROUND SQUIRRELS DURING THE HIBERNATING CYCLE
TIME or Sxm, LmO
MEDULLA OBLONOATA (wrrHPDN~
HYPo~s
DURING THE HIBERNATING
CYCLB
No.
Pefiodofacfivity Induction ~-xo per cent 48-55 per cent 95-moper cent Allperiods
6 6
7 4 8
3I
NE (~g. per g,±S.D.) I'I6 (4-0"33) 1.34 (4-O'OI) I'~9 (4-0"24) I'I7 (4-0"30 1'30 (+Oq6) X'24 (4-0"24)
No. 5* 6 6 4 8 24
CEREBELLUM
NE (gg. per g.:I:S.D.) No. 0"42 (4-0"05) 0"67 (4-oq4) o.66 (4-o'x6) o'6x (4-0'06) 0"65 (4-0"07)
0"65 (4-o',,)
NE (gg. per g.4-S.D.)
5 2 4
0.20 (+0"05) o.x8 (+0.02) 0.22 (+0.06)
X
3
o.x 9 0.26 (4-0.02)
I5
o'I9 (4-0"05)
*Because of statistical significance (P
The average concentrations of norepinephrine in the three regions of the brain at the different times of sampling are shown in Table II. Student's totest was used to compare the mean concentrations. No significant differences existed between any two sampling periods or any single sampling period and groupings of other sampling periods for concentrations of norepinephrine in the hypothalamus or cerebellum. A comparison
5t
BRAIN CATECHOLAMINES IN HIBERNATION
'970, x
each sample determination. T h e averages of these data for the different regions of the brain at the different times of sampling are shown in Table IV. Statistical evaluations of these data (analyses of regression, variance, and t-tests) indicated that the relative concentrations of the two catecholamines for each region of the brain was independent of the time of sampling. T h e total concentration of epinephrine and norepinephrine for each tissue for all
of the mean values for the medulla oblongata (with pons), however, showed that the average concentration of norepinephrine during the period of activity was significantly lower (P
Table III.--ME.~ CONCZSTI~OS OF EPINEPHRINEIN THE Bma~mo~ GROUNDSQUIRRELSDURINGTHE
I-ImERNATmGCYCLE oF SAMPLING HYPOTHALAMUS DURING THE HmERNAT~O E CYCLE No. (gg. per g.±S.D.)
MEDULLA OBLONOATA (WITH PONS)
T~
Period of activity [nduction >-,o per cent ~-55 per cent p5-xoo per cent All periods
6 6 7 4 8 31
No.
o . , o (±0"05) o . , , (±0"04) o.,o (4-0.03) o ' , 4 (±0"04) o.,x (4-0"04) o . , o (4-0"04)
CEREBELLUM
E
(gg. per g.±S.D.)
4
0"041 ('4-0"016)
6 6 4 8 =8
0"0'5 (±0"009) o'o30 (±o'oI6) 0.008 (±o'oo4)
E
No.
(gg. per g.±S.D.)
5
0.oo8 (±o-o, i) o'oo9 (±o"oo9) o.ol i (±o'oo9) o.oo6 o.oo7 (+0-007) o.oo9 (±0.008)
0
4 I
0.003 (±o'o14)
3
0.006 (4-o'o14)
'5
Table IV.--RATm O~ CONCZm~.ATmmO~ EPINEPHRINETO NOREPINEPHRINEIN THE BRAINOF GROUND
SQummzLs nuRiNo THE HmERNATINOCYCLE Tn~z oF SAMPLING
MEDULLAOBLONOATA (wrrH PONS)
HYPOTHALAMUS
DURING THE
CEREBELLUM
Hm~.RNATINO CYCLE
Period of activity [nducfion )--to per cent i~-55 per cent }5-1oo per cent MI periods
No.
N-~E(±S.D.)
6 6 7
o.ioo (±0-058) 0.080 (±o.o0o) o.mo (±0"005)
4
O'10I ( ± 0 " 0 1 8 )
8 3x
0"087 (±0"028) o'o96 (-I-o"o34)
No.
~(±S.D.)
4 6 6 4
0"078 (4-0"053) 0.008 (±o'o2o) o'o53 (±o'o35) 0"049 (±o'o16)
8
0"040 (z~O'OI8)
08
0-048 (±0"033)
measured are shown in Table I I L Statistical evaluations of these data (the same as those indicated for norepinephrine) showed no significant variation with regard to time of sampling during the hibernating cycle. T h e ratio of the concentration of epinephrine to norepinephrine was calculated for
No.
~(±S.D.)
5 " 4 x 3 I5
0"047 (-4-0-o58) 0"035 (±0"007) 0"057 (-4-o'o5x) 0"030 0"044 (±0"040) 0"047 (±0"043)
sampling times (excepting norepinephrine in the medulla oblongata (with ports) during the period of activity) showed average values as follows: hypothalamns, t.36 ttg. per g. (8.8 per cent epinephrine); medulla oblongata (with pons), o.68 ~tg. per g. (3.6 per cent epinephrine); and cerebellum, o.2o ttg. per g. (4"3 per cent epinephrine).
5~
vwz~rm ST AL.
DISCUSSION The relatively constant levels of epinephrine in the hypothalamns and medulla oblongata (with pons) found throughout the hibernating period do not support the original hypothesis of an accumulation of this monoamine. The possibility that epinephr/ne is involved with progressive irritability and with the ' t r i g g e r ' for arousal from the hibernating period is not negated, however. Progressive irritability may result from the lowering of thresholds of particular central neurons which are involved in the process which initiates arousal from the hibernating period. I f this were the case, then no change in the level of epinephrine or the rate of release would be necessary to elicit arousal, but rather an increased response to a low concentration of the transmitter would
suffice. There is also a possibilitythat cpincphrinc may reach the central nervous system from the peripheral circulation. The arousing effectof epincphrinc injectedi.p.would seem to enhance this argument (Twcntc and Twcntc, I968b ). Wcil-Malherbc, Whitby, and Axclrod (x9 6 i) demonstratcd that small amounts of epinephrine and norepincphrine pass the blood-brain barrier in the region of the hypothalamus ofthc cat,but arc cxcludcd from thc remainder of the brain. The statistically significant lower level of norepinephrine during the period of activity should be investigated more thoroughly in order to interpret the biological significance. The unequal distribution ofnorepinephrine in the brain of the golden-mantled ground squirrel is in accordance with the findings for the brain of the cat (Vogt, i954) , of the rat (Glowinski and Iversen, 1966; Manshardt and Wurtman, x968), and of man (Sano, Gamo, Kakimoto, Taniguchi, Takesada, and Nishinuma, I959). The hypothalamus was shown to contain the highest regional concentration of norepinephrinc in all of these studies. The regional distributionof epincphrinc in the brain has bccn neglected, generally, until this study, primarily because of the difficulty of measuring the low conccntra°
Comp. gen. Pl~rmac.
tions. The unequal distribution of epinephrine that parallels that of norepinephrine in these three regions of the brain does not seem to have been reported previously for any species. The unequal regional distribution of epinephrine is of interest in regard to this monoamine as a central neurotransmitter. Several of the criteria for a neurotransmitter are met when the regional distribution is considered in conjunction with the finding of the presence and unequal distribution of phenylethanolamine-N-methyl transferase (Pohorecky and others, I969). ACKNOW/.~DOEMENT$
We are indebted to Horst-Dieter Dellmann for his assistance in teaching one of us (J. W.T.) the techniques of dissection of the regions of the brain. REFERENCES DRASK6CZY, P. R., and LYMAN, C. P. (x967),
'Turnover of catecholamines in active and hibernating ground squirrels',07. P/~rmac. exp. 7~f., I 5 ~ IOI--I I I.
yon EULZR,U. S., and LmRAJKO,F. (x959), ' The estimation of catecholamlnes in urine ', Acta physiol, scand., 45t 122-x 32. FIosJcA, V. (1965), 'An improved semi-automated procedure for fluorometric determination of plasma eatecholamines ', Clinica chim. Acta, x ~ z9I-I97. GLOWn~SKI,J., and IVERSEN,L. (I966), ' Regional studies of catecholamlnes in rat brain, I. The disposition of (SH) norepinephrine and (SH) dopa in various regions of the brain ', 07. aVeurochem.,z$, 655-669. M~SHAngT, J., and WURTm~, R. J. (I968), 'Daily rhythm in the noradrenaline content of rat hypothalamus'; Nature, Lond., ax7, 574-575. POHORECKY, L. A., ZIGMOND, M. J., K~TEN, H.J., and WUgT~'% R.J. (i 969), ' Enzymatic conversion of norepinephrine to epinephrine by the brain ', 07. Pharmac. exp. Ther., x65, x9o-195. ROBINSON,R. L., and WATTS, D. T. (I965), 'An
automatcd trihydroxyindoleprocedure for the differentialanalysis of catccholamines', Clin. Chem., xx, 986--997. SnNO, I., G,~o, T., KnraMowo, Y., T,~mGUCHX,K., T,~,Y.anVA, M., and NmHm-UMA, L. (x959), ' Distribution of catechol compounds in human brain ', Biochim. biophys. Acta, 32, 586-587. TW~NTE, J. W., and TWENTY, J. A. (x965a), ' Regulation of hibernating periods by temperature ', Proc. natn. Acad. Sci. U.S.A., 54, Io58xo6x.
197o, I
BRAIN CATECHOLAMINES IN HIBERNATION
TwE~rE, J. W., and Twe~rrE, J. A. 0965b), ' Effects of core temperature upon duration of hibernation of Citellva lateralis ', 07. appl. Physiol., 2 0 , 4x x-4x6. . . . . . (I967) , 'Seasonal variation in the hibernating behaviour of Citellus iateralis ', in Mammalian Hibernation (ed. FmrmR, K. C., DAWE, A. W., LYMAN, C. P., SCH(JNBAUM,E., and SOUTH, F. E.), vol. I I I , pp. 47-63. Edinburgh: Oliver and Boyd. (I968a), 'Progressive irritability of hibernating Citdlus iateralis ', Comp. Biochem. Physiol., ~5, 467-474. (I968b), ' Effects of epinephrine upon progressive irritability of hibernating Citellus lateralis ', Ibid., 25, 475-483. UUSv~A, V. T. (x963) , ' The catecholamine content of the brain and heart of the hedgehog (Erinaceus europaeus) during hibernation and in
53
an active state ', Anals Med. exp. Biol. Finn., 4 x,
340-348.
VOOT, M. (1954) , ' The concentration of sympathin in different parts of the central nervous system under normal conditions and after the administration of drugs ', .7. Physiol., Lond., x~3,
451-481.
WV_aL-MAL~RB~, H., WHrreY, L. G., and AXELROD,J. (196i), ' The blood-braln barrier for catecholamincs in different regions of the brain ', in Regional vVeurodmnistr) (ed. KXTY, S., and ELKES, J.), pp. 284-992. Oxford: Pergamon . . . . . . . . . . K~ Word Index: Brain, catecholamlncs, cerebellum, Citdlus lateralis, duration of the hibernating period, regional distribution of epinephrine, ground squirrel, hibernation, hypothalamus, medulla oblongata, regional distribution of norepinephrine.