- Email: [email protected]
Seasonal variations of brain epinephrine, norepinephrine and 5-hydroxytryptamine associated with changes in the EEG of the toad, Bufo arenarum hensel
Comp. Biochem. Physiol.,
1967, Vol. 22, pp. 843 to 850. Pergamon Press Ltd. Printed in Great Britain
SEASONAL VARIATIONS OF BRAIN EPINEPHRINE, NOREI?INEPHRINE AND 5-HYDROXYTRYPTAMINE ASSOCIATED WITH CHANGES IN THE EEG OF THE TOAD, BUFO A R E N A R U M HENSEL* E. T. S E G U R A , t A. M. B I S C A R D I ~ and J. A P E L B A U M : ~ Instituto de Biologla y Medicina Experimental, Obligado 2490, Buenos Aires and Instituto de Anatomia y Embriologla, Facultad de Medicina, Buenos Aires, Argentina (Received
13 M a r c h 1967)
Abstrac't--1. Observations concerning the concentrations of epinephrine (E), norepinephrine (NE) and 5-hydroxytryptamine (serotonin, 5-HT) in the brain of the toad and the EEG pattern according to the seasons of the year were simultaneously performed. 2. Brain E showed marked seasonal changes, with its lowest value near the onset of the mating period. From this moment it began to increase, reaching its maximum at the middle of estivation. Thereafter a progressive and significant lowering was noted. 3. Conversely, 5-HT brain content showed its highest during hibernation, whereas a marked fall coinciding with mating was evident. 4. Accordingly, the 5-HT/E ratio varied in the course of the year from a maximtma of 18 during hibernation to 1 "3 in the middle of the estivating period. 5. Our results concerning these seasonal and opposite displacements of brain levels in E and 5-HT in toads are discussed in relation to simultaneously occurring charLges in the EEG and to the hibernation-estivation cycle in amphibians.
INTRODUCTION
THE POSSIBLErole of brain monoamines in the control of different aspects of animal behaviour has, been extensively considered in the literature. F r o m a methodological point of view the most convenient approach to this p r o b l e m seems obviously to be the search for a clear-cut causal correspondence between a given pattern of natural behaviour and its specific biochemical correlate. T h i s is not a simple task, however, and while a very long inventory of scattered analytical data is available on the subject, little is known about its actual functional meaning (Brodie & Costa, 1962; Kety, 1966). T h e permanently changing nature of animal behaviour induces one to suspect that its biochemical support m a y also * Aided by a grant from the Consejo Nacional de Investigaciones Cientificas y T6cnicas de la Argentina.. t Established Investigator, Consejo Nacional de Investigaciones Cientificas y T6cnicas de la Argentina. Present address: Thudichum Psychiatric Research Laboratory, Galesburg, Illinois, U.S.A. 843
844
E.T. SEGUP.A,A. M. BISCARDIANDJ. APELBAUM
present concomitant fluctuations. For this reason, natural circadian sleep-wakefulness and seasonal hibernation-cstivation cycles seem to furnish excellent prototypes for observational studies on simultaneously occurring behavioural, electrica, and biochemical events. For instance, a significant increase in brain serotonin coinciding with natural sleep has been reported by Mattussek & Patschke (1964) in the male hamster. Conversely, when the animals were at their highest motor activity, brain serotonin markedly diminished. On the other hand, Uusp~ia (1963a, b) found that the brain of the hedgehog (EHnaceus europaeus) contains a larger amount of NE during natural hibernation than during summer, the opposite being the case for serotonin. Seasonal changes of brain NE have also been described by BeauvaUet et al. (1962) in the rat. Furthermore, several papers concerning the EEG changes that take place coinciding with hibernation in mammals have been published (Chat-field et al., 1951 ; Kayser et al., 1951; Rohmer et al., 1951; Strumwasser, 1959a, b; Shtark, 1961), whereas in cold-blooded animals, to the best of our knowledge, the only available data are those of Segura & de Juan (1966). They found in the toad, Bufo arenarum Hensel, that brain waves changed from a "slow hibernating" pattern in winter to an "alpha-like" fusiform tracing during the estivation period, in spite of the fact that external conditions were kept constant. Simultaneous observations of both electrical and humoral changes in the brain, related to natural behavioural states are not available in the current literature. For this reason it was thought to be of interest to investigate whether or not the seasonal variations in the EEG of the toad are paralleled by significant changes in brain E, NE and 5-HT. MATERIALS AND METHODS
Throughout two consecutive years (1964-65 and 1965-66) monthly observations concerning the EEG pattern and brain E and NE were carried out. During 1964 the brain content of 5-HT was also assayed bimonthly. Adult animals of both sexes were used after they had been kept for at least 3 weeks in captivity, in an air-conditioned windowless room at a fixed temperature (22°C + 0.5), with humidity at saturation and with automatically controlled periods of light (14 hr daily). As was described in another paper (Segura & de Juan, 1966), both acute as well as chronic experiments were performed in order to detect any periodical change in brain electrical rhythms. Other series of observations concerning the variations of brain monoamine content in animals under the same experimental conditions were simultaneously carried out. For this purpose a total of almost nine hundred animals were used. Immediately after decapitation, the brain was removed and dissected in an ice-cold watchglass and rapidly weighed with a Roller-Smith Precision Balance. The following three electrically active areas were explored: olfactory bulbs, brain vesicles and mesencephalon without cerebellum.
845
EEG CHANGES IN THE TOAD B U F O A R E N A R U M
For E and NE determinations, four samples of eight brains each were tested monthly, using the method of Cohen & Goldenberg (1957), after adsorption on alumina columns (yon Euler & Lishajko, 1959). With respect to 5-HT, ten individual biological assays were performed bimonthly with the technical procedure described by Vane (1957) in the rat fundus. Statistical evaluation of the data was done with variance analysis; in the case of catecholammes, after elimination of secular variations (Lison, 1958).
RESULTS The brain concentrationof the three monoaminesstudied(E, NE and 5-HT) showed significant changes throughout the year (Tables 1 and 2; Fig. 1). At the same time, a close parallel between seasonal variations in brain potentials and the E and 5 - H T content of the electrically active explored areas was clearly noted (Fig. 1). In effect, during the hibernating period, i.e. autumn and winter (AprilAugust in the southern hemisphere), comparatively large amounts of 5 - H T were present, while E was at its lowest, together with a slow (3-5 c/s) pattern of EEG, irregular in arnplitude, without spindles and low sensitivity to external stimuli. T A B L E 1 - - - E P I N E P H R I N E AND NOREPINEPHRINE CONCENTRATIONS IN TOAD BRAIN
Catecholamine (/zg/g)* Season
Month
Year
Winter
June July August September October November December January February March April May
1964-65 1964-65 1964-65 1964t 1964--65 1964-65 1964-65 1965-66 1965-66 1965-66 1965-66 1965-66
Spring Summer Auturan
E
NE
1"04 + 0"02 0'64 + 0.02 0"64 + 0"01 0"49+ 0"03 0"73 + 0-07 0.43+ 0.006 0.66 +_0"05 0"47+ 0.001 1"21 + 0"07 1"48+ 0.15 0'99 + 0"01 0"40+_0.007 1"01 + 0"19 0"23+ 0.005 1"13 + 0.04 0"47_+0"03 1.36 + 0"08 < 0-1 0'72 + 0"07 0"60+ 0"02 0"84 + 0"04 0"93+ 0"01 0"97 + 0"18 1"73_+0"13
* Mean value of both years + standard error. No data available for September 1965. In marked synchronism with the onset of the mating period (September) significant changes in brain waves as well as in E and 5 - H T were evident. A typical "alpha-like" rhythm, ranging from 8 to 12 c/s in frequency, fusiform in shape and very sensitive to external stimulation appeared, in close coincidence with a sharp increase in brain E and a significant decrease of 5 - H T (Table 2; Fig. 1). Concurrently the 5 - H T to E ratio also showed important yearly variations. In effect, during July (winter) it reached its highest value of 18, whereas at the
846
E.T.
SEGURA, A. M . BISCARDI AND J. APELBAUM
beginning of the mating period (September) this ratio fell to 10. Thereafter, the ratio progressively decreased until December (summer) when it showed its lowest value (1-3). With the end of the estivating period and just when the animals were going into hibernation again (March-April) a marked and sudden increase in the 5-HT to E ratio was noted. The variance analysis, after elimination of secular variations, proved that the seasonal changes for brain E and NE were significant for P < 0.01; variance
]50/~V
I-2--
--6.0
I see
5.0
I.C-- I
I ~O.E
--
\ -
4-0 en
f
•
c" 5.0 o
\
o
.c_
>--0~
"~ 0.4
-
0.2--
I I I [ I I I I I d
A S 0 N D J
.0
I
F M A M
FIG. 1. O p p o s i t e changes i n b r a i n e p i n e p h r i n e (full line) a n d 5 - H T , coinciding w i t h seasonal variations of t h e E E G . Records at t h e top illustrate the p a t t e r n of t h e E E G , a c c o r d i n g to t h e season. See text. TABLE 2--COMPARISON
BETWEEN
E
AND
5-HT
C O N C E N T R A T I O N I N TOAD B R A I N *
M o n o a m i n e s (/~g/g) I" Season
Month
5-HT
Winter Spring
July September October December March April
5"77 _+0-11 1'85 _+0"12 2'19 _+0"54 1"40 _+0"16 2"53 + 0-10 3"88 + 0"10
Summer Autumn
* M e a n value + s t a n d a r d error. D a t a c o r r e s p o n d i n g to 1 yr.
E 0"32 0"18 0"68 1-08 0"28 0"30
_+0'01 _+0-05 +_0-07 + 0"19 + 0'07 + 0"04
5 - H T to E ratio 18 10 3-2 1-3 9 13
EEGCHANGESIN THE TOADBUFO ARENARUM
847
analysis in the brain content of 5-HT showed them to be significant for P < 0.05. No sexual differences were observed. DISCUSSION AND CONCLUSIONS According to the well-known hypothesis proposed by Hess (1948), basic patterns of behaviour in mammals could be under the contro] of two opposite somato-vegetative coordinating structures of the CNS. Those behavioural s t a t e s characterized by a marked expenditure of energy are postulated to be modulated by a so-called "ergotropic system", adrenergic in nature. They usually involve a high degree of responsiveness to external stimulation, sympathetic predominance and increased motor activity. On the other hand, physiological states such as natural sleep :and hibernation, whose common trait is energy storage, could be regulated by another "trophotrophic system" with primarily cholinergic mechanisms. The behavioural correlates of these last conditions are a lowering in physical activity, with progressive indifference to sensory stimuli and increased central parasympathetic output. In recent years, the works of Brodie and his group have given support to the view that NE is the possible neurotransmitter for the "ergotropic" system of Hess, while 5-HT could play a similar role for the "trophotropic" one (Brodie & Shore, 1957; Brodie et al., 1959). The well-established fact that E instead of NE is the main catecholamine present in brain as well as in peripheral tissues of frogs and toads (Bertler & Rosengren, 1959; Anton & Sayre, 1962; Bogdanski et al., 1963; Brodie et al., 1964; Brodie & Bogdanski, 1964; Segura & Biscardi, 1967), associated with some experimental evidence (Falck et al., 1963 ; Segura & Biscardi, 1967) suggests that E and not NE acts as sympathetic transmitter in amphibians. As Brodie et al. (1964) also pointed out, the brain 5-HT to catecholamines ratio seems to be of particular interest from the phylogenetical point of view; they found this relation to be about 1 : 1 in rats and rabbits, whereas in birds, reptiles and amphibia it reached an average value of 2. The possible functional correspondence between the level of some naturally occurring brain monoamines and several behavioural states, such as sleep and hibernation, has only been the subject of cross-sectional studies, while no longitudinal approaches have been reported in the literature up to date. For this reason, the close coincidence between the seasonal evolution of brain 5-HT and the yearly biological cycles in toads reported in this paper deserves to be stressed. In effect, during winter, when animal behaviour clearly denotes a predominance of energy-saving mechanisms, with lower motor activity, an important decrease in O3 consumption and general metabolic rate (Fromm & Johnson, 1955), associated witlh increased storage of carbohydrate, fat and proteins in different organs (Mazzocco, 1938a, b, c, 1940), is noted. Also a marked relative arterial hypotension with a lowering in vasomotor responsiveness has been reported in this period (Segura & D'Agostino, 1964).
848
E. T.
SEGURA,A. M. BISCARDI AND J. APELBAUM
All these features suggest prima f a d e a functional preponderance of parasympathetic mechanisms. At the same time, brain waves present their typical winter pattern, with low frequency, smooth appearance and high threshold to external stimuli. T h i s picture, observed precisely when toad brain serotonin level reaches its m a x i m u m , is quite similar to that described in m a m m a l s when receiving this substance, its precursor 5-hydroxytryptophan, or drugs that deplete brain N E without affecting 5 - H T stores (Costa et al., 1960; GlOsser & Mantegazzini, 1960; Koella et al., 1960; Brodie & Costa, 1962; Koella & Czicman, 1966). Similarly, the marked decrease in brain E observed in this period might give some support to the view that a rise in the 5 - H T to E cerebral index could play a special role in the triggering and maintenance of hibernating states in this species. Conversely, O 3 consumption and the general endocrino-metabolic rate as well as circulatory responses are greatly enhanced during mating and estivation. T h e s e facts, in phase with the significant and opposite displacement in the E and 5 - H T brain content, again induce one to consider a possible causal relationship between the seasonal cycles and the level of monoamines in the C N S of the toad. Also the E E G pattern of this period, showing a marked increase in frequency, together with the appearance of spindles and high sensitivity to afferent stimulation, suggest an increment in the sympathetic "ergotropic" tonus. REFERENCES ANTON A. H. & SAYI~ D. F. (1962) A study of the factors affecting the aluminum oxide trihydroxyindole procedure for the analysis of catecholamines. J. Pharmac. exp. Ther. 138, 360-375. BEAUVALLETM., FUGAZZAJ. & SOLIER i . (1962) Nouvelles recherches sur les variations saisonni~res de la noradr6naline c6r6brale..7. Physiol., Paris 54, 289. BERTLERA. & ROSENGRENE. (1959) Occurrence and distribution of dopamine in brain and other tissues. Experientia 15, 10-11. BOODANSKI D. F., BONOMI L. & BRODIE B. B. (1963) Occurrence of serotonin and catecholamines on brain and peripheral organs of various vertebrate classes. Life Sci. 2, 80-84. BRODIF-B. B. & BOGDANSKXD. F. (1964) Biogenic amines and drug action in the nervous system of various vertebrate classes. Prog. Brain Res. 9, 234-242. BRODIE B. B., BOGDANSKID. F. & BONOMI L. (1964) Formation, storage and metabolism of serotonin (5-hydroxytryptamine) and catecholamines in lower vertabrates. In Comparative Neuroehemistry (Edited by RICHTER D.) pp. 367-377. Pergamon Press, Oxford. BRODIE B. B. & COSTAE. (1962) Some current views on brain monoamines. In Monoamines et systbme Nerveux Central (Edited by DE AJURIAGUERRAJ.) pp. 13-49. Georg & Cie, Gen6ve. BRODIE B. B. & SHORE P. A. (1957) A concept for a role of serotonin and norepinephrine as chemical mediators in the brain. Ann. N . Y . Acad. Sci. 66, 631-642. BRODIE B. B., SPECTOR S. & SHORE P. A. (1959) Interaction of drugs with norepinephrine in the brain. Pharmac. Rev. 11, 548-564. CHATFmLOP. O., LYMANC. P. & PURPtmA D. P. (1951) The effects of temperature on the spontaneous and induced electrical activity in the cerebral cortex of the golden hamster. Electroenceph. clin. Neurophysiol. 3, 225-230.
EEG CHANGES IN THE TOAD BUFO ARENARU~VI
849
COHEN G. & (.~OLDENBERG M. (1957) T h e simultaneous fluorometric determination of adrenaline and noradrenaline in plasma. II. Peripheral venous plasma concentrations in normal subjects and in patients with pheochromocytoma. J . Neurochem. 2, 71-80. COSTA E., PSCHIZIDTG. R., METER W. G. VAN & HIMWICH H. E. (1960) Brain concentrations of biogenic amines and E E G patterns of rabbits. J. Pharmac. exp. Ther. 130, 81-88. Euta~R U. S. yon & LIS~JKO F. (1959) T h e estimation of catecholamines in urine. Acta physiol, scald. 45, 122-132. FALCK B., HAGGm,mALJ. & Owm~'~ CH. (1963) T h e localization of adrenaline in adrenergic nerves in the frog. Quart.J. exp. Physiol. 48, 253-257. FaOMM P. O. & JOHNSON J. Z. (1955) T h e respiratory metabolism of frogs as related to season, y. cell. comp. Physiol. 45, 343-359. GLOSSER A. & M~'~TEOAZZINI P. (1960) Action of 5-hydroxytryptamine and of 5-hydroxytryptophan on the cortical electrical activity of the midpontine pretrigeminal preparation of the cat with and without mesencephalic hemisection. Archo. ital. Biol. 98, 351-366. HESS W. R. (1948) Die Funktionelle Organisation des Vegetativen Nervensystems. pp. 1-226. Schwave, Basel. I~'¢SEa~ CH., ROHMER F. & HmBEL G. (1951) L ' E . E . G . de l'hibernant. L~thargie et r~veil spontan~ du spermophile. Essai de r~production de I'E.E.G. chez le spermophile et le rat blanc. Rev. Neurol., Paris 84, 570-578. KETY S. S. (1966) Catecholamines in neuropsychiatric states. Pharmac. Rev. 18, 787-798. KOELLA W. P. & CZlCM~a'~ J. H. (1966) Mechanism of the EEG-synchronizing action of serotonin. Am. J. Physiol. 211, 926-934. KOELLA W. P., SMYTHmS J. R., BULL D. M. & LEvY CH. K. (1960) Physiological fractionation of the effect of serotonin on evoked potentials. Am. v~. Physiol. 198, 205-216. LISON L. (1958) Statistique Appliqude ~ la Biologic Expdrimentale. pp. 1-346. GauthierVillars, Paris. MATTUSSEK W. & PATSCHK~ U. (1964) Beziehungen des Schlaf und Wachrhythmus zum Noradrenalin- und Serotoningehalt im Zentralnervensystem yon Hamstern. Med. Expl. 11, 81-87. MAZZOCCO P. (1938a) Variaciones estacionales de la composici6n del hlgado de sapo Burg arenarum Hensel. Revta Soc. argent. Biol. 14, 123-144. MAzzocco P. (1938b) Variaciones estacionales de la composici6n quima del musct~lo del Bufo arenarum Hensel. Revta Soc. argent. Biol. 14, 236-245. MAZZOCCO P. (1938e) Oscilaciones peri6dicas del metabolismo hidrocarbonado en el sapo BurG arenarum Hensel. Revta Soc. argent. Biol. 14, 330-334. MAZZOCCO,P. (1940) Variaciones estacionales del peso y composici6n de los cuerpos adiposos del sapo B~fo arenarum Hensel. Revta Soc. argent. Biol. 16, 129-138. ROHMER F . , H]EBEL G. & KAYSERCH. (1951) Reeherches sur le fonctionnement du syst6me nerveux de'; hibernants. Les ondes c6r6brales pendant le sommeil hibernal et le r6veil. Etude sur le spermophile. C. r. s3anc. Soc. Biol. 145, 747-752. S E G ~ E. T . & B1sc~tDt A. M. (1967) Changes in brain E and NE induced by afferent stimulation in the isolated toad head. Life Sci. 6, 1599-1603. SECU~ E. T. & D'AGosTINO S. A. (1964) Seasonal variations of blood pressure, vasomotor reactivity and plasmatic cateeholamines in the toad. Aetaphysiol. latinoam. 14, 231-237. SEOUR~ E. T . & DE JuAN A. (1966) Electroencephalographic studies in toads. Electroenceph. clin. Neurophysiol. 21, 373-380. SHT~K M. B. (1961) Electrophysiological study of hibernation. Sechenov physiol, v~. U S S R 47, 1028-1036. STRUMWASSER F. (1959a) Thermoregulatory brain and behavioral mechanisms during entrance into hibernation in the squirrel, Citellus beecheyi. Am. ~. Physiol. 196, 15-22. STRUMWASSER F. (1959b) Regulatory mechanisms, brain activity and behavior during deep hibernation in squirrel, Citellus beecheyi. Am..7. Physiol. 196, 23-30.
850
E. T. SEGURA,A. M. BlSCARDIAND J. APELBAUM
UUSP~ V. J. (1963a) The catecholamine content of the brain and heart of the hedgehog (Erinaceus europaeus) during hibernation and in an active state. Ann. ivied, exp. Biol. Fenn. 41, 340-348. UusP)L~ V. J. (1963b) The 5-hydroxytryptamine content of the brain and some other organs of the hedgehog (Erinaceus europaeus) during activity and hibernation. Experientia 19, 156. VANE S. R. (1957) A sensitive method for the assay of 5-hydroxytryptamine. Br.J. Pharmac. 12, 344-349.
1967, Vol. 22, pp. 843 to 850. Pergamon Press Ltd. Printed in Great Britain
SEASONAL VARIATIONS OF BRAIN EPINEPHRINE, NOREI?INEPHRINE AND 5-HYDROXYTRYPTAMINE ASSOCIATED WITH CHANGES IN THE EEG OF THE TOAD, BUFO A R E N A R U M HENSEL* E. T. S E G U R A , t A. M. B I S C A R D I ~ and J. A P E L B A U M : ~ Instituto de Biologla y Medicina Experimental, Obligado 2490, Buenos Aires and Instituto de Anatomia y Embriologla, Facultad de Medicina, Buenos Aires, Argentina (Received
13 M a r c h 1967)
Abstrac't--1. Observations concerning the concentrations of epinephrine (E), norepinephrine (NE) and 5-hydroxytryptamine (serotonin, 5-HT) in the brain of the toad and the EEG pattern according to the seasons of the year were simultaneously performed. 2. Brain E showed marked seasonal changes, with its lowest value near the onset of the mating period. From this moment it began to increase, reaching its maximum at the middle of estivation. Thereafter a progressive and significant lowering was noted. 3. Conversely, 5-HT brain content showed its highest during hibernation, whereas a marked fall coinciding with mating was evident. 4. Accordingly, the 5-HT/E ratio varied in the course of the year from a maximtma of 18 during hibernation to 1 "3 in the middle of the estivating period. 5. Our results concerning these seasonal and opposite displacements of brain levels in E and 5-HT in toads are discussed in relation to simultaneously occurring charLges in the EEG and to the hibernation-estivation cycle in amphibians.
INTRODUCTION
THE POSSIBLErole of brain monoamines in the control of different aspects of animal behaviour has, been extensively considered in the literature. F r o m a methodological point of view the most convenient approach to this p r o b l e m seems obviously to be the search for a clear-cut causal correspondence between a given pattern of natural behaviour and its specific biochemical correlate. T h i s is not a simple task, however, and while a very long inventory of scattered analytical data is available on the subject, little is known about its actual functional meaning (Brodie & Costa, 1962; Kety, 1966). T h e permanently changing nature of animal behaviour induces one to suspect that its biochemical support m a y also * Aided by a grant from the Consejo Nacional de Investigaciones Cientificas y T6cnicas de la Argentina.. t Established Investigator, Consejo Nacional de Investigaciones Cientificas y T6cnicas de la Argentina. Present address: Thudichum Psychiatric Research Laboratory, Galesburg, Illinois, U.S.A. 843
844
E.T. SEGUP.A,A. M. BISCARDIANDJ. APELBAUM
present concomitant fluctuations. For this reason, natural circadian sleep-wakefulness and seasonal hibernation-cstivation cycles seem to furnish excellent prototypes for observational studies on simultaneously occurring behavioural, electrica, and biochemical events. For instance, a significant increase in brain serotonin coinciding with natural sleep has been reported by Mattussek & Patschke (1964) in the male hamster. Conversely, when the animals were at their highest motor activity, brain serotonin markedly diminished. On the other hand, Uusp~ia (1963a, b) found that the brain of the hedgehog (EHnaceus europaeus) contains a larger amount of NE during natural hibernation than during summer, the opposite being the case for serotonin. Seasonal changes of brain NE have also been described by BeauvaUet et al. (1962) in the rat. Furthermore, several papers concerning the EEG changes that take place coinciding with hibernation in mammals have been published (Chat-field et al., 1951 ; Kayser et al., 1951; Rohmer et al., 1951; Strumwasser, 1959a, b; Shtark, 1961), whereas in cold-blooded animals, to the best of our knowledge, the only available data are those of Segura & de Juan (1966). They found in the toad, Bufo arenarum Hensel, that brain waves changed from a "slow hibernating" pattern in winter to an "alpha-like" fusiform tracing during the estivation period, in spite of the fact that external conditions were kept constant. Simultaneous observations of both electrical and humoral changes in the brain, related to natural behavioural states are not available in the current literature. For this reason it was thought to be of interest to investigate whether or not the seasonal variations in the EEG of the toad are paralleled by significant changes in brain E, NE and 5-HT. MATERIALS AND METHODS
Throughout two consecutive years (1964-65 and 1965-66) monthly observations concerning the EEG pattern and brain E and NE were carried out. During 1964 the brain content of 5-HT was also assayed bimonthly. Adult animals of both sexes were used after they had been kept for at least 3 weeks in captivity, in an air-conditioned windowless room at a fixed temperature (22°C + 0.5), with humidity at saturation and with automatically controlled periods of light (14 hr daily). As was described in another paper (Segura & de Juan, 1966), both acute as well as chronic experiments were performed in order to detect any periodical change in brain electrical rhythms. Other series of observations concerning the variations of brain monoamine content in animals under the same experimental conditions were simultaneously carried out. For this purpose a total of almost nine hundred animals were used. Immediately after decapitation, the brain was removed and dissected in an ice-cold watchglass and rapidly weighed with a Roller-Smith Precision Balance. The following three electrically active areas were explored: olfactory bulbs, brain vesicles and mesencephalon without cerebellum.
845
EEG CHANGES IN THE TOAD B U F O A R E N A R U M
For E and NE determinations, four samples of eight brains each were tested monthly, using the method of Cohen & Goldenberg (1957), after adsorption on alumina columns (yon Euler & Lishajko, 1959). With respect to 5-HT, ten individual biological assays were performed bimonthly with the technical procedure described by Vane (1957) in the rat fundus. Statistical evaluation of the data was done with variance analysis; in the case of catecholammes, after elimination of secular variations (Lison, 1958).
RESULTS The brain concentrationof the three monoaminesstudied(E, NE and 5-HT) showed significant changes throughout the year (Tables 1 and 2; Fig. 1). At the same time, a close parallel between seasonal variations in brain potentials and the E and 5 - H T content of the electrically active explored areas was clearly noted (Fig. 1). In effect, during the hibernating period, i.e. autumn and winter (AprilAugust in the southern hemisphere), comparatively large amounts of 5 - H T were present, while E was at its lowest, together with a slow (3-5 c/s) pattern of EEG, irregular in arnplitude, without spindles and low sensitivity to external stimuli. T A B L E 1 - - - E P I N E P H R I N E AND NOREPINEPHRINE CONCENTRATIONS IN TOAD BRAIN
Catecholamine (/zg/g)* Season
Month
Year
Winter
June July August September October November December January February March April May
1964-65 1964-65 1964-65 1964t 1964--65 1964-65 1964-65 1965-66 1965-66 1965-66 1965-66 1965-66
Spring Summer Auturan
E
NE
1"04 + 0"02 0'64 + 0.02 0"64 + 0"01 0"49+ 0"03 0"73 + 0-07 0.43+ 0.006 0.66 +_0"05 0"47+ 0.001 1"21 + 0"07 1"48+ 0.15 0'99 + 0"01 0"40+_0.007 1"01 + 0"19 0"23+ 0.005 1"13 + 0.04 0"47_+0"03 1.36 + 0"08 < 0-1 0'72 + 0"07 0"60+ 0"02 0"84 + 0"04 0"93+ 0"01 0"97 + 0"18 1"73_+0"13
* Mean value of both years + standard error. No data available for September 1965. In marked synchronism with the onset of the mating period (September) significant changes in brain waves as well as in E and 5 - H T were evident. A typical "alpha-like" rhythm, ranging from 8 to 12 c/s in frequency, fusiform in shape and very sensitive to external stimulation appeared, in close coincidence with a sharp increase in brain E and a significant decrease of 5 - H T (Table 2; Fig. 1). Concurrently the 5 - H T to E ratio also showed important yearly variations. In effect, during July (winter) it reached its highest value of 18, whereas at the
846
E.T.
SEGURA, A. M . BISCARDI AND J. APELBAUM
beginning of the mating period (September) this ratio fell to 10. Thereafter, the ratio progressively decreased until December (summer) when it showed its lowest value (1-3). With the end of the estivating period and just when the animals were going into hibernation again (March-April) a marked and sudden increase in the 5-HT to E ratio was noted. The variance analysis, after elimination of secular variations, proved that the seasonal changes for brain E and NE were significant for P < 0.01; variance
]50/~V
I-2--
--6.0
I see
5.0
I.C-- I
I ~O.E
--
\ -
4-0 en
f
•
c" 5.0 o
\
o
.c_
>--0~
"~ 0.4
-
0.2--
I I I [ I I I I I d
A S 0 N D J
.0
I
F M A M
FIG. 1. O p p o s i t e changes i n b r a i n e p i n e p h r i n e (full line) a n d 5 - H T , coinciding w i t h seasonal variations of t h e E E G . Records at t h e top illustrate the p a t t e r n of t h e E E G , a c c o r d i n g to t h e season. See text. TABLE 2--COMPARISON
BETWEEN
E
AND
5-HT
C O N C E N T R A T I O N I N TOAD B R A I N *
M o n o a m i n e s (/~g/g) I" Season
Month
5-HT
Winter Spring
July September October December March April
5"77 _+0-11 1'85 _+0"12 2'19 _+0"54 1"40 _+0"16 2"53 + 0-10 3"88 + 0"10
Summer Autumn
* M e a n value + s t a n d a r d error. D a t a c o r r e s p o n d i n g to 1 yr.
E 0"32 0"18 0"68 1-08 0"28 0"30
_+0'01 _+0-05 +_0-07 + 0"19 + 0'07 + 0"04
5 - H T to E ratio 18 10 3-2 1-3 9 13
EEGCHANGESIN THE TOADBUFO ARENARUM
847
analysis in the brain content of 5-HT showed them to be significant for P < 0.05. No sexual differences were observed. DISCUSSION AND CONCLUSIONS According to the well-known hypothesis proposed by Hess (1948), basic patterns of behaviour in mammals could be under the contro] of two opposite somato-vegetative coordinating structures of the CNS. Those behavioural s t a t e s characterized by a marked expenditure of energy are postulated to be modulated by a so-called "ergotropic system", adrenergic in nature. They usually involve a high degree of responsiveness to external stimulation, sympathetic predominance and increased motor activity. On the other hand, physiological states such as natural sleep :and hibernation, whose common trait is energy storage, could be regulated by another "trophotrophic system" with primarily cholinergic mechanisms. The behavioural correlates of these last conditions are a lowering in physical activity, with progressive indifference to sensory stimuli and increased central parasympathetic output. In recent years, the works of Brodie and his group have given support to the view that NE is the possible neurotransmitter for the "ergotropic" system of Hess, while 5-HT could play a similar role for the "trophotropic" one (Brodie & Shore, 1957; Brodie et al., 1959). The well-established fact that E instead of NE is the main catecholamine present in brain as well as in peripheral tissues of frogs and toads (Bertler & Rosengren, 1959; Anton & Sayre, 1962; Bogdanski et al., 1963; Brodie et al., 1964; Brodie & Bogdanski, 1964; Segura & Biscardi, 1967), associated with some experimental evidence (Falck et al., 1963 ; Segura & Biscardi, 1967) suggests that E and not NE acts as sympathetic transmitter in amphibians. As Brodie et al. (1964) also pointed out, the brain 5-HT to catecholamines ratio seems to be of particular interest from the phylogenetical point of view; they found this relation to be about 1 : 1 in rats and rabbits, whereas in birds, reptiles and amphibia it reached an average value of 2. The possible functional correspondence between the level of some naturally occurring brain monoamines and several behavioural states, such as sleep and hibernation, has only been the subject of cross-sectional studies, while no longitudinal approaches have been reported in the literature up to date. For this reason, the close coincidence between the seasonal evolution of brain 5-HT and the yearly biological cycles in toads reported in this paper deserves to be stressed. In effect, during winter, when animal behaviour clearly denotes a predominance of energy-saving mechanisms, with lower motor activity, an important decrease in O3 consumption and general metabolic rate (Fromm & Johnson, 1955), associated witlh increased storage of carbohydrate, fat and proteins in different organs (Mazzocco, 1938a, b, c, 1940), is noted. Also a marked relative arterial hypotension with a lowering in vasomotor responsiveness has been reported in this period (Segura & D'Agostino, 1964).
848
E. T.
SEGURA,A. M. BISCARDI AND J. APELBAUM
All these features suggest prima f a d e a functional preponderance of parasympathetic mechanisms. At the same time, brain waves present their typical winter pattern, with low frequency, smooth appearance and high threshold to external stimuli. T h i s picture, observed precisely when toad brain serotonin level reaches its m a x i m u m , is quite similar to that described in m a m m a l s when receiving this substance, its precursor 5-hydroxytryptophan, or drugs that deplete brain N E without affecting 5 - H T stores (Costa et al., 1960; GlOsser & Mantegazzini, 1960; Koella et al., 1960; Brodie & Costa, 1962; Koella & Czicman, 1966). Similarly, the marked decrease in brain E observed in this period might give some support to the view that a rise in the 5 - H T to E cerebral index could play a special role in the triggering and maintenance of hibernating states in this species. Conversely, O 3 consumption and the general endocrino-metabolic rate as well as circulatory responses are greatly enhanced during mating and estivation. T h e s e facts, in phase with the significant and opposite displacement in the E and 5 - H T brain content, again induce one to consider a possible causal relationship between the seasonal cycles and the level of monoamines in the C N S of the toad. Also the E E G pattern of this period, showing a marked increase in frequency, together with the appearance of spindles and high sensitivity to afferent stimulation, suggest an increment in the sympathetic "ergotropic" tonus. REFERENCES ANTON A. H. & SAYI~ D. F. (1962) A study of the factors affecting the aluminum oxide trihydroxyindole procedure for the analysis of catecholamines. J. Pharmac. exp. Ther. 138, 360-375. BEAUVALLETM., FUGAZZAJ. & SOLIER i . (1962) Nouvelles recherches sur les variations saisonni~res de la noradr6naline c6r6brale..7. Physiol., Paris 54, 289. BERTLERA. & ROSENGRENE. (1959) Occurrence and distribution of dopamine in brain and other tissues. Experientia 15, 10-11. BOODANSKI D. F., BONOMI L. & BRODIE B. B. (1963) Occurrence of serotonin and catecholamines on brain and peripheral organs of various vertebrate classes. Life Sci. 2, 80-84. BRODIF-B. B. & BOGDANSKXD. F. (1964) Biogenic amines and drug action in the nervous system of various vertebrate classes. Prog. Brain Res. 9, 234-242. BRODIE B. B., BOGDANSKID. F. & BONOMI L. (1964) Formation, storage and metabolism of serotonin (5-hydroxytryptamine) and catecholamines in lower vertabrates. In Comparative Neuroehemistry (Edited by RICHTER D.) pp. 367-377. Pergamon Press, Oxford. BRODIE B. B. & COSTAE. (1962) Some current views on brain monoamines. In Monoamines et systbme Nerveux Central (Edited by DE AJURIAGUERRAJ.) pp. 13-49. Georg & Cie, Gen6ve. BRODIE B. B. & SHORE P. A. (1957) A concept for a role of serotonin and norepinephrine as chemical mediators in the brain. Ann. N . Y . Acad. Sci. 66, 631-642. BRODIE B. B., SPECTOR S. & SHORE P. A. (1959) Interaction of drugs with norepinephrine in the brain. Pharmac. Rev. 11, 548-564. CHATFmLOP. O., LYMANC. P. & PURPtmA D. P. (1951) The effects of temperature on the spontaneous and induced electrical activity in the cerebral cortex of the golden hamster. Electroenceph. clin. Neurophysiol. 3, 225-230.
EEG CHANGES IN THE TOAD BUFO ARENARU~VI
849
COHEN G. & (.~OLDENBERG M. (1957) T h e simultaneous fluorometric determination of adrenaline and noradrenaline in plasma. II. Peripheral venous plasma concentrations in normal subjects and in patients with pheochromocytoma. J . Neurochem. 2, 71-80. COSTA E., PSCHIZIDTG. R., METER W. G. VAN & HIMWICH H. E. (1960) Brain concentrations of biogenic amines and E E G patterns of rabbits. J. Pharmac. exp. Ther. 130, 81-88. Euta~R U. S. yon & LIS~JKO F. (1959) T h e estimation of catecholamines in urine. Acta physiol, scald. 45, 122-132. FALCK B., HAGGm,mALJ. & Owm~'~ CH. (1963) T h e localization of adrenaline in adrenergic nerves in the frog. Quart.J. exp. Physiol. 48, 253-257. FaOMM P. O. & JOHNSON J. Z. (1955) T h e respiratory metabolism of frogs as related to season, y. cell. comp. Physiol. 45, 343-359. GLOSSER A. & M~'~TEOAZZINI P. (1960) Action of 5-hydroxytryptamine and of 5-hydroxytryptophan on the cortical electrical activity of the midpontine pretrigeminal preparation of the cat with and without mesencephalic hemisection. Archo. ital. Biol. 98, 351-366. HESS W. R. (1948) Die Funktionelle Organisation des Vegetativen Nervensystems. pp. 1-226. Schwave, Basel. I~'¢SEa~ CH., ROHMER F. & HmBEL G. (1951) L ' E . E . G . de l'hibernant. L~thargie et r~veil spontan~ du spermophile. Essai de r~production de I'E.E.G. chez le spermophile et le rat blanc. Rev. Neurol., Paris 84, 570-578. KETY S. S. (1966) Catecholamines in neuropsychiatric states. Pharmac. Rev. 18, 787-798. KOELLA W. P. & CZlCM~a'~ J. H. (1966) Mechanism of the EEG-synchronizing action of serotonin. Am. J. Physiol. 211, 926-934. KOELLA W. P., SMYTHmS J. R., BULL D. M. & LEvY CH. K. (1960) Physiological fractionation of the effect of serotonin on evoked potentials. Am. v~. Physiol. 198, 205-216. LISON L. (1958) Statistique Appliqude ~ la Biologic Expdrimentale. pp. 1-346. GauthierVillars, Paris. MATTUSSEK W. & PATSCHK~ U. (1964) Beziehungen des Schlaf und Wachrhythmus zum Noradrenalin- und Serotoningehalt im Zentralnervensystem yon Hamstern. Med. Expl. 11, 81-87. MAZZOCCO P. (1938a) Variaciones estacionales de la composici6n del hlgado de sapo Burg arenarum Hensel. Revta Soc. argent. Biol. 14, 123-144. MAzzocco P. (1938b) Variaciones estacionales de la composici6n quima del musct~lo del Bufo arenarum Hensel. Revta Soc. argent. Biol. 14, 236-245. MAZZOCCO P. (1938e) Oscilaciones peri6dicas del metabolismo hidrocarbonado en el sapo BurG arenarum Hensel. Revta Soc. argent. Biol. 14, 330-334. MAZZOCCO,P. (1940) Variaciones estacionales del peso y composici6n de los cuerpos adiposos del sapo B~fo arenarum Hensel. Revta Soc. argent. Biol. 16, 129-138. ROHMER F . , H]EBEL G. & KAYSERCH. (1951) Reeherches sur le fonctionnement du syst6me nerveux de'; hibernants. Les ondes c6r6brales pendant le sommeil hibernal et le r6veil. Etude sur le spermophile. C. r. s3anc. Soc. Biol. 145, 747-752. S E G ~ E. T . & B1sc~tDt A. M. (1967) Changes in brain E and NE induced by afferent stimulation in the isolated toad head. Life Sci. 6, 1599-1603. SECU~ E. T. & D'AGosTINO S. A. (1964) Seasonal variations of blood pressure, vasomotor reactivity and plasmatic cateeholamines in the toad. Aetaphysiol. latinoam. 14, 231-237. SEOUR~ E. T . & DE JuAN A. (1966) Electroencephalographic studies in toads. Electroenceph. clin. Neurophysiol. 21, 373-380. SHT~K M. B. (1961) Electrophysiological study of hibernation. Sechenov physiol, v~. U S S R 47, 1028-1036. STRUMWASSER F. (1959a) Thermoregulatory brain and behavioral mechanisms during entrance into hibernation in the squirrel, Citellus beecheyi. Am. ~. Physiol. 196, 15-22. STRUMWASSER F. (1959b) Regulatory mechanisms, brain activity and behavior during deep hibernation in squirrel, Citellus beecheyi. Am..7. Physiol. 196, 23-30.
850
E. T. SEGURA,A. M. BlSCARDIAND J. APELBAUM
UUSP~ V. J. (1963a) The catecholamine content of the brain and heart of the hedgehog (Erinaceus europaeus) during hibernation and in an active state. Ann. ivied, exp. Biol. Fenn. 41, 340-348. UusP)L~ V. J. (1963b) The 5-hydroxytryptamine content of the brain and some other organs of the hedgehog (Erinaceus europaeus) during activity and hibernation. Experientia 19, 156. VANE S. R. (1957) A sensitive method for the assay of 5-hydroxytryptamine. Br.J. Pharmac. 12, 344-349.