European Journal o f Pharmacology, 56 (1979) 397--401 © Elsevier/North.Holland Biomedical Press
397
EFFECT OF SEROTONIN ON CYCLIC AMP LEVEL IN RAT HYPOTHALAMUS SLICES DURING DEVELOPMENT ANNIE DASZUTA *, FRAN~OISE PONS et JEAN CADILHAC **
* Unit~ de Recherche Neurobiologique INSERM, 280 Bd. Sainte Marguerite, 13009 Marseille, and ** Laboratoire de Pathologie et Th~rapeutique Gdn~rales, Facult~ de Mddecine, 34000 Montpellier, France Received 18 October 1978, revised MS received 29 December 1978, accepted 20 March 1979
A. DASZUTA, F. PONS and JEAN CADILHAC, Effect o f serotonin on cyclic AMP level in rat hypothalamus slices during development, European J. Pharmacol. 56 (1979) 397--401. The effect of serotonin (5-HT) on cyclic AMP levels in rat hypothalamic slices was age-dependent. The sensitivity of the cyclic AMP system to 5 x 10 -s M 5-HT already existed in the foetus at 21 days of pregnancy. It reached a maximum on the 7th postnatal day and then decreased with age. In adult tissue the response was still present and was antagonized by two serotonergic antagonists (methiothepin and metergoline). When compared to the progressive increase of 5-HT level in the hypothalamus the data suggest a different evolution of serotonergic innervation and of 5-HT receptors. Serotonin
Cyclic AMP
Hypothalamus
1. Introduction Many workers have been interested in the role of cyclic AMP in the central nervous system during ontogenesis, and particularly in its synthesis and its response to biogenic amines (Schmidt et al., 1970; Weiss, 1971). Dopamine, norepinephrine and serotonin (5-HT) stimulate the activity of brain adenylate cyclase early in postnatal life (Von Hungen et al., 1974; Enjalbert et al., 1977). In newborn animals the stimulating effect of 5-HT on adenylate cyclase has been shown to be maximal in rat brain areas containing a high density of 5-HT terminals (Von Hungen et al., 1975). During development the response to serotonin seems to decrease in the midbrain, corpus striatum, hypothalamus and hippocampus. However, in the adult rat brain, 5-HT activates adenylate cyclase in synaptic membranes (Pagel et al., 1976). In fact the magnitude of the response appears to depend on the species, on the region of the brain and also on the ty_pe of preparation. For example norepinephrine receptors coupled with
Ontogenesis
adenylate cyclase were detected in brain homogenates from newborn rats (Von Hungen et al., 1974), but 5-HT did not elicit an increase in cyclic AMP level in brain slices obtained from neonatal rats (Schmidt et al., 1970). Thus we have reinvestigated the 5-HTinduced response of cyclic AMP levels in slices of the hypothalamus from very young rats. Using a highly sensitive radioimmunological technique to measure cyclic AMP (Cailla et al., 1973) we have found an increase in the cyclic AMP level in slices of hypothalamus exposed to 5-HT. These results suggest that slices can be used to study the characteristics of 5-HT receptors in the rat brain, as well as those of norepinephrine and histamine receptors.
2. Materials and methods The experiments were performed on male Wistar rats housed at a temperature of 24°C, under standard light conditions: 12 h light--
398
A. D A S Z U T A ET AL.
12 h dark. Food and water were given ad libiturn. Adult rats (250--300 g), 15 day and 7 day old rats, and foetuses from day 21 of pregnancy (first day was determined by finding sperm in vaginal smear) were used. The animals were always decapitated at the same hour to eliminate the influence of possible circadian variations in 5-HT levels. The brains were removed rapidly and placed in cold Krebs--Ringer bicarbonate buffer pH 7.4 (6.9 g/1 NaC1, 0.354 g/1 KC1, 0.186 g/l, CaCl:, 0.294 g/1 MgSO4, 0.162 g/1 K2HPO4, 2.1 g/1 NaHCO3, 1.8 g/1 glucose, 0.2 g/1 ascorbic acid, 0.05 g/1 EDTA and 10 -s M niamide). The hypothalami were dissected out and sliced by hand into cubes approximately 0.5 mm thick. These slices were then submerged in 5 ml of fresh buffer equilibrated with 9 5 : 5 % O2--CO: and shaken gently for a 20 min preincubation period at 37°C. The buffer was then removed and the incubation period started. Fresh buffer was added and various concentrations of 5-HT (Sigma) from 0 to 10 -4 M were mixed into the buffer. After 6 min (preliminary experiments demonstrated that maximal responses to 5-HT occurred at this time), the incubation was stopped by filtration and the medium separated from the tissues. The tissues were collected in 500 pl of perchloric acid and stored at --20°C to await the extraction and assay of cyclic AMP (cf. Cailla et al., 1973). The results were expressed in picomoles cyclic AMP per mg of protein recovered in the perchloric pellet (Lowry et al., 1951). Endogenous levels of 5-HT were measured
in freshly dissected hypothalami by a microradioenzymatic assay (Saavedra et al., 1973). The statistical significance of the results was determined by means of Student's t-test. Methiothepin and metergoline, two 5-HT receptors blocking agents (mixed with or without 5-HT in the incubation medium) were kindly provided by Dr. M. Hamon.
3. Results The wet weight and protein content of the hypothalamus increased rapidly for the first 15 days following birth (table 1). Although the protein/wet weight ratio increased progressively during this period, its value on the 15th day was still much lower than that found in adults. The development of the 5-HT level was also examined (table 1). At the end of gestation, the hypothalamic 5-HT level was only 24% of the adult value. Serotonergic innervation is poorly developed at birth: Bourgoin (1976) observed that 5-HT levels in the forebrain of newborn rats were only 20% of those of adults and that the developmental pattern was far more gradual for this region than for the brain-stem. Serotonergic cell bodies are mainly localised in the brain-stem and the hypothalamus presumably contains only terminals. Thus, in the latter structure the developmental pattern of 5-HT level may represent the appearance of the terminals. This appeared to occur at a slow rate, since at 15 days of age, the 5-HT concentration was only about one third that of the adult.
TABLE 1 D e v e l o p m e n t a l variations o f w e t w e i g h t , p r o t e i n and s e r o t o n i n (5-HT) levels in t h e h y p o t h a l a m u s o f the rat. Values are the m e a n s + S.E.M. o f groups o f 10 animals. Age
Wet weight (mg) P r o t e i n (mg) 5-HT (pg/g)
Foetus
7 days
15 d a y s
Adult
8.1 + 0.4 0.50 + 0.04 0.16 + 0.02
22.1 +- 0.9 1.35 -+ 0.11 0.20 + 0.03
30.0 -+ 1.3 2.28 +- 0.22 0.27 + 0.03
56.0 -+ 1.8 5.06 + 0.19 0.66 + 0.05
O N T O G E N E T I C S T I M U L A T I O N O F CYCLIC A M P BY S E R O T O N I N TABLE 2 D o s e - - r e s p o n s e in h y p o t h a l a m i c slices f r o m f o e t u s a n d a d u l t rat b r a i n s in p r e s e n c e o f s e r o t o n i n . E a c h value r e p r e s e n t s t h e m e a n ± S.E.M. f r o m 6 animals. Significantly d i f f e r e n t f r o m c o n t r o l value: 1 0.05 > P > 0 . 0 1 , 2 P < 0.01. Addition
Concentration
Cyclic AMP (pmoles/mg prot.)
(M) Foetus None 10 -7 10 -6 5 X 1 0 -6 10 -s 5 X 1 0 -s 10 -4 5 X 1 0 -4
Serotonin
Adult
20.2±1.5
96 ± i0
19.1±1.3 20.5±2.2 31.7±4.1 36.2±5.5 39.7±5.0 34.7±4.4 42.6±8.1
86 + 9 90 + 9 120 ± 11 1 3 0+ - 8 136 ± 11 t
1 1 2 1 1
/
E 100
,,~'=*i 5.HT #.,.s
17
& n
J
/,~
/
/.///
control -
,/
~E < 0
As is shown in table 2, 5-HT was able to induce an increase in cyclic AMP level in hypothalamic slices. This effect was particularly pronounced in tissues from foetal animals. Maximal stimulation was obtained with 5 X 10 -s M 5-HT, the result being that the control level was doubled. A significant enhancement of cyclic AMP already occurred with 5 X 10 -6 M 5-HT. When the same procedure was applied to tissues from adult rats, the response was generally much less than that in tissues from foetal rats. Nevertheless, the maximum cyclic AMP production again was observed with 5 X 10-SM 5-HT. Using this concentration, we examined the capacity of 5-HT to stimulate cyclic AMP production
i~'~
E
.U_
120 ± 13 116 ± 15
399
50
(97-+1)
(187+28) b
(64+9)c
(42_'7)a
w
_1
?
Is AGE
(days)
90 AOULT
Fig. 1. Cyclic A M P r e s p o n s e t o s e r o t o n i n in t h e d e v e l o p i n g rat h y p o t h a l a m u s . C h o p p e d tissue was p r e p a r e d f r o m t h e b r a i n s o f rats aged as i n d i c a t e d a n d was i n c u b a t e d for 6 m i n w i t h or w i t h o u t 5 x 10 -s M s e r o t o n i n . Values are t h e m e a n s ± S.E.M. f r o m 5 animals, * S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s 0.05 > P > 0.01. ** S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s P < 0.01. I n p a r e n t h e s e s , p e r c e n t a g e s t i m u l a t i o n ± S.E.M. a S i g n i f i c a n t l y d i f f e r e n t from f o e t u s value P < 0.01. b Significantly d i f f e r e n t f r o m f o e t u s value P < 0.02. c S i g n i f i c a n t l y d i f f e r e n t f r o m f o e t u s value P < 0.05.
400
at various ages (fig. 1). On foetal day 21, the basal cyclic AMP level was 20% of that measured in the adult and 5-HT induced a doubling of the cyclic nucleotide level in hypothalamic slices. The stimulating effect of 5-HT was far more pronounced in tissues from 7 day old rats, since the cyclic AMP level was tripled in the presence of indolamine. The specific activity of the serotonin sensitive adenylate cyclase attained a maximum at this period, and decreased to the adult activity 15 days after birth. Whereas the increase due to 5-HT went up 2-fold from birth to adulthood, the basal level went up 5-fold. In order to check the specificity of the 5-HT-induced response in adult hypothalamic slices, the effects of methiothepin and metergoline, two potent 5-HT antagonists (Fuxe et al., 1975; Hamon et al., 1977) on the cyclic AMP level were analysed. In the absence of indolamine, the basal level of cyclic AMP (96_+ 10 pmoles cAMP/mg prot.) was not changed by the addition of 5 X 10 -s M of each drug (97.3 _+ 10 pmoles cAMP/mg prot. with methiothepine; 94 _+ 12 pmoles cAMP/ mg prot. with metergoline). However the stimulating effect of 5 X 10 -s M 5-HT (136 _+ 16 pmoles cAMP/mg prot.) was totally abolished by methiothepine (98 _+ 9 pmoles cAMP/mg prot.) or metergoline (84.4_+ 12 pmoles cAMP/mg prot.). 4. Discussion The present investigation showed that brain slices could be used to study the 5-HT receptors coupled with an adenylate cyclase in the rat brain. As already observed with homogenates, the effect of 5-HT on cyclic AMP formation in rat hypothalamus slices was age
397
EFFECT OF SEROTONIN ON CYCLIC AMP LEVEL IN RAT HYPOTHALAMUS SLICES DURING DEVELOPMENT ANNIE DASZUTA *, FRAN~OISE PONS et JEAN CADILHAC **
* Unit~ de Recherche Neurobiologique INSERM, 280 Bd. Sainte Marguerite, 13009 Marseille, and ** Laboratoire de Pathologie et Th~rapeutique Gdn~rales, Facult~ de Mddecine, 34000 Montpellier, France Received 18 October 1978, revised MS received 29 December 1978, accepted 20 March 1979
A. DASZUTA, F. PONS and JEAN CADILHAC, Effect o f serotonin on cyclic AMP level in rat hypothalamus slices during development, European J. Pharmacol. 56 (1979) 397--401. The effect of serotonin (5-HT) on cyclic AMP levels in rat hypothalamic slices was age-dependent. The sensitivity of the cyclic AMP system to 5 x 10 -s M 5-HT already existed in the foetus at 21 days of pregnancy. It reached a maximum on the 7th postnatal day and then decreased with age. In adult tissue the response was still present and was antagonized by two serotonergic antagonists (methiothepin and metergoline). When compared to the progressive increase of 5-HT level in the hypothalamus the data suggest a different evolution of serotonergic innervation and of 5-HT receptors. Serotonin
Cyclic AMP
Hypothalamus
1. Introduction Many workers have been interested in the role of cyclic AMP in the central nervous system during ontogenesis, and particularly in its synthesis and its response to biogenic amines (Schmidt et al., 1970; Weiss, 1971). Dopamine, norepinephrine and serotonin (5-HT) stimulate the activity of brain adenylate cyclase early in postnatal life (Von Hungen et al., 1974; Enjalbert et al., 1977). In newborn animals the stimulating effect of 5-HT on adenylate cyclase has been shown to be maximal in rat brain areas containing a high density of 5-HT terminals (Von Hungen et al., 1975). During development the response to serotonin seems to decrease in the midbrain, corpus striatum, hypothalamus and hippocampus. However, in the adult rat brain, 5-HT activates adenylate cyclase in synaptic membranes (Pagel et al., 1976). In fact the magnitude of the response appears to depend on the species, on the region of the brain and also on the ty_pe of preparation. For example norepinephrine receptors coupled with
Ontogenesis
adenylate cyclase were detected in brain homogenates from newborn rats (Von Hungen et al., 1974), but 5-HT did not elicit an increase in cyclic AMP level in brain slices obtained from neonatal rats (Schmidt et al., 1970). Thus we have reinvestigated the 5-HTinduced response of cyclic AMP levels in slices of the hypothalamus from very young rats. Using a highly sensitive radioimmunological technique to measure cyclic AMP (Cailla et al., 1973) we have found an increase in the cyclic AMP level in slices of hypothalamus exposed to 5-HT. These results suggest that slices can be used to study the characteristics of 5-HT receptors in the rat brain, as well as those of norepinephrine and histamine receptors.
2. Materials and methods The experiments were performed on male Wistar rats housed at a temperature of 24°C, under standard light conditions: 12 h light--
398
A. D A S Z U T A ET AL.
12 h dark. Food and water were given ad libiturn. Adult rats (250--300 g), 15 day and 7 day old rats, and foetuses from day 21 of pregnancy (first day was determined by finding sperm in vaginal smear) were used. The animals were always decapitated at the same hour to eliminate the influence of possible circadian variations in 5-HT levels. The brains were removed rapidly and placed in cold Krebs--Ringer bicarbonate buffer pH 7.4 (6.9 g/1 NaC1, 0.354 g/1 KC1, 0.186 g/l, CaCl:, 0.294 g/1 MgSO4, 0.162 g/1 K2HPO4, 2.1 g/1 NaHCO3, 1.8 g/1 glucose, 0.2 g/1 ascorbic acid, 0.05 g/1 EDTA and 10 -s M niamide). The hypothalami were dissected out and sliced by hand into cubes approximately 0.5 mm thick. These slices were then submerged in 5 ml of fresh buffer equilibrated with 9 5 : 5 % O2--CO: and shaken gently for a 20 min preincubation period at 37°C. The buffer was then removed and the incubation period started. Fresh buffer was added and various concentrations of 5-HT (Sigma) from 0 to 10 -4 M were mixed into the buffer. After 6 min (preliminary experiments demonstrated that maximal responses to 5-HT occurred at this time), the incubation was stopped by filtration and the medium separated from the tissues. The tissues were collected in 500 pl of perchloric acid and stored at --20°C to await the extraction and assay of cyclic AMP (cf. Cailla et al., 1973). The results were expressed in picomoles cyclic AMP per mg of protein recovered in the perchloric pellet (Lowry et al., 1951). Endogenous levels of 5-HT were measured
in freshly dissected hypothalami by a microradioenzymatic assay (Saavedra et al., 1973). The statistical significance of the results was determined by means of Student's t-test. Methiothepin and metergoline, two 5-HT receptors blocking agents (mixed with or without 5-HT in the incubation medium) were kindly provided by Dr. M. Hamon.
3. Results The wet weight and protein content of the hypothalamus increased rapidly for the first 15 days following birth (table 1). Although the protein/wet weight ratio increased progressively during this period, its value on the 15th day was still much lower than that found in adults. The development of the 5-HT level was also examined (table 1). At the end of gestation, the hypothalamic 5-HT level was only 24% of the adult value. Serotonergic innervation is poorly developed at birth: Bourgoin (1976) observed that 5-HT levels in the forebrain of newborn rats were only 20% of those of adults and that the developmental pattern was far more gradual for this region than for the brain-stem. Serotonergic cell bodies are mainly localised in the brain-stem and the hypothalamus presumably contains only terminals. Thus, in the latter structure the developmental pattern of 5-HT level may represent the appearance of the terminals. This appeared to occur at a slow rate, since at 15 days of age, the 5-HT concentration was only about one third that of the adult.
TABLE 1 D e v e l o p m e n t a l variations o f w e t w e i g h t , p r o t e i n and s e r o t o n i n (5-HT) levels in t h e h y p o t h a l a m u s o f the rat. Values are the m e a n s + S.E.M. o f groups o f 10 animals. Age
Wet weight (mg) P r o t e i n (mg) 5-HT (pg/g)
Foetus
7 days
15 d a y s
Adult
8.1 + 0.4 0.50 + 0.04 0.16 + 0.02
22.1 +- 0.9 1.35 -+ 0.11 0.20 + 0.03
30.0 -+ 1.3 2.28 +- 0.22 0.27 + 0.03
56.0 -+ 1.8 5.06 + 0.19 0.66 + 0.05
O N T O G E N E T I C S T I M U L A T I O N O F CYCLIC A M P BY S E R O T O N I N TABLE 2 D o s e - - r e s p o n s e in h y p o t h a l a m i c slices f r o m f o e t u s a n d a d u l t rat b r a i n s in p r e s e n c e o f s e r o t o n i n . E a c h value r e p r e s e n t s t h e m e a n ± S.E.M. f r o m 6 animals. Significantly d i f f e r e n t f r o m c o n t r o l value: 1 0.05 > P > 0 . 0 1 , 2 P < 0.01. Addition
Concentration
Cyclic AMP (pmoles/mg prot.)
(M) Foetus None 10 -7 10 -6 5 X 1 0 -6 10 -s 5 X 1 0 -s 10 -4 5 X 1 0 -4
Serotonin
Adult
20.2±1.5
96 ± i0
19.1±1.3 20.5±2.2 31.7±4.1 36.2±5.5 39.7±5.0 34.7±4.4 42.6±8.1
86 + 9 90 + 9 120 ± 11 1 3 0+ - 8 136 ± 11 t
1 1 2 1 1
/
E 100
,,~'=*i 5.HT #.,.s
17
& n
J
/,~
/
/.///
control -
,/
~E < 0
As is shown in table 2, 5-HT was able to induce an increase in cyclic AMP level in hypothalamic slices. This effect was particularly pronounced in tissues from foetal animals. Maximal stimulation was obtained with 5 X 10 -s M 5-HT, the result being that the control level was doubled. A significant enhancement of cyclic AMP already occurred with 5 X 10 -6 M 5-HT. When the same procedure was applied to tissues from adult rats, the response was generally much less than that in tissues from foetal rats. Nevertheless, the maximum cyclic AMP production again was observed with 5 X 10-SM 5-HT. Using this concentration, we examined the capacity of 5-HT to stimulate cyclic AMP production
i~'~
E
.U_
120 ± 13 116 ± 15
399
50
(97-+1)
(187+28) b
(64+9)c
(42_'7)a
w
_1
?
Is AGE
(days)
90 AOULT
Fig. 1. Cyclic A M P r e s p o n s e t o s e r o t o n i n in t h e d e v e l o p i n g rat h y p o t h a l a m u s . C h o p p e d tissue was p r e p a r e d f r o m t h e b r a i n s o f rats aged as i n d i c a t e d a n d was i n c u b a t e d for 6 m i n w i t h or w i t h o u t 5 x 10 -s M s e r o t o n i n . Values are t h e m e a n s ± S.E.M. f r o m 5 animals, * S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s 0.05 > P > 0.01. ** S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s P < 0.01. I n p a r e n t h e s e s , p e r c e n t a g e s t i m u l a t i o n ± S.E.M. a S i g n i f i c a n t l y d i f f e r e n t from f o e t u s value P < 0.01. b Significantly d i f f e r e n t f r o m f o e t u s value P < 0.02. c S i g n i f i c a n t l y d i f f e r e n t f r o m f o e t u s value P < 0.05.
400
at various ages (fig. 1). On foetal day 21, the basal cyclic AMP level was 20% of that measured in the adult and 5-HT induced a doubling of the cyclic nucleotide level in hypothalamic slices. The stimulating effect of 5-HT was far more pronounced in tissues from 7 day old rats, since the cyclic AMP level was tripled in the presence of indolamine. The specific activity of the serotonin sensitive adenylate cyclase attained a maximum at this period, and decreased to the adult activity 15 days after birth. Whereas the increase due to 5-HT went up 2-fold from birth to adulthood, the basal level went up 5-fold. In order to check the specificity of the 5-HT-induced response in adult hypothalamic slices, the effects of methiothepin and metergoline, two potent 5-HT antagonists (Fuxe et al., 1975; Hamon et al., 1977) on the cyclic AMP level were analysed. In the absence of indolamine, the basal level of cyclic AMP (96_+ 10 pmoles cAMP/mg prot.) was not changed by the addition of 5 X 10 -s M of each drug (97.3 _+ 10 pmoles cAMP/mg prot. with methiothepine; 94 _+ 12 pmoles cAMP/ mg prot. with metergoline). However the stimulating effect of 5 X 10 -s M 5-HT (136 _+ 16 pmoles cAMP/mg prot.) was totally abolished by methiothepine (98 _+ 9 pmoles cAMP/mg prot.) or metergoline (84.4_+ 12 pmoles cAMP/mg prot.). 4. Discussion The present investigation showed that brain slices could be used to study the 5-HT receptors coupled with an adenylate cyclase in the rat brain. As already observed with homogenates, the effect of 5-HT on cyclic AMP formation in rat hypothalamus slices was age
