218
Brain Research, 577 (1992) 218-225 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17601
Serotonin may play a role. in the anorexia induced by amphetamine injections into the lateral hypothalamus M a r c o A. P a r a d a a, Luis H e r n ~ n d e z a and E m i l D e g o m a b ULaboratorio de Fisiolog[a de la Conducta, Facultad de Medicina, Universidad de Los Andes, Merida (Venezuela) and bLawrenceville School, Lawrenceville, NJ 08648 (USA)
(Accepted 19 November 1991) Key words: Serotonin; Dopamine; Lateral hypothalamus: Feeding behavior; Amphetamine anorexia
Amphetamine (AMPH) injections into the lateral hypothalamus (LH) are known to inhibit feeding and this effect has been shown to be mediated by the release and the reuptake blockade of catecholamines. LH serotonin (5-HT) has been suggested to be involved in feeding inhibition and a recent study showed that LH amphetamine infusion increases extracellular dopamine (DA), norepinephrine (NE) and 5-HT, which suggests that 5-HT might also be involved in amphetamine anorexia. The present study investigated this possibility. A correlational study was performed between the anorectic effect of LH unilateral microinjections of each monoamine and the anorectic effect of AMPH. Six groups of male rats were used. The rats in each group were submitted to 2 series of 6 experimental sessions. Each session consisted of one microinjection in 24 h food-deprived rats, followed by the measurement of food intake 30 rain later. The first series was similar for all groups and explored the AMPH effect (difference between the mean food intake after 3 AMPH injections (40 ~g/0.5/A) and the mean food intake after 3 saline injections (0.5 ~1)). The second series explored the effects of DA (40/~g), NE (25/lg), EPI (25 ~g), 5-HT (25/~g) or AMPH again, in a similar way as described for AMPH in the first series. Linear regression analysis on the first series AMPH effect and the amine effect of the 2nd series showed a positive correlation between both series of AMPH, AMPH and DA, and AMPH and 5-HT. This last correlation was replicated in a different group. No correlation was found between AMPH and NE or AMPH and epinephrine (EPI). These results suggest that LH AMPH anorexia could be mediated through DA and 5-HT release. INTRODUCTION
responsive place in the brain for amphetamine to induce its suppressive effect on feeding 26. The most compelling
The perifornical region of the lateral hypothalamus has been involved in feeding regulation 1's'15'4°'49. This
evidence favoring the involvement of D A and N E or perhaps EPI, in the anorectic effect of A M P H injected in the LH, comes from the fact that this effect can be blocked by the concomitant administration of dopaminergic or/3-adrenergic antagonists27'29.
area contains a dense catecholaminergic innervation which includes dopamine ( D A ) , norepinephrine (NE), and epinephrine (EPI) as evidenced by fluorescence microscopy and biochemical detection 2°'21'44'53. Different detection methods have also shown the presence of serotonin (5-HT) in this area ls'24'41'45'5°. Serotonin levels in the lateral hypothalamus (LH) are even larger than the levels of the other monoamines44'5°, and the perifornical region of the LH has been shown to be one of the hypothalamic areas with the largest content of 5-HT 5°. A great deal of experimental evidence suggests that perifornical lateral hypothalamic D A 32'33, N E or more probably EPI 3x'32, as well a s 5 - H T 2'19'22"52 play an inhibitory role in food intake regulation, and catecholaminergic mechanisms have been postulated to be responsible for the anorectic effect of intraperifornically applied amphetamine ( A M P H ) 27'29. This last drug is a potent anorexigenic agent which is known to release and block the reuptake of catecholamines 6'7'9'13'43 and 5-HT 4'16. The perifornical LH has been shown to be the most
A recent report as combining microdialysis in the lateral hypothalamus with simultaneous A M P H infusion through the same probe, showed a significant increase in the extracellular levels of 5-HT, D A and NE. A M P H infusion decreased also dihydroxyphenylacetic acid ( D O P A C ) and 5-hydroxyindoleacetic acid (5-HIAA). Thus, it seems evident from those experiments that A M P H in the L H potentiates, through the release and the reuptake blockade, not only endogenous D A and NE but also 5-HT. Those findings suggest also that any of these m o n o a m i n e s could mediate the behavioral changes induced by local administration of the drug into the LH. In the present study we have focused on the possible involvement of the four m o n o a m i n e s in the anorectic effect of A M P H . In fact, and as previously stated, catecholaminergic mechanisms seem to be involved in this A M P H effect 2v'2~, but a possible participation of
Correspondence : M.A. Parada, (present address) Department of Psychology, Princeton University, Princeton, NJ 08544, USA.
219 5 - H T can n o t b e discarded. T h e a p p r o a c h used in this study was to get the m e a n a n o r e c t i c effect o f i n t r a p e r i f o r n i c a l (i.p.f.) A M P H injections and the m e a n a n o r e c t i c effect o f i.p.f, injections of e a c h o n e of the m o n o a m i n e s r e l e a s e d by A M P H .
The
d e g r e e of c o r r e l a t i o n b e t w e e n t h o s e m e a n s was e x p l o r e d
breakdown of the monoamines potentiating thereby their synaptic actions and allowing the use of lower doses. Nevertheless, MAO inhibitors might have an effect on their own, and its use would have introduced an additional variable. Therefore it was decided not to use such compounds in the present experiments and to use moderate high doses of the amines instead. Among the biogenic amines tested in this study, DA seems to be the most susceptible to metabolic breakdown 28 so a larger dose of DA was used.
using linear r e g r e s s i o n analysis. T h e results of t h e s e tests and analysis indicate that the a n o r e c t i c A M P H c o r r e l a t e s well with the a n o r e c t i c effects o f D A
effect and
5 - H T but n o t with t h o s e o f N E o r E P I .
MATERIALS AND METHODS The subjects used were 152 adult male rats, weighing between 300 and 360 g at the time of the surgery. The animals were unevenly distributed in 6 groups, maintained at room temperature (22 + 2°C), under normal light-dark cycles (12 h-12 h) and fed ad libitum with lab chow pellets and tap water except for 24 h of food deprivation before each experimental session.
Statistical and histological analysis For each group the correlation between the AMPH effect on food intake in the first series of injections and the monoamine effect in the second series was tested by means of linear regression analysis. Selection of the animals according to their response to the intrahypothalamic injections was not performed. All the animals tested were included in the correlation study, even if they did not respond to AMPH. At the end of the experiments, the rats were sacrificed, and the brains perfused with formaline. After at least 5 days of fixation, the brains were sliced, and the tips of the injector cannulas were localized histologically according to the atlas of K6nig and Klippe124. RESULTS AND DISCUSSION
Surgery Under ketamine anesthesia each rat was implanted with one guide cannula (26 gauge, 20 mm long) aimed 2 mm above the perifornical area of the LH. The coordinates for the cannula, with the incisor bar placed 3.5 mm below the interaural line, were: 6.5 mm anterior to the interaural line, 1.6 mm lateral to the midsagittal sinus, and 5.7 mm perpendicularly below the surface of the cortex. The experimental manipulations began at least one week after postsurgical recovery.
Figs. 1 - 6 s u m m a r i z e b o t h the results of t h e c o r r e l a tional studies and the histological findings for e a c h g r o u p of rats. Fig. 1 shows a significant c o r r e l a t i o n b e t w e e n the a n o r e c t i c effects of a first and a s e c o n d series o f a m p h e t a m i n e injections (r = 0.72; P < 0.001). This finding is r e l e v a n t to this study since it suggests that r e p e a t e d inj e c t i o n s into the L H did n o t substantially d a m a g e the
Drugs The following drugs from Sigma Chemical Co. were examined: D-amphetamine sulfate (AMPH), dopamine hydrochloride (DA), L-arterenol bitartrate (NE), L-epinephrine bitartrate (EPI), serotonin creatinine sulfate (5-HT).
n e r v o u s tissue, and m o n o a m i n e r g i c t e r m i n a l s in this a r e a w e r e still able to r e s p o n d to the last 3 injections of A M P H in a similar way as t h e y did after the first 3 amp h e t a m i n e injections. In a p r e v i o u s r e p o r t 17 it was s h o w n
Test procedure
that r e p e a t e d i n t r a h y p o t h a l a m i c A M P H
Four groups of animals were used to investigate the correlation between the anorectic effect of AMPH and the anorectic effect of each monoamine; the experiment with the fifth group was intended to replicate the result obtained with AMPH and 5-HT; and the experiment with the sixth group explored the correlation between two series of AMPH injections and was used as a control to assess a possible damage caused by the repeated intracerebral drug administration. The animals in each group were submitted to 2 series of 6 experimental sessions. The sessions were separated by 4-day intervals. Each session started with one unilateral i.p.f, microinjection in 24-h food deprived rats. After the injections the animals were placed in their cages with a measured amount of food and total intake was measured 30 min later. In the first series, that was similar for all rats and explored the AMPH effect, 3 sessions in which the animals received saline (0.5 pl) alternated with 3 sessions in which they received AMPH (40 pg/0.5 pl). The AMPH effect was the difference between the mean food intake after the 3 AMPH injections and the mean food intake after the 3 saline injections. The second series was specific for each group and explored the effects of DA (40 pg), NE (25 pg), EPI (25 pg), 5-HT (25 pg) or a second series of AMPH (40 pg) injections. In this series 3 saline sessions alternated with 3 monoamine sessions. The monoamine effect for each group was calculated by subtracting the mean food intake of the saline sessions from the mean food intake of the monoamine sessions, in a similar way as described above. Prior systemic administration of monoamine oxidase (MAO) inhibitors is a resource commonly employed in experiments exploring the effects of intracerebrally applied monoamine agonists. Those drugs retard the
/~g, Q I D , for 3 days) c a u s e d d e p l e t i o n of h y p o t h a l a m i c
injections (50
N E , and i n c r e a s e d f o o d i n t a k e and b o d y weight. H o w e v e r , such a n e u r o c h e m i c a l a l t e r a t i o n can be d i s r e g a r d e d in the p r e s e n t study, d u e to the fact that t h e doses of AMPH
e m p l o y e d w e r e l o w e r and a d m i n i s t e r e d with a
t i m e i n t e r v a l of at least 10 days. F r o m Figs. 2 and 3 it is e v i d e n t that t h e r e is a lack o f c o r r e l a t i o n b e t w e e n the A M P H
effect o n f o o d i n t a k e
and the effects of N E and E P I . This n e g a t i v e finding is surprising since b o t h o f t h e m are b e l i e v e d to be i n v o l v e d in the a n o r e c t i c effect of A M P H directly a p p l i e d in the p e r i f o r n i c a l r e g i o n 27'29. O f course, a n e g a t i v e result d o e s not p r e c l u d e
a role for t h o s e c a t e c h o l a m i n e s
in the
A M P H a n o r e x i a , especially for E P I , which s e e m s to be the m o r e clearly i n v o l v e d in an inhibitory r e g u l a t i o n of f e e d i n g 3L32. N E has b e e n c l a i m e d to h a v e a m o r e relev a n t role in p r o m o t i n g f e e d i n g , acting t h r o u g h a - a d r e n ergic r e c e p t o r s in the p a r a v e n t r i c u l a r nucleus ( P V N ) 2s'
37.
F u r t h e r m o r e , w h a t e v e r the role of e n d o g e n o u s N E
in the f e e d i n g s u p p r e s s i o n i n d u c e d by i n t r a h y p o t h a l a m i c A M P H injections, it should be e x e r t e d after 30 min ( t i m e i n t e r v a l used in this study) since the N E release
220
2"
•
al_
,~
o
i m.jtf"
•
Nil
• •
--
----
•
•
'1
Brain Research, 577 (1992) 218-225 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17601
Serotonin may play a role. in the anorexia induced by amphetamine injections into the lateral hypothalamus M a r c o A. P a r a d a a, Luis H e r n ~ n d e z a and E m i l D e g o m a b ULaboratorio de Fisiolog[a de la Conducta, Facultad de Medicina, Universidad de Los Andes, Merida (Venezuela) and bLawrenceville School, Lawrenceville, NJ 08648 (USA)
(Accepted 19 November 1991) Key words: Serotonin; Dopamine; Lateral hypothalamus: Feeding behavior; Amphetamine anorexia
Amphetamine (AMPH) injections into the lateral hypothalamus (LH) are known to inhibit feeding and this effect has been shown to be mediated by the release and the reuptake blockade of catecholamines. LH serotonin (5-HT) has been suggested to be involved in feeding inhibition and a recent study showed that LH amphetamine infusion increases extracellular dopamine (DA), norepinephrine (NE) and 5-HT, which suggests that 5-HT might also be involved in amphetamine anorexia. The present study investigated this possibility. A correlational study was performed between the anorectic effect of LH unilateral microinjections of each monoamine and the anorectic effect of AMPH. Six groups of male rats were used. The rats in each group were submitted to 2 series of 6 experimental sessions. Each session consisted of one microinjection in 24 h food-deprived rats, followed by the measurement of food intake 30 rain later. The first series was similar for all groups and explored the AMPH effect (difference between the mean food intake after 3 AMPH injections (40 ~g/0.5/A) and the mean food intake after 3 saline injections (0.5 ~1)). The second series explored the effects of DA (40/~g), NE (25/lg), EPI (25 ~g), 5-HT (25/~g) or AMPH again, in a similar way as described for AMPH in the first series. Linear regression analysis on the first series AMPH effect and the amine effect of the 2nd series showed a positive correlation between both series of AMPH, AMPH and DA, and AMPH and 5-HT. This last correlation was replicated in a different group. No correlation was found between AMPH and NE or AMPH and epinephrine (EPI). These results suggest that LH AMPH anorexia could be mediated through DA and 5-HT release. INTRODUCTION
responsive place in the brain for amphetamine to induce its suppressive effect on feeding 26. The most compelling
The perifornical region of the lateral hypothalamus has been involved in feeding regulation 1's'15'4°'49. This
evidence favoring the involvement of D A and N E or perhaps EPI, in the anorectic effect of A M P H injected in the LH, comes from the fact that this effect can be blocked by the concomitant administration of dopaminergic or/3-adrenergic antagonists27'29.
area contains a dense catecholaminergic innervation which includes dopamine ( D A ) , norepinephrine (NE), and epinephrine (EPI) as evidenced by fluorescence microscopy and biochemical detection 2°'21'44'53. Different detection methods have also shown the presence of serotonin (5-HT) in this area ls'24'41'45'5°. Serotonin levels in the lateral hypothalamus (LH) are even larger than the levels of the other monoamines44'5°, and the perifornical region of the LH has been shown to be one of the hypothalamic areas with the largest content of 5-HT 5°. A great deal of experimental evidence suggests that perifornical lateral hypothalamic D A 32'33, N E or more probably EPI 3x'32, as well a s 5 - H T 2'19'22"52 play an inhibitory role in food intake regulation, and catecholaminergic mechanisms have been postulated to be responsible for the anorectic effect of intraperifornically applied amphetamine ( A M P H ) 27'29. This last drug is a potent anorexigenic agent which is known to release and block the reuptake of catecholamines 6'7'9'13'43 and 5-HT 4'16. The perifornical LH has been shown to be the most
A recent report as combining microdialysis in the lateral hypothalamus with simultaneous A M P H infusion through the same probe, showed a significant increase in the extracellular levels of 5-HT, D A and NE. A M P H infusion decreased also dihydroxyphenylacetic acid ( D O P A C ) and 5-hydroxyindoleacetic acid (5-HIAA). Thus, it seems evident from those experiments that A M P H in the L H potentiates, through the release and the reuptake blockade, not only endogenous D A and NE but also 5-HT. Those findings suggest also that any of these m o n o a m i n e s could mediate the behavioral changes induced by local administration of the drug into the LH. In the present study we have focused on the possible involvement of the four m o n o a m i n e s in the anorectic effect of A M P H . In fact, and as previously stated, catecholaminergic mechanisms seem to be involved in this A M P H effect 2v'2~, but a possible participation of
Correspondence : M.A. Parada, (present address) Department of Psychology, Princeton University, Princeton, NJ 08544, USA.
219 5 - H T can n o t b e discarded. T h e a p p r o a c h used in this study was to get the m e a n a n o r e c t i c effect o f i n t r a p e r i f o r n i c a l (i.p.f.) A M P H injections and the m e a n a n o r e c t i c effect o f i.p.f, injections of e a c h o n e of the m o n o a m i n e s r e l e a s e d by A M P H .
The
d e g r e e of c o r r e l a t i o n b e t w e e n t h o s e m e a n s was e x p l o r e d
breakdown of the monoamines potentiating thereby their synaptic actions and allowing the use of lower doses. Nevertheless, MAO inhibitors might have an effect on their own, and its use would have introduced an additional variable. Therefore it was decided not to use such compounds in the present experiments and to use moderate high doses of the amines instead. Among the biogenic amines tested in this study, DA seems to be the most susceptible to metabolic breakdown 28 so a larger dose of DA was used.
using linear r e g r e s s i o n analysis. T h e results of t h e s e tests and analysis indicate that the a n o r e c t i c A M P H c o r r e l a t e s well with the a n o r e c t i c effects o f D A
effect and
5 - H T but n o t with t h o s e o f N E o r E P I .
MATERIALS AND METHODS The subjects used were 152 adult male rats, weighing between 300 and 360 g at the time of the surgery. The animals were unevenly distributed in 6 groups, maintained at room temperature (22 + 2°C), under normal light-dark cycles (12 h-12 h) and fed ad libitum with lab chow pellets and tap water except for 24 h of food deprivation before each experimental session.
Statistical and histological analysis For each group the correlation between the AMPH effect on food intake in the first series of injections and the monoamine effect in the second series was tested by means of linear regression analysis. Selection of the animals according to their response to the intrahypothalamic injections was not performed. All the animals tested were included in the correlation study, even if they did not respond to AMPH. At the end of the experiments, the rats were sacrificed, and the brains perfused with formaline. After at least 5 days of fixation, the brains were sliced, and the tips of the injector cannulas were localized histologically according to the atlas of K6nig and Klippe124. RESULTS AND DISCUSSION
Surgery Under ketamine anesthesia each rat was implanted with one guide cannula (26 gauge, 20 mm long) aimed 2 mm above the perifornical area of the LH. The coordinates for the cannula, with the incisor bar placed 3.5 mm below the interaural line, were: 6.5 mm anterior to the interaural line, 1.6 mm lateral to the midsagittal sinus, and 5.7 mm perpendicularly below the surface of the cortex. The experimental manipulations began at least one week after postsurgical recovery.
Figs. 1 - 6 s u m m a r i z e b o t h the results of t h e c o r r e l a tional studies and the histological findings for e a c h g r o u p of rats. Fig. 1 shows a significant c o r r e l a t i o n b e t w e e n the a n o r e c t i c effects of a first and a s e c o n d series o f a m p h e t a m i n e injections (r = 0.72; P < 0.001). This finding is r e l e v a n t to this study since it suggests that r e p e a t e d inj e c t i o n s into the L H did n o t substantially d a m a g e the
Drugs The following drugs from Sigma Chemical Co. were examined: D-amphetamine sulfate (AMPH), dopamine hydrochloride (DA), L-arterenol bitartrate (NE), L-epinephrine bitartrate (EPI), serotonin creatinine sulfate (5-HT).
n e r v o u s tissue, and m o n o a m i n e r g i c t e r m i n a l s in this a r e a w e r e still able to r e s p o n d to the last 3 injections of A M P H in a similar way as t h e y did after the first 3 amp h e t a m i n e injections. In a p r e v i o u s r e p o r t 17 it was s h o w n
Test procedure
that r e p e a t e d i n t r a h y p o t h a l a m i c A M P H
Four groups of animals were used to investigate the correlation between the anorectic effect of AMPH and the anorectic effect of each monoamine; the experiment with the fifth group was intended to replicate the result obtained with AMPH and 5-HT; and the experiment with the sixth group explored the correlation between two series of AMPH injections and was used as a control to assess a possible damage caused by the repeated intracerebral drug administration. The animals in each group were submitted to 2 series of 6 experimental sessions. The sessions were separated by 4-day intervals. Each session started with one unilateral i.p.f, microinjection in 24-h food deprived rats. After the injections the animals were placed in their cages with a measured amount of food and total intake was measured 30 min later. In the first series, that was similar for all rats and explored the AMPH effect, 3 sessions in which the animals received saline (0.5 pl) alternated with 3 sessions in which they received AMPH (40 pg/0.5 pl). The AMPH effect was the difference between the mean food intake after the 3 AMPH injections and the mean food intake after the 3 saline injections. The second series was specific for each group and explored the effects of DA (40 pg), NE (25 pg), EPI (25 pg), 5-HT (25 pg) or a second series of AMPH (40 pg) injections. In this series 3 saline sessions alternated with 3 monoamine sessions. The monoamine effect for each group was calculated by subtracting the mean food intake of the saline sessions from the mean food intake of the monoamine sessions, in a similar way as described above. Prior systemic administration of monoamine oxidase (MAO) inhibitors is a resource commonly employed in experiments exploring the effects of intracerebrally applied monoamine agonists. Those drugs retard the
/~g, Q I D , for 3 days) c a u s e d d e p l e t i o n of h y p o t h a l a m i c
injections (50
N E , and i n c r e a s e d f o o d i n t a k e and b o d y weight. H o w e v e r , such a n e u r o c h e m i c a l a l t e r a t i o n can be d i s r e g a r d e d in the p r e s e n t study, d u e to the fact that t h e doses of AMPH
e m p l o y e d w e r e l o w e r and a d m i n i s t e r e d with a
t i m e i n t e r v a l of at least 10 days. F r o m Figs. 2 and 3 it is e v i d e n t that t h e r e is a lack o f c o r r e l a t i o n b e t w e e n the A M P H
effect o n f o o d i n t a k e
and the effects of N E and E P I . This n e g a t i v e finding is surprising since b o t h o f t h e m are b e l i e v e d to be i n v o l v e d in the a n o r e c t i c effect of A M P H directly a p p l i e d in the p e r i f o r n i c a l r e g i o n 27'29. O f course, a n e g a t i v e result d o e s not p r e c l u d e
a role for t h o s e c a t e c h o l a m i n e s
in the
A M P H a n o r e x i a , especially for E P I , which s e e m s to be the m o r e clearly i n v o l v e d in an inhibitory r e g u l a t i o n of f e e d i n g 3L32. N E has b e e n c l a i m e d to h a v e a m o r e relev a n t role in p r o m o t i n g f e e d i n g , acting t h r o u g h a - a d r e n ergic r e c e p t o r s in the p a r a v e n t r i c u l a r nucleus ( P V N ) 2s'
37.
F u r t h e r m o r e , w h a t e v e r the role of e n d o g e n o u s N E
in the f e e d i n g s u p p r e s s i o n i n d u c e d by i n t r a h y p o t h a l a m i c A M P H injections, it should be e x e r t e d after 30 min ( t i m e i n t e r v a l used in this study) since the N E release
220
2"
•
al_
,~
o
i m.jtf"
•
Nil
• •
--
----
•
•
'1
