Hypothalamic infusion of amphetamine increases serotonin, dopamine and norepinephrine

Physiology& Behavior,Vol. 44, pp. 607-610. Copyright©PergamonPress plc, 1988.Printed in the U.S.A.

0031-9384/88$3.00 + .00

Hypothalamic Infusion of Amphetamine Increases Serotonin, Dopamine and Norepinephrine MARCO PARADA,* LUIS HERNANDEZ, DAVID SCHWARTZ AND BARTLEY G. HOEBEL Department o f Psychology, Princeton University, Princeton, N J 08544 *Laboratorio de Fisioiogia de la Conducta, Facultad de Medicina Universidad de los Andes, Merida, Venezuela, 5101-A

PARADA, M., L. HERNANDEZ, D. SCHWARTZ AND B. G. HOEBEL. Hypothalamic infusion of amphetamine increases serotonin, dopamine and norepinephrine. PHYSIOL BEHAV 44(4/5) 607--610, 1988.--Microinjoctions of amphetarnine into the lateral hypothalamus are known to cause anorexia and hypodipsia. These effects are thought to be mediated by an action of amphetamine on the catocholaminergic terminals to release dopamine and norepinephrine and block reuptake. Direct evidence of neurochemical release was lacking; therefore microdialysis was used to measure monoamines and their metabolites in the extracelhilar fluid of the lateral hypothalamus while amphetamine diffused out through the microdialysis probe. Amphetamine infusion significantly increased serotonin, dopamine and norepinephrine; it decreased dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA), and did not change homovanillic acid (HVA). These results suggest that amphetamine releases dopamine, norepinephrine and serotonin and blocks reuptake which thereby retards neurotransmitter breakdown. The net effect was a quadrupling of extracellular monoamines which could react with postsynaptic receptors. This supports the hypothesis that the behavioral effects of amphetamine injections into the lateral hypothalamus are mediated by dopamine, norepinephrine and suggests, in addition, serotonin. Amphetamine

Lateral hypothalamus

Dopamine

Norepinephrine

Serotonin

Microdialysis

METHOD

F E E D I N G behavior can be either suppressed or enhanced by microinjection of amphetamine into the perffornical region of the lateral hypothalamus depending on the dose. Amphetamine injections in doses ranging from 0.8/~g to 51.4 /zg decreased feeding by 20% to 88%, and this effect was blocked by dopamine (DA) and norepinephrine (NE) receptor blockers (2,12). Dopamine blockers injected in the lateral hypothalamus caused hyperphagia, an effect predicted by the anorectic action of intrahypothalamic amphetamine (14,20). Repeated intrahypothalamic injections of amphetamine at high doses cause hyperphagia and obesity, probably due in part to the resultant depletion of hypothalamic NE (7). Lesions of the ventral noradrenergic bundle also produce hyperphagia, obesity and hypothalamic depletion of NE (1). In these animals intrahypothalamic amphetamine loses its anorectic potency (13). All of these results strongly suggest that catecholamines mediate the behavioral effects of intrahypothalamic injections of amphetamine (9). However, this proposition has never been subjected to direct experimental examination. In the present report we used microdialysis in the lateral hypothalamus combined with simultaneous amphetamine infusion in freely moving rats. Interestingly, amphetamine increased extracellular serotonin (5-HT) as well as DA and NE.

Subjects Eight Sprague-Dawley male rats weighing between 350 g and 400 g were individually housed on a 15 hr-9 hr light-dark cycle with food and water ad lib. The rats were anesthetized with 20 mg/kg of pentobarbital combined with 40 mg/kg of ketamine for stereotaxic placement of an implanted 21 ga stainless steel guide shaft aimed at the perifornical region of the lateral hypothalamus. The stereotaxic coordinates were: 6.5 mm anterior to the interaural line, 1.5 mm lateral to the midsagittal sinus and 3.0 mm perpendicularly ventral to the level surface of the cerebral cortex (21). Microdialysis probes to be inserted later would protrude 5 mm. One week was allowed for recovery before starting experiments.

Microdialysis Probes were made of a concentric 36 ga stainless steel tube inside of a 26 ga stainless steel tube ending in a tip of 0.2 mm diameter cellulose dialysis tubing with a 6,000 molecular weight cutoff. The probes used in this experiment had a 3 mm long tip and a relative recovery of neurochemicals from the extracellular space of 6% to 10% depending on the neurochemical (8).

607

608

PARADA, HERNANDEZ, SCHWARTZ AND HOEBEL

A Ringer's solution containing 189 mM NaCI, 3.9 mM KC1, and 3.37 mM CaCI2 was warmed to 38°C and flushed with nitrogen to partially remove oxygen. A gas tight syringe loaded with this solution was placed in a pump with the flow rate set at 1/zl/min. The syringe was connected by polyethylene (PE-20) tubing, through a swivel joint to the inlet tube of a microdialysis probe. The outlet tube of the probe was connected by PE-10 tubing to a collection vial placed 2 cm above the head of the rat. The vial contained 10/zl of 0.1 N HCI and 100/~M EDTA to prevent catecholamine degradation. The microdialysis probe was inserted in the guide shaft and secured to the headpiece of the rat with tape. This procedure prevented movement of the microdialysis probe during perfusion and provided a stable baseline. The animal was placed in a 18x 30x 30 cm Plexiglas cage, and sample collection was started two hours after insertion of the probe. Samples were collected every 20 minutes. A separate section of PE-20 tubing was preloaded with 20/~l of amphetamine solution (10/xg//zl in Ringer' s solution= 200/~g amphetamine total). After collecting 4 samples showing a stable baseline, this preloaded tubing was intercalated in the PE-20 flow line leading to the probe. This procedure applied the drug by outward diffusion from the probe without disturbing the brain tissue. Simultaneously neurochemicals diffused into the probe and were carded into the vial to be assayed. Assuming amphetamine diffused out of the probe at about the same rate that monoamines diffuse in (8), roughly 10% (or 20 /zg) of the drug diffused out of the microdialysis probe in 20 min.

30"

AMPHETAMINE INFUSION *

.~.

20

160

200

MINUTES

ill

20-

Z nill

_z

10

Ug O Z

0

0

4

0

i

80

|

120

|

i

160

200

160

200

MINUTES

Analysis of Dialysates The dialysates were analyzed by injecting the content of the vial directly into an HPLC (Model 400, BAS Co.) equipped with a 10.0 cm long, 3.2 mm bore, 3 /zm ODS reverse phase column. The catecholamines were oxidized on a glassy carbon electrode at 0.71 V relative to a Ag/AgCI reference electrode. The order of elution of the monoamines and their metabolites was NE, DOPAC, 5-HIAA, DA, HVA and 5-HT. The retention times were 1.7, 3.3, 6.1, 7.7, 11, and 22.6 minutes respectively. The concentration of these neurochemicals in the samples was measured by the ratio of sample to standard peak heights, and the results expressed as pg/20/xl of sample.

200~t

150-

Z O

_ = ~¢~

gg IM (/)

100"

5o-

40

80

120

MINUTES

Histology After the experiments the rats were anesthetized with pentobarbital; brains were perfused with formalin, sectioned and stained with cresyl violet.

FIG. 1. Intrahypothalamic amphetamine infusion increases extracellular dopamine, F(5,35)=4.94, p<0.002, norepinephrine, F(5,35)= 9.88,p<0.001, and serotonin, F(5,35)= 16.7,p<0.001 (*p<0.01 after Newman-Keuls test; ND=not detected).

Statistical Analysis The data were analyzed by one-way ANOVA followed by least significant difference related t-test. RESULTS

Basal and peak levels of monoamines and their metabolites are shown in Figs. 1 and 2. During amphetamine infusion, DA, NE and 5-HT increased, DOPAC and 5-HIAA decreased, and HVA did not change. In basal conditions the order of concentrations was 5-HIAA > > DOPAC > HVA > NE > DA > 5-HT. Amphetamine infusion changed this order to 5-HIAA > 5-HT > HVA > DOPAC > DA > NE. Although all three monoamines increased significantly, 5-HT increased most. The time course of the effect is shown in Figs. 1 and 2.

Serotonin reached a peak during the infusion, then DA (second sample), and then NE (fourth sample). DOPAC reached its lowest level during the third sample, and 5-HIAA during the second sample after amphetamine infusion. The tracks of the probes were located in the medial forebrain bundle, lateral to the fornix. DISCUSSION

Amphetamine infusion into the lateral hypothalamus increased extracellular 5-HT, DA and NE. The fastest effect was on 5-HT, then DA, and then NE. These temporal patterns could be explained by different mechanisms of action of amphetamine on different intracellular pools of transmit-

AMPHETAMINE, LH MONOAMINES AND MICRODIALYSIS AMPHETAMINE INFUSION

120 100

.~

8o •

20 0

i

|

|

!

i

40

80

120

160

200

MINUTES

120 100

,<

--1

80

>

~

40 20 0 ~o

so

,;o

|

i

,~o

2oo

MINUTES 800'

600'


400"

200-

0

m

0

40

i

~

!

|

80

1 0

1 60

200

MINUTES FIG. 2. Intrahypothalamic amphetamine infusion decreases DOPAC, F(5,35)=16.7, p<0.001, and 5-HIAA, F(5,35)=10.5, p<0.001, but does not change HVA (*p<0.01 after Newman-Keuls test).

ters. Amphetamine releases DA preferentially by exocytosis from a vesicular newly synthesized releasable pool (17, 25, 27). Then dopamine declines fast because the transfer of DA from the storage pool to the releasable pool is relatively slow (15). In the case of norepinephrine, amphetamine does not seem to release norepinephrine by exocytosis. It is believed that amphetamine is transported into the noradrenergic terminals by a carrier mechanism and releases norepinephrine from the storage vesicular pool into the cytoplasm. NE is released from this cytoplasmic pool into the extracellular compartment (26). The transfer of NE from the storage pool to the releasable pool is fast enough to prolong increased levels of NE longer than the increased levels of DA. The quick increase in serotonin is interesting because a mul-

609

ticompartment distribution of serotonin has also been suggested. A readily-releasable pool of newly synthesized serotonin as well as a more tightly bound storage pool has been suggested as in the case of dopamine and norepinephfine (10,16). If this view is correct then amphetamine has a dramatic effect on the releasable pool of serotonin and is less effective in transferring serotonin from the storage to the releasable pool. These different mechanisms of action on the different 5-HT pools would explain the rapid release followed by a rapid decline of serotonin after amphetamine infusion. Interestingly, two other drugs, phencyclidine and fenfluramine, directly applied on the serotonergic terminals (6,24) increase extracellular serotonin with the same time course as amphetamine. This indicates that serotonin increases by the same mechanism when the serotonergic terminals are challenged with different pharmacological agents. Therefore amphetamine might be revealing profound differences in the way monoamines are distributed, released and metabolized as has been previously suggested by several authors. The magnitude of the 5-HT increase was greater than the DA or the NE increase. This phenomenon parallels the fact that the 5-HT content of the perifornical hypothalamns (30 ng/mg of protein) is almost twice as large as the NE content (17 ng/mg of protein), and five times as large as DA content (6 ng/mg of protein) (18,22). Interestingly, this situation was not reflected in the basal extracellular levels of neurotransmitters because NE and DA were more concentrated than 5-HT. Therefore, it is intriguing that serotonin was very often undetectable in basal conditions. One possible explanation is that the long retention time of serotonin (22.6 rain) flattens the peak and lowers the sensitivity for serotonin in the HPLC system. Another explanation is based upon the fact that serotonin neurons show a clear correlation with arousal. When the animal is awake serotonin neurons are active, and when the animal is sleeping serotonin neurons are quiescent (3). Since our experiments were done during the day, when the rats sleep, we would expect low, undetectable serotonin levels in the extracellular compartment. DOPAC and 5-HIAA significantly decreased, indicating that the action of amphetamine was not due to unspecific irritation of the terminals, and also suggesting that amphetamine acted as a reuptake blocker for DA and 5-HT. HVA levels did not vary, presumably because in contrast to DOPAC and 5-HIAA which are formed inside dopaminergic and serotonerglc neurons, HVA is formed in nondopaminergic neurons and in glial cells (11). In this situation amphetamine should have increased HVA because there was more substrate available in the extracellular compartment. However, since amphetamine also inhibits monoamine oxidase (4), it is possible that this inhibition compensated for the increase in substrate, leaving the concentration of HVA essentially unchanged. As for NE we could not detect its main metabolite, 4hydroxy-3-methyl-phenyl(ethylene)glycol(MOPEG), because most of it exists in the sulfated form in the rat brain (23) and sulfated compounds can not be oxidized at 0.71 V. Other experiments suggest that intracerebral injections of amphetamine also block reuptake of NE (5). The present findings suggest that the behavioral changes induced by local applications of amphetamine into the lateral hypothalamus are mediated by 5-HT, DA and NE. The relative behavioral role of each still has to be determined. In other experiments, anorexia caused by local LH injection of serotonin and dopamine was well correlated with anorexia

610

PARADA, H E R N A N D E Z , SCHWARTZ AND H O E B E L

by intrahypothalamic injection of amphetamine (19). Judging by the temporal course of the increase of the three monoamines in Fig. 1, it appears that each of them might dominate the behavioral events in a time sequence.

ACKNOWLEDGEMENTS This research was supported by the Campbell Soup Company and PHS grant DA-03597. The help of Dawn Davidson and Kathleen McGeady is gratefully acknowledged.

REFERENCES 1. Ahlskog, J. E.; Hoebel, B. G. Overeating and obesity by damage to a noradrenergic system in the brain. Science 182:166-169; 1973. 2. Booth, D. A. Amphetamine anorexia by direct action on the adrenergic feeding system of the rat hypothalamus. Nature 217:869-970; 1968, 3. Fornal, C. A.; Jacobs, B. L. Physiological and behavioral correlates of serotonergic single unit activity. In: Osborne, N. N.; Hamon, M., eds. Neuronal serotonin. New York: John Wiley & Sons, Inc.; 1988. 4. Glowinsky, J.; Axelrod, J.; Iversen, L. L. Regional studies of catecbolamines in rat brain. IV. Effects of drugs in the disposition and metabolism of [3H]NE and [3H]DA. J. Pharmacol. Exp. Ther. 155:30-41; 1966. 5. Hernandez, L.; Hoebel, B. G. Overeating after midbrain 6-hydroxydopamine: prevention by central injectionof selective blockers. Brain Res. 245:333-343; 1982. 6. Hernandez, L.; Hoebel, B. G. Phencyclidine (PCP) injected in the nucleus accumbens increases extracellular dopamine and serotonin as measured by microdialysis. Life Sci. 42:1713-1723; 1988. 7. Hernandez, L.; Parada, M.; Hoebel, B. G. Amphetamine induced hyperphagia and obesity caused by intraventricular or lateral hypothalamic injections in rats. J. Pharmacol. Exp. Ther. 227:524-530; 1983. 8. Hernandez, L.; Stanley, B. G.; Hoebel, B. G. A small, removable microdialysis probe. Life Sci. 39:2629-2637; 1986. 9. Hoebel, B. G.; Leibowitz, S. F. Brain monoamines in the regulation of self-stimulation, feeding and body weight. In: Weiner, H.; Hofer, M. A.; Stunkard, A. J., eds. Brain, behavior and bodily disease. New York: Raven Press; 1981. 10. Kleven, M. S.; Dwoskin, L. ~P.; Sparber, S. B. Pharmacological evidence for the existence of multiple functional pools of brain serotonin: analysis of brain perfusate from conscious rats. J. Neurochem. 41:1143-1149; 1983. 11. Kopin, I. J. Catecholamine metabolism: Basic aspects and clinical significance. Pharmacol. Rev. 37:334-358; 1985. 12. Leibowitz, S. F. Catecholaminergic mechanisms of the lateral hypothalamus: Their role in the mediation of amphetamine anorexia. Brain Res. 98:529-545; 1975. 13. Leibowitz, S. F.; Brown, S. L. Histochemical and pharmacological analysis of catecholaminergic projections to the perifornical hypothalamus in relation to feeding inhibition. Brain Res. 201:315-345; 1980.

14. Leibowitz, S. F.; Miller, N. Unexpected adrenergic effect of chlorpromazine: eating elicited by injection into rat hypothalamus. Science 165:609-611; 1969. 15. McMillen, B. A.; German, D. C.; Shore, P. A. Functional and pharmacological significance of brain dopamine and norepinephrine storage pools. Biochem. Pharmacol. 29:3045--3050; 1980. 16. Mennini, T.; Borroni, E. ; Samanin, R. ; Garattini, S. Evidence of the existence of two different intraneuronalpools from which pharmacological agents can release serotonin. Neurochem. Int. 3:289-294; 1981. 17. Miller, H. H.; Shore, P. A. Effects of amphetamine and amphonelic acid on the disposition of striatal newly synthesized dopamine. Eur. J. Pharmacol. 78:33-44; 1982. 18. Palkovitz, M.; Brownstein, M.; Saavedra, J. M.; Axelrod, J. Norepinephrine and dopamine content in hypothalamic nuclei in rat. Brain Res. 107:137-149; 1974. 19. Parada, M. A.; Hernandez, L. Correlacion significativa entre la anorexia por anfetamina, serotonina y dopamine intrahipotalamicas. XXXII Convencion anual de ASOVAC, Caracas, Venezuela; 1982. 20. Parada, M. A.; Hernandez, L.; Hoebel, B. G. Sulpiride injections in the lateral hypothalamus induce feeding and drinking in rats. Pharmacol. Biochem. Behav. 30:917-923; 1988. 21. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates. Orlando: Academic Press; 1986. 22. Saavedra, J. M.; Palkovitz, M.; Brownstein, M. J.; Axelrod, J. Serotonin distribution in the nuclei of the rat hypothalamus and preoptic region. Brain Res. 107:157-165; 1974. 23. Schanberg, S. M.; Breese, G. R.; Schildkraut, J. J.; Gordon, E. K.; Kopin, I. J. 3-Methoxy-4-hydroxyphenylgiycol sulfate in brain and cerebrospinal fluid. Biocbem. Pharmacol. 17:247-254; 1968. 24. Schwartz, D.; Kloecker, J.; Hernandez, L.; Hoebel, B. G. Fenfluramine increases extracellular serotonin in the lateral hypothalamus: A microdialysis study. Brain Res.; in press. 25. Shore, P. A.; Dorris, R. L. On a prime role for newly synthesized dopamine in striatal function. Eur. J. Pharmacol. 30:315318; 1975. 26. Trendelenburg, U. Release induced by phenethylamines. In: Paton, D. M., ed. The release of catecholamines from adrenergic neurons. New York: Pergamon Press; 1979. 27. Walker, R. J. Biosynthesis, storage and release of dopamine. In: Winlow, W.; Markstein, R., eds. The neurobioiogy of dopamine systems. Manchester: Manchester University Press; 1986.