Ingestion in the satiated rat: Role of alpha and beta receptors in mediating effects of hypothalamic adrenergic stimulation

Physiology and Behavior, Vol. 14, pp. 743-754. Brain Research Publications Inc., 1975. Printed in the U.S.A.

Ingestion in the Satiated Rat: Role of Alpha and Beta Receptors in Mediating Effects of Hypothalamic A drenergic Stimulation I S A RA H F R Y E R LEIBOWlTZ

The Rockefeller University, N e w York N Y 10021

(Received 30 November 1973) LEIBOWlTZ, S. F. Ingestion in the satiated rat: role o f alpha and beta receptors in mediating effects o f hypothalamic adrenergic stimulation. PHYSIOL. BEHAV. 14(6) 743-754, 1975. - Anterior perifornical hypothalamic injection of l-norepinephrine in satiated rats elicits a brief, vigorous drinking response followed within a minute or two by a vigorous feeding response. These adrenergically elicited responses, which bear striking similarities to a rat's naturally motivated ingestive behaviors, were examined in the present series of experiments. It was found that: (1) Both responses could be elicited by perifornical hypothalamic injection of l-epinephrine, which was actually found to be more potent than l-norepinephrine. In contrast, only feeding could be elicited by the alpha-stimulant metaraminol, and neither feeding nor drinking could be elicited by hypothalamic injection of d-norepinephrine, 1-isoproterenol, or dopamine. (2) The threshold doses of/-epinephrine for eliciting reliable ingestive responses were quite low, namely, 0.8 nmole (0.15 /~g) for drinking and 0.2 nmole (0.04 ~g) for feeding. (3) Pharmacological analysis of the ingestive behaviors induced by l-norepinephrine or /-epinephrine indicated that the eating response was mediated by alpha-adrenergic receptors, whereas the drinking response involved the synergistic action of both alpha- and beta-adrenergic receptors. No evidence for the involvement of dopaminergic or cholinergic (muscarinic) receptors was obtained. (4) A third adrenergically elicited phenomenon, namely, a suppression of drinking, was observed during and after the period of induced feeding. Analysis of this effect revealed its dependence solely upon alpha-adrenergic receptor activity. Drinking behavior Feeding behavior Hypothalamus Alpha- and beta-adrenergic receptors Epinephrine Isoproterenol Dopamine Norepinephrine

next 10 to 20 min, consuming an amount of food (generally between 2 and 4 g) that is positively correlated with the amount of water previously consumed. Additional water consumption is almost never observed during this period of feeding, nor is it generally observed for a period of time after feeding stops. This subsequent interval of no drinking amounts to a suppression of drinking relative to what uninjected rats normally consume (1.5 to 2.0 ml) under conditions of no feeding. This sequence of behavioral changes reveals three reliable effects of hypothalamic noradrenergic stimulation on ingestion: an initial stimulation of drinking followed by a stimulation o f feeding and a suppression of drinking. The present investigation focused on the problem of identifying the

THE ingestive behavior of food- and water-satiated rats can be dramatically altered by hypothalamic injection o f the agonist norepinephrine [4, 7, 13, 25, 30, 40]. A consistent pattern o f water and food consumption has been observed after administration of this noradrenergic agonist into the perifornical region of the anterior hypothalamus [30]. This pattern, which has striking similarities to the pattern of feeding and associated drinking normally exhibited by rats under laboratory conditions [8,19], can be described as follows. Within a minute or two after drug injection, the rat starts to drink water. He drinks vigorously during the next 2 to 3 min, during which time he consumes between 1 and 4 ml. After a minute or two o f no ingestive behavior, the rat then starts to eat food. He eats vigorously during the

1This research was supported by NIH research grant MH 13189 and by funds from the Grant Foundation, Hoffman-La Roche, and Smith Kline and French. The author gratefully acknowledges the excellent technical assistance of Mrs. Ruth Hechinger and Messrs. Alan Katz, Steven Feiertag, and Kevin Chang. The author wishes to thank the following Companies for their generous supplies of drugs: Amend Drug and Chemical Co. (atropine), Ayerst Laboratories (1- and d-propranolol), CIBA Pharmaceutical Co. (phentolamine and tolazoline), McNeil Laboratories (haloperidol), Mead Johnson and Co. (sotalol), Merck and Co. (metaraminol), Sandoz Pharmaceuticals (LB-46), Smith Kline and French Laboratories (phenoxybenzamine), and Winthrop Laboratories (isoproterenol). Portions of these findings were reported at the meeting of the Eastern Psychological Association held in Boston, April 1972, and at the Vth International Conference on Physiology of Food and Fluid Intake, held in Israel, October 1974. 743

744

LEIBOWlTZ

type(s) of receptors mediating these three effects, each of which can occur independently of each other. In these experiments, the effectiveness of a variety of drugs in eliciting the ingestive responses, and the threshold doses for the effective drugs, were examined. In addition, the effects of selective receptor antagonists on the effects of central adrenergic stimulation were tested. The results obtained in these experiments demonstrate that the above changes in ingestive behavior can be induced by l-epinephrine, as well as by l-norepinephrine, at remarkably low doses. The results also provide evidence for the involvement of both alphaand beta-adrenergic receptors in the mediation of the centrally elicited effects, but not for the involvement of either dopaminergic or cholinergic receptors. METHOD

Animals A total of 204 male albino Sprague-Dawley rats were used, weighing approximately 350 g at the start of the experiment. The rats were housed individually and were maintained and tested, in their home cages, on Purina lab chow pellets and tap water. All rats were tested while foodand water-satiated.

Surgery A chronic unilateral cannula was stereotaxicaUy implanted in each rat, according to the procedure described earlier [30]. All cannulas were aimed for the perifornical region of the anterior hypothalamus. The coordinates used for this region were at the frontal level of bregma, 1.3 mm lateral, and 8.2 mm below the skull surface. The top of the upper incisor bar of the stereotaxic instrument was placed 3.1 mm above the center of the aural bars. Previous histological examination of this cannula placement [30,40] has shown it to be located close to the fornix, at the frontal level of the paraventricular nucleus. Analysis of the brains of the rats used in the present study confirm this location, as illustrated in Fig. 1.

General Test Procedure The experimental tests were started 1 to 2 weeks after surgery. Each rat was tested in the morning every 2 to 3 days. To ensure maximal satiation at the start of the test, each rat was given fresh food and water 60 min beforehand. During this time each animal was handled and mockinjected at least once, to adapt him to the test procedure. In all experiments, only one goal object, food or water, was available at a given time. Water was made available through calibrated tubes with drinking nozzles. Measured food (lab chow pellets) was placed in a corner of the cage, and the remainder removed and weighed at the end of each test interval. The food lost through spillage was added to the nonconsumed total. There was no apparent spillage of water. The tests began immediately after injection of the adrenergic agonist, and the rats' behavior was observed for up to 60 min. Except where otherwise indicated, statistical evaluation of the results was based on a two-tailed t-test for dependent means.

Drug Injection The drugs used [ 17] were: (1) the noradrenergic agonist l-norepinephrine-d-bitartrate (NE); (2) the d-isomer of nor-

epinephrine bitartrate (d-NE); (3) The adrenergic agonist l-epinephrine bitartrate (EPI); (4) the relatively pure betaadrenergic agonist l-isoproterenol-d-bitartrate (ISOP); (5) the relatively pure alpha-adrenergic stimulant metaraminol bitartrate (MET); (6) the catecholamine agonist dopamine hydrochloride (DA); (7) the alpha-adrenergic receptor blockers, phentolamine hydrochloride (PHT), tolazoline h y d r o c h l o r i d e (TOL), and phenoxybenzamine hydrochloride (PHB); (8) the beta-adrenergic receptor blockers, l-propranolol hydrochloride (/-PROP), LB-46, and sotalol hydrochloride (MJ 1999); (9) the dopaminergic receptor blocker haloperidol (HAL); and (10) the cholinergic (muscarinic) receptor blocker atropine sulfate (ATP). In order to determine whether the effects of /-PROP were caused by its local anesthetic action [33], as opposed to its beta-blocking action, the d-isomer of this compound, which has potent local anesthetic action but relatively weak betareceptor blocking action, was also tested. Both LB-46 and MJ 1999, in contrast to/-PROP, appear to have little local anesthetic activity [ 11,33, 38]. All drugs except PHB, HAL, and LB-46 were dissolved in sterile physiological saline (0.9 percent) or distilled water. Because of their relative insolubility in water, PHB was dissolved in a 10 percent ethanolic saline solution, and HAL and LB-46 were dissolved in a dilute solution of tartaric acid. All drugs were injected directly into the perifornical hypothalamus through the chronically implanted cannulas. The volume of the injection was 0.5 ul in all cases. Fresh solutions of each drug were prepared immediately before the start of the test. Doses of the injections are stated below. Each experiment involved the treatment of the same rats with a variety of drug doses or drug combinations. The sequence in which the rats received the different treatments, which included at least one vehicle control injection, was determined by a Latin square design. The different treatments of the receptor blocker experiments always consisted of the following paired injections: blocker plus agonist, blocker vehicle plus agonist, and blocker vehicle plus agonist vehicle. (The fourth combination, blocker plus agonist vehicle, was tested in separate experiments described below.) The first injection of each pair, blocker or its vehicle, was administered 5 min before the second injection, the agonist or its vehicle. In these experiments, moderate to high doses of the adrenergic agonists (20 to 30 nmoles) were used to assure obtaining robust effects. The doses of the blocking agents were selected so as to be adequate for effective blockade, yet sufficiently low so that the blocker itself would have no effect of its own on the rats' food or water consumption. Previous central studies carried out in this [22, 25, 31 ] and other [5, 32, 3 9 - 4 1 ] laboratories have already determined the blocker doses required to effectively antagonize the activity of alpha-adrenergic, beta-adrenergic, dopaminergic, and cholinergic receptors. With regard to possible direct effects of the blockers themselves on ingestive behavior, this laboratory conducted some initial experiments involving injections of the antagonists alone in counterbalanced order with their vehicle. These tests, carried out in satiated as well as in thirsty and hungry rats, failed to reveal any blocker-induced effects at any of the doses used in the present study. The main adrenergic effects examined in this article are not uniformly exhibited by all rats with perifornical hypothalamic cannulas. Approximately 70 percent of the rats

MEDIATING ROLE OF ADRENERGIC RECEPTORS IN INGESTION tested respond reliably to adrenergic stimulation. (This variability of responsiveness is very possibly due to variability of cannula placements. See [ 3 0 , 4 0 ] . ) F o r the purposes of the present investigation, it was necessary to have a high, stable baseline for examining the agonists' effects in combination with the adrenergic receptor blockers. To provide this baseline, preselection tests with NE or EPI (25 nmoles) were performed to screen out nonresponders. A rat was considered a nonresponder if, over 3 tests, he failed to exhibit: (1) an average eating response of at least 1.5 g during the first 30 min after injection, (2) an average drinking response of at least 1.0 ml during the first 5 rain after injection, or (3) an average drinking suppression of at least 1.0 ml during the subsequent 55 min of a 60 min test. Such screening tests were employed in all but Experiment 5. Histology To confirm their cannula placements, the rats were sacrificed under Nembutal anesthesia and perfused with saline and a 10 percent Formalin solution. Frozen sections of 50 were cut and stained, using either cresyl violet or a modified K1Bver and Barrera technique [43]. The location of the cannula tip was determined according to the atlas of K~Snig and Klippel [20]. The results of this analysis, for the rats used in Experiment 2, are illustrated in Fig. 1. These results reveal an injection site representative of all rats used in this study.

745 TABLE 1

EFFECTS OF DRUGS ON DRINKING AND FEEDINGIN SATIATED RATS

Group 1-4

Drug NE

d-NE

2

MET

ISOP

3

DA

Dose (nmoles)

Water Intake (ml in 10 min)

Food Intake (g in 60 min)

saline

0.0

0.3

25

3.0~

3.5~

saline

0.0

0.4

10

0.1

0.5

25

0.2

0.9

saline

0.1

0.2

50

0.1

1.0"

100

0.5

1.9t

saline

0.1

0.5

10

0.5

0.1

100

0.4

0.1

saline

0.2

0.4

10

0.3

0.4

25

0.8

1.0

saline

0.1

0.4

EXPERIMENT 1: TESTS WITH d-NE, MET, ISOP, DA, AND EPI This experiment tested the effectiveness of various catecholamine agonists, as well as a non-catecholamine sympathomimetic drug, in eliciting the ingestive responses observed after perifornical hypothalamic injection of NE. A total of 40 satiated rats with perifornical hypothalamic cannulas were used in this experiment. The rats were first tested with NE (25 nmoles) or saline to establish their baseline performance with respect to elicited drinking and feeding. After this initial test, they were randomly assigned to one of 4 groups (10 animals per group) and then tested, according to a Latin square sequence, with saline and 2 doses of d-NE (Group 1), MET and ISOP (Group 2), DA (Group 3), and EPI (Group 4). Each group received 2 series of 60 min tests: one having water only with measurements taken at 10 and 60 min after injection, and one having food only with measurements taken just at 60 rain. Subsequent to these tests, Group 4 was used in an additional series of tests in which both NE and EPI (10 nmoles of each) were injected in balanced order with saline. These tests, which had both water and food simultaneously present, were conducted for the purpose of directly comparing the effectiveness of the two adrenergic agonists, as well as the sequence of their elicited effects. Results and Discussion The results of these tests (Tables 1 and 2) indicate that the ingestive responses elicited by perifornical hypothalamic injection of NE are stereospecific and that they cannot be obtained with the agonists ISOP or DA, at least at the doses tested. The results also demonstrate that the alpha-adrenergic stimulant MET can produce reliable eating but not drinking, whereas the agonist EPI can produce both drinking and eating and, in fact, is even more potent than NE in eliciting these responses.

4

EPI

10

2.3t

3.1t

50

3.6:~

4.2:~

are the mean scores, N = 10 per group. Two-tailed dependent t-tests comparing saline and NE scores: *p<0.05 tP<0.01 Sp<0.001 Given

TABLE 2 COMPARISON OF NE AND EPI EFFECTS ON DRINKING AND FEEDING

Drug

Dose (nmoles)

Water Intake (ml in 5 min) 0.0

Food Intake (g in 30 min) 0.3 + 0.1

Saline

-

0.0 ±

NE

10

1.4 ± 0.5*

2.2 ± 0.6t

EPI

10

2.4 + 0.4 t

3.4 ± 0.6:~

Given are the means + standard error of the means, N = 10. Two-tailed dependent t-tests comparing saline and drug scores: *p<0.05 tp<0.01 ~p<0.001

746 When the/-isomer of NE (25 nmoles) was injected into the perifornical hypothalamus of these satiated rats, the expected increase in consumption of both water and food was reliably obtained (Table 1). In the water only tests, the rats started to drink within a minute or so after injection. This elicited drinking was a vigorous response, continuing without interruption for the next 2 to 3 min. At approximately 5 min after injection, the drinking stopped, and essentially no further water consumption was observed during the remainder of the test. In the food only tests, the rats started to eat at 5 to 6 min after injection. Their feeding response continued for around 20 min and then stopped completely. As shown in Table 1, perifornical hypothalamic injection of the d-isomer of NE was found to be ineffective in eliciting either of these responses obtained with I-NE injection, as were the beta-adrenergic agonist ISOP and another catecholamine agonist, DA. Earlier studies, which measured feeding but not drinking behavior, obtained results similar to these [5,40] and, in addition, found the agonist ISOP to produce a strong suppression of feeding in hungry rats [ 12,22]. In pigs, a similar suppression of food consumption was observed with this beta-adrenergic agonist [18], while in sheep both a stimulation and a suppression of feeding have been demonstrated [ 1]. In a study on drinking behavior, in which ISOP was injected into the lateral hypothalamus of the rat, this agonist was found to produce a reliable increase in water consumption [24,25]. This drinking observed with ISOP was quite different from the food-associated response obtained with NE injection into the more anterior perifornical hypothalamic site. The ISOP response started after a relatively long latency of approximately 10 min, it occurred sporadically over a period of 2 to 3 hr, and, as stated above, it was not associated with enhanced feeding. In the present study, in which ISOP was injected into the perifornical hypothalamus, this prolonged drinking response was not observed. Measurements taken at the end of the 60 min test failed to reveal a reliable increase in water consumption. In contrast to the beta-adrenergic agonist ISOP, the alpha-adrenergic stimulant MET (Group 2) was found to produce a reliable increase in food consumption. The feeding response was not large, however, and it required higher doses than were needed with NE. (In the peripheral nervous system, MET has similarly been found to be less potent than NE [17].) As with the agonists ISOP and DA, a reliable increase in water consumption was not observed with MET. Epinephrine (Group 4), in contrast to all other drugs tested, was found to mimic the stimulatory effect of NE on water consumption, as well as its stimulatory effect on food consumption. In the first series of tests in which water and food were presented separately (Table 1), EPI at 10 and 50 nmoles reliably produced both drinking and feeding. As with NE, the drinking response occurred during the first few minutes after injection and the feeding response during the first 30 rain. These results are consistent with earlier reports of EPI-induced feeding in satiated rats [5, 13, 40]. In the second series of tests in Group 4, the effects of both EPI and NE were examined with water and food simultaneously present. The results of these tests (Table 2) revealed a sequence of ingestive behaviors with EPI injection which was similar to that previously obtained with injection of NE [30]. Within 1 to 2 min after injection of either agonist, the rats started to drink water. They drank for approximately 2 min and then, after a minute or so,

LEIBOWlTZ started to eat and continued eating for around 15 rain. After the cessation of eating, no further ingestive behaviors were observed. Further analysis of the results obtained with the two adrenergic agonists revealed a strong positive correlation between their effectiveness in a particular rat. This correlation was obtained for water intake (r = +0.89, p < 0 . 0 0 1 ) a s well as for food intake (r = +0.92, p<0.001). Direct comparisons between the two agonists' scores for drinking and feeding, using dependent t-tests, showed that EPI was reliably more potent (at p<0.05) than NE in eliciting both responses. These findings are consistent with the results of Slangen and Miller [40] and of peripheral studies [17] which have generally found EPI to be the more potent neurohumoral agent.

EXPERIMENT 2: THRESHOLD DOSES This experiment was designed to determine the threshold doses, of both NE and EPI, that are required for perifornical hypothalamic injection to elicit reliable drinking and feeding. Thirty rats, each with a perifornical hypothalamic cannula, were first tested with a moderate dose (20 nmoles) of NE and EPI. Eight of these rats were found to be nonresponders (see Method section) and therefore were'eliminated from the group and their brains prepared for histology. The remaining 22 rats (responders) were carried through a series of tests where the doses of the agonists were lowered progressively, in half-steps, until a reliable response was no longer observed. At each dose level, starting at 6.4 nmoles, NE, EPI, or saline were injected into each rat according to a Latin square design. Half of the rats received 5 min tests with only water available, and the other half received 30 min tests with only food available. At the end of this test series, these 22 rats were injected once again with 20 nmoles of NE and EPI, to determine whether their sensitivity had changed during the period of testing for threshold dose levels. Subsequent to this final test, the rats were perfused and their brains prepared for histology. Results and Discussion

In aggreement with the finding of Experiment 1, EPI was found here to be consistently more potent than NE at every dose level (Table 3). Furthermore, EPI's threshold doses for eliciting reliable ingestive responses were somewhat lower than those of NE. The threshold doses for eliciting a reliable drinking response were at least as low as 1.6 nmoles (0.27/~g free base) for NE and 0.8 nmole (0.15 ug free base) for EPI. The threshold doses for eliciting a reliable feeding response were somewhat lower, 0.8 nmole (0.14 ug) and 0.2 nmole (0.04 ug), respectively. The rats' responsiveness to the moderate (20 nmole) dose of the agonists remained unchanged at the end of the test series. (No significant differences were obtained, at p<0.05, between the first test and last test scores shown in Table 3.) When the brains of the rats in this experiment were examined histologically (Fig. 1), it was seen that all of the cannulas belonging to the responders were located either near to or medial to the fornix at the frontal level of the paraventricular nucleus. The injection sites more than 0.5 mm lateral, ventral, or dorsal to the fornix belonged to nonresponders. These findings agree with earlier studies [4, 7, 30, 40, 44] in suggesting that an effective region for

MEDIATING ROLE OF A D R E N E R G I C RECEPTORS IN INGESTION

747

TABLE 3 THRESHOLD DOSES FOR DRINKING AND FEEDING ELICITED BY PERIFORNICAL HYPOTHALAMIC INJECTIONS OF NE AND EPI Drug Dose (nmoles)

Water Intake (ml in 5 min)

Food Intake (g in 30 min)

0.0

0.2

0.0 (saline) NE

EPI

NE

EPI

3.1~

3.4~

3.4~

4.0~

6.4

2.1t

2.8~

2.7¢

3.4¢

3.2

1.5"

1.9t

2.1t

2.8~

1.6

1.1"

1.2"

1.8"

2.1t

0.8

0.3

1.0"

1.1"

2.0t

0.4

-

0.3

0.5

1.3"

-

-

-

-

-

-

20

(first test)

0.2 0.1 20

(last test)

2.8~t

3.3~t

3.5~

1.1" 0.2 3.6~

Given are the means. (Total N = 22, with 11 rats used in water intake tests and I 1 rats in food intake tests.) Two-tailed dependent t-test comparing saline and drug scores: *p<0.05 tP<0.01 ~p<0.001 eliciting drinking and/or feeding with adrenergic stimulation is located at the level of the anterior hypothalamus. This effective region may involve the bed nucleus of the fomix, or possibly the paraventricular nucleus which lies medial to the fornix ([7,29] and unpublished observations).

EXPERIMENT 3: ANALYSIS OF RECEPTORS MEDIATING FEEDING ELICITED BY NE OR EPI This experiment examined the nature of the neuronal receptors mediating the feeding response induced by perifornical hypothalamic injection of NE or EPI. The effects of the different blocking agents on NE- and EPI-elicited eating were investigated in 4 groups of rats. Each group, consisting of 10 to 12 animals each with a perifornical hypothalamic cannula, was tested on at least one blocker in combination with NE (20 nmoles) and EPI (20 nmoles). For each series of tests on each of the agonists, the group was tested in balanced order on the vehicle alone, the agonist alone (for determining baseline performance), and one or more of the blocker plus agonist combinations. The specific tests carried out in each group are outlined in Table 4. Immediately after injection of the agonist or its vehicle, the rats were given food (but no water). Their food consumption was then measured during the next 30 min. Results and Discussion The results of these receptor-blocker studies (Table 4

~/

I

C- I I I I I I

FIG. 1. Injection sites illustrated on frontal sections of the KSnig and Klippel atlas [20]. All rats had unilateral cannulas aimed for the perifornical region of the anterior hypothalamus. Filled circles (e) indicate injection sites for responders and open circles (o) for nonresponders (see text). Abbreviations: fornix (F), paraventricular nucleus (pvn), anterior hypothalamus (ha), and lateral hypothalamus (hi).

and Fig. 2) demonstrate that the eating response induced by either NE or EPI can be antagonized only by the alphaadrenergic receptor blockers. Specifically, perifornical hypothalamic injection of NE alone elicited a reliable feeding response, of between 3 and 4 g, in each of the groups tested. This response was found to be totally blocked by the three alpha blockers, PHT, TOL, and PHB. The antagonism induced by PHT was found to increase in magnitude with increase in dose. In contrast, the beta blockers/-PROP, LB-46, and MJ 1999 each failed to suppress NE-elicited feeding and, in fact, /-PROP and LB-46 tended to potentiate the effect. The d-isomer of PROP, the dopaminergic blocker HAL, and the cholinergic blocker ATP all failed to have any effect on NE-elicited feeding. These results indicate that alpha-adrenergic receptors, as opposed to beta-adrenergic, dopaminergic, or cho°

LEIBOWITZ

748 TABLE 4

Food Intake (g) in 30 min

EFFECTS OF RECEPTOR BLOCKERS ON FEEDING ELICITED BY NE OR EPI

Group 1

2

3

4

Drug Injections (nmoles) First Second

O

q

Food Intake (g) NE EPI

Vehicle

Vehicle

0.2 :t:

0.3 $

Vehicle

Agonist (20)

3.2

3.6 2.5

PHT (30)

Agonist (20)

1.6"

PHT (60)

Agonist (20)

0.55

1.3t

/-PROP (120)

Agonist (20)

4.0

4.9*

d-PROP (120)

Agonist (20)

3.3

3.6

Vehicle

Vehicle

0.3 $

0.6 $

Vehicle

Agonist (20)

3.3

3.9

TOL (100)

Agonist (20)

0.4:~

1.4 t

LB-46 (140)

Agonist (20)

3.9

-

MJ 1999 (400)

Agonist (20)

3.1

4.0

Vehicle

Vehicle

0.3 ~:

0.3 $

Vehicle

Agonist (20)

3.5

3.6

PHB (15)

Agonist (20)

0.65

-

HAL (3)

Agonist (20)

-

2.8

HAL (27)

Agonist (20)

3.0

3.6

Vehicle

Vehicle

0.2 :~

0.4 $

Vehicle

Agonist (20)

3.1

4.0

ATP (6.5)

Agonist (20)

3.0

3.7

Given are the mean scores, N = 10-12 per group. Two-tailed dependent t-tests comparing Vehicle plus Agonist score with each of the other scores: *p<0.05 tp<0.01 :~p<0.001

linergic (muscarinic) receptors, mediate the feeding response induced by NE. These findings obtained with combined injections of NE and various receptor blockers are, in part, a replication of the work of other investigators. Booth [5] and Slangen and Miller [40] examined the blockers PHT, PHB, and PROP, and their evidence revealed a block of NE-induced eating with the two alpha blockers but not with the beta blocker PROP. These results are consistent with the results of the present study which, in addition, revealed a similar pattern with the adrenergic receptor blockers TOL, LB-46, and MJ 1999 and also demonstrated the ineffectiveness of the dopaminergic blocker HAL. Our failure to observe a blocking effect with the cholinergic blocker ATP contrasts with the small but reliable suppression of NE-elicited eating observed by Lonowski and Levitt [35] with this drug. Our results with the agonist EPI demonstrate that the eating response induced by this agonist, like that of NE, can be reliably reduced by the two alpha blockers PHT and T•L. The EPI response, however, in contrast to the NE response, was not totally abolished by these antagonists.

I

2

3

I

I

I

Saline

Adrenergic agonist (NE or EPI)

I Alpho-adrenergic blockers Receptor blockers +

Beta-adrenergic blockers

adrenergic agonist

Dopaminergic blocker Cholinergic blocker

[ I ]

FIG. 2. Summary of blocker-agonist results presented in Table 4. This greater resistance of EPI to alpha-receptor blockade may simply reflect the fact that EPI's alpha-receptor action is somewhat more potent than that of NE (Experiments 1 and 2 and references [17,40]). In contrast to the alphareceptor blockers, the two beta-receptor blockers /-PROP and MJ 1999 failed to reduce the EPI eating response. In fact, /-PROP reliably enhanced this effect. The d-isomer of PROP failed to affect the EPI response, which indicates that the more potent beta-blocking action of/-PROP is the source of this isomer's effectiveness and not the local anesthetic property shared by both isomers. The failure of MJ 1999 to mimic /-PROP's potentiating effect may be due to the fact that MJ 1999 is a less effective beta blocker [38]. Haloperidol, the dopaminergic blocker, and ATP, the cholinergic blocker, failed to have any effect on the EPIinduced eating response. It appears from these results that the feeding elicited by EPI, as well as that produced by NE, is an alpha-receptor phenomenon. The finding that beta-receptor blockade tends to enhance this alpha inducement of feeding suggests that beta receptors may actually have an antagonistic effect on food intake. From the results obtained with the blockers of dopaminergic and cholinergic receptors, it appears that these types of receptors are not directly involved in mediating the induced feeding response. EXPERIMENT 4: ANALYSIS OF RECEPTORS MEDIATING FOOD-ASSOCIATED DRINKING ELICITED BY NE OR EPI This experiment focused on the brief drinking response which is also induced by perifornical hypothalamic injection of NE or EPI. Tests with the receptor blockers were carried out on this food-associated drinking phenomenon, to determine the nature of its mediating receptors. These tests were similar to those conducted in Experiment 3. Four groups of satiated rats were used, each group having 10 to 12 animals with perifornical hypothalamic cannulas. A variety of blockers was tested in combination

MEDIATING ROLE OF ADRENERGIC RECEPTORS IN INGESTION

749

Water intake (ml) in IOmin

TABLE 5

0

EFFECTS OF RECEPTOR BLOCKERS ON DRINKING ELICITED BY NE OR EPI

Group 1

2

3

4

Drug Injections (nmoles) First Second Vehicle

0.0~

-

Vehicle

Agonist (30)

3.2

-

PHT (30)

Agonist (30)

1.4"

-

PHT (60)

Agonist (30)

0.3 ¢

-

/-PROP (100)

Agonist (30)

0.6t

-

d-PROP (100)

Agonist (30)

1.5 *

-

Vehicle

Vehicle

0.0~:

0.2~t

Vehicle

Agonist (30)

3.3

3.8

PHT (60)

Agonist (30)

-

0.8t

LB-46 (140)

Agonist (30)

0.25

0.4¢

Vehicle

Vehicle

0.2t

0.1 ~t

Vehicle

Agonist (30)

2.9

3.2

TOL (100)

Agonist (30)

0.6t

0.4~t

MJ 1999 (400)

Agonist (30)

0.9~"

1.5"

Vehicle Vehicle

Vehicle Agonist (30)

0.0:~ 3.2

0.05 3.6

HAL (3)

Agonist (30)

-

3.7

HAL (9) ATP (6.5)

Agonist (30) Agonist (30)

3.0

2.9 -

Given are the mean scores, N = 10-12 per group. Two-tailed dependent t-tests comparing Vehicle plus Agonist score with each of the other scores: *p<0.05 tP<0.01 Sp<0.001 with NE or EPI (30 nmoles). The organization of each test series, and the drugs and doses used, are presented in Table 5. In contrast to the tests of Experiment 3, the tests here had water but no food. The rats were given water tubes immediately after injection of the agonist or its vehicle, and their water consumption was measured during the next 10 min. Results and Discussion

These receptor-blocker tests demonstrate that the foodassociated drinking response elicited by NE or EPI injection can be antagonized by both alpha- and beta-adrenergic receptor blockers. Specifically, the results (Table 5 and Fig. 3) show that in each group a vigorous drinking response of between 3 and 4 ml was reliably observed during the first 10 min after perifornical hypothalamic injection of NE or EPI. The involvement of alpha receptors in the mediation of this adrenergically elicited drinking is shown by the finding that PHT and TOL, two alpha-receptor antagonists, can totally eliminate the response. Furthermore, the antagonism

2

I

I

3

Saline

Water Intake (ml) NE EPI

Vehicle

I

•A.drenergic agonist (NE or EPI)

Alpha-adrenergic blockers Receptor blockers +

Beto-adrenergic blockers

adrenergic ogonist

Dopaminergic blocker Cholinergic blocker

I ]

FIG. 3. Summary of blocker-agonist results presented in Table 5. imposed by PHT is found to increase with the dose of the blocker. Thus, it appears that elicited drinking, like eating, may require the activity of neuronal receptors which are alpha-adrenergic in nature. In contrast to the eating response, however, the results obtained here with the blockers of beta-adrenergic receptors suggest that this type of receptor may also be involved in mediating the drinking response. Rats pretreated with either LB-46 or MJ 1999 failed to exhibit a reliable response after injection of the adrenergic agonists, l-Propranolol, another beta blocker, also abolished the drinking, although part of this block may be attributed to/-PROP's local anesthetic action. (d-Propranolol reliably suppressed but did not abolish the drinking response. Interestingly, in Experiment 3, d-PROP was not found to have any effect on the feeding response.) There was no evidence to suggest the involvement of dopaminergic or cholinergic (muscarinic) receptors, since pretreatment with HAL and ATP had no effect on the drinking. These findings differentiate the adrenergic drinking response from the angiotensin-induced drinking which can be blocked by HAL [9] and the carbachol-induced drinking which can be blocked by ATP [13,32,41]. In support of the above results obtained with central injection of NE or EPI, there is considerable evidence in the periphery that these adrenergic agonists can act on both alpha and beta receptors [17]. Epinephrine, however, is generally found to be the more potent neurohumoral agent, which is consistent with the finding that hypothalamic EPI is more effective than NE in eliciting food-associated drinking (Experiments 1 and 2). The additional tests of Experiment 1, with drugs which selectively stimulate either alpha or beta receptors, have shown that these particular agents, in contrast to EPI and NE which act on both types of receptors, are ineffective in producing a reliable drinking response, although they are effective in altering feeding behavior (see also reference [22]). These findings, in conjunction with the blocker results of the present experiment,

750

LEIBOWITZ

suggest that combined alpha- and beta-receptor stimulation, as opposed to separate stimulation of either alpha or beta receptors, is a necessary and sufficient condition for producing drinking. EXPERIMENT 5:

4

NE-ELICITED DRINKING FACILITATED BY ISOP

The present experiment was conducted to further examine the hypothesis, generated by Experiment 4, that both alpha and beta receptors are involved in the foodassociated drinking response elicited by adrenergic stimulation of the perifornical hypothalamus. In Experiment 1, it was shown that the potent and relatively pure beta-receptor agonist ISOP, when injected alone, was ineffective in producing the initial drinking observed with NE injection. In view of the evidence of Experiment 4, which indicates the additional involvement of alpha receptors in the elicited response, it seems possible that an effect of ISOP on drinking might be revealed in rats primed with an agonist, such as NE, which provides the necessary alphareceptor stimulation. To test this possibility, 2 groups of 12 satiated rats with perifornical hypothalamic cannulas were tested for the effects of a solution containing both NE and ISOP. The rats in Group 1 received the following 6 treatments in a balanced order: saline, NE (20 nmoles) plus ISOP (80 nmoles) simultaneously injected, NE alone at 20 and 100 nmoles, and ISOP alone at 80 and 100 nmoles. (A total dose of 100 nmoles was chosen on the basis of a dose-response study with NE [30] which revealed the greatest amount of drinking at this particular dose.) The rats in Group 2 received the same drug injections but at different dose levels: saline, NE (50 nmoles) plus ISOP (50 nmoles), NE alone at 50 and 100 nmoles, and ISOP alone at 50 and 100 nmoles. Immediately after injection, the rats were given water (but no food), and their water consumption was measured during the next 10 min.

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t, Dose 0 (nm01es)

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I00

80

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20 + 80

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r-h Dose (nmoles)

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Saline

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50 + 50

/ NE+ISOP

Results and Discussion

The results of this experiment, presented in Fig. 4, demonstrate that the beta agonist ISOP, although ineffective when injected alone, has a pronounced enhancing effect on the NE-elicited drinking response and even assists NE in producing significant drinking in individual rats that were apparently unresponsive to NE alone. These results provide further confirmation that both alpha and beta receptors participate jointly in the adrenergic elicitation of food-associated drinking. When treated with saline or with ISOP alone, the rats in both groups exhibited essentially no drinking during the 10-rain test period. Norepinephrine alone, however, produced a reliable drinking response at each of the doses tested, and in both Group 1 and Group 2, simultaneous injection of ISOP was found to markedly enhance the response elicited by NE. Direct comparisons between ISOP plus NE scores and the NE alone or the ISOP alone scores (at any dose level) revealed reliable differnces (at least at p<0.01). The strength of this enhancing effect of ISOP on drinking became particularly striking when the individual rat scores were examined. Such an analysis revealed that, after injection of NE alone, 38 percent of the rats drank 1 ml or less. However, when ISOP was injected in combination with NE, only 8 percent of the rats drank 1 ml or less.

FIG. 4. Water consumption observed in satiated rats after perifornical hypothalamic injection of saline, norepinephrine (NE) alone, isoproterenol (ISOP) alone, or NE in combination with ISOP. EXPERIMENT 6: ANALYSIS OF RECEPTORS MEDIATING LONG-TERM DRINKING SUPPRESSIONPRODUCED BY NE OR EPI The brief initial drinking response elicited by perifornical hypothalamic adrenergic stimulation (Experiments 4 and 5) has been shown to be followed by a long period (at least 60 min) during which the rats' normal sporadic drinking behavior is totally suppressed [30]. This subsequent suppressive phenomenon is not simply a consequence of the initial drinking, as these effects can be observed independently of each other. The present experiment examined this suppression in an effort to identify the nature of the receptors involved. Two groups of 10 satiated rats, each with a perifornical hypothalamic cannula, were carried through a series of tests similar to those in Experiment 4, except that the test interval was extended to 60 min. In Group 1, the following drugs were tested: the alpha blocker PHT (60 nmoles), the beta blocker LB-46 (100 nmoles), and the dopaminergic

MEDIATING ROLE OF ADRENERGIC RECEPTORS IN INGESTION blocker HAL (20 nmoles), all in combination with the agonist NE (30 nmoles). In Group 2, the alpha blocker TOL (100 nmoles), the beta b l o c k e r / - P R O P (100 nmoles), and the cholinergic blocker ATP (6.5 nmoles) were tested in combination with the agonist EPI (30 nmoles). Immediately after injection of the agonist, the rats were given water, and their consumption was measured every 5 min over the course o f the next 60 min. No food was available during the test.

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GENERAL DISCUSSION The above results show that the agonist EPI can elicit the same pattern of ingestive behaviors induced by NE injection into the perifornical hypothalamus of satiated

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Results and Discussion

As in Experiment 4, both the alpha and the beta blockers were found to abolish the initial adrenergic drinking response. Only the alpha blockers, however, were able to eliminate the subsequent suppression of drinking. The patterns of water consumption observed during the 60-min test interval can be seen in Fig. 5. Both NE and EPI elicited a drinking response (3.2 and 3.5 ml, respectively) during the first 5 min after injection. This 5 rain period of water consumption was then followed by a 55 min period during which the rats exhibited no further drinking. In contrast to this pattern of water consumption seen after adrenergic stimulation, the same rats, when treated with just saline, drank nothing during the first 5 min after injection but then drank a total of 1.7 ml during the remaining 55 min of the test. Comparisons between the first 5 min scores for the agonists and saline revealed a highly reliable drinking response (p<0.001) after injection of either NE or EPI. When the total scores for the subsequent 55 min period were compared, the NE and EPI scores were each found to be significantly lower than the saline scores (at p<0.01). Pretreatment with the adrenergic receptor blockers markedly altered these patterns of water ingestion (Fig. 5). As shown in Experiment 4, both the alpha- and beta-blocking agents entirely eliminated the initial drinking elicited by NE or EPI. However, only the alpha blockers were found to eliminate the subsequent suppression of drinking. When pretreated with the alpha blockers PHT and TOL, the NEor EPI-injected rats drank at their normal level during the entire 60-min test. In contrast, when pretreated with the beta blockers LB-46 or/-PROP, these rats failed to exhibit any drinking at all, either during the first 5 min or the subsequent 55 min. This shows that the long-term suppressive effect of adrenergic stimulation on drinking survives beta-receptor blockade. It also demonstrates that this suppression can occur in the absence of the initial elicited drinking response, which confirms earlier indications that these two effects on water consumption are independent phenomena [30]. Unlike the alpha and beta blockers, the dopaminergic (HAL) and cholinergic (ATP) blockers had no reliable effect on either the initial drinking response or the subsequent long-term suppression of drinking. It appears from these results that, in addition to eliciting food-associated drinking behavior by interacting with alpha and beta receptors, NE and EPI can have a suppressive effect, through alpha-receptor action, on the rat's normal longer-term water ingestion.

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1.5

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A T P + EPI

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o

2

4 r

i

1

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6

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12

Successive 5-min intervcls FIG. 5. Patterns of water consumption observed during the 60-min test period after injection of saline (o), an adrenergic agonist (o) (either norepinephrine [NE] or epinephrine [EPI] ), or a receptor blocker plus agonist combination. The blockers tested in combination with NE (top graph) were the alpha-adrenergic blocker phentolamine (PHT; u), the beta-adrenergic blocker LB-46 (m), and the dopaminergic blocker haloperidol (HAL; a). In combination with EPI (bottom graph) were tested the alpha-adrenergic blocker tolazoline (TOL; n), the beta-adrenergic blocker 1-propranolol (/-PROP; m), and the cholinergic blocker atropine (ATP; zx). rats. This pattern consists of vigorous drinking, which occurs during the first few minutes after injection, followed within 1 to 2 min by vigorous feeding, which lasts approximately 20 min, and by a drinking suppression, which lasts at least 60 min. In previous studies [5, 30, 36], the drinking and feeding responses elicited by NE injection have been found to increase in magnitude with increase in dose of the noradrenergic agonist. In Experiment 2 of the present study, in which the effectiveness of EPI and NE were directly compared, a similar dose-response relationship was revealed for both agonists which, in addition, were found to produce reliable ingestive responses at much lower doses than were previously thought to be effective. For EPI, which was reliably more potent than NE, the threshold doses were 0.8 nmole (0.15 #g) for the induced drinking

752 response and 0.2 nmole (0.04 ug) for the induced feeding response. These doses are approximately 100-fold smaller than have generally been used in studies on ingestive behavior. The finding that such low doses are reliably effective lends support to the suggestion that central adrenergic systems act physiologically to induce drinking and feeding behavior in the rat. This hypothesis is further strengthened by the findings that the ingestive responses elicited by exogenous adrenergic stimulation are specific to the biologically active /-isomer (Experiment 1) and furthermore, that they have striking similarities to the ingestive responses exhibited by laboratory rats under normal conditions [8, 19, 30]. The temporal patterns of the elicited and natural behaviors are similar, and in both cases there exists a fairly constant ratio between the amount of food and the amount of water that a rat ingests at a meal. While the magnitudes of the adrenergic responses clearly depend upon the agonist dose used, they still fall within the bounds of the rat's natural ingestive responses. As demonstrated above, the adrenergic agonists NE and EPI are both found to be effective in eliciting feeding and drinking responses in satiated rats. This evidence with exogenous agonists leads us to the question of which neurohumor, NE or EPI, might be active in mediating the rat's ingestive responses exhibited under normal conditions. Unfortunately, the available evidence does not provide a clear choice. In the present study, both agonists were found to reliably alter ingestive behavior at remarkably low doses, and in histochemical studies [2, 6, 10, 15, 37, 42] both have been found to exist naturally in the rat brain. In fact, the site of injection used in the present study, namely, the perifornical region of the anterior hypothalamus, has been found to be particularly dense with both NE-containing [ 10,34] and EPI-containing [ 15] nerve terminals. The sensitivity of this area to adrenergic stimulation has also been demonstrated in monkeys [44], which, like rats, respond by increasing their food intake. A major focus of the present series of experiments was to determine the nature of the postsynaptic receptors mediating the effects of central adrenergic stimulation on ingestive behavior. With the use of sympathomirnetic stimulants and receptor-blocking drugs, evidence was obtained to support the suggestion that central adrenergic (alpha and/or beta) receptors were critically involved in producing these effects, in contrast to either dopaminergic or cholinergic receptors. Analysis of the vigorous drinking response observed during the first few minutes after NE or EPI injection yielded evidence to suggest that both alpha- and beta-adrenergic receptors participate in the initiation of this behavior (Fig. 6). The nature of the involvement of these two types of receptors appears to be one of synergism, where apparently the combined activation of both receptors is required to produce a demonstrable effect. The basis for this suggestion can be found in the results of Experiment 1 in which selective alpha and beta stimulants were tested, Experiment 4 in which receptor blockers were tested in combination with NE or EPI, and Experiment 5 in which combined injections of ISOP and NE were given. Our finding that adrenergically induced drinking can be antagonized by centrally administered alpha-receptor blockers provides the first evidence that central alpha-adrenergic receptors may be involved in the elicitation of water consumption in the rat. The possibility that central beta recep-

LEIBOWITZ SEQUENCE OF ADRENERGIC AND TYPE OF RECEPTORS

HYPOTHALAMIC ADRENERGIC STIMULATION

EFFECTS INVOLVED

alpha

alpha and beta receptors 1.5 min t ...... pause DRINK

2 rnin pause

for 3 min

receptors EAT

for 20 min

"

~

alpha receptors SUPPRESSIONof DRINKING for 60 min

FIG. 6. Proposed receptor mediation of drinking and feeding effects produced by injection of norepinephrine or epinephrine into the anterior perifornieal hypothalamus of the rat.

tors may be involved in this process has been proposed by Lehr e t al. [21] on the basis of their finding that systemically injected ISOP, through the activation of beta receptors, produces an increase in water consumption. It would appear, however, that the beta receptors involved in mediating this response, or a similar response elicited by central ISOP injection [24,25], are not the same as those involved in mediating the food-associated drinking response elicited by central NE or EPI injection. These two responses are very different, with respect to their latency (ISOP elicits drinking after 5 to 10 min) and duration (ISOP-elicited drinking lasts 2 to 3 hr). Furthermore, the drinking induced by ISOP is associated with a suppression of feeding [ 12, 22, 23, 2 5 ] , in contrast to NE- or EPI-elicited drinking which is associated with enhanced feeding. Finally, there are results demonstrating the dependency of ISOP drinking on the kidneys [16], in contrast to NE- and EPI-elicited drinking which are unaffected by removal of the kidneys ([30] and Leibowitz, unpublished observations). These basic differences between the drinking elicited by ISOP alone and the food-associated drinking elicited by NE or EPI lead us to propose the existence of two separate beta-adrenergic receptor mechanisms for the mediation of these two responses. As described above, the drinking response elicited by perifornical hypothalamic injection of NE or EPI is essentially always followed within a minute or two by a vigorous feeding response which lasts approximately 20 rain (Fig. 6). This feeding response has been the subject of several investigations [5, 14, 40] which, through tests with NE and adrenergic receptor blockers, have obtained evidence suggesting that alpha-adrenergic, but not beta-adrenergic, receptors participate in mediating the response. Experiment 3 of the present study, which tested additional alpha- and beta-adrenergic blockers as well as dopaminergic and cholinergic blockers, confirmed these earlier findings with NE, and in addition provided suggestive evidence that dopaminergic and cholinergic receptors were not directly involved. Further support for the hypothesis that feeding stimulation is specific to alpha-receptor activity was also obtained in Experiment 1 in which the selective alphaadrenergic stimulant MET, but not the selective betaadrenergic stimulant ISOP, was effective in producing an increase in food consumption, and in Experiment 3 in which eating induced by the agonist EPI, like that induced by NE, was blocked only by alpha-receptor antagonists. While it is clear that beta-receptor activity in the rat does

MEDIATING ROLE OF ADRENERGIC RECEPTORS IN INGESTION not enhance feeding behavior (Experiment 1 and references [5,40] ), there is some evidence, obtained in Experiment 3, that it may actually have the opposite effect, that of suppressing the feeding response. The basis for this suggestion is the finding that hypothalamic beta-receptor blockade can potentiate the feeding induced by EPI or NE in satiated rats. (A similar effect was noted by Berger e t al. [3] who used the ventricular route of injection.) This evidence is consistent with results obtained in hungry rats [ 12, 22, 23] showing that hypothalamic beta-adrenergic stimulation can markedly suppress the feeding induced by deprivation. Thus, it appears that in contrast to the food-associated drinking response which involves synergistic action of alpha and beta receptors, the feeding response is found to be antagonistically affected by alpha- and beta-receptor activity. These synergistic and antagonistic effects on behavior of presumed central alpha and beta receptors have their counterpart in peripheral adrenoceptor control of physiological responses [17]. In studies conducted in the periphery, NE and EPI (the latter being generally more potent) have both been shown to act on alpha and beta receptors of the peripheral nervous system. Depending upon the system being investigated, stimulation of these two types of adrenergic receptors has been found to yield either similar or opposite effects. In the control o f intestinal motility, for example, alpha- and beta-receptor stimulation both produce a decrease in activity. Similarly, in their effects on skeletal muscle function or respiratory activity, synergistic facilitation has been demonstrated. Examples of the antagonistic effects of peripheral alphaand beta-receptor stimulation can be found in the vascular system where, respectively, constriction and dilatation are observed, and in the control of urinary bladder activity where contraction and relaxation are produced. In our earlier analyses of the effects produced by central drug administration [30], we found that, as with naturally motivated ingestive responses [ 8,19], adrenergically elicited drinking and feeding were closely associated behaviors, with respect to both time and magnitude of the responses. A possible neurochemical basis for this close association between drinking and feeding behavior is suggested by our receptor-blocker results of Experiments 3 and 4. In these experiments, central alpha-receptor blockade was found to antagonize both adrenergically elicited drinking and adrenergically elicited feeding, thus implicating a similar underlying mechanism. If one assumes that the alpha receptors active during the food-associated drinking response are the same as those involved in eliciting the eating, it would seem possible that this set of alpha receptors might provide the necessary link for coordinating the two ingestive behaviors. This coordinating function, if it indeed occurs, appears to be specific to receptors located at the level of the anterior hypothalamus ( [ 2 9 , 3 0 ] , unpublished observations). This particular brain region is the only area where we have found adrenergic stimulation to elicit both drinking and feeding, in contrast to other areas where adrenergic stimulation

753

elicits feeding without drinking. In light of the evidence that food-associated drinking is antagonized by beta-receptor as well as alpha-receptor blockade, one may conjecture that the presence of beta receptors constitutes the crucial difference between those brain regions which elicit feeding plus drinking and those regions which elicit only feeding. It is important to note, however, that the threshold for eliciting feeding was found in Experiment 2 (and in reference [30] ) to be lower than the threshold for eliciting drinking. This lower threshold could result in a wider area of effective drug diffusion, which in turn would lead to our finding that feeding can be elicited from a larger number of sites than drinking. In addition to the above evidence suggesting a role of central alpha- and beta-adrenergic receptors in the elicitation of ingestive responses, our analyses of a third adrenergic phenomenon, namely, the suppression of drinking, suggest that central alpha-adrenergic receptors may also be involved in the inhibition of an ingestive response. This suppression of water intake, observed in tests with either NE or EPI, becomes apparent immediately after cessation of the initial food-association drinking response (Fig. 6). It lasts for approximately 1 hour, during which time no drinking is observed, in contrast to the sporadic short bursts o f drinking normally exhibited by satiated rats during vehicle control tests. The receptor-blocker studies of Experiment 6 demonstrate that this suppression of drinking can be antagonized by alpha-adrenergic blockers but not by beta-adrenergic, dopaminergic, or cholinergic blockers. These findings in water-satiated rats are in agreement with those obtained in thirsty rats in which an even more dramatic alphaadrenergic inhibition of drinking can be observed [13, 24, 26, 28]. In some preliminary studies in which we tested the adrenergic agonists at low dose levels, we obtained evidence that this alpha-adrenergic suppression of drinking phenomenon can be produced by doses at least as low as 0.005 nmole (0.9 ng) [28]. The effectiveness of these unusually low doses suggests the possibility that central alpha-receptor mechanisms may indeed have a physiological function in controlling (inhibiting) water consumption in the rat. It is possible that the mechanism activated by the exogenous stimulation may, for example, provide a satiety signal for the termination of drinking which normally precedes a meal. Rather than directly inhibiting drinking behavior, however, it is alternatively possible that the alpha-receptor mechanism provides a neurochemical basis for switching, or facilitating a switch, from one response (drinking) to another response (eating). A third possibility is that the suppression of drinking observed with alpha stimulation is simply an indirect consequence of the enhancement of feeding also induced by alpha stimulation. Evidence counter to this latter possibility is provided by the finding [23,27] that the drug amphetamine produces a suppression of drinking, through alpha-receptor activity, in the absence of a feeding-enhancement effect.

REFERENCES

1. Baile, C. A., C. W. Simpson, L. F. Krabill and F. H. Martin. Adrenergic agonists and antagonists and feeding in sheep and cattle. Life Sci. l l : 661-668, 1972. 2. Barchas, J. D., R. D. Ciaranello and A. M. Steinman. Epinephrine formation and metabolism in mammalian brain. Biol. Psychiat. 1: 31-48, 1969.

3. Berger, B. D., C. D. Wise and L. Stein. Norepinephrine: Reversal of anorexia in rats with lateral hypothalamic damage. Science 172: 281-284, 197l. 4. Booth, D. A. Localization of the adrenergic feeding system in the rat diencephalon. Science 158: 515-517, 1967.

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LEIBOWlTZ

5. Booth, D. A. Mechanism of action of norepinephrine in eliciting an eating response on injection into the rat hypothalamus. J. Pharmac. exp. Ther. 160: 336-348, 1968. 6. CiaraneUo, R. D., R. E. Barchas, G. S. Byers, D. W. Stemmle and J. D. Barchas. Enzymatic synthesis of adrenaline in mammalian brain. Nature, Lond. 221: 368-369, 1969. 7. Davis, J. R. and R. E. Keesey. Norepinephrine-induced eating: Its hypothalamic locus and an alternate interpretation of action. J. comp. physiol. Psychol. 77: 3 9 4 - 4 0 2 , 1971. 8. Fitzsimons, J. T. and J. Le Magnen. Eating as a regulatory control of drinking in the rat. Z comp. physiol. Psychol. 67: 273-283, 1969. 9. Fitzsimons, J. T. and P. E. Setler. Catecholaminergic mechanisms in angiotensin-induced drinking. J. Physiol. Lond. 218: 43P-44P, 1971. 10. Fuxe, K. IV. Distribution of monoamine nerve terminals in the central nervous system. Acta physiol, scand. 64: Suppl, 247, 3 7 - 1 0 2 , 1965. 11. Giudicelli, J. F., H. Schmitt and J. R. Boissier. Studies on dl-4-(2-hydroxy-3-isopropylaminopropoxy)-indole (LB-46), a new potent beta-adrenergic blocking drug. J. Pharmac. exp. Ther. 168: 116-126, 1969. 12. Goldman, H. W., D. Lehr and E. Friedman. Antagonistic effects of alpha- and beta-adrenergically coded hypothalamic neurones on consummatory behaviour in the rat. Nature, Lond. 231: 4 5 3 - 4 5 5 , 1971. 13. Grossman, S, P. Direct adrenergic and cholinergic stimulation of hypothalamic mechanisms. Am. J. Physiol. 202: 8 7 2 - 8 8 2 , 1962.

14. Grossman, S. P. Effects of adrenergic and cholinergic blocking agents on hypothalamic mechanisms. Am. Z Physiol. 202: 1230-1236, 1962. 15. H~kfelt, T., K. Fuxe, M. Goldstein and O. Johansson. Immunohistochemical evidence for the existence of adrenaline neurons in the rat brain. Brain Res. 66: 235-251, 1974. 16. Houpt, K. A. and A. N. Epstein. The complete dependence of beta-adrenergic drinking on the renal dipsogen. Physiol. Behav. 7: 897-902, 1971. 17. Innes, I. R. and M. Nickerson. Drugs acting on post-ganglionic adrenergic nerve endings and structures innervated by them (sympathomimetic drugs). In: The Pharmacological Basis o f Therapeutics, edited by L. S. Goodman and A. Gilman, 4th edition. New York: Macmillan, 1970, chapter 24. 18. Jackson, H. M. and D. W. Robinson. Evidence for hypothalamic adrenergic receptors involved in the control of food intake of the pig. Br. Vet. J. 1 2 7 : 5 1 - 5 3 passim, 1971. 19. Kissileff, H. R. Food-associated drinking in the rat. J. comp. physiol. Psychol. 67: 284-300, 1969. 20. KSnig, F. R. and R. A. Klippel. The Rat Brain. A Stereotaxic Atlas. Baltimore: Williams and Wilkins, 1963. 21. Lehr, D., J. Mallow and M. Krukowski. Copious drinking and simultaneous inhibition of urine flow elicited by beta-adrenergic stimulation and contrary effect of alpha-adrenergic stimulation. J. Pharmac. exp. Ther. 158: 150-163, 1967. 22. Leibowitz, S. F. Hypothalamic /3-adrenergic "satiety" system antagonizes an a-adrenergic "hunger" system in the rat. Nature, Lond. 226: 9 6 3 - 9 6 4 , 1970. 23. Leibowitz, S. F. Reciprocal hunger-regulating circuits involving alpha- and beta-adrenergic receptors located, respectively, in the ventromedial and lateral hypothalamus. Proc. natn. Acad. Sci. U. S. A. 67: 1063-1070, 1970.

24. Leibowitz S. F. Hypothalamic alpha- and beta-adrenergic systems regulate both thirst and hunger in the rat. Proc. natn. Acad. ScL U. S. A. 68: 332-334, 1971. 25. Leibowitz S. F. Central adrenergic receptors and the regulation of hunger and thirst. In: Neurotransmitters, edited by I. J. Kopin. Res. Publ. ARNMD, Vol, 50, 1972, pp. 327-358. 26. Leibowitz S. F. Hypothalamic alpha-adrenergic suppression of drinking: Effects on several types of thirst. Proc. 80th a. Convn. Am. psychol. Ass. 1972, pp. 845-846. 27. Leibowitz S. F. Alpha-adrenergic receptors mediate suppression of drinking induced by hypothalamic amphetamine injection. Fedn Proc. 32: 754, 1973. 28. Leibowitz S. F. Adrenergic receptor mechanisms in eating and drinking. In: The Neurosciences: Third Study Program, edited by F. O. Schmitt and F. G. Worden. MIT Press: Cambridge Mass. 1973, pp. 713-719. 29. Leibowitz, S. F. Paraventricular nucleus: A primary site mediating adrenergic feeding-elicitation. Paper presented at meeting of Eastern Psychological Association, Philadelphia, 1974. 30. Leibowitz, S. F. Pattern of drinking and feeding produced by hypothalamic norepinephrine injection in the satiated rat. Physiol. Behav. 14: 7 3 1 - 7 4 2 , 1975. 31. Leibowitz, S. F. Catecholaminergic mechanisms of the lateral hypothalamus: Their role in the mediation of amphetamine anorexia, Brain Research, in press. 32. Levitt, R. A. and A. E. Fisher. Anticholinergic blockade of centrally induced thirst. Science 154: 520-522, 1966. 33. Levy, J. V. Myocardial and local anesthetic actions of 13-adrenergic receptor blocking drugs: Relationship to physiochemical properties. Eur. J. Pharmac. 2: 250-257, 1968. 34. Lindvall, O., A. Bj6rklund, A. Nobin and U. Stenevi. The adrenergic innervation of the rat thalamus as revealed by the glyoxylic acid fluorescence method. J. comp. Neurol. 154: 317-348, 1974. 35. Lonowski, D. J. and R. A. Levitt. Inhibition of chemically elicited feeding and drinking by autonomic blocking agents. Behav. Biol. 8: 251-259, 1973. 36. Miller, N. E., K. S. Gottesman and N. Emery. Dose response to carbachol and norepinephrine in rat hypothalamus. Am. J. Physiol. 206: 1384-1388, 1964. 37. Pohorecky, L. A., M. Zigmond, J. Karten and R. J. Wurtman. Enzymatic conversion of norepinephrine to epinephrine by the brain. J. Pharmac. exp. Ther. 165: 190-195, 1969. 38. Raper, C. and J. Wale. Propranolol, MJ 1999 and CIBA 39089 Ba in ouabain and adrenaline induced cardiac arrhythmias. Eur. J. Pharmac. 4: 1-12, 1968. 39. Singer, G. and J. Kelly. Cholinergic and adrenergic interaction in the hypothalamic control of drinking and eating behavior. Physiol. Behav. 8: 8 8 5 - 8 9 0 , 1972. 40. Slangen, J. L. and N. E. Miller. Pharmacological tests for the function of hypothalamic norepinephrine in eating behavior. Physiol. Behav. 4: 5 4 3 - 5 5 2 , 1969. 41. Stein, L. and J. Seifter. Muscarinic synapses in the hypothalamus. Am. J. Physiol. 202: 7 5 1 - 7 5 6 , 1962. 42. Ungerstedt, U. Stereotaxic mapping of the monoamine pathways in the rat brain. Acta physiol, scand. Suppl. 367: 1-48, 1971. 43. Wolf, G. and J. S. Yen. Improved staining of unembedded brain tissue. Physiol. Behav. 3: 2 0 9 - 2 1 0 , 1968. 44. Yaksh, T. L. and R. D. Myers. Hypothalamic "coding" in the unanesthetized monkey of noradrenergic sites mediating feeding and thermoregulation. Physiol. Behav. 8: 251-257, 1972.