Pattern of drinking and feeding produced by hypothalamic norepinephrine injection in the satiated rat

Physiology and Behavior, Vol. 14, pp. 731-742. Brain Research Publications Inc., 1975. Printed in the U.S.A.

Pattern of Drinking and Feeding Produced by Hypothalamic Norepinephrine Injection in the Satiated Rat' SARAH FRYER LEIBOWlTZ

The Rockefeller University, N e w York N Y 10021

(Received 30 November 1973) LEIBOWlTZ, S. F. Pattern o f drinking and feeding produced by hypothalamic norepinephrine injection in the satiated rat. PHYSIOL. BEHAV. 14(6) 731-742, 1975. - Injection of norepinephrine into the perifornical region of the anterior hypothalamus elicited both drinking and feeding in satiated rats. Analysis of these behaviors revealed the following: (1) Both responses were dose-dependent, occurring at doses at least as low as 0.5 t~g. (2) The drinking response (1-4 ml) had a latency of around 1.5 min and a duration of 2-3 min. It was followed within a minute or two by eating (2-4 g) that lasted approximately 20 rain. It was also followed by a period of drinking suppression that lasted approximately 60 min. (3) Satiation from the ingestion process appeared to be a primary factor in terminating the elicited feeding response, whereas a time-related factor was important in terminating the elicited drinking. (4) These ingestive responses produced by noradrenergic stimulation of the anterior perifornical hypothalamus were found to bear striking similarities to the rat's natural feeding behavior and premeal component of his natural food-associated drinking behavior. (5) These noradrenergically elicited responses could not be observed with lateral hypothalamic stimulation, while only feeding was elicited by ventromedial hypothalamic stimulation. (6) The drinking induced by central noradrenergic stimulation, in contrast to that induced by peripheral beta-adrenergic stimulation, was unaffected by nephrectomy. Drinking behavior

Feeding behavior

Hypothalamus

Norepinephrine

respectively, lateral hypothalamic and hippocampal injection of isoproterenol. With ventromedial hypothalamic injection of the agonist epinephrine, Myers [33] observed a small drinking response in some of the rats tested. Furthermore, Slangen and Miller [35], in their studies on nore p i n e p h r i n e - i n d u c e d feeding, noticed that some rats injected with this agonist drank water before starting to eat. These findings, however, have not been subjected to systematic examination. Studies which have examined the ingestive behavior of rats under normal conditions have found that naturally motivated drinking and feeding are closely associated behaviors [8,16]. In the present series of experiments, these two ingestive responses, and their interrelation, were systematically investigated under conditions of central noradrenergic stimulation. Satiated rats, each implanted with a unilateral brain cannula aimed at one of three hypothalamic sites, were treated with norepinephrine and were then tested with either water alone, food alone, or with both water and food simultaneously present. Experiment 1

IN 1962, Grossman [ 11] found that hypothalamic injection of norepinephrine can elicit eating in satiated rats. This discovery has stimulated a great deal of research focused on the possibility that central adrenergic systems mediate natural hunger. The idea that a physiologically active alphaadrenergic feeding-stimulation system exists in the rat brain is supported by the finding that eating behavior is stimulated by hypothalamic injection of drugs which increase the concentration of endogenous norepinephrine in the synaptic cleft [7,35] and by results showing that the introduction of alpha-receptor blockers leads to a suppression of both norepinephrine-elicited feeding [3, 12, 35] and deprivation-induced feeding [ 12,20]. The possibility that central adrenergic systems play a role in the elicitation of drinking behavior has received somewhat less attention. Lehr et al. [ 18] found that peripheral injection of isoproterenol, a beta-adrenergic agonist, stimulated a prolonged drinking response which could be inhibited by a beta-receptor antagonist. Leibowitz [21] and Mountford [32] observed a similar effect with,

tThis research was supported by NIH research grant MH 13189 and by funds from the Grant Foundation, Hoffmann-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. 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. 731

732

LEIBOWITZ

focused on norepinephrine's effects as a function of dose; Experiment 2 on the duration of the effects; Experiment 3 on their temporal sequence; Experiment 4 on their interrelation; Experiment 5 on their site specificity; and Experiment 6 on a possible peripheral mediating component. The results obtained in these experiments demonstrate that noradrenergic stimulation of the hypothalamus, specifically the anterior perifornical region, can elicit both drinking and feeding in satiated rats and that these behaviors, which are dose-dependent, are similar in many respects to the rat's naturally motivated feeding behavior and pre-meal component of drinking behavior. METHOD

Animals The animals were 111 male albino Sprague-Dawley rats weighing 3 5 0 - 4 0 0 g. 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 food- and water-satiated.

Surgery Each rat was stereotaxically implanted with a chronic unilateral cannula under Nembutal anesthesia. The cannulas were constructed from a 23 g stainless steel hypodermic needle cut to a length of 9.5 mm. The hub of the needle was threaded to receive a protective cap with a stainless steel wire stylette which extended into the cannula and terminated flush with the cannula's tip. The outer cannula was fixed in place on top of the skull with acrylic cement and stainless steel hooks penetrating the bone. Each of the implanted cannulas was aimed at 1 of 3 hypothalamic areas which are known to be particularly responsive to adrenergic stimulation [2, 5, 19, 20, 35, 38] and/or to be involved in the regulation of ingestive behavior [14]. These 3 areas (and their respective coordinates and skull orientation) were: (1) the perifornical region of the anterior hypothalamus (at the frontal level of bregma, 1.3 mm lateral, and 8.2 mm below skull surface, with the top of the incisor bar 3.1 mm above the center of the aural bars); (2) the ventromedial hypothalamus caudal to the perifornical hypothalamic placement (2.5 mm behind bregma, 0.3 mm lateral, and 9.5 mm below the surface of the skull, with bregma height equal to lambda height); and (3) the lateral hypothalamus at the same frontal level as the ventromedial nucleus (2.5 mm behind bregma, 1.7 mm lateral, and 8.5 mm below skull surface, with bregma height equal to lambda height).

General Test Procedure The rats were given at least 1 week of postoperative recovery before testing. During this period, they were frequently handled and mock-injected in order to adapt them to the test procedure. Each rat was then tested in the morning every 2 to 3 days while food- and water-satiated. The rats were given fresh food and water 1 hr before testing, in order to ensure maximal satiation. During this pretest period, each animal was handled and mock-injected at least once, to minimize any spontaneous bursts of drinking and feeding induced by the test procedure. In most cases, the tests were started immediately after drug injection, at which time measured water and/or food was pre-

sented to the rats. 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 drug used in each of the 6 experiments was the noradrenergic agonist l-norepinephrine-d-bitartrate (NE). This agonist was dissolved in sterile physiological saline (0.9 percent) immediately before the start of each test. In most experiments, it was injected directly into the hypothalamus through the implanted cannula, in a volume of 0.5 ul. (Doses of the injections are stated below.) Control tests with the saline (0.9 percent) vehicle alone were given in balanced order (according to a Latin square design) with each NE test. Additional control tests were conducted with solutions adjusted for pH and osmolarity or with solutions containing sodium nitrite or vasopressin (to control for NE's vasomotor effects) or tartaric acid alone (to control for NE's counter ion, bitartrate). Except where indicated, statistical evaluations of the results were based on a two-tailed t-test for dependent means.

Histology To determine the precise placements of the brain cannulas, the rats were sacrificed under Nembutal anesthesia and perfused with saline and then a 10 percent Formalin solution. Frozen sections of 50 u were cut and then stained, using either cresyl violet or a modified Kifiver and Barrera technique [37]. The location of the cannula tip was localized by direct projection of the sections onto the atlas of KSnig and Klippel [ 17]. EXPERIMENT 1: DRINKING AND FEEDING ELICITED BY NE In this experiment, we examined the effects of NE on both food and water consumption at a wide range of doses. For this dose-response study, a group of 21 rats was used, each with a cannula aimed at the perifornical region of the anterior hypothalamus. (This region had been found by Booth [2] to be particularly effective for obtaining an eating response in rats with local administration of NE.) Each rat was injected, according to a Latin square design, with either saline or one of 6 doses (3.1, 6.3, 12.5, 25, 50 or 100 nmoles) of NE. (1.0 nmole of NE is equivalent to 0.17 ~g free base.) At each close level, a given rat received two 60 rain tests, one with only water present and one with only food present. The rats were carefully observed throughout the test interval, and the time course (latency and duration) of each observed behavior was recorded. In addition to the above dose-response study, a series of control tests for pH, osmolarity, NE's bitartrate counter ion, and its vasomotor effects were also conducted. In a group of 10 rats, also with perifornical hypothalamic cannulas, we examined the effects of specific control solutions, each of which was administered in balanced order (according to a Latin square design) with NE (25 nmoles). These control solutions were: (1) the saline vehicle with adjusted pH and osmolarity equal to that of the NE solution; (2) tartaric acid with the same concentration and pH as the NE solution; and (3) the vasodilator sodium nitrite ( 2 0 - 4 0 percent solutions) and the vasoconstrictor vaso-

NOREPINEPHRINE-ELICITED DRINKING AND F E E D I N G

The drinking behavior elicited by NE injection was a vigorous and continuous response, lasting between 1 and 3 min. This response started very soon after injection, its latency decreasing to as low as 1.5 min as the NE dose increased to 100 nmoles. (At this and the lower doses, the rats' individual latency scores generally fell within a relatively short range of between 0.8 and 3.0 min.) Both the magnitude and the duration of the drinking response increased with increasing doses of NE. At the lowest dose (3.1 nmoles), the rats drank 1.1 ml over a 1 min period, while at the highest dose (100 nmoles), they drank 3.6 ml over a 3 min period. During the remaining period (approximately 55 min) of each NE test, no further drinking was observed. The feeding behavior elicited by NE was a vigorous and continuous response which, in comparison with the drinking response, was long-lasting and had a relatively long latency. The feeding response started approximately 5 min (generally between 2.5 and 8.0 min) after injection. Both the magnitude and the duration of this response were found to increase as the dose of NE increased, which confirms the finding of earlier studies [3,31] that also found NE-elicited feeding to be dose-dependent. At a dose of 3.1 nmoles, the rats are 1.8 g in a 10 min period; at 100 nmoles, they ate 4.1 g in a 25 min period. During the remaining period of each NE test (30 to 40 min), no further eating was observed. These patterns of water and food consumption observed during the NE tests contrast markedly with those observed during the saline control tests. When injected with just saline, the rats drank nothing during the first 5 min o f the test (in contrast to 1 - 4 ml after NE) but drank an average of 1.5 ml during the remaining 55 rain (in contrast to 0.0 ml after NE). Direct comparisons between the saline and NE scores for the entire 60 min test revealed a significant

pressin (5 and 10 mU), to test for possible effects of local vasomotor changes.

Results and Discussion The results of the dose-response experiment are presented in Table 1 and Fig. 1. They show that perifornical hypothalamic injection of NE can independently elicit both drinking and eating behavior in satiated rats and that the magnitude and latency of these ingestive responses are dependent upon the dose injected. 4

Foo

.c_ ,E

~

Sotine

I

I

I

I

I

I

5.1

6.5

12.5

25

50

I00

733

Dose of NE in nmoles

FIG. 1. Drinking and feeding elicited by l-norepinephrine (NE) as a function of dose. Norepinephrine was injected into the perifornical region of the anterior hypothalamus of satiated rats. (Based on data presented in Table 1).

TABLE 1 NE-ELICITED DRINKING AND FEEDING AS A FUNCTION OF DOSE

NE Dose (nmoles) 0.0 (saline)

Mean Latency* (min)

-

Water Intake Drinking Response (ml in 5 min) 0.0 +- 0.0

Mean Duration* (min)

-

Mean Latency* (min)

-

Food Intake Feeding Response (g in 60 min)

0.2

±

Mean Duration* (rain)

0.1

3.1

2.2

1.1 ± OAt

1.0

6.5

1.8 ± 0.4~t

10

6.3

2.2

1.4 ± 0.5t

1.6

6.2

2.2 -+ 0.55

13

12.5

2.0

2.1 -+ 0.65

1.8

5.6

3.1 ± 0.5§

21

25

1.6

3.1 ± 0.7§

2.8

4.9

3.3 ± 0.4§

22

50

1.7

3.5 -+ 0.5§

2.9

4.7

3.9 ± 0.6§

26

100

1.5

3.6 ± 0.7§

3.0

5.3

4.1 ± 0.8§

25

Given are the means -+ standard error of the means, N = 21. *Non-responders (rats failing to drink within first 5 rain or to eat within first 15 min) were not included in the means. Two-tailed dependent t-tests comparing saline and NE response scores: tp<0.05 :~p<0.01 §p<0.001

734

LEIBOWlTZ

NE-induced increase in water consumption (at least at p<0.05) at doses above 12.5 nmoles. Direct comparisons between the water intake scores for the first 5 rain of the test (Table 1) showed a significant increase with all doses tested. Very little food consumption (0.2 g) was observed during the saline control test. This contrasts with the 2 - 4 g of food consumed during the first 30 min after NE injection. Direct comparisons between the NE and saline food intake scores (Table 1) demonstrated reliable differences at all doses tested. Results obtained with the different control solutions indicated that NE's effects on ingestive behavior were not caused by NE's bitartrate counter ion, nor by local changes in pH, osmolarity, or vasomotor activities. In the 10 rats tested with these control solutions, the NE bitartrate solution was found to reliably elicit both drinking (3.0 ml during the first 10 min, p<0.001) and eating (3.5 g during first 30 min, p<0.001). Neither response was observed after injection of the saline vehicle appropriately adjusted for pH and osmolarity, nor after injection of the bitartrate solution, sodium nitrite, or vasopressin. With these solutions, the mean water and food intake scores were, respectively, 0.1 ml and 0.3 g. The finding of the dose-response experiment, that a 3.1 nmole dose of NE is reliably effective in eliciting both drinking and feeding, was somewhat unexpected in light of other studies which have reported insignificant feeding at 5.0 nmoles of NE [35] and a relatively poor feeding response at 7.2 nmoles [3]. Most studies on the NE-elicited feeding response have used relatively high doses of NE, between 20 and 100 nmoles. From the present results, however, it appears that such high doses may not be necessary, at least in the brain region tested in this investigation. (See also [34] ). Further analysis of each individual rat's drinking and feeding scores at each of the NE dose levels revealed the following relationship between the two elicited ingestive responses. It was found that at a given dose of NE, there was a positive correlation between the amount of water a rat drank in the water only test and the amount of food he ate in the food only test. This correlation between each rat's water and food intake scores was observed at each dose level, with the lowest correlation (r = +0.46, p<0.05) being observed at 3.1 nmoles and the highest correlation (r = +0.76, p<0.001) being observed at 50 nmoles. E X P E R I M E N T 2: D E T E R M I N A N T S

OF RESPONSE DURATION

Experiment 1 showed that perifornical hypothalamic injection of NE can elicit a brief, short-latency drinking response as well as a more prolonged, longer-latency feeding response. Both responses were found to be vigorous and to continue generally without interruption until approximately 5 min (for drinking) and 30 min (for eating) after injection, at which point they stopped completely. The question arises as to why the rats stopped responding at these particular times. Was it because of a decrease in the effectiveness of NE, or was it a result of some negative feedback arising out of the response itself, perhaps satiation? By introducing delays in the availability of the water or the food, the present experiment examined the processes involved in terminating the noradrenergically induced drinking and feeding. Two series of 4 tests were carried out in a group of 12 satiated rats, each with a perifornical hypothalamic can-

nula. For the first series, only food was made available, and the treatments (given according to a Latin square sequence) consisted of an injection of saline or NE (25 nmoles) followed by a 0 min versus a 30 rain delay in presenting the food to the rat. Food consumption was measured at 60 min after presentation of the lab chow pellets. For the second series of tests, only water was made available, and the treatments here consisted of an injection of saline or NE (25 nmoles) followed by a 0 min versus a 5 min delay in presenting the water to the rats. Water consumption was measured at two points after presentation of the water tubes: at 5 rain, to examine the effects of delay on the initial NE drinking response, and at 60 min, to examine the effects of delay on the rats' overall long-term drinking pattern. Results and Discussion

The results indicate that the ingestion process, perhaps satiation from the ingested food, is a primary factor in terminating the elicited feeding response, whereas some other factor, one which develops as a function of time, is important in terminating drinking. In Table 2 it can be seen that a 30 min delay in presenting food to the rats failed to have any effect on the magnitude of the NE-elicited feeding response. This finding suggests that the cessation of feeding after 20 to 30 min is not due to a decrease in NE's effectiveness over time but rather is due possibly to negative feedback arising from the ingestion process. In contrast to these results on feeding, the results on drinking show that the first 5 min after NE injection is a critical period for the elicitation of water consumption. A 5 min delay in presenting water to the rats caused a significant reduction, from 2.9 to 0.2 ml (p<0.O1), in the amount of water consumed during the first 5 rain after water was available. TABLE 2 NE-ELICITED

FEEDING AND DRINKING AFTER PRESENTING FOOD OR WATER

Food intake (g in 60 min) Water intake (ml in 5 min)

Post-injection Delay (min)

Saline

D E L A Y IN

NE

0

0.3 -+ 0.1

3.3 +- 0.6t

30

0.2 -+ 0.1

3.4 ÷ 0.4 t

0 5

0.0 -+ 0.0 0.0 -+ 0.0

2.9 -+ 0.7* 0.2 ÷ 0.1

Given are the means _+standard error of the means, N = 12. Two-tailed dependent t-tests comparing saline and NE response scores:

*p<0.01 tp<0.001 Analysis of the rats' long-term pattern of water ingestion (over a period of 60 min) has yielded additional results which are illustrated in Fig. 2. As in Experiment 1, the pattern of drinking produced by NE injection under conditions of immediate presentation of water was an initial brief drinking response (lasting approximately 5 min) followed by a long period (lasting 55 min) of essentially no drinking

NOREPINEPHRINE-ELICITED DRINKING AND FEEDING (Fig. 2a). This pattern contrasts with that observed during the saline test, in which the rats drank nothing during the first 5 min but subsequently showed sporadic bursts of drinking which by the end of the 60-min test added up to 1.8 ml. Direct comparisons between the NE and saline scores for water intake revealed a reliable increase (p< 0.01) during the first 5 min after NE injection, followed by a reliable decrease (p<0.01) during the subsequent 55 min period. From the temporal sequence of these effects on water ingestion, it might appear that the subsequent suppression is simply a consequence of supersatiation produced by the initial elicited drinking response. Results obtained in the present experiment, however, under conditions of d e l a y e d presentation of water (Fig. 2b) indicate that the long-term suppression of drinking can occur independently of the initial response and therefore that it may alternatively reflect a direct effect of NE on a central regulatory mechanism. The evidence in support of this possibility is the finding that rats given water 5 min after injection of NE failed to exhibit the brief initial drinking behavior and, despite this lack of initial water intake, they also failed to exhibit, during the remainder of the test, the sporadic drinking which they exhibited following treatment with saline. During the entire 60 min, these rats drank an average of 0.5 ml in the NE test, which contrasts with the 1.7 ml (p<0.05) which they drank in the comparable saline control test. These results demonstrated that after producing an initial drinking response, NE has an independent suppressing effect on the rats' normal longer-term ingestion of water. This suppressive phenomenon, observed here in satiated rats, has also been observed in thirsty rats [ 11, 2 I, 24].

b

o O-rain delay

3o -

5 - m i n deloy

2.0" E

1,5

o c 1.0

I

o

--~

0.5 o/ ~aline 2

NE \ 4 6 8 I0 12

JE] oI ~line

NE 2 4

6 8 I0 12

Successive 5 - m i n inlervols

FIG. 2. Patterns of drinking observed after hypothalamic injection of l-norepinephrine (NE) or saline, under conditions of immediate (a) or delayed (b) presentation of water. Values given represent mean water consumed (in ml) during successive 5 min intervals by 12 rats used in Experiment 2.

735 EXPERIMENT 3: SEQUENCE OF NE-ELICITED INGESTIVE BEHAVIOR

The tests of Experiments 1 and 2 were all carried out with only one goal object, water or food, available at a given time. In this experiment, a series of tests was conducted in which both food and water were present during each test. The purpose of this experiment was to determine the sequence, with free access to food and water, of the two ingestive behaviors elicited by hypothalamic noradrenerglc stimulation. A total of 16 satiated rats, with cannulas aimed at the anterior perifornical hypothalamus, were injected with saline or a moderate dose of NE (25 nmoles) in counterbalanced (ABBA) order. Immediately after injection, the rats were given both food and water, and the magnitude and time course of their ingestive behaviors were recorded for 60 min. Results and Discussion

In the presence of both water and food, perifornical hypothalamic injection of NE reliably elicited a strikingly consistent sequence of independent effects: a short burst of drinking followed by more prolonged feeding and a continuing suppression of drinking (Fig. 3). This response sequence of drinking before feeding is the same as that noted by Slangen and Miller [35]. At a mean of 1.5 min after injection of NE, the rats started to drink from the water tubes. They drank vigorously and continuously (2.1 ml versus 0.0 ml after saline) for the next 2 min, followed by 1 to 2 min of grooming and searching but no ingestive behavior. At a mean of 5.5 min after injection, the rats started to eat the lab chow. The response (3.4 g versus 0.2 g after saline) occurred vigorously and continuously during the next 20 min and then stopped completely. During this feeding period and for the remainder of the test, there was no drinking. This actually amounted to a suppression of 1.7 ml relative to the water these rats consumed during the same period of the saline control test. This pattern of ingestion was observed in all of the rats (N = 13) that exhibited each of the three NE effects. In one rat, the feeding and suppression of drinking effects were obtained in the absence of an initial drinking response, and in the remaining two rats no elicited drinking or feeding was observed. In these tests in which water and food were simultaneously available, the amount of water that a particular rat drank during the first few minutes after NE injection was found to be positively correlated (r = +0.68, p<0.01) w i t h the amount of food which that rat subsequently consumed. This finding is consistent with the results of Experiment 1 in which a positive correlation was found to exist between the amount of water a NE-injected rat consumed in a water only test and the amount of food he consumed in a food only test.. From these results and the results of the preceding experiments, it can be seen that the drinking response elicited by perifornical hypothalamic injection of NE has very specific characteristics, namely, it occurs rapidly after injection, is quite brief, and is closely associated with feeding behavior. These characteristics distinguish it from other drinking responses which have been observed after d r u g administration. Drinking induced by carbachol [11,31] and angiotensin [6] are considerably larger re-

736

LEIBOWlTZ

SEQUENCE OF ADRENERGIC EFFECTS HYPOTHALAMIC NOREPINEPHRINE INJECTION

1 . 5 rain

DRINK .,, for 2 rnin pause ( + 2 . 1 ml)

2 rain • pause

EAT For 20 rain ( + 3 . 2 g)

SUPPRESSION of I DRINKING I for 55 min ( - 1 . 7 ml

FIG. 3. Temporal sequence of effects on rat ingestive behavior induced by perifornical hypothalamic injection ofl-norepinephrine. sponses of between 5 and 15 ml at a high dose range, and they appear to be accompanied by a suppression in feeding [11,30]. The drinking elicited by histamine [13,26] is also a relatively large response, one which appears, if anything to be accompanied by a potentiation of feeding [25]. Finally, the drinking response induced by injection of isoproterenol [18, 21, 23] contrasts dramatically with NEelicited drinking, in that it has a long latency (at least 5 to 10 min), is very prolonged (occurring sporadically over a period as long as 3 hr), and is associated with feeding suppression [ 10, 19, 20, 23]. This isoproterenol-elicited drinking response also appears to involve the kidneys, as it was found to be abolished by nephrectomy [15]. In Experiment 6 of the present study, the drinking elicited by hypothalamic NE injection is shown to be unaffected by removal of the kidneys.

Histological Findings Figure 4 illustrates the anterior perifornical hypothalamic injection sites for the 16 rats used in the present experiment, as well as the 21 rats used in the dose-response study of Experiment 1. (The cannula tips for the remaining 22 perifornical hypothalamic rats of Experiments 1 and 2 were similarly located but were not plotted, for purposes of clarity of presentation.) Figure 5 ( a - c ) presents 3 brain photomicrographs showing the amount of tissue damage that typically occurs at the tip of the implanted cannula. It can be seen from the illustrated findings that the tips of the anterior perifornical hypothalamic cannulas were in most cases located near to the fornix at the frontal level of the paraventricular nucleus (Levels 5340 to 5780 of KSnig and Klippel [17]), 0.8 to 1.3 mm lateral to the midline, and 6.9 to 7.6 mm below the dorsal surface of the brain. In most cases, the fornix appeared to remain relatively or totally intact (Fig. 5a), although frequently being somewhat displaced as shown in Fig. 5b. In a few cases, a nearly total disruption of the fornix was observed (Fig. 5c). To relate these histological findings to the rats' behavioral data, the rats were grouped according to their responsiveness to NE (25 nmoles). They were classified as responders if after NE injection they consistently drank at least 1.0 ml of water, ate at least 1.5 g of food, or did both. On the basis of this classification, 31 of the 37 rats were considered responders (28 met both criteria, 2 only ate significantly, and 1 only drank significantly), and the remaining 6 were considered nonresponders. Histological analysis of these rats' brains (Fig. 4) showed that the cannula tips of all the responders were, with two exceptions located close to the fornix, no more than 0.3 mm away. The exceptions were two cannulas which fell medial to the

FIG. 4. Injection sites illustrated on frontal sections of the K6nig and Klippel atlas [17]. All rats had unilateral cannulas aimed for the perifomical region of the anterior hypothalamus. Filled circles (.) 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 (hl). fornix (by 0.5 mm). These cannulas both belonged to responders. With regard to the points more than 0.5 mm lateral or ventral to the fornix, these belonged to nonresponders. Thus, it appears that the perifornical region at the frontal level of the paraventricular nucleus is indeed an effective site for eliciting eating and drinking with noradrenergic stimulation. This particular brain region has been found to be especially dense with noradrenergic nerve terminals [9,29]. On the basis of the location of the cannula tips belonging to nonresponders, it appears that the effective region for eliciting ingestive behavior may not extend far ventral or lateral, in agreement with the histological results of Slangen and Miller [35]. While the two medial points indicate possible sensitivity in this direction (see [27 and 28]), the lack of points more than 0.5 mm dorsal to the fornix precludes any statement regarding the sensitivity of this brain area.

NOREPINEPHRINE-ELICITED

DRINKING AND FEEDING

737

FIG. 5. Photomicrographs of frontal sections of the rat brain showing representative injection sites (indicated by arrows) for animals with unilateral cannulas aimed for the anterior perifornical hypothalamus (a-c), the ventromedial hypothalamus (d and e), or the lateral hypothalamus (f).

738

LEIBOWlTZ

EXPERIMENT 4: INTERACTION OF NE-ELICITED DRINKING AND FEEDING The results of Experiments 1 and 3 demonstrate that drinking and feeding induced by noradrenergic stimulation are closely associated, both with respect to time and to the magnitude of the responses. To investigate a possible interaction between these two noradrenergically elicited ingestive behaviors, the present experiment examined the latency and magnitude of the responses of NE-injected rats under a one- versus a two-goal object test condition. The two questions addressed here are: (a) does the presence of food influence drinking? and (b) does the presence of water influence feeding? In the preceding experiments, the rats were tested in either a single or a double goal-object condition, but not in both. The present experiment tested a single group of rats (N = 12) under all three conditions of goal object availability, namely, water only, food only, and water plus food. The rats received a total of 6 tests according to a Latin square design. For these tests the rats, each with a perifornical hypothalamic cannula, were treated with saline or NE (25 nmoles) and were given either water, food, or water plus food. The magnitude and latency of the observed behaviors were recorded. Results and Discussion

The results, presented in Table 3, reveal two statistically reliable differences between the single and the double goalobject conditions. First, the presence of food clearly had an inhibitory influence on the magnitude of the NE-elicited drinking response. With food absent, the rats drank 3.2 ml (p<0.001 versus saline score of 0.0 ml) during the first 5 min after injection. However, with food present, they drank only 1.8 ml during this period (p<0.01). One possible explanation for this reliable reduction in drinking in the presence of food is that the physical act of eating interfered with continuation of the drinking response. This does not appear to be the case, however, since with both food and water present the rats generally did not start to eat until 2 min after they had stopped drinking (see Fig. 3). This gap in time between the two responses suggests the intriguing

possibility that an anticipatory stimulus arising out of the sight or smell of food initiated a process which led to the suppression of drinking well before the start of eating. The second interaction occurred in the latency of the feeding response. While the magnitude of the NE-elicited feeding response (over 3 g, significant at p<0.001) remained unaffected by the presence of water (and therefore drinking), its latency relative to time of injection was reliably increased. This observation is interesting in light of the questions some investigators have raised regarding the relatively long latency of NE-elicited feeding. It has often been suggested that this long latency may be due to the time required for the drug to diffuse to the effective site or to be taken up presynaptically and then released to produce eating. While these factors very possibly play a role, the present study suggests that another factor also contributes to this rather long latency. This factor is the interference which results from the initial elicitation of a prior drinking response which delays the feeding response. When the drinking response is prevented from occurring, by having no water available, the latency of the feeding response is reliably reduced. EXPERIMENT 5: NE INJECTION INTO DIFFERENT HYPOTHALAMIC SITES As shown in the first four experiments, the perifornical region of the anterior hypothalamus is reliably responsive to NE injection with regard to both elicited drinking and elicited feeding. To obtain information on the site-specificity of these NE-induced responses, the present experiment tested the effects of NE injection into two additional hypothalamic sites, the ventromedial hypothalamus and the lateral hypothalamus, two areas known to participate in the control of food and water ingestion [ 14]. In this experiment, 2 groups of 12 satiated rats were used, one group with cannulas aimed for the ventromedial hypothalamus and the other with cannulas aimed for the lateral hypothalamus. Both groups were simultaneously given a series of tests in which the effects of NE on both drinking and feeding were examined at a variety of doses. The animals in both groups were each injected with the

TABLE 3 NE-ELICITED DRINKING AND FEEDING AS A FUNCTION OF GOAL OBJECT AVAILABLE

Water intake

Food intake

Goal Object Available

Mean Latency min (Range)

Mean Response ml or g (_+ SEM)

water

1.5 (1.0-2.5)

3.2 -+ 0.6*

water and food

1.4 (0.8-2.4)

1.8 -+ 0.5

food

4.5 (2.0--7.0) t

3.3 _+0.4

water and food

6.4 (4.5-7.8)

3.4 ÷ 0.6

N=12 *p<0.05 compared with water and food response score (two-tailed dependent t-test) tp<0.01 compared with water and food latency score (Wilcoxen matched-pairs, signed-ranks test)

NOREPINEPHRINE-ELICITED DRINKING AND FEEDING

739

TABLE 4 INJECTION OF NE INTO THE VENTROMEDIAL AND LATERAL HYPOTHALAMLIS: EFFECTS ON INGESTIVE BEHAVIOR Ventromedial Hypothalamus Dose (nmoles)

Lateral Hypothalamus

Water Intake*

Food Intaket

Water Intake*

Food Intaket

0.0 -+ 0.0

0.3 -+ 0.1

0.2 -+ 0.I

0.1 -+ 0.1

10

0.0 -+ 0.0

1.7 -+ 0.6~

0.1 -+ 0.1

0.3 -+ 0.2

30

0.5 -+ 0.2

3.1 -+ 1.0§

0.3 +- 0.1

0.0 +- 0.0

90

0.2 -+ 0.1

3.2 -+ 0.9§

0.0 -+ 0.0

0.8 -+ 0.3

0.0 (saline)

Given are the means -+ standard error of the means, N = 12 for each cannula placement. *ml in 5 min ~g in 60 min Two-tailed dependent t-tests comparing saline and NE scores: ~p<0.01 §p<0.001 saline vehicle, or with 10, 30, or 90 nmoles of NE, according to a Latin square design. Immediately after injection, they were given both food and water, and their consumption was measured during the next 60 min. Results and Discussion From the results presented in Table 4, it is apparent that site of injection is indeed an important factor for eliciting drinking and feeding with NE. In contrast to the perifornical hypothalamus where NE injection elicited both responses, the lateral hypothalamus appears to be almost totally unresponsive, whereas the ventromedial hypothalamus responds by eliciting only feeding. The feeding response induced by ventromedial hypothalamic injection of NE was dose-dependent and could be reliably observed at a dose at least as low as 10 nmoles. The lateral hypothalamus, in contrast, failed to respond reliably to a dose as high as 90 nmoles. These findings confirm, at a wider dose range, the results obtained in earlier studies [20, 22, 27] which actually found lateral hypothalamic adrenergic stimulation to produce a suppression of feeding in hungry rats. The drinking elicited by perifornical hypothalamic injection of NE was not observed with injection into either the ventromedial or the lateral hypothalamus. If anything, a decrease in water consumption during the 60 min test was evident. This result indicates that NE-induced drinking, like feeding, is a site-specific p h e n o m e n o n and, furthermore, that these two ingestive responses, which are closely associated in the case of perifornical hypothalamic injection, can become dissociated with ventromedial hypothalamic injection. Histological examination of the brains of the 24 rats used in this experiment (results are presented in Fig. 5 d - f and Fig. 6) showed that the cannulas of these rats were generally located between 1.0 and 1.5 mm caudal to the perifornical hypothalamic placement (Fig. 4 and Fig. 5 a - c ) . All cannula tips were found to be at the frontal levels of 4230 to 4620 according to the atlas of KSnig and

Klippel [17]. The ventromedial hypothalamic cannulas (Fig. 5 d - e ) were located medial to the fornix at the depth of the ventromedial nucleus. Damage to this nucleus appeared to range from slight to perhaps 30 percent, depending upon the position of the cannula. (Figure 5e shows significant damage to the anterior portion of the ventromedial nucleus. Caudal to this site, 60 to 70 percent of the nucleus remained intact.) The lateral hypothalamic cannulas (Fig. 5f) were in all cases located lateral to the fornix, dorsolateral to the ventromedial nucleus.

EXPERIMENT 6: NEPHRECTOMY AND CENTRAL NORADRENERGICALLY ELICITED DRINKING In natural hunger and thirst, several peripheral as well as central physiological mechanisms are known to be activated. The question of peripheral involvement in behavior elicited by central noradrenergic stimulation was recently addressed by Carmona and Slangen [4] who found that, in addition to eliciting feeding, hypothalamically injected NE caused an increase in the acid output of the stomach. With regard to possible peripheral mediation of elicited drinking behavior, it has been shown that water ingestion produced by peripheral beta-adrenergic stimulation involves some mechanism of the kidneys [15]. The present experiment focused on the question of whether the feeding-associated drinking induced by central noradrenergic stimulation similarly involves the kidneys. In this series of tests, the effect of nephrectomy on the elicitation of this response was examined. In addition, the effects of peripherally administered NE were explored. A total of 16 satiated rats was used, each with a cannula aimed at the perifornical hypothalamic region. These rats were first tested with central injections of NE (25 nmoles). After these tests, the rats were given, according to a Latin square design, intraperitoneal injections (0.2 ml) of saline or of 20, 100, or 500 nmoles of NE. Their consumption of water (in the absence of food) was measured during the next 10 min.

740

LEIBOWlTZ

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4580~ I ~

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......

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FIG. 6. Injection sites illustrated on frontal sections of the K6nig and Klippel atlas [17]. Filled circles (e) indicate injection sites for rats with cannulas aimed at the ventromedial hypothalamus, and open circles (o) for cannulas aimed at the lateral hypothalamus. Abbreviations: fornix (F), ventromedial nucleus (hvm), and lateral hypothalamus (hi). For the nephrectomy tests, these rats were then randomly assigned to one of two groups (8 rats per group). On Days 1 and 3 of this test series, they were centrally injected with either physiological saline (Group 1) or with 25 nmoles of NE (Group 2), and their water intake was recorded 10 rain after injection. On the afternoon of Day 2, all of the rats were bilaterally nephrectomized under ether anesthesia. Their kidneys were passed through a paralumbar incision, carefully stripped of their capsules, and removed after ligation of the renal pedicles. Results and Discussion

Peripherally administered NE, at all doses tested, failed to have any effect on water intake during the 10-min test period. The saline baseline score of these satiated rats (0.1

ml) was unaltered by peripheral NE injection, although in the same rats it was reliably enhanced (to 3.4 ml; p<0.001) by perifornical hypothalamic injection of NE (25 nmoles). This result would appear to argue against the possibility that the drinking elicited by central NE injection is mediated through diffusion of the drug into the periphery. The site-specificity obtained in Experiment 5 provides further evidence along these lines. The results of the nephrectomy tests demonstrate that this drinking response induced by central noradrenergic stimulation, unlike the drinking elicited by peripheral isoproterenol injection [15], is not dependent upon the kidneys. Bilateral nephrectomy clearly failed to interfere with the NE-elicited response. Direct comparisons (using the t-test for independent means) between the water intake scores of the saline-injected rats and those of the NEinjected rats revealed a reliable drinking response both before (2.8 ml, p<0.01) and after (3.4 ml, p<0.01) removal of the kidneys. It would appear therefore that the underlying mechanisms for the brief drinking response produced by central noradrenergic stimulation and the quite different (prolonged) drinking response elicited by peripheral betaadrenergic stimulation are not the same. Similar studies into the effects of nephrectomy on the drinking responses induced by other drugs have shown that carbachol- and angiotensin-elicited drinking are also unaffected by this operation [1,15], while histamine-elicited drinking is suppressed by 50 percent [ 13]. GENERAL DISCUSSION The above experiments demonstrate that perifornical hypothalamic injection of NE can independently elicit, in a dose-dependent fashion, both drinking and feeding behavior in satiated rats. Detailed examination of these effects reveals striking similarities, both in magnitude and temporal characteristics, between the responses elicited by central noradrenergic stimulation and the ingestive behaviors exhibited normally by laboratory rats [ 8,16]. In the laboratory, rats usually take between 6 and 10 well-separated meals each day. The size of each meal generally varies between 2 and 3 g. A few minutes before or after each meal, the rats frequently drink a small amount of water, usually between 0.5 and 2.5 ml, but sometimes as much as 5.0 ml. The amount of water which each rat ingests at a meal has been found to be positively correlated with the size of the meal [8]. This drinking behavior, because of its link to eating behavior, has been called "food-associated" drinking [ 16], and it appears to account for at least 70 percent of a rat's water intake under normal conditions. The initial drinking response observed in the present study after perifornical hypothalamic injection of NE is similar in magnitude and duration to the drinking bouts observed in rats under normal food-associated conditions. The amount of water ingested after noradrenergic stimulation usually varied between 1 and 4 ml, while only a few animals drank more than 5 ml. This drinking response lasted 2 to 3 min and was vigorous and continuous throughout this interval. Whenever both food and water were simultaneously available, the drinking elicited by NE was always found to precede, by just a minute or two, the feeding response also induced by this agent. Moreover, the amount of water ingested by individual rats was found to be positively corre-

NOREPINEPHRINE-ELICITED DRINKING AND FEEDING lated with the size of his subsequent meal. This indicates that, as with most drinking bouts observed in the rat under normal conditions, the initial drinking response induced by central noradrenergic stimulation is closely associated with feeding behavior. For this reason, I shall refer to this chemically elicited effect as the food-associated drinking response. With regard to the induced feeding response, the meals triggered by NE were similar in size to the discrete meals which laboratory rats normally take several times a day. After injection o f the noradrenergic agonist, a great majority o f the rats ate between 1 and 4 g, while only a few ate more than 5 g. As with natural eating, the feeding behavior elicited by drug injection was continuous throughout the meal, with only occasional interruptions by short bursts of grooming. After exhibiting the pattern of preprandial drinking described above, NE-injected and normal rats are both found to enter a second phase during which drinking behavior is rarely observed. This period of drinking cessation begins with the initiation of eating and extends throughout the course of the meal. In view of the frequent sporadic bursts of drinking which rats normally exhibit in the absence of food, this lack of prandial drinking might actually reflect a food-related suppression of water consumption. In NE-injected rats, this suppression of drinking appeared to extend beyond the interval of feeding, at least to the end o f the 60-min test. (See Experiments 2 and 3, and discussion below.) In normal rats, drinking frequently resumes within 20 min to an hour after completion o f the meal. The possibility that the NE-injected rats also exhibit this post-prandial drinking response, some time after the 60 min point, will have to be determined in tests longer in duration than those of the present study. The analysis of response duration conducted in Experiment 2 has provided some insight into the possible mechanisms involved in terminating a rat's ingestive responses. Norepinephrine-elicited feeding, which stops well before NE's effectiveness diminishes, appears to be terminated by internal satiety cues resulting from the ingested food or the ingestion process. The similarity between the termination points for NE-elicited feeding and for a normal meal suggests that the satiety mechanism involved in stopping these responses may be similar. With regard to termination of the brief NE-elicited drinking response, the time elapsed following drug administration appears to be a far more critical factor. By approximately 5 min after injection, the point at which drinking normally stops, NE was no longer found to be effective in inducing this behavior. The question is, why in the case of drinking as opposed to eating is the effectiveness o f NE so short-lasting? Since the drug itself is still active as much as 30 min after injection, when it still elicits a strong eating response, it would appear that NE's ineffectiveness in eliciting drinking at 5 min after injection does not reflect a decrease in its potency but rather may reflect the development of an additional factor which exerts an inhibitory effect on NE's inducement of water consumption. This inhibitory influence does not appear to result from the eating response itself since NE-elicited drinking terminates in the absence of food. However, the inhibition may in

741 some way be associated with eating, as suggested by the finding of Experiment 4 that the drinking response can be prematurely terminated simply by the presence (sight or smell) of food. The additional possibility that the drinking elicited by NE injection is terminated by a direct effect of NE on a central mechanism for regulating water consumption is suggested by evidence obtained in Experiments 2 and 3 (see Figs. 2 and 3). In each of these experiments, NE-injected rats were found to be in a state of drinking inhibition immediately after the initial elicited drinking had stopped. This subsequent inhibition of water intake, which lasted for at least 60 min, was not due to supersatiation resulting from the initial ingestion of water, since it could occur i n the absence o f the initial drinking response. Nor was it due to interference from eating, as it was observed long after eating had stopped and could occur in the absence of food. While additional experiments are necessary to determine the relationship between this inhibitory effect of NE on water intake and its stimulatory effects on feeding and food-associated drinking, it is intriguing to speculate that a central neurochemical such as NE not only elicits a particular response but also produces the conditions for terminating this response at the appropriate time. The numerous similarities which appear to exist between the ingestive responses elicited by exogenous NE injection and those observed in normal rats (see above) provide a basis for the hypothesis that a noradrenergic system in the brain has a physiological function in the initiation of both food consumption and food-associated water consumption in the rat. Anatomical evidence provided in this and other studies suggests that, if such a system does indeed exist, its focus o f action may be at the frontal level of the anterior hypothalamus, apparently involving the perifornical region and/or structures medial to the fornix. In the present study, the perifornical hypothalamic region was one o f the sites tested. This site, which in the rat as well as the m o n k e y has previously been described as a particularly sensitive site for eliciting eating [2, 5, 35, 38], and which has been found to be particularly dense with NE=containing nerve terminals [9,29], was found here to be an effective site for producing both the feeding and the food-associated drinking responses. In contrast to the perifornical region of the anterior hypothalamus, the more caudal ventral and lateral hypothalamic placements were found to be considerably less responsive. That is, only feeding could be obtained with ventromedial hypothalamic injection (see also [ 3 6 ] ) a n d neither feeding nor drinking could be obtained with lateral hypothalamic injection. In a more extensive series of localization experiments conducted in the author's laboratory ( [ 2 7 , 2 8 ] , unpublished observations), this medial-lateral differentiation for noradrenergic stimulation of feeding has been confirmed and, in hungry rats, extended to other anterior-posterior levels of the hypothalamus. With regard to the drinking response elicited by noradrenergic stimulation, the evidence once again indicates a strong sensitivity of the medial hypothalamus. In contrast to feeding, however, this sensitivity to elicited drinking appears to be restricted to the frontal level of the anterior hypothalamic region.

742

LEIBOWlTZ

REFERENCES 1. Antelman, S. M., A. K. Johnson and A. E. Fisher. Survival of cholinergic drinking following nephrectomy. Physiol. Behav. 8: 1169-1170, 1972. 2. Booth, D. A. Localization of the adrenergic feeding system in the rat diencephalon. Science 158: 5 1 5 - 5 1 7 , 1967. 3. Booth, D. A. Mechanism of action of norepinephrine in eliciting an eating response on injection into the rat hypothalamus. J. Pharmac. exp. Ther. 160: 3 3 6 - 3 4 8 , 1968. 4. Carmona, A. and J. L. Slangen. Effects of chemical stimulation of the hypothalamus upon gastric secretion. Physiol. Behav. 10: 6 5 7 - 6 6 1 , 1973. 5. Davis, J. R. and R. E. Keesey. Norepinephrine-induced eating: Its hypothalamic locus and an alternate interpretation of action. Z comp. physiol. Psychol. 77: 394-402, 1971. 6. Epstein, A. N., J. T. Fitzsimons and B. Rolls (n6e Simons). Drinking induced by injection of angiotensin into the brain of the rat. J. Physiol., Lond. 210: 4 5 7 - 4 7 4 , 1970. 7. Evetts, K. D., J. T. Fitzsimons and P. E. Setler. Eating caused by 6-hydroxydopamineqnduced release of noradrenaline in the diencephalon of the rat. J. Physiol., Lond. 223: 3 5 - 4 7 , 1972. 8. Fitzsimons, J. T. and J. Le Magnen. Eating as a regulatory control of drinking in the rat. J. comp. physiol. Psychol. 67: 273-283, 1969. 9. 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. 10. 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. 11. Grossman, S. P. Direct adrenergic and cholinergic stimulation of hypothalamic mechanisms. Am. J. Physiol. 202: 8 7 2 - 8 8 2 , 1962. 12. Grossman, S. P. Effects of adrenergic blocking agents on hypothalamic mechanisms. ArrL Z Physiol. 202: 1230-1236, 1962. 13. Gutman, Y. and M. Krausz. Drinking induced by dextran and histamine: Relation to kidneys and renin. Eur. J. Pharmac. 23: 256-263, 1973. 14. Hoebel, B. G. Feeding: Neural control of intake. A. Rev. Physiol. 33: 5 3 3 - 5 6 8 , 1971. 15. Houpt, K. A. and A. N. Epstein. The complete dependence of beta-adrenergic drinking on the renal dipsogen. Physiol. Behav. 7: 8 9 7 - 9 0 2 , 1971. 16. Kissileff, H. R. Food-associated drinking in the rat. J. eomp. physiol. Psychol. 67: 2 8 4 - 3 0 0 , 1969. 17. KiSnig, F. R. and R. A. Klippel. The Rat Brain. A Stereotaxic Atlas. Baltimore: Williams and Wilkins, 1963. 18. 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. Z Pharmac. exp. Ther. 158: 150-163, 1967. 19. Leibowitz, S. F. Hypothalamic #-adrenergic "satiety" system antagonizes an a-adrenergic "hunger" system in the rat. Nature, Lond. 226: 9 6 3 - 9 6 4 , 1970. 20. 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.

21. Leibowitz, S. F. Hypothalamic alpha- and beta-adrenergic systems regulate both thirst and hunger in the rat. Proc. natn. Acad. Sci. U.S.A. 68: 3 3 2 - 3 3 4 , 1971. 22. Leibowitz, S. F. Hypothalamic norepinephrine as an alpha- and beta-adrenergic neurotransmitter active in the regulation of hunger. Proc. 79th a. Convn. Am. psychol. Ass. 1971, pp. 741-742. 23. 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. 24. Leibowitz, S. F. Hypothalamic alpha-adrenergic suppression of drinking: Effects on several types of thirst. Proc. 80th a. Convn. Am. psychol. Asg 1972, pp. 845-846. 25. Leibowitz, S. F. Central histaminergic control of ingestive behavior in the rat. Proc. 81st a. Convn. Am. psychol. Ass. 1973, pp. 1049-1050. 26. Leibowitz, S. F. Histamine: A stimulatory effect on drinking behavior in the rat. Brain Res. 63: 4 4 0 - 4 4 4 , 1973. 27. Leibowitz, S. F. Brain norepinephrine and ingestive behavior. In: Frontiers in Catecholamine Research, edited by E. Usdin and S. Snyder. Oxford: Pergamon Press, 1973, pp. 711-713. 28. Leibowitz, S. F. Paraventricular nucleus: A primary site mediating adrenergic feeding-elicitation. Paper presented at meeting of Eastern Psychological Association, Philadelphia, 1974. 29. 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. 30. McFarland, D. J. and B. Rolls. Suppression of feeding by intracranial injections of angiotensin. Nature, Lond. 2 3 6 : 1 7 2 - 1 7 3 , 1972. 31. 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. 32. Mountford, D. Alterations in drinking following isoproterenol stimulation of hippocampus. Physiologist 12: 309, 1969. 33. Myers, R. D. Modification of drinking patterns by chronic intracranial chemical infusion. In: Thirst, edited by M. J. Wayner. New York: Pergamon Press, 1964, pp. 533-551. 34. Ritter, R. C. and A. N. Epstein. Brain noradrenergic receptors may control meal size. Paper presented at Vth International Conference on Physiology of Food and Fluid Intake, held in Israel, October 1974. 35. Siangen, 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. 36. Wagner, J. W. and J. deGroot. Changes in feeding behavior after intracerebral injections in the rat. Am. Z Physiol. 204: 4 8 3 - 4 8 7 , 1963. 37. Wolf, G. and J. S. Yen. Improved staining of unembedded brain tissue. Physiol. Behav. 3: 209-210, 1968. 38. 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.