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Chronic infusion of norepinephrine and clonidine into the hypothalamic paraventricular nucleus
0361-9230184 $3.00 + .OO
Brain Research BuNetin, Vol. 13, pp. 591-595, 1984. 0 Ankho International Inc. Printed in the U.S.A.
BRIEF COMMUNICATION
Chronic Infusion of Norepinephrine and Clonidine into the Hypothalamic Paraventricular Nucleus SAUL S. LICHTENSTEIN, AND SARAH The Rockefeller
CONSTANTIN F. LEIBOWITZ’
MARINESCU
University, New York, NY 10021
Received
11 June 1984
LICHTENSTEIN, S. S., C. MARINESCU AND S. F. LEIBOWITZ. Chronic infusion ofnorepinephrine and clonidine into rhe hypothalamic pamventricular nucleus. BRAIN RES BULL 13(4) 591-595, 1984.-Previous experiments have shown that acute injection of NE and CLON into the PVN initiates a short-term feeding response in satiated rats. This study examined, in brain-cannulated rats, the impact of remote, chronic injections of NE, CLON, or saline on daily food intake and body weight gain. Over a period of 14 days, NE was infused into the PVN, either continuously at a rate of 12 nmipllhr, or discretely at a rate of 6 nm/@ec. In addition, the cY,-adrenergic agonist CLON was infused into the PVN discretely at a rate of 3 nm/0.5 ~1130sec. Relative to saline infusion, chronic (continuous or discrete) stimulation of the PVN with either of these drugs was effective in potentiatingdaily food intake by 12-l% and in increasing body weight gain, from approximately 1.5 g/day to 3.3 g/day. This evidence indicates that medial hypothalamic NE, especially within the PVN, is sufficiently robust to alter long-term feeding patterns and body weight regulation. Feeding behavior Hypothalamus Chronic drug infusion Body weight
Paraventricular nucleus Drinking behavior
EVIDENCE has accumulated over the past decade to suggest that a-noradrenergic stimulation of the medial hypothalamus, in particular the paraventricular nucleus (PVN), is effective in potentiating eating behavior [4,11]. This phenomenon has been demonstrated numerous times with acute hypothalamic injections of I-norepinephrine (NE) and of other cy-noradrenergic, in contrast to /3-adrenergic, agents [2, 7, 20, 271. Recent studies with the selective on-noradrenergic agonist clonidine (CLON) have also substantiated the sensitivity of the PVN to injection of this agonist and have demonstrated that the cr,-type receptor, in contrast to (Y,, is specifically involved in mediating the eating response to NE and CLON [15,17]. Several biochemical studies have revealed site-specific changes in endogenous NE levels and turnover, particularly within the medial hypothalamus, in response to food deprivation [4, 16, 23, 241. This evidence greatly strengthens the hypothesis that noradrenergic innervation of this brain area has a physiological role in controlling normal eating behavior. Further support for this possibility is provided by a recent study that has demonstrated a significant increase in daily food intake with repeated manual injections of NE into the PVN [ 141. With 4-7 injections given over an 8-hr period, into rats maintained on ad lib or restricted feeding schedules,
Norepinephrine
Clonidine
has revealed a reliable enhancement of food intake, meal size, and body weight. Although these results clearly indicate that PVN a,-noradrenergic stimulation is sufficiently robust to override normal food intake and body weight regulation, the procedures utilized have several limitations which preclude a full appreciation of this study’s potential for identifying a noradrenergic regulatory mechanism. The primary limitations are that the animals, and consequently their eating patterns were periodically disturbed with repeated manual injections; they received NE injections only during daytime hours; and their sequence of repeated injections lasted only for a total of 5 consecutive days. In addition to establishing a possible function of hypothalamic NE in regulating normal feeding behavior, the demonstration of a significant behavioral change with chronic NE infusion may help us to understand possible neurochemical substrates of human eating disorders. The present study advanced our initial efforts with repeated NE administration, by utilizing an infusion pump set-up which permitted us to administer NE remotely, either continuously or discretely, over a full 24-hr period and for a period of several weeks. The results of this study, presented in preliminary form at the Society for Neuroscience [ 111, demonstrate once again that chronic NE infusion into the PVN reliably enhances
this study
‘Requests for reprints should be addressed to S. F. Leibowitz.
591
food intake and body weight, over a period of at leas1 14 days. without any evidence for tolerance. A similar effect is produced by infusion of the ~~-noradrenergic agonist CLON into the PVN. Three experiments were conducted, utilizing IX male albino Sprague-Dawley rats (Charles River Labs). initially weighing 3.50-400 g. The rats were maintained ad lib and tested on a milk-mash diet consisting of lab chow powder mixed with sweetened condensed milk and then shaped into a ball for easy access. A 12: 12 light-dark cycle, with lights on at 7:30 a.m. was used throughout these experiments. Animals were housed individually during the experiments in wire-mesh cages covered by open-top wooden infusion chambers, described by Myers [l9], which were approximately 30 inches in height. At the start of the experiments. each rat. while under pentobarbital anesthesia, was stereotaxically implanted with a chronic Khavari-type cannula-swivel assembly 151, aimed at the dorsal surface of the PVN and cemented permanently to skull surface. The coordinates used were: 0.2 mm caudal to Bregma, 0.4 mm lateral to midline, and 8.2 mm ventral to skull surface, with the tooth bar placed 3.0 mm dorsal to intraaural line. The drugs and saline vehicle (with ascorbic acid) were chronically infused directly into the PVN with a Hamilton gastight IOO-~1syringe mounted onto a Sage Compact Infusion Pump (Model 355), according to the procedures described hy Myers 1191. The fluids were infused either continuously, OI discretely with the help of a variable speed solid-state timer which controlled the functioning of the pump. For continuous infusion, the pump was set to deliver the solution steadily at a rate of 1 ~I160 min. For discrete infusion, the timer activated the pump every 30 min, at which time it was set to deliver a 0.5~1 solution over a 30-set interval. Thus, the volume of solution delivered over a 24-hr period (approximately 24 ~1) was similar for all experiments. Approximately 1 week after stereotaxic surgery, the animals were placed in their testing cages and permitted to adapt to the infusion procedure for 7-10 days. During this period, they were infused with saline, and daily food intake and body weight measures were taken every afternoon between 3:00 and 5:OOp.m. The actual infusion experiment was initiated when stable measures were obtained. Body weights at this time ranged between 400 and 500 g. Both ~-norepinephrine-~~-bita~rate (Sigma) and clonidine HCI (Boehringer Ingelheim, Ltd.) were dissolved in a 0.04% ascorbic acid-sterile physiological saline vehicle, in concentrations of 10 nmolesipl for NE and 6 nmolesipl for CLON. Two groups of rats were tested with NE; one group (n--6) received a W~U~~WOI~.S infusion of NE, whereas the other (n=6) received a dis~~tr infusion of NE. In both cases, 10 nmoles of NE were infused into the PVN each hour, providing a total daily dose of 240 nmoles. The third group was infused discretely with CLON, 6 nmolesi60 min, or a total daily dose of 144 nmoles. Each rat received two ICday infusion sequences, one with NE or CLON and one with the saline (ascorbic acid) vehicle. (Routine tests comparing results obtained with vehicle versus no vehicle infusion have shown no effect of this ascorbic acid vehicle on food intake or body weight.) Half the rats received the drug sequence first, and the other half received the saline sequence first. A 2-3 day adjustment period separated the two 14-day sequences. Between 3:00 and 5:00 p.m. each day, all drug solutions were changed. Measurements of food and water intake, as well as of body weight, were taken, and then fresh diet and water were supplied. After completing their drug-
4~_
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NOREPINEPHRINE I
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NOREPINEPHRINE (Continuous, IOnm/60 mid
1.2
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10
5
15
DAYS FIG. I. The effect of chronic continuous infusion ofnorepinephrine (NE) into the PVN, on total daily food intake and body weight gain. Rat\ (n=6) were maintained ad lib on a milk-mash diet. Norepinephrine or its saline (ascorbic acid) vehicle was continuously infused at a rate of 10 nmihr. over the 14 days of PVN infusion. NE significantly increased daily food intake by IF{ (~~iO.(KfIt. This effect was associated with a significant enhancement of daily body weight gain (p~O.005).
saline or saline-drug sequences, lasting 30-35 days, the rats were sacrificed, their brains removed and histologicaIly analyzed and, in all cases, the placement of the cannula tip was confirmed to be within the vicinity of the PVN. The daily food intake, water intake, and body weight measures were analyzed using two-factor analyses of variance. Continous (Fig. I) as well as discrete (Figs. 2) infusion of NE into the PVN of rats fed ad lib produced a reliable increase in daily food intake and body weight gain. These results were replicated with discrete infusion of the c+noradrenergic agonist CLON (Fig. 3), supporting recent f’indings of a stimulatory feeding effect of this drug. similar to that of NE, after acute PVN injection [15,17f. Daily food intake with continuous NE was increased by approximately 12%, F(1,5)=43.36, p
CHRONIC
INFUSION
OF NE AND CLON
593
NOREPINEPHRINE (Discrete. 5nrMDminl
CLONIDINE (Discrete. 3nm0Omin) Meon Doily Food lntoke (g) 38.2
32.1 L
25
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I
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1
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1
1 5
1
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I
60SALINE
1 5Os
Mean Weight Goin (g/day)
NOREPINEPHRINE (Discrete, 5nmI3Omin)
3.5
Z$ 40W !z ”
30-
g
1.7
$ 20ED W
L
IO
5
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0
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-10 1 0
-IO 0
DAYS
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1
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1 15
DAYS
FIG. 2. Stimulation of feeding and body weight gain produced by chronic discrete infusion of norepinephrine (NE) into the PVN of rats (n=5) maintained ad lib on a milk-mash diet. Norepinephrine or saline vehicle was infused twice each hour at a rate of 5 nm130 set for 14 days. Over this period, both daily food intake cl%, p
period, as reflected by an insignificant drug x day interaction. Stimulatory effects on body weight were also observed with chronic PVN infusion of NE and CLON (Figs. l-3). Under drug as well as saline vehicle conditions, a steady and significant growth in body weight for all rats was observed over the 14-day sequence, F(1,470)=52.62, p
FIG. 3. Reliable increase in food intake (12%, p
gained between 1.2 and 1.7 g/day, this rate increased substantially to 3.1-3.5 g/day with chronic infusion of either NE or CLON. This effect was statistically reliable whether the raw scores for body weight or the derived body weight gain values (Figs. l-3) were analyzed. This enhancement of body weight occurred consistently over the entire 1Cday infusion period. Water intake measurements recorded during the discrete infusion experiments with NE and CLON revealed a small increase, F( 1,4)= 11.47, pcO.028, with NE infusion (41.2k4.1 ml) relative to saline infusion (36.823.6 ml). This effect was not observed in the case of CLON (28.Ok2.1 ml) compared with saline (31.0a 1.1 ml), F(1,3)= 1.87,~>0.10. In both cases, daily water intake, during either saline or drug infusion, remained relatively stable over the 14-day sequence of infusion. The enhancement of daily food intake and the small changes in water intake observed with both noradrenergic drugs were generally associated with an enhanced food/water ratio, which was statistically significant @<0.005) in the case of CLON.
594
These results, showing a stimulatory effect of chronic NE and CLON on daily food intake and body weight gain, confirm the sensitivity of the PVN to ff~-noradrenerg~c stimulation j&9] and reveal the power of noradrenergic control and the robustness of its response. They are consistent with a recent study, which examined the effect of repeated manual injections of NE into the PVN, and demonstrated, over a one-day or a five-day period, a reliable enhancement of 24-hr food intake and body weight gain 1141. In that study, NE was injected four times daiiy at a relatively high dose of 20 nmoles per injection, and the injections were given every other hour during daytime hours. Under these manual conditions, the magnitude of the NE effect, an increase in food intake of g-12 g, was somewhat larger than the 4 g increase observed here in these studies may be due to the difference in length of these tests (l-5 days versus 14 days), dosage, or mode of injection (manual versus remote). Given the greater number of test days in the present infusion experiment, it is possible that tolerance may have developed, consequently reducing NE’s effectiveness. This does not appear to be the case, however, since NE proved to be equally effective throughout the entire ICday sequence. These results obtained with chronic NE and CLON infused into the PVN are in agreement with the findings of Schelmmer ef crl. 1211, showing consistent overeating and obesity with chronic peripheral CLON administration in the monkey. It is interesting to note that, in humans, increased body weight has been observed as a side effect of CLON administration for treatment of hypertension (see Physicians’ Desk Reference).
The finding that NE is sufficiently robust to aher Icmp~ term feeding patterns and body weight regulation is con+ tent with the hypothesis that endogenous NE in the PVN has a physiological function in the control of normal feeding pat-. terns (e.g., [X. 10. 14, 161). Further supporting evidence i\ provided by the findings that: (a) PVN-injected NE is effective in altering feeding at near-physiological do$es 19. 10. XI]: (b) The meal pattern observed with PVN NE in.iection closely resembles that of spontaneous feeding ]hj; (CI in freely-feeding rats, exogenous NE is selective in it\ effect vn the maintenance rather than the initiation of’ feeding 1 I, 14, 201; (d) Electrolytic lesions of the PVN, the area most sensi.. tive to NE stimulation, significantly enhance daily food intake and body weight gain. suggesting that exogenous NE may act similarly, through its inhibitory effect on PVN “\;rtiety” neurons [12-141; tef Under food-depriv~lti~)n condi-
tions, significant alterations occur in endogenous NE and in a-adrenergic receptor activity, specficiaily within the PVN [4]; (f) At the onset of a rat’s eating bout, an increase in the efflux of W-labeled NE occurs in the periventricular area of the hypothalamus [ 161; (g) lntragastric infusion elf carbo hydrate nutrients. which normally induces satiety. is azso. ciated with an inhibitio~l of NE release. specifically within the PVN 1181. ACKNOWLEDGEMBN'lS
This research was supported by grant MH 22X79 and from the Whitehall Foundation, We thank Paul Roos\in assistance
in setting up the infusion
bq fun&, for hi\
timer
REFERENCES 1. Grinkler, J., C. Marinescu and S. F. Leibowitz. Effects of central injections of neurotransmitters on drugs on freely feeding rats. Sot Nearosci Abstr 8: 604, 1982. 2. Grossman, S. P. Direct adrenergic and cholinergic stimulation of hy~th~~~c mechanisms. Am J Physiol202: 872-8821962. 3. Hoebel, B. G. and S. F. Leibowitz. Brain monoamines in the modulation of self-stimulation, feeding, and body weight. In: Brain, Behavior, and Bodily Disease, edited by H. Weiner, M. A. Hofer and A. J. Stunkard. New York: Raven Press, 1981, pp. 103-142. 4. Jhanwar-Uniyal, M., F. Fleischer, B. E. Levin and S. F. Leibowitz. Impact of food deprivation on hypothalamic a-adrenergic receptor activity and norepinephrine (NE) turnover in rat brain. Sot Neurosci Abstr 8: 711, 1982. 5. Khavari, K. A. Chemical microinjections into brain of freemoving small laboratory animals. Physio/ Behav 5: I 187-I 189, 1970. 6. Leibowitz, S. F. Pattern of drinking and feeding produced by hypothalamic norepineph~ne injection in the satiated rat. Physiof Behav 14: 731-742, 1975. 7. Leibowitz, S. F. Ingestion in the satiated rat: Role of alpha and beta receptors in mediating effects of hypothalamic adrenergic stimulation. Physiol Behav 14: 743-754, 1975. 8. Leibowitz, S. F. Paraventricular nucleus: A primary site mediating adrenergic stimulation of feeding and drinking. P~la~rna~of Biochem Behav 8: 163-175, 1978. 9. Leibowitz, S. F. Adrenergic stimulation of the p~avent~cular nucleus and its effects on ingestive behavior as a function of the drug dose and time of ingestion in the light-dark cycle. Bruin Res Bull 3: 357-363, 1978. 10. Leibowitz, S. F. Neurochemical systems of the hypothalamus: Control of feeding and drinking behavior and water-electrolyte excretion. In: Handbook ofthe Hypothalamus, ~013, Part A, Behavioral Studies of the Hypotha~am~~.~,edited by P. J. Morgane and J. Panksepp. New York: Marcel Dekker, 1980, pp. 299-437.
1 I. Leibowitz, S. F., C. Marinescu and S. Lichtenstein. Continuous and phasic infusion of norepinephrine (NE) into the hypothalamic paraventricular nucleus (PVN) increases daily food intake and body weight in rat. Srtr .~~~~f~(~s~i Abstr 8: 71 I. 1982. 12. Leibowitz, S. F., N. J. Hammer and K. Chang. Hy~tbalam~~ paraventricular nucleus lesions produce overeating~and obesity in the rat. Phvsiol Behav 27: 1031-1040. 1981. 13. Leibowitz, S:F., N. J. Hammer and K. Chang. Feeding behavior induced by central norepinephrine injection is attenuated by discrete lesions in the hypothalamic paraventricular nucleus. Pharmacol Biochem Behat 1% 945-950. 1983. 14 Leibowitz, S. F., P. Roossin and M. Rosenn. Chronic norepinephrine injection into the hypothalamic paraventricular nucleus produces hyperphagia and increased body weight in the rat. Pharmacol Biorhem Behau 21: 801-808, 1984. 15 Marino, L. A., M. D. De Bellis and S. F. LeibowitL. Lu,-Adrenergic receptors in the paraventricular nucleus mediate feeding induced by norepinephrine and clonidine. Sor, Nrarr>rri Abstr 9: 467, 1983. 16, Martin, G. E. and R. 0. Myers. Evoked release of [‘*C]norepinephrine from the rat hypothalamus during feeding. Am J Physiol 229: 1547-1555, 1975. McCabe, J. ‘I., M. De Bellis and S. F. Leibowitz. Clonidineinduced feeding: Analysis of central sites of action and fiber projections mediating this response. Brain Res. in press, 1984. MeCaleb, M. L., R. D. Myers, G. Singer and G. Wiliis. Hypothaiamic norepinephrine in the rat during feeding and push-~11 perfusion with gbtcose, 2-M;. or insulin. Am J Phvsiol 236: 312-321, 1978. Myers, R. D. Chronic methods: Intraventricular infusion, cerebrospinal fluid sampiing, and push-pull perfusion. In: Methods in Psychobiology. vol 3, edited by R. D. Myers. New York: Academic Press, 1977, pp. 281-315.
CHRONIC
INFUSION
OF NE AND CLON
20. Ritter, R. C. and A. N. Epstein. Control of meal size by central noradrenergic action. Proc Natl Acad Sci USA 72: 2740-3743, 1975. 21. Schlemmer, R. I?., R. C. Casper, N. Narasimhachari and J. M. Davis. Clonidine-induced hyperphagia and weight gain in monkeys. Psychophurmu~~~logy {Eerlin) 61: 233-234, 1979. 22. Slangen, J. I.,. and N. E. Miller. Pha~acolo~~al tests for the function of hypothalamic norepineprhine in eating beahvior. Physioi Behav 4: 543-552, 1969.
595
23. Stachowiak, M., J. Bialowas and M. Jurkowski. Catecholamines in some hypothalamic and telencephalic nuclei of fooddeprived rats. Acta Neurobiol Exp 38: 157-165, 1978. 24. Van der Gugten, J. and J. L. Slangen. Release of endogenous catecholamines from rat hypothalamus in vivo related to feeding and other behaviors. Pharmacof Biochem Behav 7: 211-219, 1977.
Brain Research BuNetin, Vol. 13, pp. 591-595, 1984. 0 Ankho International Inc. Printed in the U.S.A.
BRIEF COMMUNICATION
Chronic Infusion of Norepinephrine and Clonidine into the Hypothalamic Paraventricular Nucleus SAUL S. LICHTENSTEIN, AND SARAH The Rockefeller
CONSTANTIN F. LEIBOWITZ’
MARINESCU
University, New York, NY 10021
Received
11 June 1984
LICHTENSTEIN, S. S., C. MARINESCU AND S. F. LEIBOWITZ. Chronic infusion ofnorepinephrine and clonidine into rhe hypothalamic pamventricular nucleus. BRAIN RES BULL 13(4) 591-595, 1984.-Previous experiments have shown that acute injection of NE and CLON into the PVN initiates a short-term feeding response in satiated rats. This study examined, in brain-cannulated rats, the impact of remote, chronic injections of NE, CLON, or saline on daily food intake and body weight gain. Over a period of 14 days, NE was infused into the PVN, either continuously at a rate of 12 nmipllhr, or discretely at a rate of 6 nm/@ec. In addition, the cY,-adrenergic agonist CLON was infused into the PVN discretely at a rate of 3 nm/0.5 ~1130sec. Relative to saline infusion, chronic (continuous or discrete) stimulation of the PVN with either of these drugs was effective in potentiatingdaily food intake by 12-l% and in increasing body weight gain, from approximately 1.5 g/day to 3.3 g/day. This evidence indicates that medial hypothalamic NE, especially within the PVN, is sufficiently robust to alter long-term feeding patterns and body weight regulation. Feeding behavior Hypothalamus Chronic drug infusion Body weight
Paraventricular nucleus Drinking behavior
EVIDENCE has accumulated over the past decade to suggest that a-noradrenergic stimulation of the medial hypothalamus, in particular the paraventricular nucleus (PVN), is effective in potentiating eating behavior [4,11]. This phenomenon has been demonstrated numerous times with acute hypothalamic injections of I-norepinephrine (NE) and of other cy-noradrenergic, in contrast to /3-adrenergic, agents [2, 7, 20, 271. Recent studies with the selective on-noradrenergic agonist clonidine (CLON) have also substantiated the sensitivity of the PVN to injection of this agonist and have demonstrated that the cr,-type receptor, in contrast to (Y,, is specifically involved in mediating the eating response to NE and CLON [15,17]. Several biochemical studies have revealed site-specific changes in endogenous NE levels and turnover, particularly within the medial hypothalamus, in response to food deprivation [4, 16, 23, 241. This evidence greatly strengthens the hypothesis that noradrenergic innervation of this brain area has a physiological role in controlling normal eating behavior. Further support for this possibility is provided by a recent study that has demonstrated a significant increase in daily food intake with repeated manual injections of NE into the PVN [ 141. With 4-7 injections given over an 8-hr period, into rats maintained on ad lib or restricted feeding schedules,
Norepinephrine
Clonidine
has revealed a reliable enhancement of food intake, meal size, and body weight. Although these results clearly indicate that PVN a,-noradrenergic stimulation is sufficiently robust to override normal food intake and body weight regulation, the procedures utilized have several limitations which preclude a full appreciation of this study’s potential for identifying a noradrenergic regulatory mechanism. The primary limitations are that the animals, and consequently their eating patterns were periodically disturbed with repeated manual injections; they received NE injections only during daytime hours; and their sequence of repeated injections lasted only for a total of 5 consecutive days. In addition to establishing a possible function of hypothalamic NE in regulating normal feeding behavior, the demonstration of a significant behavioral change with chronic NE infusion may help us to understand possible neurochemical substrates of human eating disorders. The present study advanced our initial efforts with repeated NE administration, by utilizing an infusion pump set-up which permitted us to administer NE remotely, either continuously or discretely, over a full 24-hr period and for a period of several weeks. The results of this study, presented in preliminary form at the Society for Neuroscience [ 111, demonstrate once again that chronic NE infusion into the PVN reliably enhances
this study
‘Requests for reprints should be addressed to S. F. Leibowitz.
591
food intake and body weight, over a period of at leas1 14 days. without any evidence for tolerance. A similar effect is produced by infusion of the ~~-noradrenergic agonist CLON into the PVN. Three experiments were conducted, utilizing IX male albino Sprague-Dawley rats (Charles River Labs). initially weighing 3.50-400 g. The rats were maintained ad lib and tested on a milk-mash diet consisting of lab chow powder mixed with sweetened condensed milk and then shaped into a ball for easy access. A 12: 12 light-dark cycle, with lights on at 7:30 a.m. was used throughout these experiments. Animals were housed individually during the experiments in wire-mesh cages covered by open-top wooden infusion chambers, described by Myers [l9], which were approximately 30 inches in height. At the start of the experiments. each rat. while under pentobarbital anesthesia, was stereotaxically implanted with a chronic Khavari-type cannula-swivel assembly 151, aimed at the dorsal surface of the PVN and cemented permanently to skull surface. The coordinates used were: 0.2 mm caudal to Bregma, 0.4 mm lateral to midline, and 8.2 mm ventral to skull surface, with the tooth bar placed 3.0 mm dorsal to intraaural line. The drugs and saline vehicle (with ascorbic acid) were chronically infused directly into the PVN with a Hamilton gastight IOO-~1syringe mounted onto a Sage Compact Infusion Pump (Model 355), according to the procedures described hy Myers 1191. The fluids were infused either continuously, OI discretely with the help of a variable speed solid-state timer which controlled the functioning of the pump. For continuous infusion, the pump was set to deliver the solution steadily at a rate of 1 ~I160 min. For discrete infusion, the timer activated the pump every 30 min, at which time it was set to deliver a 0.5~1 solution over a 30-set interval. Thus, the volume of solution delivered over a 24-hr period (approximately 24 ~1) was similar for all experiments. Approximately 1 week after stereotaxic surgery, the animals were placed in their testing cages and permitted to adapt to the infusion procedure for 7-10 days. During this period, they were infused with saline, and daily food intake and body weight measures were taken every afternoon between 3:00 and 5:OOp.m. The actual infusion experiment was initiated when stable measures were obtained. Body weights at this time ranged between 400 and 500 g. Both ~-norepinephrine-~~-bita~rate (Sigma) and clonidine HCI (Boehringer Ingelheim, Ltd.) were dissolved in a 0.04% ascorbic acid-sterile physiological saline vehicle, in concentrations of 10 nmolesipl for NE and 6 nmolesipl for CLON. Two groups of rats were tested with NE; one group (n--6) received a W~U~~WOI~.S infusion of NE, whereas the other (n=6) received a dis~~tr infusion of NE. In both cases, 10 nmoles of NE were infused into the PVN each hour, providing a total daily dose of 240 nmoles. The third group was infused discretely with CLON, 6 nmolesi60 min, or a total daily dose of 144 nmoles. Each rat received two ICday infusion sequences, one with NE or CLON and one with the saline (ascorbic acid) vehicle. (Routine tests comparing results obtained with vehicle versus no vehicle infusion have shown no effect of this ascorbic acid vehicle on food intake or body weight.) Half the rats received the drug sequence first, and the other half received the saline sequence first. A 2-3 day adjustment period separated the two 14-day sequences. Between 3:00 and 5:00 p.m. each day, all drug solutions were changed. Measurements of food and water intake, as well as of body weight, were taken, and then fresh diet and water were supplied. After completing their drug-
4~_
daily
NOREPINEPHRINE I
tCont~nuous,lOom~60mini *
Menr3301iy
-G
‘;; 35
i
3O
30 6
LL 25
+--v------_r-.-
NOREPINEPHRINE (Continuous, IOnm/60 mid
1.2
-1011 0
10
5
15
DAYS FIG. I. The effect of chronic continuous infusion ofnorepinephrine (NE) into the PVN, on total daily food intake and body weight gain. Rat\ (n=6) were maintained ad lib on a milk-mash diet. Norepinephrine or its saline (ascorbic acid) vehicle was continuously infused at a rate of 10 nmihr. over the 14 days of PVN infusion. NE significantly increased daily food intake by IF{ (~~iO.(KfIt. This effect was associated with a significant enhancement of daily body weight gain (p~O.005).
saline or saline-drug sequences, lasting 30-35 days, the rats were sacrificed, their brains removed and histologicaIly analyzed and, in all cases, the placement of the cannula tip was confirmed to be within the vicinity of the PVN. The daily food intake, water intake, and body weight measures were analyzed using two-factor analyses of variance. Continous (Fig. I) as well as discrete (Figs. 2) infusion of NE into the PVN of rats fed ad lib produced a reliable increase in daily food intake and body weight gain. These results were replicated with discrete infusion of the c+noradrenergic agonist CLON (Fig. 3), supporting recent f’indings of a stimulatory feeding effect of this drug. similar to that of NE, after acute PVN injection [15,17f. Daily food intake with continuous NE was increased by approximately 12%, F(1,5)=43.36, p
CHRONIC
INFUSION
OF NE AND CLON
593
NOREPINEPHRINE (Discrete. 5nrMDminl
CLONIDINE (Discrete. 3nm0Omin) Meon Doily Food lntoke (g) 38.2
32.1 L
25
I
I
I
I
1
I
I
1
1 5
1
I IO
I
I I5
I
60SALINE
1 5Os
Mean Weight Goin (g/day)
NOREPINEPHRINE (Discrete, 5nmI3Omin)
3.5
Z$ 40W !z ”
30-
g
1.7
$ 20ED W
L
IO
5
z z
0
I
-10 1 0
-IO 0
DAYS
I
1
5
IO
1 15
DAYS
FIG. 2. Stimulation of feeding and body weight gain produced by chronic discrete infusion of norepinephrine (NE) into the PVN of rats (n=5) maintained ad lib on a milk-mash diet. Norepinephrine or saline vehicle was infused twice each hour at a rate of 5 nm130 set for 14 days. Over this period, both daily food intake cl%, p
period, as reflected by an insignificant drug x day interaction. Stimulatory effects on body weight were also observed with chronic PVN infusion of NE and CLON (Figs. l-3). Under drug as well as saline vehicle conditions, a steady and significant growth in body weight for all rats was observed over the 14-day sequence, F(1,470)=52.62, p
FIG. 3. Reliable increase in food intake (12%, p
gained between 1.2 and 1.7 g/day, this rate increased substantially to 3.1-3.5 g/day with chronic infusion of either NE or CLON. This effect was statistically reliable whether the raw scores for body weight or the derived body weight gain values (Figs. l-3) were analyzed. This enhancement of body weight occurred consistently over the entire 1Cday infusion period. Water intake measurements recorded during the discrete infusion experiments with NE and CLON revealed a small increase, F( 1,4)= 11.47, pcO.028, with NE infusion (41.2k4.1 ml) relative to saline infusion (36.823.6 ml). This effect was not observed in the case of CLON (28.Ok2.1 ml) compared with saline (31.0a 1.1 ml), F(1,3)= 1.87,~>0.10. In both cases, daily water intake, during either saline or drug infusion, remained relatively stable over the 14-day sequence of infusion. The enhancement of daily food intake and the small changes in water intake observed with both noradrenergic drugs were generally associated with an enhanced food/water ratio, which was statistically significant @<0.005) in the case of CLON.
594
These results, showing a stimulatory effect of chronic NE and CLON on daily food intake and body weight gain, confirm the sensitivity of the PVN to ff~-noradrenerg~c stimulation j&9] and reveal the power of noradrenergic control and the robustness of its response. They are consistent with a recent study, which examined the effect of repeated manual injections of NE into the PVN, and demonstrated, over a one-day or a five-day period, a reliable enhancement of 24-hr food intake and body weight gain 1141. In that study, NE was injected four times daiiy at a relatively high dose of 20 nmoles per injection, and the injections were given every other hour during daytime hours. Under these manual conditions, the magnitude of the NE effect, an increase in food intake of g-12 g, was somewhat larger than the 4 g increase observed here in these studies may be due to the difference in length of these tests (l-5 days versus 14 days), dosage, or mode of injection (manual versus remote). Given the greater number of test days in the present infusion experiment, it is possible that tolerance may have developed, consequently reducing NE’s effectiveness. This does not appear to be the case, however, since NE proved to be equally effective throughout the entire ICday sequence. These results obtained with chronic NE and CLON infused into the PVN are in agreement with the findings of Schelmmer ef crl. 1211, showing consistent overeating and obesity with chronic peripheral CLON administration in the monkey. It is interesting to note that, in humans, increased body weight has been observed as a side effect of CLON administration for treatment of hypertension (see Physicians’ Desk Reference).
The finding that NE is sufficiently robust to aher Icmp~ term feeding patterns and body weight regulation is con+ tent with the hypothesis that endogenous NE in the PVN has a physiological function in the control of normal feeding pat-. terns (e.g., [X. 10. 14, 161). Further supporting evidence i\ provided by the findings that: (a) PVN-injected NE is effective in altering feeding at near-physiological do$es 19. 10. XI]: (b) The meal pattern observed with PVN NE in.iection closely resembles that of spontaneous feeding ]hj; (CI in freely-feeding rats, exogenous NE is selective in it\ effect vn the maintenance rather than the initiation of’ feeding 1 I, 14, 201; (d) Electrolytic lesions of the PVN, the area most sensi.. tive to NE stimulation, significantly enhance daily food intake and body weight gain. suggesting that exogenous NE may act similarly, through its inhibitory effect on PVN “\;rtiety” neurons [12-141; tef Under food-depriv~lti~)n condi-
tions, significant alterations occur in endogenous NE and in a-adrenergic receptor activity, specficiaily within the PVN [4]; (f) At the onset of a rat’s eating bout, an increase in the efflux of W-labeled NE occurs in the periventricular area of the hypothalamus [ 161; (g) lntragastric infusion elf carbo hydrate nutrients. which normally induces satiety. is azso. ciated with an inhibitio~l of NE release. specifically within the PVN 1181. ACKNOWLEDGEMBN'lS
This research was supported by grant MH 22X79 and from the Whitehall Foundation, We thank Paul Roos\in assistance
in setting up the infusion
bq fun&, for hi\
timer
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INFUSION
OF NE AND CLON
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