Feeding induced by opiates injected into the paraventricular hypothalamus

Peptides, Vol. 4, pp. 287-292, 1983. ~ AnkhoInternationalInc. Printed in the U.S.A.

Feeding Induced by Opiates Injected Into the Paraventricular Hypothalamus S T A F F O R D M c L E A N " A N D B A R T L E Y G. H O E B E L ~

Department o f Psychology, Princeton University, Princeton, N J 08544 R e c e i v e d 10 D e c e m b e r 1982 McLEAN, S. AND B. G. HOEBEL. Feeding induced by opiates injected into the paraventricular hypothalamus. PEPTIDES 4(3) 287-292, 1983.--Injection of morphine or d-ala-2-met-5-enkephalinamide (DALA) into the paraventricular nucleus of the hypothalamus (PVN) produced a dose dependent increase in feeding in rats. DALA increase d feeding within 45 minutes; morphine within 90 minutes. Naloxone injected into the PVN diminished the effect. DALA increased water intake only when food was available, suggesting the primary effect was on feeding. In summary, an enkephalin analogue induced feeding, and an opiate receptor blocker attenuated it; therefore the PVN may contain opiate receptors that facilitate feeding. Opiates

Enkephalin

Feeding

Paraventricular nucleus

OPIATE peptides have been found in cell bodies and nerve terminals in areas of the brain not involved in encoding pain; this has focused attention on the role of opiates in other behaviors [30, 31, 32]. Opiate involvement in energy regulation was suggested by Margules et al. [25] when they found that genetically obese animals had elevated levels of pituitary beta-endorphin and that naioxone reduced their food intake [36]. Opiate antagonists also reduced feeding in rats eating spontaneously [4, 8, 24] or in response to deprivation [4, 6, 8, 14, 15, 16, 34, 35], tail-pinch [23, 29, 34], VMH lesions [18], or 2-DG [23, 34, 40], but not insulin ([23], see however [34]). Opiate antagonists can also reduce drinking in response to water deprivation [6, 14, 15, 16, 33, 41] but not scheduleinduced polydipsia [5]. Thus naioxone has suppressive effects on some, but not all, ingestive behaviors. This suggests the presence of endogenous opiate receptors for some types of feeding. Some of these receptors may be in the brain, as suggested by studies in which opiate agonists were injected centrally. Intraventricular injections of morphine, d-ala-d-leu enkephalinamide or beta-endorphin increased food intake; whereas other peptides, somatostatin, substance P and neurotensin had no effect [2,17]. Injections of morphine or beta-endorphin into the ventromedial or paraventricular hypothalamus (PVN) elicited eating [!1, 21, 45], but it was not known whether enkephalin would have a similar effect, nor whether an effect would be specific to feeding. We first confirmed the feeding effect with injections of morphine into the PVN, and then found a similar effect with d-ala-2-met-5-enkephalinamide(DALA) [26]. The present report gives the details of DALA-induced feeding, including dose-response effects, unilateral as well as bilateral tests, effects on water intake, and reversal with a receptor antagonist.

Hypothalamus G E N E R A L METHOD SURGERY

Male Sprague-Dawley rats (350-450 g) were housed and tested in individual cages in a colony room with lights on from 7:30 a.m. to 10:30 p.m. They were implanted with 26 gauge stainless steel guide cannulas positioned 0.5 mm above the PVN. Coordinates for the actual injection site were 0.5 mm posterior to bregma, 0.5 mm lateral to the midsagittal sinus, and 7.7 mm ventral to the surface of the skull with the incisor bar 3.0 mm above the interaural line. The guide cannulas were kept patent by 33 gauge obturators ground flush with the end of the cannulas. For injection, the obturators were replaced by a 33 gauge inner cannula that protruded 0.5 mm beyond the guide cannula. During a 10 day recovery period following surgery the animals were handled and mock injected. PROCEDURE AND FEEDINGTEST To assure that animals were satiated, extra Purina rat chow pellets were placed on the floor of the cage 30 rain before each test. Injectors were connected to a 10/zl syringe via P.E. 20 tubing. Infusions were delivered by a syringe pump at the rate of 0.3 p.l/min for a total volume of 0.3 txl. After infusing the drug, injectors remained in place for an additional 1 min to allow diffusion away from the tip. For bilateral injections one side was injected I min after the other. Following injection the obturators were replaced, and the animal was returned to its home cage with a premeasured amount of food and water. The first series of experiments focused on morphine and were designed to confirm that bilateral PVN norepinephrine and morphine would induce feeding (Experiment 1), show

~This research was supported by USPHS Grant MH 35740, E. R. Squibb and Sons, Inc., and a predoctoral fellowship to Stafford McLean. zCurrent address: Adult Psychiatric Branch, NIMH, Building 10, Bethesda, MD 20205. :~Requests for reprints should be addressed to Bartley G. Hoebel, Department Psychology, Princeton University, Princeton, NJ 08544.

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that naloxone would reverse the effect of morphine (Experiment 2), and determine a dose-response effect using unilateral morphine (Experiment 3). In the second series of experiments we tested DALA-induced feeding using bilateral injections (Experiment 4), a dose-response effect with unilateral injections (Experiment 5), naloxone reversal (Experiment 6), and D A L A ' s effect on water intake without food available (Experiment 7). E X P E R I M E N T 1: B I L A T E R A L I N J E C T I O N S OF N O R E P I N E P H R I N E AND M O R P H I N E

In a within subjects design, 8 animals received bilateral infusions of 0.9% saline vehicle on the first day. Subsequent infusions of 6.4/xg norepinephrine bitartrate (Sigma) or 2/xg morphine sulfate (Mallinkrodt) were delivered in a counterbalanced order with three days between tests. Food intake was measured to the nearest 0.5 g at i, 2, 4, and 8 hours after each infusion.

E X P E R I M E N T 7: TEST OF DALA ON WATER I N T A K E A L O N E

Eight animals with unilateral PVN cannulas were infused with 4 p~g DALA or saline in counterbalanced order. Water intake to the nearest 0.5 ml was measured 45, 90, and 135 min post-injection.

Histology and Statistics Brains were perfused with formalin, frozen, cut in 50/xm sections, stained with cresyl violet and examined microscopically to determine the location of cannula tips. Data were analyzed by an analysis of variance for repeated measures and post hoc by Duncan's Multiple Range Test. In experiments using more than one injection of saline as a control, mean saline scores were used in the statistical anfilysis.

RESULTS

E X P E R I M E N T 2: I N J E C T I O N OF MORPHINE AND N A L O X O N E

E X P E R I M E N T I: PVN MORPHINE I N D U C E S F E E D I N G

To test for naloxone reversal the same 8 animals were left for seven days, drug free, then bilaterally infused with 2 p~g morphine into the PVN followed 40 min later by infusions of 10/xg naloxone hydrochloride, or saline vehicle. Five days later the procedure was repeated with the saline group receiving naloxone, and the naloxone group, saline. Food intake was measured 1, 2, and 4 hours after the morphine injection.

As shown in Fig. 1, both norepinephrine and morphine increased food intake. It is evident that morphine had a delayed effect. Norepinephrine elicited significantly greater feeding than saline (p<0.001) during the first hour. Morphine, on the other hand, elicited a significant increase in food intake compared to saline at 2 hr (p<0.005) and 4 hr (p<0.001). By the eighth hour all rats had eaten about the same amount (Fig. 1).

E X P E R I M E N T 3: DOSE-RESPONSE TEST OF MORPHINE

Sixteen animals with unilateral cannulas aimed at the PVN received infusions of saline, or 0.1, 1, 5, or 10/xg of morphine sulfate at the rate of 0.6 pd/min for a total volume of 0.3/xl. Injections were given in a randomized order at least one week apart. Food intake was recorded 45, 90, and 135 min after injection.

E X P E R I M E N T 2: N A L O X O N E A T T E N U A T E S M O R P H I N E - I N D U C E D FEEDING

Figure 2 shows that bilateral naloxone given 40 min after bilateral morphine greatly reduced the feeding which normally occurs in the second hour. Analysis of variance indicated a significant effect, F(I,7)=2.08, p<0.03, which post hoc analysis revealed to be in the second and fourth hours (p<0.05).

E X P E R I M E N T 4: B I L A T E R A L I N J E C T I O N S OF DALA

To test an analogue of a naturally occurring enkephalin, 8 animals received bilateral infusions of 0.9% saline vehicle followed two days later with bilateral infusion of 2/~g d-ala2-met-5-enkephalinamide (Calbiochem). Several days later, the same animals were tested with 4 or 8/xg DALA in counterbalanced order with a saline test between each drug test. All infusions were separated by at least three days. Food intake was measured l, 2, and 4 hours after infusions. E X P E R I M E N T 5: DOSERESPONSE TEST OF DALA

Unilateral injections were used to determine the lowest dose necessary to elicit feeding. Sixteen animals with unilateral cannulas received 1, 2, 4, 8, and 16 p.g DALA in a randomized order with saline tests between each DALA test. Three days separated each injection. Food and water intake were measured 45, 90, and 135 min after each injection. E X P E R I M E N T 6: INJECTION OF D A L A A N D N A L O X O N E

At the completion of the dose-response study l0 animals were used to assess naloxone's effect on DALA-induced feeding. Naloxone (10 p~g) or saline was administered to the PVN 20 min before unilateral infusion of 2 p~g DALA. Food intake was recorded 45 min later.

E X P E R I M E N T 3: D O S E - D E P E N D E N T E F F E C T OF U N I L A T E R A L MORPHINE

A dose-dependent increase in food intake after unilateral morphine is shown in Fig. 3. Analysis of variance gave a significant effect for dose, F(4,148)=9.54, p<0.0001, and a significant effect for dose x time, F(8,96)=6.42, p<0.0001. Analysis of variance for order of administration was not significant, F(3,12)=2.37, p<0.12. As shown in Fig. 3, within the first 45 min only the I/zg dose of morphine gave a significant increase in food intake (p<0.01). At 90 and 135 min all doses of morphine resulted in a significant increase. Doses of 1, 5, and 10 /xg were not significantly different from each other but all resulted in significantly greater intake than 0.1 /zg morphine (p<0.05 at least). E X P E R I M E N T 4: B I L A T E R A L DALA I N D U C E S DOSE-DEPENDENT FEEDING

Figure 4 shows that three different doses of DALA given bilaterally caused a dose-dependent increase in food intake. Analysis of variance gave a significant effect for dose, F(3,21)=18.97, p<0.0001, and for dose by hour, F(6,42)=2.72, p<0.025. A post hoc Duncan indicated 8/zg DALA elicited more food intake than 4/xg (p<0.001), and 4 /xg elicited more than 2/.tg (p<0.005); however 2 ~g was not significantly different from saline.

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FIG. 5. Unilateral infusion of DALA increased food intake in a dose-dependent manner, tp<0.05, p<0.005, *p<0.001 indicate a significant difference from saline. Although not indicated on the graph, 8/xg resulted in significantly greater food intake than 1 /zg at 45 min, 90 min, and than 1 and 2/xg at 125 min. Two and 4/xg were significantly greater than 1 p.g at all time periods, while 16 ~g was significantly less than 2, 4, and 8 p.g at 45 min. E X P E R I M E N T 5: U N I L A T E R A L D A L A I N D U C E S D O S E - D E P E N D E N T EFFECT

As can be seen in Fig. 5 unilateral DALA also caused a dose-dependent increase in food intake. Analysis of variance gave a significant effect for dose, F(5,75)=4.61, p<0.001, and for the interaction of dose × time, F(10,150)=5.65, p<0.0001. A post hoc Duncan (on the first 45 min) indicated 2, 4, and 8/zg elicited significantly greater food intake than either 1 /xg or saline. Figure 5 shows this effect was still present at 135 min when all but the lowest dose elicited significantly more food intake than saline. A significant increase in water intake 135 min after the infusion is seen in Fig. 6. Analysis of variance yielded a significant effect for dose, F(5,75)=3.3, p<0.009, and the interaction of dose with time, F(10,150)=5.0, p<0.0001. As can be seen in Fig. 6, the highest dose suppressed water intake significantly at 90 min (p<0.001). The three lower doses, 2, 4, and 8 ~g, elicited significantly greater water intake than saline after 135 min (p<0.001). E X P E R I M E N T 6: N A L O X O N E A T T E N U A T E S D A L A F E E D I N G

Figure 7 indicates that 10 p~g of naloxone injected into the PVN attenuated DALA-induced feeding, F(2,14)=4.82, p<0.02. E X P E R I M E N T 7: W I T H O U T F O O D TO EAT, D A L A DOES N O T INCREASE WATER INTAKE

Figure 8 shows that when 4 p.g of DALA was given to animals with only water available, there was no significant increase in water intake, F(I,6)=0.59, p<0.47. G E N E R A L DISCUSSION Within the range of doses tested, injection of morphine or

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DALA into the region of the PVN increased feeding in a dose-dependent manner; naloxone partially reversed it. Morphine, DALA and norepinephrine induced feeding with dissimilar time courses. Feeding after norepinephrine generally occurred within the first 10 min as reported by Leibowitz [19]. The opiates had a slower onset time. Morphine's effects were not seen for at least 90 min, confirming the reports of others [21,45]. We find that DALA is faster than morphine; it usually induced food intake within 30 to 45 minutes. Although infusions of DALA into the PVN increased intake of water as well as food when both were available, the results of Experiment 7 indicate the increased water intake was probably secondary to the increased food intake. The animals drank because they were eating dry food. This is supported by a comparison of the time course for feeding (Fig. 5) with that of water intake (Fig. 6). A significant increase in food intake occurred within 45 min, while water intake was not significantly different from control until 135 min. This finding is consistent with work demonstrating that enkephalins do not cause thirst and will even inhibit drinking induced by either water deprivation [7,9] or angiotensin [43]. This lack of direct effect on water intake can be contrasted with the preprandial drinking following injection of NE into the PVN. With NE all drinking occurs within the first 5 min, and the presence of food decreases the amount of water consumed [18]. The opiates increased water intake only in the presence of food and only following increased food intake. The observed dose-dependent increases in food intake with D A L A and the reversal by naloxone argue that the phenomenon is receptor mediated and not due to some nonspecific receptor interaction. This is consistent with the finding that agonists which are active, but not inactive isomers of

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opiate agonists, increase food intake in rats [46]. We note that doses of naloxone (10-40 ~g) which reversed the enkephalin-induced feeding do not affect the food intake of mildly food deprived animals when injected unilaterally. This is consistent with reports that up to 50 tzg naloxone given unilaterally into the lateral ventricle failed to affect food or water intake of 24 hr food deprived animals [14,48]. However, when naloxone was injected bilaterally in the ventricles food and water intake were decreased [16]. Injection of morphine into the medial hypothalamus releases A C T H [22], which is secreted concommitantly with B-endorphin by the pituitary [12]. Therefore it is possible that B-endorphin released from the pituitary is partially responsible for the feeding following microinjection of morphine or D A L A into the PVN. However, results from our laboratory show that an injection of dexamethasone that blocks stress-induced B-endorphin release [38] does not affect feeding elicited by injection of D A L A into the PVN [27]. These results agree with Pittman's [35] report that opiates

have relatively little effect on PVN neurons that project to the neurohypophysis, but do inhibit parvocellular neurons. As a large number of parvocellular neurons project to the brainstem [1,44] the opiates may be acting to disinhibit a medullary center involved in food intake, normally under the inhibitory control of the PVN. This hypothesis is buttressed by studies demonstrating that lesions of the PVN efferents [10] or of the nucleus itself 120] result in increased food intake. Although both norepinephrine and D A L A increase food intake when infused into the PVN, we do not think the opiates are acting through the release of endogenous NE because of the difference in their effects on water intake and the fact that manipulation of the pituitary-adrenal axis affected NE feeding but not D A L A feeding [27]. In summary, these results suggest that there are opiate receptors in the PVN with the function of releasing feeding from inhibition. They influence feeding more than drinking, and respond to enkephalin as well as to larger opiate molecules.

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