Factors influencing feeding elicited by intracranial noradrenaline in rats

Brain Research, 141 (1978) 119-128 © Elsevier/North-Holland Biomedical Press

FACTORS I N F L U E N C I N G F E E D I N G N O R A D R E N A L I N E IN RATS

119

ELICITED

BY

INTRACRANIAL

J. W. MATTHEWS, D. A. BOOTH and I. P. STOLERMAN* Departments of Pharmacology and Psychology and MRC Neuropharmacology Unit, University of Birmingham (Great Britain)

(Accepted May 18th, 1977)

SUMMARY An improved design of microcannula was used to inject noradrenaline into discrete areas of the rat hypothalamus. The area of the paraventricular nucleus was shown to be a more effective site than the midlateral anterior hypothalamus for eliciting feeding with noradrenaline (4.8/~g). The magnitude but not the direction of the effect of noradrenaline on feeding was influenced by the day-night cycle. The facilitating effect of the drug reached significance in the light period but not in the dark period, possibly because the baseline food intake was higher by night than by day. The facilitation of feeding occurred whether the rats were maintained and tested on a diet of solid or liquid food.

INTRODUCTION Intrahypothalamic administration of (-)noradrenaline can elicit feeding in satiated rats a. Neither (÷)noradrenaline nor several other drugs elicited feeding, and other control injections established that the feeding elicited by (-)noradrenaline was not due to osmotic stimulation, pH changes or vasomotor effects4,s,2n. However, Margules has consistently found that intrahypothalamic noradrenaline suppresses feeding in rats 17. A number of factors might be responsible for this contrasting result and these include: dose, the neuroanatomical site of injection, the stage of the rat in its day-night cycle, and the use of solid or liquid food. Our experiments have been designed to examine some of these variables, and thus attempt to explain why injections of the same drug, noradrenaline, can have different effects in different circumstances. * Address for reprints: I. P. Stolerman, MRC Neuropharmacology Unit, The Medical School, Birmingham BI5 2TJ, England.

120 Leibowitz 14 has suggested that there are two adrenergic systems in the hypothalamus, a medial one concerned with the facilitation of feeding and a lateral one concerned with the inhibition of feeding. Possibly Margules injected into a more lateral site than most other workers and thus affected mainly the inhibitory system. However, if the site of action of the drug is to be implicated as a factor, tissue damage and the spread of drug must be minimised by using the smallest practicable injection cannulae. Early work s,3° attempting to identify an area particularly sensitive to the facilitatory effects of noradrenaline on feeding suggested that the caudal region of the lateral hypothalamus was the best site. These workers used large sizes of guide cannulae with large volumes or high concentrations of drug. Using guide cannulae half the size of those used previously, Booth found the most effective sites to be in the anterior hypothalamus a, a conclusion subsequently confirmed by Grossman 9. A later study by Davis and Keesey 6 also showed that the anterior medial hypothalamic region was most effective. However, whereas the perifornical area at the level of the anterior hypothalamic nucleus, and more anteriorally the area around the stria medullaris were the effective sites in Booth's study, Davis and Keesey 6 and Leibowitz 14 have suggested, after sampling more medial regions than Booth, that the paraventricular nucleus might be the focal area for the effects on feeding of noradrenaline injected into this region. However, larger cannulae and volumes of drug were used in the studies by Davis and Keesey and by Leibowitz. Our first experiment compared the feeding elicited by intracranial noradrenaline at midlateral 3 and medial 6 anterior hypothalamic sites using an improved version of Booth's microcannula 4. A second difference between the procedures used by Margules and by others relates to the rats' day-night cycle. Margules obtained evidence that noradrenaline suppressed feeding in the dark and facilitated feeding in the light is. In our second experiment, rats were tested in the light and in the dark using a more appropriate cannula size, injection site and dose of drug. A third difference was that Margules tested with milk rather than with solid food 18. On the basis of results showing a suppression of water consumption by intracranial noradrenaline, Leibowitz 13 has suggested that Margules was affecting a system concerned with thirst rather than hunger. Our third experiment tested the hypothesis 13 that the water content of milk influenced the effects of noradrenaline on feeding. METHODS Animals" Male, hooded rats (OLAC, Oxfordshire) weighing approximately 300 g at the time of surgery were used throughout. The rats were housed under conditions of controlled temperature and humidity on a 12 hr/12 hr light and dark cycle, switched from bright white light to dull red light at 17.00 hr. Microcannula system The cannulae used were close to the dimensions recommended by Booth 4 and by

121 Routtenberg ~4 as the smallest practicable for repeated injections in awake rats. A modified design was developed to be more robust, inexpensive and to have interchangeable parts which could be made easily*. The cannula guides were made from stainless steel tubing with an outer diameter of either 0.41 mm (experiments 1 and 2) or 0.51 mm (experiment 3). Injections were made through the guide cannulae with stainless steel needles of outside diameter 0.18 mm (experiments 1 and 2) or 0.23 mm (experiment 3). The tips of the injection needles were level with the tips of the guide cannulae. The procedure for injecting a rat involved a visual check to ensure that no air bubbles were present; the filled injection needle and 10-/~1syringe were then centred in a Hamilton repeating dispenser which was used to deliver 0.44 #1 of solution in about 6 sec.

Surgery Cannulae were implanted into the left side of the brain under pentobarbitone anaesthesia using a K o p f stereotaxic instrument. In the first experiment two sites were chosen; cannulae implanted in rats of group I were aimed at a perifornical site at which Booth 3 found frequent facilitation of feeding by noradrenaline, whereas cannulae in group II were aimed at the more medial site at which Davis and Keesey 6 found their greatest facilitation of feeding by noradrenaline. The coordinates chosen according to the atlas of Pellegrino and Cushman ~2 were anterior 6.6 mm, medial 1.2 mm and ventral 1.6 mm (group 1) or anterior 6.2 mm, medial 0.7 mm and ventral 1.7 mm (group II). In experiments 2 and 3, all cannulae were aimed at the latter site.

Experimental Procedures Rats were maintained on food pellets (Laboratory Small Animals Diet, Spratts, Barking, Essex) with water available at all times. Following surgery the rats were given one week for recovery, during which time they were handled each day. One week after surgery, the animals were given sham injections for three days, including all of the manipulations which would occur during an actual test, except for the injection of solutions. Tests were then performed on alternate days. On test days, the usual procedure was to remove the rat and its food supply from the cage, and inject with either drug or saline. The rat was then replaced in its cage with a weighed quantity of fresh food pellets. The food was reweighed after one hour together with any food spilt, which was collected on absorbent paper placed beneath the cage. Experiment 1. The procedure varied from that described above in that the fresh food pellets were presented for 15 min prior to injection. This was to minimise the influences of novelty, and also to produce a degree of satiation against which facilitations of feeding behaviour by noradrenaline could be seen more clearly. Drug and saline injections were given twice to each rat in a sequence which was counterbalanced within groups I and Ii (4 injections per rat). All tests started at the seventh hour of the light period. * Details of cannula construction can be obtained from the authors.

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Experiment 2. Drug or saline in the light or dark was given once to each rat in a random sequence, resulting in a total of 4 injections per rat. Thus, on every alternate day each rat was tested in either the light or dark period, and was always subjected to a sham procedure when an injection was not required in order to balance across rats factors such as handling. The tests were started at the ninth hour of the light period and the second hour of the dark period; these were the times at which Margules found the greatest facilitation and inhibition respectively of feeding by injected noradrenaline is. Experiment 3. Following recovery from surgery the rats were placed on a liquid diet of milk (50 parts by volume of sweetened condensed milk (Nestle) ~- 50 parts of water + 1 part of 10 ~ solution of choline chloride), with water available ad libitum. After two weeks of habituation to this diet, tests were carried out with drug and saline given twice to each rat in a balanced design (4 injections per rat). The milk was presented in calibrated cylinders and readings were taken immediately prior to, and at one hour after injection. The rats were then returned to the solid maintenance diet and, after two weeks of habituation, each rat was tested again twice with drug and twice with saline in a balanced design. All tests started at the seventh hour of the light period. Histology Following testing, the rats were decapitated under pentobarbitone anaesthesia and the brains were fixed in formol saline. The brains were cut into 50 #m sections close to the plane of the stereotaxic atlas of K6nig and Klippe11% Placements of the cannulae tips were recorded according to the K6nig and Klippel atlas.

Drugs Saline was 155 m M NaC1. Noradrenaline was prepared as 65 mM (-)noradrenaline bitartrate (Sigma) in 90 m M NaCI solution. Both solutions were injected in volumes of 0.44 /~1 and the dose of noradrenaline base was therefore 4.8 #g4,Z0. Pentobarbitone sodium (50 mg/kg) was administered as Nembutal Veterinary solution (Abbot Laboratories, Queenborough, Kent). RESULTS

Experiment 1 The amounts of food eaten after saline injections were 0.31 i 0.15 g and 0.41 ± 0.16 g for rats in groups I and II respectively (means -t- S.E.M.). After injections of noradrenaline, rats in group II consumed 3.18 -4- 0.44 g, whereas rats in group 1 consumed only 0.83 -4- 0.42 g. Facilitation of feeding by injected noradrenaline was therefore significantly greater in group II (paraventricular) than in group I (mid-lateral anterior hypothalamus) (Mann-Whitney U = 2, P < 0.05). Fig. 1 (A) shows the placements of the 10 cannulae which, with one exception, were in the intended areas. The exception was a cannula aimed at the paraventricular nucleus which was in fact situated more rostrally, close to the perifornical site. This rat showed the smallest

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(B) Experiment 2

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500~ Fig. 1. Daytime facilitations of feeding by noradrenaline. For each experiment, two hypothalamic sections are shown, from the atlas of K6nig and Klippel; anterior-posterior coordinates are 6060 ttm (upper sections) and 5660 pm (lower sections). Anterior-posterior spread of placements was 5910-6360/~m (upper sections) and 5340-5780/~m (lower sections). Circles denote sites and average diameters of placements. Numbers within circles give mean noradrenergic facilitations of feeding (drug-saline differences to nearest gram). PS, periventricular system; fp, nucleus paraventricularis pars parvocellularis; f, fornix; fm, nucleos paraventricularis pars magnoeellularis; V, third ventricle; ha, anterior hypothalamic nucleus; re, nucleus reuniens; TO, optic tract; ZI, zona incerta; FMP, medial forebrain bundle.

facilitation of feeding by noradrenaline in group II. Furthermore, with the single exception of a placement situated dorsally in the perifornical region, the facilitation of feeding by noradrenaline became greater the closer the placements were to the paraventricular nucleus. Fig. 2 shows an histological preparation from a rat in which noradrenaline markedly increased food intake; the size of the lesion at the tip of the guide cannula is similar to that shown previously 4.

Experiment 2 After saline injections, the mean amounts of food consumed were 0.74 ± 0.22 g and 2.77 ± 0.30 g in the light and dark respectively (Wilcoxon T = 7.5, P < 0.05). The larger intake in the dark than in the light can be attributed to the typical feeding pattern of the rat eS. In the light, the mean food intake after noradrenaline injection was 3.71 q- 0.39 g, which was significantly greater than the food intake after saline injection (T = 1.5, P < 0.01). However, after noradrenaline injection in the dark, food intake was 2.99 -q- 0.45 g, which was not significantly greater than that after saline injection in the dark (T = 43). It is possible, although not established by these data,

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Fig. 2. Section of brain stained by the Kl(Jver-Barrera method and showing location of placement where noradrenaline increased food intake. The lesion at the tip of the cannula can be seen on the medial aspect of the paraventricular nucleus. that noradrenaline did not increase feeding in the dark because of the high baseline of food intake during that part of the day-night cycle. Fig. 1 (B) presents the histology and the feeding data (drug-saline differences in the light) for 13 of the 14 rats in experiment 2 (the other placement was more anterior in the third ventricle). As in experiment 1, the feeding effect was largest at placements close to the paraventricular nucleus.

Experiment 3 Milk and water consumption following noradrenaline and saline injections are presented in Fig. 3(A). Noradrenaline significantly increased milk intake (T = 4, P < 0.02) and the concurrent water intake (T = 1.5, P < 0.01) as compared with the intakes after saline injection. Furthermore, the rats in which the drug facilitated milk consumption also tended to be those in which the drug facilitated water consumption (Spearman rs = 0.68, P < 0.05). Fig. 3(B) presents the results for food pellet and water consumption following noradrenaline and saline injections. Noradrenaline increased pellet intake (T = 9, P < 0.05) but failed to increase the water intake significantly (T = 15). Rats in which noradrenaline facilitated milk consumption also tended to show a drug-induced facilitation of food pellet consumption (rs -- 0.69, P < 0.05). Similarly, those rats in which the drug facilitated water consumption during the milk test also

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Fig, 3. Effects of noradrenaline on food and water intake (experiment 3). A: effects of noradrenaline on milk and water intake (the amounts consumed after saline injections have been subtracted). The data for milk consumption are ranked so that for any rat, the greater the facilitation of feeding by the drug the more to the left of the diagram the score appears. The same ranking is used in the data for water consumption. B: effects of noradrenaline on solid food and water intake. The ranking is the same as that used in A. tended to show a drug-induced facilitation of water consumption in the food pellet test (rs = 0.75, P < 0.05). The histology and volumes of milk consumed for 9 of 11 rats tested are presented in Fig. I(C). One rat in which the drug not facilitate milk consumption had a placement situated rostrally in the region of the basal ganglia. Another placement was lost before an histological examination could be made. The results confirm that noradrenaline injected into the region of the paraventricular nucleus can elicit feeding reliably. There was also evidence that an area in the dorsal region of the perifornical, hypothalamic area also mediated effects of injected noradrenaline on feeding (Fig. I(C)). DISCUSSION

These experiments have demonstrated, using the smallest practicable size of cannula, that noradrenaline can stimulate feeding when injected into an area coincident with or bordering on the paraventricular nucleus of the hypothalamus. This effect of noradrenaline depended upon the day-night cycle, being present at the end of the light period but absent at the beginning of the dark period, possibly because food intake was already at a high level in the dark. Noradrenaline elicited feeding irrespective of whether the rats' food was solid or liquid. The site where noradrenaline was found to elicit the strongest feeding was the lateral aspect of the paraventricular nucleus. Leibowitz t4 has also suggested that the paraventricular nucleus is a primary site mediating the facilitatory effects of injected noradrenaline on feeding. Recent studies have shown that this nucleus contains some of the highest concentrations of catecholamines of all the hypothalamic nuclei21, 2a, and that it is an area densely innervated by ascending catecholaminergic pathways 11,

126 15. This evidence is consistent with the view that the injected drug is affecting feeding via endogenous catecholamine systems. Supporting this hypothesis are other results showing, first, that the putative noradrenergic feeding system may be a-adrenergic in character~,la, 26, and secondly that the activity of hypothalamic noradrenergic systems varies with the feeding pattern of the rat16,19,28. Ahlskog has shown that lesions in the ventral noradrenergic pathway can produce hyperphagial; whether this effect involves the paraventricular nucleus is uncertain since noradrenaline in that nucleus was not depleted reliably by the lesions. The emphasis on noradrenergic feeding in the present experiments does not preclude the possibility that the same substance might inhibit feeding when injected into other parts of the brain. The histological data for experiments 1 and 3 also provide evidence that an area in the dorsal perifornical region, rostral and lateral to the paraventricular nucleus, may mediate the effects of injected noradrenaline on feeding. Lindval115 has described an ascending catecholamine fibre system, probably noradrenergic in nature, which innervates the paraventricular nucleus and then ascends rostrally and laterally into the interstitial nucleus of the stria terminalis, an area where noradrenaline injections are effective in stimulating feeding~,10. It seems possible therefore that this pathway mediates effects of noradrenaline on feeding. On present information this would seem a more firmly based suggestion than Booth's original proposal that anterior hypothalamic and thalamic elicitation of feeding by injected noradrenaline was mediated by primarily habenular pathways z. Experiment 2 demonstrated a facilitation of feeding by noradrenaline in the light, and this finding is in agreement with the results of most of the work in this area. The finding that noradrenaline injections had no effect on the amount of food eaten in the dark contrasts with other reports18, 27 which have shown a suppressive effect of the drug on feeding in the dark. The suppression of feeding seen by other workers could have resulted from the use of higher doses of drug, or from the differing sites at which the drug has been injected. Stern and Zwick '27 reported that the suppressive effects of injected noradrenaline on feeding in the dark tended to occur at higher doses than the facilitatory actions of the drug in the light. Leibowitz 14 reported that two injection sites in the hypothalamus, medial and lateral in position, mediate noradrenergic facilitation and suppression of feeding. Also Booth a found that lateral hypothalamic injections of noradrenaline at the rostrocaudai level of the ventromedial hypothalamic nucleus elicited feeding much less often than mid-lateral anterior hypothalamic injections. It seems possible that more lateral and caudal hypothalamic injection sitesl7, is, or the high dosages of drug injected intraventricularlyz7, would have been more effective in stimulating a lateral inhibitory system than the smaller doses of drug injected into medial and mid-lateral anterior hypothalamic sites in this study. Another explanation of the apparent suppression during the night is suggested by the similarity of the food intakes following noradrenaline injection in the light and dark. This could indicate a 'ceiling effect' i.e. that a high baseline of food intake at night following injection of saline might have precluded further facilitation of feeding following injected noradrenaline. Baseline food intake can be manipulated inde-

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pendently of the day-night cycle by depriving the rat of food. However, deprivation does not reverse the effect of injected noradrenaline2,7. The determinants of high baseline food intake however might well differ between night time and food deprivation. Furthermore, measuring food intake over a fixed period of time fails to discriminate between two variables which can vary independently: meal size and interval between meals. Intrahypothalamic infusions of very low doses of noradrenaline during meals can increase meal size without affecting the time between meals23. Future experiments may therefore be more productive if the pattern of feeding is studied rather than the total amount of food consumed. The third experiment confirmed and extended to the paraventricular nucleus earlier reports that noradrenaline can facilitate milk consumption in rats 2°,30. It further demonstrated a positive correlation between food pellet and milk consumption, suggesting that both increases in consumption may be mediated by the same mechanism. It follows that the use of milk is not likely to be the factor responsible for the suppressive effects of noradrenaline observed by Margules18. Margules also maintained his rats on solid food and presented milk only during test periods, whereas in our experiments milk was available continuously; this factor could also be responsible for the observed suppression of intake is, although a preliminary report 14 suggests that this is unlikely. In conclusion, these results have strengthened the evidence that injected noradrenaline elicits feeding through actions in areas of the hypothalamus which are rich in endogenous catecholamines. This effect of noradrenaline is facilitatory without regard to the form of the food, but depends on the time of day at which the test is performed. ACKNOWLEDGEMENTS The authors wish to t h a n k Professor P. B. Bradley for his e n c o u r a g e m e n t of this work. J.W.M. was a Medical Research Council Scholar, 1973-6.

REFERENCES 1 Ahlskog, J. E., Food intake and amphetamine anorexia after selective forebrain norepinephrine loss, Brain Research, 82 (1974) 211-240. 2 Armstrong, S. and Singer, G., Effects of intrahypothalamic administration of norepinephrine on the feeding response of the rat under conditions of light and darkness, Pharmacol. Biochem. Behav., 2 (1974) 811-815. 3 Booth, D. A., Localization of the adrenergic feeding system in the rat diencephalon, Science, 158 (1967) 515-517. 4 Booth, D. A., Mechanism of action of norepinephrine in eliciting an eating response on injection into the rat hypothalamus, J. Pharmacol. exp. Ther., 160 (1968) 336-348. 5 Broekkamp, C. and Van Rossum, J. M., Clonidine induced intrahypothalamic stimulation of eating in rats, Psychopharmacologia (Berl.), 25 (1972) 162-168. 6 Davis, J. R. and Keesey, R. E., Norepinephrine induced eating: its hypothalamic locus and an alternative interpretation of action, J. comp. physiol. Psychol., 77 (1971) 394-402. 7 Dragovich, J. A. and Margules, D. L., Circadian sensitivity of the hypothalamus to norepinephrine HCI in rats with an altered feeding pattern, Int. J. Chronobiol., 3 (1975) 73-80.

128 8 Grossman, S. P., Direct adrenergic and cholinergic stimulation of hypothalamic mechanisms,., Amer. J. Physiol., 202 (1962) 872-882. 9 Grossman, S. P., Hypothalamic and limbic influences on food intake, Fed. Proc., 27 (1968) 1349-1360. 10 Herberg, L. J. and Stephens, D. N., Cyclic AMP and central noradrenaline receptors: failure to activate diencephalic adrenergic feeding pathways, Pharmacol. Biochem. Behav., 4 (1976) 107-110. 11 H6kfelt, T., Fuxe, K., Goldstein, M. and Johansson, O., Immunohistochemical evidence for the existence of adrenaline neurons in the rat brain, Brain Research, 66 (1974) 235-251. 12 K6nig, J. F. R. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas, Williams and Wilkins, Baltimore, 1963. 13 Leibowitz, S. F., Central adrenergic receptors and the regulation of hunger and thirst, Res. Publ. Ass. Res. herr. merit. Dis., 50 (1972) 327-358. 14 Leibowitz, S. F., Brain catecholaminergic mechanisms for control of hunger. In D. Novin, W. Wyrwicka and G. Bray (Eds.), Hunger: Basic Mechanisms and Clinical Implications, Raven Press, New York, 1976, pp. 1-18. 15 Lindvall, O. and Bj6rklund, A., The organisation of the ascending catecholamine neuron systems in the rat brain, Actaphysiol. scand., Suppl. 412 (1974) 1-48. 16 Manshardt, J. and Wurtman, R. J., Daily rhythm in the noradrenaline content of rat hypothalamus, Nature (Lond.), 217 (1968) 574-575. 17 Margules, D. L., Alpha-adrenergic receptors in hypothalamus for the suppression of feeding behavior by satiety, J. comp. physiol. Psychol., 73 (1970) 1-12. 18 Margules, D. L., Lewis, M. J., Dragovich, J. A. and Margules, A. S., Hypothalamic norepinephrine: circadian rhythms and the control of feeding behavior, Science, 178 (1972) 640-643. 19 Martin, G. E. and Myers, R. D., Evoked release of [14C]norepinephrine from the rat hypothalamus during feeding, Amer. J. Physiol., 229 (1975) 1547-1555. 20 Miller, N. E., Gottesman, K. S. and Emery, N., Dose response to carbachol and norepinephrine in rat hypothalamus, Amer. J. Physiol., 206 (1964) 1384-1388. 21 Palkovits, M., Brownstein, M., Saavedra, J. M. and Axelrod, J., Norepinephrine and dopamine content of hypothalamic nuclei of the rat, Brain Research, 77 (1974) 137-149. 22 Pellegrino, L. J. and Cushman, A. J., A stereotaxic Atlas o f the Rat Brain, Appleton Century Crofts, New York, 1967. 23 Ritter, R. C. and Epstein, A. N., Control of meal size by central noradrenergic action, Proc. nat. Acad. Sci. (Wash.), 72 (1975) 3740-3743. 24 Routtenberg, A., Intracranial chemical injection and behavior: a critical review, Behav. Biol., 7 (1972) 601-641. 25 Seigel, P. S. and Stuckey, H. L., The diurnal course of water and food intake in the normal mature rat, J. comp. physiol. Psychol., 40 (1947) 365-370. 26 Slangen, J. L. and Miller, N. E., Pharmacological tests for the function of hypothalamic norepinephrine in eating behavior, Physiol. Behav., 4 (1969) 543-552. 27 Stern, J. J. and Zwick, G., Effects of intraventricular norepinephrine and estradiol benzoate on weight regulatory behavior in female rats, Behav. Biol., 9 (1973) 605-612. 28 Van Der Gugten, J. and Slangen, J. L., Norepinephrine uptake by bypothalamic tissue from the rat related to feeding, Pharmacol. Biochem. Behav., 3 (1975) 855-860. 29 Van Der Gugten, J., Palkovits, M., Wijnen, H. L. J. M. and Versteeg, D. H. G., Regional distribution of adrenaline in rat brain, Braiu Research, 107 (1976) 171-175. 30 Wagner, J. W. and De Groot, J., Changes in feeding behavior after intracerebral injections in the rat, Amer. J. PhysioL, 204 (1963) 483-487.