Feeding and Drinking in Sheep Following Hyphothalamic Injections of Carbachol

Feedingand Drinking in SheepFollowingHypotholmic Injections of Corbochol J. M. FORBES ~ AND C. A. BAILE Smith Kline Animal Health Products 1600 Paoli Pike, West Chester, PA 19380 University of Pennsylvania Philadelphia, Pennsylvania 19104

Abstract

Carbachol (carbamycholine chloride) (28 nmol) was injected on 77 occasions into 69 loci in the hypothalamus of 13 sheep. Of the injections, 31% elicited feeding, 14% elicited drinking, and 4% elicited both feeding and drinking responses. One carbachol feeding locus in each of eight sheep was injected with carbachol at doses from 1.75 to 112 nmol. Feeding was elicited by 3.5 nmol or more carbachol. At carbachol-bound feeding loci in eight sheep, the effect of carbachol was blocked by atropine, a cholinergic antagonist, but not by phenoxybenzamine or LB-46, a and fladrenoceptor antagonists, respectively; atropine alone had no effect. Increases in water intake accompanying carbachoIinduced feeding may be due to increased dry matter intake. There were no significant effects on intraperi,toneal temperature. Cholinergic pathways passing through the hypothalamus of sheep is proposed as an explanation for the feeding. Introduction

In recent years interest has been focused on the dipsogenic effects of intracranial placements of carbachol (13, 20). It is possible that carbachol may stimulate a cholinergie pathway normally involved in the control of water intake (19, 20). Although injections into several parts of the hypothalamus of rats depressed feed intake (16, 28) or had no effect (22), Antunes-Rodriquez and McCann (1) elicited feeding in rats by injections of 11 nmol of carbachol into Received December 10, 1973.

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the third ventricle. Feeding was also increased in rabbits following iniections of carbachol (.3 to 5.8 nmol) into lateral hypothalamic and preoptic areas (29); low doses (.5 nmol) injected into the preoptic area of cats may also stimulate feeding (23). Higher doses depressed intake in both of these experiments. Lateral hypothalamic (21) and preoptie (18) treatment with 27.5 nmol carbachol in rats caused hypothermia which was not merely the result of the increased drinking (17). The effects of adrenoceptor stimulation of the hypothalamus on feeding (15, 16), drinking (15), and temperature regulation (24) ot laboratory animals have been widely studied. Similar approaches have now been made towards understanding the control of feed intake in ruminants, using injections of adrenoeeptor agonists and antagonists into the cerebral ventricles and hypothalamus (2, 3, 4, 5, 6, 9, 11). We have now extended this work to include the effects of cholinergic agonists and antagonists on feeding, water intake, and body temperature in sheep. This species is of particular interest, not only because of the economic importance of ruminants, but also because of the large differences in digestion and intermediary metabolism between these animals which possess the large forestomach where partial fermentation of food occurs and those which do not (4). Methods

Thirteen castrate male sheep (45 to 65 kg) were penned individually in a room at 20 to 22 C. A pelleted complete feed (5) was offered ad libitum with fresh feed being added at the same time each morning; water was continuously available from buckets. Animals were prepared surgically with three cannula guides aimed for the ventromedial hypothalamus and, eontralaterally, three guides aimed for the lateral hypothalamus, using a modification (9) of the X-ray stereotaxic method of Tarttelin (30). The three guides in each area were in

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and water buckets were weighed at time of injection and 30, 60, and 120 rain later; 24-h feed intakes were also recorded. Body temperatures were taken at 0, 30, 60, and 120 rain in most sheep by placing thermistor probes into blind silastic tubes surgically implanted into the peritoneal cavity between liver and rumen (9). At the end of the experiments each animal was sacrificed by an overdose of anesthetic. Two hypothalamic loci, one on each side of the midline, were marked with injections of 1 /~liter ink, and the brain was removed from the cranium, fixed in formalin, and later cut into 2 mm cross sections. The positions of those loci not marked with ink were calculated from the

line in an anterior-posterior direction and were 1 m m apart. Hypothalamic iniections were begun at least 10 days after surgery when animals had recovered their pre-operative feed intake. They were injected with 1 p.liter of synthetic cerebrospinal fluid (CSF) (25) containing the drug. This was delivered by a microliter syringe through a 28 gauge cannula inserted through a guide to the desired depth. At least 24 h elapsed between iniections and 48 h elapsed between iniections through any one guide. Iniections were given 80 to 100 min after fresh feed was added to the buckets; this initial feeding period helped to synchronize feeding of animals at time of iniection. Feed

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FIc. 1. Placement of intracranial iniections. Cross sections are planes A31 to A26 of Richard's arias (26), i.e., 31 to 9.6 mm anterior to the ear bars. 1, 2, 3, carbachol induced a feeding response; loci marked 2 and 3 were those used in Experiments II and III, respectively. Q, a drinking response; (), both feeding and drinking; ~ , neither feeding nor drinking. All injection loci are on the left. Scales in ram. Superscript I, 2, 3 indicates 1, 2, and 3 mm

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austral to A31; superscript 4 indicates 1 ram caudal to A26. AHA, anterior hypothalamic area; AHD, dorsomedial hypothalamic area; AHL, lateral hypothalamic area; APO, prex~tie area; C. Int, inteaual capsule; FMT, mamillothalamie tract; FX, £ornix; MV, medial nucleus; NHA, anterior hypothalamic nucleus; NHDM, dorsomedial hypothalamic nucleus; NHVM, ventromedial hypothalamic nucleus; Ret, reticular nucleus; viii, third ventric.~e. JOURNAL OF DAIRY SCIENCE VOL.

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rostrocaudal distance of the guide through which ink was injected and the difference between the length of needle used for each injection of drug and that used for injecting the ink. This technique ensured that the points marked on Fig. 1 are not more than .5 mm from the actual point of injection; i.e., the diameter of the circles indicates the maximum error in the location of injection loci. In cases where ink spots could not be seen, 7 / a n sections of imbedded hypothalamic tissue were mounted and stained and injection loci were identified histologically. Means in Experiment I were compared by t-test. Data from Experiments II and I I I were subieeted to analyses of variance from which significant differences were determined by Duncan's multiple range test (12).

Experiment I. Identification of carbachol sensitive loci. Procedure. Injections of 28 nmol of earbachol were first given 4 mm above the ventral dura mater utilizing each guide. In some animals injections were also given at loci 3 and 5 mm above the ventral dura mater. Results. In previous experiments under the same conditions (9, 11) sheep seldom ate more than 100 g of feed in the 60 rain following control or sham injections. A feeding response in this experiment is defined, therefore, as a feed intake of 100 g or more in the 60 min following injection of carbachol. Fig. 1 shows the results of 77 injections at a total of 69 loci in 13 sheep; a feeding response followed 25 injections (mean intake, 175 +_ 15 g, SE, in 60 min compared with 44 ___ 6 g for mean no response intake). Eleven injections resulted in a drinking response, which is defined as an intake of 500 ml or more of water in 60 rain, and three injections were followed by both feeding ( > 1 0 0 g) and drinking (>1,000 ml). Thirty-nine loci gave no response. There were no significant differences in feed or water intakes between first and second injections in the seven loci which were injected twice. Intraperitoneal temperature increased during feeding responses by .14 __+ .07 C during the 60 min following injection, compared with a decrease of .02 ___ .06 C when feeding was not elicited; drinking and feeding-with-drinking responses were accompanied by decreases of .33 _ .14 and .40 __+ .42 C, respectively. None of the differences between these means was significant.

Experiment H. Dose-response ~elationships. JOURNAL OF DAIRY SCIENCE VOL. 57, NO. 8

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Procedure. Eight sheep from Experiment I were injected with the following doses of carbachol at loci (marked "2" in Fig. 1) in which carbachol previously elicited feeding but not drinking: 14, 7, 3.5, 0, 56, 112, and 1.75 nmol (in order of injection). Data collected in Experiment I were used for the 28 nmol dose. Results. Feed intakes during the 60 rain after injection of the various doses of carbachol are in Fig. 2. Intakes were greater than control with 3.5, 112 ( P < . 0 5 ) , and 28 (P<.01) nmol. There were no significant differences at 120 min or 24 h. Water intake (Fig. 2) was increased above control by 14, 56, and 112 nmol carbachol ( P < . 0 5 ) . There were no significant differences between treatments either in the ratio water intake:feed intake at 60 min or in intraperitoneal temperature. Experiment 1H. Effects of antagonists and agonists. Procedure. Eight sheep were injected with the following drugs at loci (marked "3" in Fig. 1) in which carbaehol previously elicited feeding but not drinking: earbaehol, 28 nmol; atropine sulphate, 28 nmol, a cholinergie blocker, followed 3 min later by carbachol, 28 nmol; synthetic CSF; atropine sulphate, 28 nmol; phenoxybenzamine (PILE), 120 nmol, an a adrenoceptor antagonist, followed 3 rain later by ,carbaehol, 28 nmol; LB-46, 120 nmol, a fladrenoceptor antagonist, followed 3 min later

CARBACHOL E L I C I T E D F E E D I N G AND DRINKING

bachol treatment was followed by a water intake at 60 rain which was almost ten times that following CSF, Table 1. There were no significant differences between water to feed intake ratios or intraperitoneal temperatures (Table

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Fie. 3. Feed intakes (with SE) during 30, 60, and 120 min following iniections of carbachol and antagonists at the loci marked ® in Fig 1. (n=8). At each of the times of weighing, columns with the same subscript letter do not differ significantly. by carbaehol, 28 nmol (in order of injection). All eight sheep were given the same treatment on any one day. Results. The carbachol treatment caused significant increases in feed intake at 30, 60, and 120 min (Fig. 3). This effect was almost completely blocked by a pre-injeetion of atropine but was not altered by pre-injections of PHE or LB-46. Atropine injections alone had no significant effect on feeding. There were no differences in 24 h intakes (Table 1). Water intake was variable between sheep. There were no significant differences between treatment means although the LB-46/car-

The parts of the brain investigated in the screening study (Experiment I) were somewhat more dorsal than intended and ranged from the internal capsule at 34 mm to the mammillothalamic t r a ~ at 25 m m rostral to the internal auditory canal, with most injections 29 to 31 mm anterior to the internal auditory canal. Of five injections in the ventromedial nucleus of the hypothalamus, four caused a feeding response which in two cases was accompanied by a drinking response. The other loci at which 28 nmol of carbaehol elicited feeding were scattered throughout the areas studied. Only one drinking locus was found caudal to plane A29 (Fig. 1), which agrees with results from work with rats where anterior hypothalamic and preoptic placements of earbachol were more effective in eliciting drinking than were more posterior injections (22). Similar to responses with carbachol in rats (18) and adrenoceptor agonists in sheep (5, 7, 8, 9), feeding and drinking loci in this experiment seemed to be discrete in that injections only 1 mm away often had no effect. T h e repeatability of responses to injections in the same locus suggests that feeding or drinking occurred following the stimulation of specific areas of the limbic system and was not merely a random occurrence. Also, because of the localization of the responsive loci, it is unlikely

TABLE 1. Water intake, body temperature change, and 24-h feed intakes following intraeranial injections of carbaehol with atropine, phenoxybenzamine, or LB-46. Treatment (nmol)

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a Because the mean of this ratio for individual sheep was in some cases biased by one or two anlreals with low feed intakes or highwater intakes, group totals for each treatment were used for calculating ratios in this column. No differences between treatments were significant. CSF ---- eerebrospinal fluid, Carb = carbaehol, Atr -- atropine, PHE ---- phenoxybenzamine. JOURNAL OF DAIRY SCIENCE VOL. 57, NO. 8:

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that the effect of intrahypothalamic injections of carbachol is via its diffusion into the ventric~alar system and transport by the CSF t9 other Jreceptors, as envisaged by Routtenberg (27). ~In a separate experiment, h3wever, we observed a small increase in feed intake following lateral ventricular iniections of 28 nmol carbachol (6). When feeding occurred, it was accompanied, as expected (10), by increases in body temperature. Drinking, on the other hand, was followed by declines in temperature, and it seems likely that this was caused by ingestion of large volumes of water, although a controlled experiment would be necessary to demonstrate this dearly. The dose of carbachol selected for the initial screening (28 nmol) was based on results with other species (e.g., 20), bearing in mind the larger size of sheep. As explained earlier, this dose was omitted from the dose-response curve experiment because data had previously been obtained with 28 nmol at the same loci in the initial screening. Unfortunately, there is a discrepancy in the results which shows up clearly in Fig. 2 but which cannot be explained. With the exception of this 28 nmol dose, water intake increased with feed intake, as is usually the case with sheep (14), and the water to feed intake ratio was not affected by carbachot dose. There was no preprandial drinking at these feeding loci such as was usually seen at drinking loci. The increase in water intake with increasing dose of carbachol is presumed to be a result of the greater intake of dry feed. This is in contrast to results of a similar experiment with rabbits (29) where, in the same locus, water intake was stimulated by low doses of carbachol (up to 5.9 nmol) but depressed by the higher doses (over 11.8 nmoI) which stimulated feeding. The optimum intrahypothalamic dose of carbachol to elicit drinking in rats (22) and feeding in rabbits (31) is 5.5 nmol per kg of body weight; 28 nmol is about .5 nmol/kg for a sheep. The largest dose used in this experiment (112 nmol) is still only 2 nmol/kg body weight; feed intake was stimulated to a similar extent by doses ranging from 3.5 to 113 nmol (with the exception of the 28 nmol treatment). Both in the rabbit (30) and eat (23) hypothalamic doses of carbachol above the optimal dose were followed by less than maximal feeding, and it will be necessary to use higher doses in sheep to establish whether a similar inverted-U dose-response curve holds true for this species. The specificity of the carbachol stimulus is JOURNAL OF DAIRY SCIENCR VOL. 57. NO. 8

illustrated in Experiment III. Feed intakes following atropine injections were similar t3 control whether or not this eholinergic antagonist was followed by carbaehol. To test atropine for an effect depressing feed intake, a fast should precede the test to allow a more sensitvie measure of depression. Adrenergie blockers, whether ez or t , did not antagonize the feeding elicited by carbachol. This is evidence that carbachol was not affecting feeding indirectly by affecting adrenoceptor pathways. Adrenoceptor agonists elicit feeding when injected into the anterior medial hypothalamus of sheep (2, 5, 7, 8, 9, 11). Feeding to norepinephrine and drinking to carbachol can be induced in identical loci in the rat (15). Experiments described here do not show whether carbaehol is stimulating a physiological feeding circuit in the brain by supplementing endogenous acetylcholine or whether it is only a pharmacological effect. There are two pieces of incomplete evidence which suggest that the latter might be true: 1) Atropine alone did not depress feed intake below that following control injections, the opposite of carbachol. 2) Injections of eserine, an anticholinesterase which would be expected t~ increase endogenous acetylcholine, had no significant effect on feeding at c~rbaeholbound feeding loci (Forbes and Baile, unpublished data). Other doses of atropine and eserine need to be used before conclusions can be firm on these two points. In favor of a physiological role for a cholinergie feeding pathway are: 1) Carbachol-induced feeding was specific, both chemically and anatomically; carbachol-induced drinking was anatomically specific but chemical specificity has yet to be established in the sheep. 2) Feeding and drinking behaviors induced by carbaehol appeared normal and only in two eases was vocalization and excitement observed. The slight depression in body temperature following carbaeholinduced drinking was probably a result of loss of body heat used in warming the water to body temperature, although a specific effect on the temperature regulating center of the hypothalamus cannot be ruled out (17). Acknowledgments This work was supported in part by a grant from the National Science Foundation (GB-

CARBACHOLELICITED FEEDING AND DRINKING 9.8836). Chemical sources: carbachol - carcholin (carbachol, MSD) - Merck Sharpe & Dohme, atropine sulphate-CalBiochem, phenoxybenzamine HC1 - SK&F, LB-46 -- Sandoz Pharmaceuticals. References

(1) Antunes-Rodriguez, J., and S. M. MeCann. 1970. Water, sodium chloride, and food intake induced by injections of cholinergie and adrenergic drugs into the third ventricle of the rat brain. Prec. See. Exp. Biol. Med. 133:1464. (2) Baile, C. A. 1974. Putative neurotransmitters in the hypothalamus and feeding. Fed. Prec. 33:1166. (3) Baile, C. A., and J. M. Forbes. 1973. Diffusion of noradrenaline between hypothalamus and cerebral ventricles of sheep. J. Physiol. 234:44. (4) Baile, C. A., and 1. M. Forbes. 1974. Control of feed intake and the regulation of energy balance in nuninants. Physiol. Rev. 54:160. (5) Baile, C. A., L. F. Krabill, and C. W. Simpson. 1973. Feeding elicited by a and adrencceptor agonists in sheep and cattle. Pharrn. Biochem. Behav. 1:531. (6) Baile, C. A., and F. H. Martin. 1974. Parotid secretion and feeding in sheep following intraventrieular iniections of l-norepinephrine, dl-isoproterenol, pentobarbital, and carbachol. J. Dairy Sci. 57:308. (7) Baile, C. A., and F. H. Martin. 1973. Relationship between prostaglan~n E~, polyphloretin phosphate and a and ~ adrenoceptor-bound feeding loci in the hypothalamus of sheep. Pharm. Biochem. Behav. 1:539. (8) Baile, C. A., F. H. Martin, J. M. Forbes, R. L. Webb, and W. Kingsbury. 1974. Intrahypothalamie injections of prostaglandins and prostaglandin antagonists and feeding in sheep. J. Daiz~¢Sci. 57:81. (9) Baile, C. A., F. H. Martin, C. W. Simpson, J. M. Forbes, and J. S. Beyea. 1974. Feeding elicited by ~ and /3 adrenoceptor agouists injected intrahypothalamieally in sheep. J. Dairy Sci. 57:68. (10) Baile, C. A., and J. Mayer. 1968. Hypothalamic temperature and regulation of feed intake in goats. Amer. J. Physiol. 214:677. (11) Baile, C. A., C. W. Simpson, L. F. Krabill, and F. H. Martin. 1972. Adrermrgie agonists and antagonists and feeding in sheep and cattle. Life Sei. 11:661. (12) Duncan, D. ]3. 1955. Multiple range and multiple F tests. Biometrics 11:1. (13) Fisher, A. E., and J. N. Coury. 1962. Cholinergie tracing of a central neural circuit underlying the thirst drive. Science 138: 691.

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(14) Forbes, J. M. 1968. The water intake of ewes. Brit. J. Nutr. 22:33. (15) Grossman, S. P. 1962. Direct adrencrgie and cholinergie stimulation of hypothalamie mechanisms. Amer. J. Physiol. 202:872. (16) Grossman, S. P. 1968. Hypothalamie and limbie irdtuences on food intake. Fed. Prec. 27:1349. (17) Hulst, S. G. T. 1972. Intracerebral implantation of carbaehol in the rat: its effect on water intake and body temperature. Physiol. Behav. 8:865. (18) Hulst, S. G. T., and D. de Wied. 1967. Changes in body temperature and water intake following intracerebral implantation of carbachol in rats. Physiol. Behav. 2:367. (19) Krikstone, B. J., and R. A. Levitt. 1970. Interactions between water deprivation and chemical brain stimulation. J. Comp. Physiol. Psychol. 71:334. (20) Levitt, R. A., and R. P. Boley. 1970. Drinking elicited by injection of eserine or carbachol into rat brain. Physiol. Bchav. 5:693. (21) Miller, N. E. 1965. Chemical coding of behavior in the brain. Science 148:328. (22) Miller, N. E., K. S. Gottesman, and N. Emery. 1964. Dose response to earbachol and norepinephrine in rat hypothalamus. Amer. J. Physiol. 206:1384: (23) Milner, .|. S., D. M. Nance, and D. E. Sheer. 1971. Effects of hypothalamic and amygdaloid chemical stimulation on appetitive behavior in the cat. Psychon. Sci. 23:25. (24) Myers, R. D. 1969. Temperature regulation: Neurochemical systems in the hypothalamus. Page 506 in The Hypothalamus. W. Haymaker and C. C. Thomas, eds. Springfield, Illinois. (25) Pappenheimer, J. R., S. R. Heisy, E. F. Jordon, and J. De C. Downer. 1962. Perfusion of the cerebral ventricular system in unanesthetised goats. Amer. J. Physiol. 203:763. (26) Richard, P. 1967. Stereotaxique du cervean de Brebis "Prealpes-du-Sud". Paris: InstXtut National de la Recherche Agronomique. (27) Routtenberg, A., and J. B. Simpson. 1971. Carbachol-induced drinking at ventrienlar and subfornieal organ sites of application. Life Sci. 10:481. (28) Singer, G., and J. Kelly. 1972. Cholinergic ,and adrenergie interactkm in the hypothalamie control of drinking and eating behavior. Physiol. Behav. 8:885. (29) Sommer, S. R., D. Novin, and M. Levirto. 1967. Food and water intake after intrahypothalamie injections of earbachol in the rabbit. Science 156:983. (30) Tarttelin, M. F. 1971. A radiographic method for accurately locating deep-seated structures in the brain stem of sheep. Physiol. Behav. 7:789. JOURNAL OF DAIRY SCIENCE VOL. 57, NO. 8'