Cholecystokinin acts through catecholaminergic mechanisms in the hypothalamus to influence ingestive behaviour in the rat

0028-3908/84 $3.00 f 0.00 Copyright :w? 1984 Pergamon Press Ltd

Neuropitarmacoio~y Vol.23,No. 1I, pp. 1351-1356, 1984 Printed in Great Britain. All rights reserved

CHOLECYSTOKININ

ACTS ~ROUGB

CATECHOLAMINERGIC ~CHANIS~S

INGESTIVE

BEHAVIOUR

S.H. Tsai, E. Passaro

Department

of Surgery,

Defence

Medical

Medical

Center

**Department

Tri-Service

Centre,

Taipei,

(Wadsworth

of Physiology Centre,

Jr *.and M.T. Lin**

General

Taiwan,

& Biophysics,

(Accepted

TO INFLUENCE

IN THE RAT

Hospital

R.O.C.;

Div.), Los Angeles,

Taipei,

IN THE HYPOTHAL~US

*Surgical

California,

National

Taiwan,

and National

Defence

Service

VA

USA; and Medical

R.O.C.

17 Oc.tobeti1984)

Summare- Administration of cholecystokinin (CCK)(O.2 - 0.6 pg in a volume of 2 ~1) into the lateral cerebral ventricle caused a decrease in intake of food but a relative increase in intake of water (or waterto-food ratio) in rats, To determine whether the anorexic actions of CCK were mediated through the hypothalamic nuclei, rats were infused with CCK (0.02 - 0.12 pg in a volume of 0.5 pl) through previously implanted hypothalamic cannulae. Administration of CCK into the lateral hypothalamus, but not the anterior hypothalamus or ventromedial caused decreased intake of food and a relative increased hypothalamus, by injection of CCK intake of water. In addition, the responsesinduced into the hypothalamus were completely abolished by selective depletion of catecholamines in the hypothalamus (eg. noradrenaline and dopamine) with intra-hypothalamic injection of 6-hydroxydopamine. Intraperitoneal administration of 0.12 pg of CCK had no effect on the intake of food and water in rats. The data indicate that CCK acts through catecholaminergic mechanisms in the hypothalamus to influence feeding behaviour

Recently, much evidence has accumulated to suggest that cholecystokinin (CCK) has a neuroregulatory role in the central nervous system in addition to its well known hormonal functions in controlling digestion (Gibbs, Young and Smith, 1973; Dockray, 1982). For example, administration of CCK has been shown to produce satiety (Gibbs et al., 1973), suppressed exploratory behaviour (Crawley, Hays and Paul, 1981) and analgesia (Zetler, 1980) in rats. When administered peripherally, the gut-brain peptide, CCK, suppressed intake of food and sham feeding in rats (Gibbs et al.,1973; Grinker, Schneider, Ball, Cohen, Strochmayer administration of CCK was found to suppress feeding in and Hirsch, 1980). Intraventricular sheep but failed to decrease feeding in rats (Della-Fera and Baile, 1979). However, the intake of food in rats deprived of food for 4 hours has been reduced by a bolus intraventricular injection of caerulein, which has been shown to decrease the intake of food when administered systemically (Stern, Cudillo and Krupper, 1976). Thus, it appears that the mode and site of action of CCK in producing satiety is uncertain. the effects of administration of CCK into the cerebral In the present investigation, ventricles and various nuclei of the hypothalamus, on the intake of food and water, were assessed in normal rats and in rats with selective depletion of catecholamines in the hypothalamus.

METHODS were performed on male Sprague-Dawley rats, weighing Experimental animals: Experiments between 230 and 265 g. The animals were fed with a dry powder chow that is commonly used for chickens (Taiwan Sugar Co.). They were housed in wire-mesh cages in a room maintained at 22+2.G°C with natural light-dark cycles. The feeding and drinking experiments were performed on unrestrained conscious animals. Intake of food and water, and body weight were measured daily at 10.00 h. sodium (6 mg/lOO Implantation of cannulae: Each animal was anaesthetized with pentobarbital and a burr hole was drilled in the ealvarium above the lateral cerebral ventricle 8.7 i.p.) 1351

1352

Preliminary

Notes

or the hypothalamus. For the direct injection of drugs into the lateral cerebral ventricle, the lateral hypothalamus, the anterior hypothalamus or the ventromedial hypothalamus, a stainless steel cannula consisting of a guide tube with a snug-fitting trocar and a cannula insert were introduced into the tube at the time of the injection (Lin, Chu and Leu, 1983; Lin, Wu, Chandra and Tsay, 1982). The cannula guide tube with trocars was implanted using the stereotaxic atlas and coordinates of Kiinig and Klippel (1963). The cannula insert was connected to a lo-u1 Hamilton microsyringe by PE 10 polyethylene tubing. The volume of injection down each cannula was 2 ~1 for the cerebral ventricle or 0.5 1~1 for the hypothalamus. A period of 2 weeks was allowed to permit the animal to recover from the operation Measurement of intake of food and water: Dry powdered chow was dispensed from a special spillage-reducing cup and water from a graduated cylinder with spout, and the intake measured. Either control vehicle or drugs were administered into the lateral cerebral ventricle or the hypothalamus about 1 min before the presentation of food or water to the animal. Normally, a rat drinks water in an amount proportional to the food ingested, ie. the amount of food ingested influences the amount of water consumed (Fitzsimons and Magnen, 1969 Lin, Yin and Chai, 1972). In the present investigation the relative intake of water and the water-to-food ratio were used to measure the change in intake of water. Drug solution:Rats were injected intraventricularly or intrahypothalamically with a dose of synthetic C-terminal octapeptide (CCK) (Sincalide, Squibb). A solution of 6-hydroxydopamine (B-OHDA) hydrobromate (Sigma Chemical Co., St. Louis, Missouri, USA) was made by dissolving the drug in 0.9% saline plus 0.1% ascorbic acid. Histological verification: After the experiments were completed, the animals were killed with an overdose of sodium pentobarbital and the cerebral circulation was perfused with 0.9% saline, followed by 10% (v/v) formalin solution. Later, sections of the fixed brain were cut at 40 urn and stained with thionin so that the stereotaxic coordinates of the cannula could be verified. Assay of monoamines: In these experiments the animals were prepared for the biochemical assay of the monoamine content of the hypothalamus. A total of 8 rats with intrahypothalamic injections of 30 ug of 6-hydroxydopamine were used. Five days after the treatment, the animals were killed and the brains removed and assayed for noradrenaline, dopamine and 5-hydroxytryptamine as described previously (Lin, 1979;1980). Statistical analysis: The significance of the diferences between Student's t-test. A value of piO.05 was taken to be significant.

means was determined

by

RESULTS Effects of intraventricular administration of CCK on intake of food and water. Figure 1 summarises the results of the present series of experiments, in which the intake of food and water was measured or calculated for the 9 animals after the intraventricular injection of normal saline or CCK (0.2 - 0.6 ug). Intraventricular administration of normal saline had no effect on the feeding or drinking behaviour of the rats. However, a temporary decrease in the intake of food and a temporary relative increase in in rats by the intraventricular injection of CCK. the intake of water were both provoked The intake of food and water returned to pre-injection levels about 1 - 3 days after the injection of the drug. It was evident that the ingestive responses induced by CCK were dose-dependent (see Figure l), Effects of intrahypothalamic administration of CCK on intake of food and water. Figure 2 shows the daily changes in intake of food and water before an after an intrahypothalamic injection of either 0.9% saline or various doses of CCK for the 8 animals. of CCK (0.02 - 0.12 pg) into the lateral hypothalamis caused a Again, administration decrease in intake of food and an increase in the intake of water. Intraperitoneal injection of 0.12 pg of CCK had no effect on the intake of food and water in 6 animals tested. The site of microinjection of CCK in the hypothalamus was determined in 17 rats and are shown in Figure 3. Sites denoted by open circles indicate no ingestive response; those denoted by solid circles indicate that hypophagia and hyperdipsia were produced. The figure shows that the lateral hypothalamus is a specific locus for the ingestive action of CCK. Administration of the same amount of CCK into the anterior hypothalamus or the ventromedial hypothalamus had no effect on the intake of food and water in rats. Administration of CCK (0.12 up) into the lateral hypothalamus caused no sedation. Effects of depletion of catecholamines in the hypothalamus on ingestive responses to CCK. The concentrations of noradrenaline, dopamine and 5-hydroxytryptamine in normal rats and in rats injected in the lateral hypothalamus with 6-hydroxydopamine (30 pg) are shown in Table 1. An intrahypothalamic injection of 30 pg of 6-hydroxydopamine caused a significant depletion of noradrenaline to 37.1% of control and of dopamine in the hypothalamus to 33.3% of control, while the concentration of 5-hydroxytryptamine in the hypothalamus was not significantly reduced at 94.7% of control. In addition, it was found that selective depletion of nor-adrenaline and dopamine in the hypothalamus abolished the ingestive

Prel

Sallne

,” 270 i .Y? ; 250

iminary

Notes

1353

0.2pg

0.4/.Lg

0.6pg

CCK

CCK

CCK

<

I

I

I

0

5

IO

I

15

I

I

20

25

I

I

30

35

Day Figure 1. The changes in body weight, food intake and relative water intake both before and after an intracerebroventricular injection of 0.9% saline or cholecystokinin (CCK) in 9 rats at an ambient temperatSignificant differences from the pre-injection value are ure of 22°C. indicated by an asterisk (*) (p
60ng 270,

Yne

JCCK

120 ng CCK

b

20ng CCK

I

120ng 3O/rg 6- OHDA CCK

b

&

250 230’

t 20

30

40

50

Day Figure 2.The changes in body weight, food intake and relative water intake before and after an intrahypothalamic injection of 0.9% saline, cholecystokinin (CCK) or 6-hydroxydopamine (B-0HDA)for 6 rats at an ambient temperature of 22OC. Significant differences from pre-injection values are indicated by an asterisk (*) (p
responses to intrahypothalamic administration of CCK is shown in Figure 2. In a comparable (30 ng) alone into the lateral hypostudy, a unilateral injection of 6-hydroxydopamine thalamus had little or not effect on the intake of food and water in rats (n=4).

1354

Preliminary

Notes

5 '6mm8

I

! : 76543210

!

:

:

:

:q

Figure 3. Outline section of the brain stem showing the sites of Sites denoted by solid cicles (01 injection of 0-CCK (20 ng in 0.5@). indicate that hypophagia was produced; thosedenoted by open circles (0) indicate no ingestive response. Anatomical abbreviations: CAIR = capsula interna, pars retiolenticularis; FMT = fasciculus mamillothalamicus; AH = nucleus anterior hypothalami; LH = nucleus lateral hypothalami; 3V = ventriculi tertii; IZ = incerta zona; OT = opticus tractus.

Table

1. Effects

of 6-hydroxydopamine

and 5-hydroxytryptamine

Treatment

Control

vehiclea

6-Hydroxydopaminea (30 Pig)

Animals (n)

on the concentration

of noradrenaline,

dopamine

in the rat hypothalamus. Noradrenaline (UP/P)

Dopamine (lJg/g)

5-Hydroxytryptamine (vg/g) ____~_

8

1.05f0.09

1.61f0.21

1.32kO.14

8

0.39+0.07*

0.54f0.11*

1.24f0.12 -__-

-...._

The values are expressed as the mean + SE. aRats killed 5 days after the intrahypo*Significantly different from thalamic injection of saline or 6-hydroxydopamine. corresponding control values at ~~0.05 using Student's t-test.

DISCUSSION Cholecystokinin was oroginally described as a gut hormone. The main hormonal function of CCK seems to be to control the contraction of the gall bladder and the secretion of pancreatic enzyme. Recently, peripheral administration of CCK was shown to produce a satiety effect in the rat (Gibbs et al., 1973). This observation led to the suggestion that circulating CCK, released from the small intestine by the entry of nutrients after a meal, not only exerted an effect on the digestive organs, but also on the brain, inhibiting feeding. However, it is important to know whether CCK, administered intraperitoneally or is acting at a peripheral site or is penetrating the blood brain barrier intravenously, and acting centrally. It has been demonstrated in the rat that vagotomytiolished the satiety effect of CCK (Smith, Jerome, Cushin, Eterno and Simansky, 1981). In addition, administration of CCK into the carotid artery in sheep was found to be no more effective in inducing satiety than injection into the jugular vein (Grovum, 1982). In a more recent report, it was demonstrated that CCK suppressed intake of food in monkeys by inhibiting gastric

Preliminary

Notes

emptying (Moran and McHugh, 1982). These observations indicate a peripheral the effect of CCK on satiety. This is not supported by the present results.

of action

for

On the other hand, CCK-immunoreactivity has been shown to be present in brain (Innis, Correa, Uhl, Schneider and Snyder, 1979). The amount of CCK found in the brain is comparable to that found in the gastrointestinal tract, but it is of a different molecular size. In addition, histochemical localization has shown that the greatest concentration of CCK occurred in the periaqueductal gray matter and in the hypothalamus (Innis et al., 1979). In fact, both the present and previous results (Della-Fera and Baile, 1979) showed that administration of CCK into the lateral cerebral ventricles had a potent satiety effect in both rats and sheep. The present results also demonstrated that direct administration of CCK, but not of the control vehicle, into the lateral hypothalamus (the feeding centre) suppressed the intake of food in rats. Microinjection of CCK into regions of the brain outside the lateral hypothalamus had little or no effect on feeding. Furthermore, peripheral administration of the same amount of CCK did not affect the intake of food in rats.Thus, it is unlikely that CCK, administered into the cerebral ventricles or the lateral hypothalamus, leaks into the blood stream and acts peripherally. These observations, together with the evidence that CCK can still produce satiety in the presence of lesions of the ventromedian hypothalamus (Kulkovsky, Breckenridge, Krinsky and Woods, 1976), suggest that CCK may act as a putative neurotransmitter in the lateral hypothalamus to mediate the satiety effect in rats.This idea is not supported by the findings of other investigators (Stern et al., 1976). These investigators showed that injections of caerulein into the ventromedial, but not the lateral hypothalamus of the rat, caused a decrease in intake of food. In the present experiments, sedation was not observed after intrahypothalamic administration of CCK. Therefore, at present, it is not possible to account for the fact that satiety was found in the experiments reported here when CCK was injected into the hypothalamus, while others have not seen this effect. One of the areas of appetite control which has been most vigorously investigated concerns the role of monoamines in the hypothalamus. According to the review by Morley (Morley, 1980), the feeding centre in the lateral hypothalamus is thought to be under dopaminergic control. Dopamine agonists facilitate the intake of food, whereas dopamine antagonists suppress feeding induced by deprivation or by injection of 2-deoxy-D-glucose. Also, depletion of dopamine in the nigrostriatal pathway duplicates most of the syndromes induced by lesions of the lateral hypothalamus. On the other hand, a beta-adrenergic system is thought to induce satiety by acting through the lateral hypothalamus.and inhibition of intake of food by hungry rats occurs when the beta-adrenergic agonist isoproterenol is injected into the lateral hypothalamus. Furthermore, selective depletion of noradrenaline in the hypothalamus resulted in a reduction of food intake. In fact, the present results show that the satiety effect induced by administration of CCK into the lateral hypothalamus was completely abolished by selective depletion of noradrenaline and dopamine in the hypothalamus. Therefore, it is thought that CCK, administered into the lateral hypothalamus, may enhance the release of endogenous noradrenaline and/or suppress the release of endogenous dopamine in the lateral hypothalamus to produce the satiety effect. Moreover, the present results showed that the relative intake of water (or water-tofood ratio) was greatly enhanced by intracerebral injection of CCK in rats. It has been suggested that food is an important stimulus for drinking, accounting for the normally close relationship between drinking and feeding in rats(Fitzsimons and Magnen, 1969). Food may induce drinking by activating peripheral nervous mechanisms (eg. the taste of food, its smell, or its presence in the mouth or stomach) in the oropharynx or stomach before it is digested and absorbed. In addition, following the ingestion of food, both the reduction in extra-cellular space and cellular dehydration may stimulate primary thirst which induces drinking. In the present experiments, intracerebral administration of CCK caused a mark reduction in intake of food with little or no change in the absolute level of water intake. The increased relative intake of water induced by CCK in the rat is due to the reduction in food intake.

Acknowledgements:

Supported by the National Science Council of the Republic of China. The authors wish to thank Mr Y.F. Chern and Miss S.Y. Chern for their excellent technical assistance.

1356

Preliminary

Notes

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