ACTA NEUROBIOL. EXP. 1978, 38: 157-165

CATECHOLAMINES IN SOME HYPOTHALAMIC AND TELENCEPHALIC NUCLEI OF FOOD DEPRIVED RATS

Michal STACHOWIAK, Jacek BIAEOWqS and Marek JURKOWSKI

Department of Physiology, Department of Anatomy and Department of General Chemistry

Institute of Medical Biology, School of Medicine Gdadsk, Poland

Abstract. The influence of 48 h food deprivation on the levels of catecholamines was investigated in different hypothalamic and telencep- halic nuclei of the rat brain. There were on changes in the levels of noradrenaline (NA) and dopamine (DA) in the lateral hypothalamic area, dorsomedial nucleus, medial preoptic area and in nucleus of diagonal band (septum). On the other hand we observed a statistically significant decrease of NA and DA in the ventromedial nucleus and the decrease of NA in the arcuate nucleus.

INTRODUCTION

There is a great body of evidence suggesting that lateral hypothalamus (LH) and ventromedial hypothalamic nuclei (N.VM) play an important role in the regulation of food intake. Destruction of lateral hypothalamic "feeding system" caused aphagia and adipsia, whereas large destructions of the ventromedial nucleus resulted in hyperphagia and obesity (2, 6, 19, 20, 26). The most recent findings suggest that lesions restricted to the ventromedial nucleus of hypothalamus were neither necessary nor suffi- cient for, and did not contribute to, the production of hypothalamic obesity (10). Hypothalamic lesions and knife cuts that to produce obesity, damage the nearby ventral noradrenergic bundle or its terminals

(I). Electrostimulation of LH elicited feeding, but electrostimulation of N.VM reduced it (3, 16, 18). Gastric distention reduced ingestive behavior and increased the activity of the ventromedial "satiety center" (3).

Ungerstedt has shown that the catecholaminergic system is essential in the regulation of food intake (32). Destruction of dopaminergic neurons originating in substantia nigra and passing through LH to caudato- putamen, caused by local application of 6-hydroxydopamine into zona compacta of substantia nigra, or ventral tegmentum or LH, resulted in every case in a typical lateral hypothalamic syndrome (34). He concluded that the syndrome was caused by desintegration of the nigrostriatal bundle. Application of 6-hydroxydopamine caudally to substantia nigra has resulted only in a depletion of noradrenaline (NA) in the telencephalon, but has not produced hyperphagia. Ungerstedt asserted that the essential transmitter was dopamine (DA) in the nigrostriatal bundle. Stricker and Zigmond (27) strongly supported this assumption. An intraventricular injection of 6-hydroxydopamine after an earlier treatment with desmethylimipramine and pargyline caused a depletion of telencephalic DA and a very slight reduction of NA concentration. And this depletion of DA was noted as the cause of aphagia and adipsia.

On the other hand, a local application of DA to the "feeding system" in the rat brain has not elicited an increased food intake (5, 11, 25) , whereas NA or its u-agonists have resulted in the stimulation of feeding (4, 5, 11, 24, 25, 37). The data presented suggest the importance of catecholaminergic neurons in the hypothalamic satiety system as well as in the feeding one. It should be noted however, that the problem whether DA or NA or both are crucial in the regulation of food intake is still to be solved.

If activity of hypothalamic adrenergic system is involved in the regulation of feeding in the rat, the activity of such a system and, consequently, catecholamine concentrations should change in relation to the need of food. Van Der Gugten and Slangen observed a positive correlation between in vitro uptake of exogenous NA by caudal hypotha-

Fig. 1. The sites of samples superimposed on 4 even-numbered plates A, B, C, D, modified from the Konig and Klippel rat brain atlas (15). E, microphotograph of the typical sites of samples. Cresyl violet ( X 20). Abbreviations used: ARC, arcuate nucleus; CA, anterior commissure; DM, dorsomedial hypothalamic nucleus; DS, Ciorsal septal nucleus; F, fornix; HA, anterior hypothalamic area; LS, lateral septal nucleus; MFB, medial forebrain bundle (lateral hypothalamic area); (a), anterior; (p), posterior; MT, mamillo-thalamic fascicle; POL, lateral preoptic area; POM, medial preoptic area; ST, stria terminalis and its nucleus; STR, striatum; TD, nucleus of the diagonal band (septum); VM, ventromedial hypothalamic nucleus.

lamus and feeding rate, and found that food deprivation changed the rate of this uptake (36). Glick et al. observed the depletion of hypothalamic content of NA (approx. 20°/o) in 48 h food deprived female rats (9).

Recently, the microdissection technic developed by Palkovits (21) enabled the removal of individual hypothalamic nuclei for biochemical analysis. Using this method, we were able to study the concentrations of NA and DA in discrete hypothalamic regions which can be involved in the regulation of food intake and those which can react to some stressfull factors.

MATERIAL AND METHODS

Wistar male rats, weighing 200-250 g were housed one per cage in diurnal lighting conditions. All animals were allowed free access to water. Control rats were given standard pelleted diet ad lib. and the food deprived ones received no food for 2 days, or were fed only limited amounts of food for 3 days to obtain 12-18010 loss of body weight. The rats were decapitated between 9-11 a. m., brains were quickly removed and frozen in a powdered dry ice and sectioned in the frontal plane with a freezing microtome. Particular nuclei were removed from 600 pm thick sections with stainless steel punches (200-500 pm i.d.) according to Palkovits (21). In order to determine the actual sites of the removed samples, the remaining parts of the brain slices were fixed in 10°/o formol-calcium with the addition of sucrose and further dissected on the freezing microtome to obtain 60-90 pm thick sections. These sections were stained with cresyl violet and observed under microscope (see Fig. 1).

Biochemical analysis. The samples of the same nuclei from three to four rats were pooled together to obtain a portion of approximately 150 ug of protein. The tissue was homogenized in microhomogenizers containing 150 p1 of 0.1 M perchloric acid with 7 mM MgSO, and O.lO/o EGTA. 20 pl of homogenate were removed for determination of protein by the method of Lowry et al. (15), using bovine serum albumin as a standard. The microhomogenizers were then placed in adaptors and centrifuged at 15,000 X g for 20 min. The catecholamines were determined in the supernatant by means of enzymatic-isotopic method of Coyle and Henry in Van Der Gugten's et al. modification (35).

Chemicals. All commercial reagents used were of analytical grade. S-adenosyl- (methyl 3H) -methionine from Amersham Radiochemical Cen- tre, noradrenaline, dopamine, adrenaline, normetanephrine, methoxyty- ramine, metanephrine, dithiothreitol, Tris from Sigma Chemical Company were used. The rest of the chemicales were obtained from Koch Light Ltd, and P.O.Ch. - Gliwice.

RESULTS AND CONCLUSIONS

The levels of catecholamines in control animals all investigated nuclei (Fig. 2 and 3) were very similar to those found by Palkovits et al. (22). Food deprivation reduced the concentrations of NA and DA in IV. VM and NA in arcuate nucleus above 36, 39 and 27010 respectively. Those changes were statistically significant (see Fig. 2 and 3).

After acute stress (20 min. of immobilization) Kvetnansky el al. observed a depletion of NA levels in N.VM and supraoptic nucleus, and decrease of DA concentration in dorsomedial hypothalamic nucleus (14). Three hours of immobilization or exposure to cold caused a decrease of catecholamines only in arcuate nucleus (23). Repeated stress enhanced NA and DA concentrations and activity of tyrosine hydroxylase in hy- pothalamus (14, 30, 31). These experimental results, as well as ours,

0-CONTROL a- FOOD-DEPRIVED

TO 3TR POM ARC VM DM MFB(a)MFB(p) P < 0,05

x x p < 0,02 x x x P ' < 0,Ol

Fig. 2. Effect of 48 h food deprivation on noradrenaline content in individual hypothalamic nuclei and septum. Results are expressed as mean values of (n) groups of individual determinations k SDM. Abbreviations - see explanations

of Fig. 1.

suggest the existence of different neuronal pathways involved in the transmission of stress and hunger stimuli.

Knott et al. observed that food deprivation caused elevation of b r a k tryptophan level without affecting tyrosine concentration (12). If their results can be extrapolated to particular brain nuclei, it may be assumed that the depletion of catecholamines in N.VM and arcuate nucleus observed by us was not due to a lowered brain tyrosine content. One of the possible explanations is that the depletion of biogenic amines was caused by their release from synaptic endings, but the possibility that it was caused by the decrease of the tyrosine hydroxylase activity can not be excluded.

In spite of normally low concentration of DA in N.VM, undetectable with hstofluorimetric method (33), the marked DA changes in this

0- CONTROL

B- FOOD -DEPMVED

TD STR POM ARC VM DM MFB(o) MFB(p) x x p< 0.02

Fig. 3. Effect of 48 h food deprivation on dopamine content in individual hypothalamic nuclei and septum. Results are expressed as mean values of (n) groups

of individual determinations k SDM. Abbreviations - see Fig. 1.

region suggest that DA can play some important role in N. VM. It is interesting that in food deprived animals changes in catecholamines concentrations occurred only in the medial region of hypothalamus (N.VM and arcuate nucleus), whereas in the lateral hypothalamic area, commonly consder as a "feeding centre", no changes were found. These facts seem to suggest that there are no direct adrenergic pathways from N.VM to lateral hypothalamus involved in the transmission of hunger stimuli. Recent experimental anatomical studies made with one or another of the variations of the Nauta or Golgi techniques led to a conclusion that the projections of N.VM beyond its border in a lateral direction are relatively few (17, 28, 29).

Brownstein et al. (7) in experiments with surgical isolation of the rat hypothalamus proved that NA in N.VM and arcuate nucleus is loca- lized mainly (90010) in axons originating in brain stem or other extra- hypothalamic regions, whereas the neurons with produce DA are situ- ated in the hypothalamus itself. According to this position, the depletion of NA that we found in those nuclei may be related to increased neuro- nal activity of some extrahypothalamic regions, whereas DA decrease in N.VM may result from changes of activity of intrahypothalamic dopaminergic neurons.

Preliminary results of our recent experiments suggest that intraperi- toneal injection of glucose 1.5 h before sacrifice in food deprived rats elevated the concentration of catecholamines in arcuate nucleus to the level of control animals.

At the present stage of our investigations it is difficult to interpret exclusively the results of our finding. However the results so-far obtained may be a step to further experiments which are at the moment con- tinued.

This investigation was supported by Project 10.4.1.01 of the Polish Academy of Sciences.

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Accepted 1 March 1978

Michal STACHOWIAK, JACEK BIAtOWqS and Marek JURKOWSKI, Institute of Medical Biology, School of Medicine, Debinki 1, 80-211 Gdansk, Poland.