Electrical activity of hypothalamus during activation of pituitary-adrenocortical system

Brain Research, 156 (1978) 67-74 ~ Elsevier/North-Holland Biomedical Press

67

E L E C T R I C A L A C T I V I T Y OF H Y P O T H A L A M U S D U R I N G A C T I V A T I O N OF P 1 T U I T A R Y - A D R E N O C O R T I C A L SYSTEM

A. A. FILARETOV and LJUBOV V. VASSILEVSKAYA Laboratory of Experimental Endocrinology, 1. P. Parlor Institute of Physiology, Academy of Sciences of the U.S.S.R., Leningrad (U.S.S.R.)

(Accepted February 23rd, 1978)

SUMMARY Multiunit activity in anterior (AHA), medial (MHA) and lateral (LHA) areas of hypothalamus during activation of the pituitary-adrenocortical system was measured in chronic experiments on rabbits. Immobilization causing blood-corticosteroid levels to rise was followed by the excitation of most pools of neurons in MHA, and by inhibition in A H A and LHA. When immobilization failed to induce stimulation of M H A (as a result of pretreatment with large doses of cortisol), it did not cause blood-corticosteroid levels to rise, either. The switching offactivity in A H A and M H A is considered to be a manifestation of functional changes which are responsible for stress-induced activation of the adrenocortical system.

INTRODUCTION The problem of functional organization of the hypothalamus involved in the regulation of adenohypophyseal functions is one of the essential ones in the field of neuroendocrinology. Functional organization presents a definite relationship of hypothalamic areas which form a centre regulating a particular function. Successes scored in research during the last decade or so have thrown some light on this problem. Of major importance in this respect are the findings on the localization of releasing factor-producing neurons, clarification of intrahypothalamic fiber connections and data on differentiation of influences of some areas of the hypothalamus on the adenohypophysis. Being the regulation centre of the pituitary-adrenocortical system, the hypothalamus inhibits it by feedback mechanisms4,5,9,a0, 22 or activates it during stress 2,3, 11,16,23. The differentiated participation of hypothalamic areas in adrenocortical

68 regulation was shown by local lesions or stimulation (for refs. see 18) and electrical activity changes when the pituitary adrenocortical system was subjected to the influence of some factors 8,8,1~,15A9,~°. In an earlier publication by one of the authors 12 it was reported that anterior (AHA), medial (MHA) and lateral (LHA) hypothalamic areas show different responses when midbrain is stimulated resulting in activation of the adrenal cortex, and it was suggested that the changes in AHA and MHA activity are associated with the regulation of the pituitary-adrenocortical system. The present study is concerned with changes in AHA, MHA and LHA activity during stress-induced activation of the pituitary-adrenocortical system. METHODS

Multiunit activity and corticosteroid level variation, after immobilization, were followed in chronic experiments on Chinchilla male rabbits weighing 2.5-3 kg.

Multhmit activity recording Nickel-chromium semimicroelectrodes, tip diameter 50/~m, were introduced into the hypothalamus 1.5-3 weeks before experiments. The following areas of the hypothalamus were studied2t : (1) AHA A 2; V from --2 to --3.5; S, D from 2 to 3, (2) MHA AP 0; V from --3.2 to --6.3; S, D from 0.4 to 1.4, (3) LHA AP 0; V from --3.8 to --5.2; S, D from 2 to 3, Fig. I. Electrodes were implanted in different regions in any one animal. After the operation 7-10 days were allowed for the animals to recover from surgery and acclimatize to experimental conditions. During the experiment the animals were partially restrained by collars. Discharge rate of neuronal pools in AHA, MHA and LHA during immobilization was studied in 32 rabbits. The animals were spread flat with fore- and hindlegs and body fixed. In experiments on 7 rabbits (some of them were used in the previous series), the response of AHA and MHA neuronal pools to immobilization was measured in the animals with the pituitary-adrenocortical system blocked. Blocking was induced by -O-

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69 cortisol administration 3-10 days prior to experiment. Cortisol-acetate (Richter, Hungary), 10 mg/kg, was injected intraperitoneally twice a day, for 2 days. Experiments were conducted not more than once or twice a week; each animal was immobilized not more than three times. The electrical activity of neuronal pools was recorded on 1 or 2 channels at the same time, beginning from 30 min before and to 60 min after onset of immobilization. Electric signals from the microelectrodes were fed into a cathode follower and via an amplifier to a standardized pulse former. Then, signals were delivered to a pulse counter. Pulse rate was measured for 15 sec at least once during 2-min intervals. Spikes were photographed. At the end of the experiments animals were sacrificed, the brain was removed and the localization of the electrode tips was established on frontal sections.

Measurements of pituitary-adrenocortical system activity Corticosteroid levels in blood plasma were measured in chronic experiments on rabbits. Blood samples were taken from the auricular vein, which was cut 50-60 rain before the first blood sample was drawn. Stimulation commenced 1-2 rain after the first sample, another 4 samples were taken during 60 min when the animal was immobilized. Measurements ofcorticosteroid levels were carried out on 4 intact animals which were immobilized and 5 immobilized rabbits with pituitary-adrenocortical system blocked. Corticosteroid levels were established fluorimetrically 24.

Statistical analysis Discharge rates before (during 30 min) and after the beginning of immobilization (during not less than 10 min) were compared for each pool of neurons. Statistical significance of changes in discharge rates of neuronal pools and corticosteroid levels was established on the basis of the t-criterion of Student. When summarized findings on responses were assessed (see Tables I and II), the d-criterion was employed a. The use of the latter criterion gave the significance of deviation of the number of pools characterized by a given reaction (excitation, inhibition, biphasic response, no changes) from random distribution. RESULTS Immobilization resulted in the activation of the pituitary-adrenocortical system as indicated by a rise in blood-corticosteroid levels (Fig. 2). It became apparent as early as after 5 min and the situation persisted throughout the whole experiment. Immobilization stress led to the development of changes in the discharge rate of neuronal pools in AHA, M H A and LHA (Fig. 3). As shown in Table I, discharge rate decreased in most neuronal pools of A H A , while in the majority of them in M H A it increased. Most of the pools in L H A were inhibited during immobilization.

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Fig. 3. Neurogrammes of responses of neuronal pools in 3 hypothalamic areas to stress, The discharge rate decreases in A H A and LHA and increases in MHA.

TABLE I

Responses of neuronal pools of hypothalamus to stress Hypothalamic area

AHA MHA LHA Total

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Fig. 4. Averaged responses (mean -L S.E.) of neuronal pool groups (which form the majority) in AHA, MHA and LHA to immobilization. Discharge rates for each interval of measurement (6 rain) are expressed as a percentage ratio to mean background frequency for the whole period of registration (30 rain) taken as 100K. - . . - , responses of neuronal pool groups which have reactions opposite to those forming the majority in AHA, MHA and LHA. * Significant difference from any measured value of background, P < 0.05. Arrow indicates onset of stress.

TABLE lI

Responses o f neuronal pools' o f hypothalamus to stress in animals with pituitary-adrenocortical system block Hypothalamic area

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72

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Fig. 5. Responses of two groups of neuronal pools (mean 4- S.E.) to immobili~fion in an animal with pituitary-adrenocorticalsystem block. Principle of graph plofing is the same as in Fig. 4. * Significant difference from any measured value of background, P < 0.05. For comparison, response to stressin intact animal is shown; cuGc (- - -) is copied from Fig. 4. The values of P are ~Vcn for differencesin responses of intact animals and those with pituitary-adrcnocorticalsystem block.

Fig. 4 demonstrates mean values for changes in discharge rate for those groups of neurons which form the majority (asterisks in Table I) in AHA, MHA and LHA. Injection of 10 mg/kg of cortisot (twice a day, for 2 days) resulted in blocking of the response of the pituitary-adrenocortical system to immobilization (Fig. 2). Immobilization of animals with pituitary,adrenocortical block was followed by a decrease in discharge rate of most neuronal pools in AHA as well as in intact rabbits (Table II). Fig. 5 demonstrates the reactions of those neuronal pools of AHA which showed a decrease in discharge rate under stress conditions. The differences in the responses of intact animals and rabbits with the pituitary-adrenocortical system blocked were not considerable. In MHA, however, the reaction to immobilization was different from that of intact animals. In the animals with block, inhibition prevailed in MHA (Table II). A comparison of groups of neuronal pools in MHA, which became activated under stress conditions, showed the increment in discharge rate during the blocking of the pituitary-adrenocortical system to be less than in unblocked animals (Fig. 5).

73 DISCUSSION

Stress-induced activation of the adrenal cortex involves changes in the activity of the 3 hypothalamic areas. In MHA, most of the neuronal pools respond to stress by a rise in discharge rate, while in AHA they respond by a decrease. It may be supposed that such a pattern of activity, i.e. inhibition of AHA and excitation of MHA, is responsible for activation of the pituitary-adrenocortical system. This is in line with the data showing that activation of the adrenal cortex induced by stimulation of the midbrain is accompanied by the excitation of M H A and inhibition of AHA lz. Stressinduced excitation of MHA is consistent with the data showing that the medial hypothalamus is responsible for activation of pituitary-adrenocortical systemV,13,z3. Evidence in support of the hypothesis that immobilization-induced excitation of MHA is responsible for the activation of the adrenal cortex rather than other vegetative reactions is provided by the experiments involving the blocking of the pituitary-adrenocortical system. Under blocking conditions immobilization induces neither excitation of MHA nor a rise in corticosteroid level. When the pituitary-adrenocortical system is blocked, the activity of neuronal pools in AHA reveals changes similar to those in intact animals. It may be supposed that the effect of block-inducing cortisol (in large doses) is restricted to medial hypothalamus. It may be suggested that, under blocking conditions, impulses induced by immobilization are delivered to AHA, thus inhibiting this area. These impulses, however, do not reach M H A or they fail to activate this site in the manner observed in intact animals. As a result excitation of CRF-producing neurons does not occur. The conclusion that excitation of MHA and inhibition of AHA results in the activation of the adrenocortical system is in good agreement with our previous data 14. It was reported that inhibiting of pituitary-adrenocortical system results in excitation of AHA and inhibition of MHA 14. These changes are opposite to those observed in the present investigation during activation of the adrenocortical system. Unlike AHA and MHA, which give opposite reactions during inhibition 14 and activation of the pituitary-adrenocortical system, LHA shows inhibition in both cases. It is probable that LHA is more heterogeneous and characterized by less specificity with respect to adrenal-cortex regulation than AHA and MHA are. Our results confirm an earlier hypothesis on the antagonism of anterior and medial hypothalamus involved in the regulation of pituitary-adrenocortical system 1~. ACKNOWLEDGEMENTS

The authors are grateful to Dr. Vera V. Rakitskaya for making histological preparations, Miss Nadezhda Vjun for assistance in statistical treatment of results, Mrs. Tatyana V. Filaretova for advice in preparation of diagrams and Mr. Anatoli I. Bogdanov for technical assistance.

74 REFERENCES 1 Bailey, N. T. J., Statistical Methods in Biology, John Wiley and Sons, New York, 1959. 2 Bouilld, C., Herbutd, S. and Bayld, J. D., Effects of hypothalamic deafferentation on basal and stress-induced adrenocortical activity in the pigeon, J. Endocr., 66 (1975) 413-419. 3 Brodish, A., Effect of hypothalamic lesions on the time course of corticosterone secretion, Neuroendocrinology, 5 (1969) 33-47. 4 Chowers, I., Conforti, N. and Feldman, S., Effects of corticosteroids on hypothalamic corticotropin-releasing factor and pituitary A C T H content, Neuroendocrinology, 2 (1967) 193-199. 5 Corbin, A., Mangili, G., Motta, M. and Martini, L., Effect of hypothalamic and mesencephalic steroid implantation on ACTH feedback mechanism, Endocrinology, 76 0965) 811-818. 6 Dafny, N.,Phillips, M.I.,Taylor, A. N. and Gilman, S. , Dose effects ofcortisol on single unit activity in hypothalamus, reticular formation and hippocampus of freely behaving rats correlated with plasma steroid levels, Brain Research, 59 (1973) 257-272. 7 EndrSczi, E. and Liss~ik, K., Effect of hypothalamic and brain stem structure stimulation on pituitary-adrenocortical function, Acta physiok Acad. Sci. hung., 24 (1963) 67-77. 8 EndrSczi, E., Liss~k, K.,Koranyi, L. and Nyakas, Cs., Influence of corticosteroids on the hypothalamic control of sciatic evoked potentials in the brain stem reticular formation and the hypothalamus in the rat, Aeta physiol. Acad. Sci. hung., 33 (1968) 375-382. 9 EndrSczi, E., Liss~tk, K. and Tekeres, M., Hormonal 'feedback' regulation of pituitary-adrenocortical activity, A cta physiol. ,4 cad. Sci. hung., 18 ( 1961 ) 291-299. l0 Feldman, S., Conforti, N. and Chowers, I., Effect of dexamethasone on adrenocortical responses in intact and hypothalamic deafferented rats, Acta endocr. (Kbh.), 73 (1973) 660-664. II Feldman• S.• C•nf•rti• N. and Ch•wers• •. • C•mp•ete inhibiti•n •f adren•c•rtical r•sp•nses f•l••wing sciatic nerve stimulation in rats with hypothalamic islands, Acta endocr. (Kbh.), 78 (t975) 539544. 12 Filaretov, A. A., The afferent input and functional organization of the hypothalamus in reactions regulating pituitary-adrenocortical activity, Brain Research, 107 (1976) 39-54. 13 Filaretov, A. A. and Rakitskaya, V. V., The role of medial hypothalamus in the regulation of the adrenocortical system, Sechenov physiol. J. U.S.S.R., 63 (1977) 1407-1410. 14 Filaretov, A. A. and Vassilevskaya, L. V., The hypothalamic mechanisms ofcorticosteroid feedback regulation, Sechenov physiol. J. U.S.S.R., 63 (1977) 1256-1260. 15 Kawakami, M., Kimura, F., lshida, S. and Yanase, M., Changes in the activity of the limbic neural pathways under the repeated immobilization stress, Endocr. Jap., 18 (1971) 469-476. 16 Makara, G. B., Stark, E., Marton, J. and Meszaros, T., Corticotrophin release induced by surgical trauma after transection of various afferent nervous pathways to the hypothalamus, J. Endocr., 53 (1972) 389-395. 17 Mandelbrod, l., Feldman, S. and Verman, R., Inhibition of firing is the primary effect of microelectrophoresis of cortisol to units in the tuberal hypothalamus, Brain Researeh, 80 (1974) 303-315. 18 Mangili, G., Motta, M. and Martini, L., Control of adrenocorticotrophic hormone secretion. In L. Martini and W. F. Ganong (Eds.), Neuroendocrinology, Vol. l, Academic Press, New York, 1966, pp. 297-370. 19 Ondo, J. G. and Kitay, J. I., Effects of dexamethasone and stressful stimuli on hypothalamic electrical activity in rats with diencephalic islands, Neuroendocrinology, 9 (1972) 215-227. 20 Porter, R. W., Alteration in electrical activity of the hypothalamus induced by stress stimuli, Amer. J. Physiol., 169 (1952) 15-20. 21 Sawyer, C. H., Everett, J. W. and Green, J. D., The rabbit diencephalon in stereotaxic coordinates, J. comp. Neurol., 101 (1954) 801-824. 22 Smelik, P. G. and Sawyer, C. H., Effects of implantation ofcortisol into the brain stem or pituitary gland on the adrenal response to stress in the rabbit, Acta endocr. (Kbh.), 41 (1962) 561-570. 23 Tonutti, E., Experimente zur Localization der hypothalamischen Steurung der ACTH-Sekretion, Acta Neuroveget. (Wien), 23 (1961) 35-49. 24 Van der Vies, J.,Bakker, R. F. M. and De Wied, D., Correlated studies on plasma free corticosterone and on adrenal steroid formation rate in vitro, Acta endocr. (Kbh.), 34 0960) 5t3-523.