EFFECT OF HYPOTHALAMIC LESIONS ON PITUITARY MELANOCYTE-STIMULATING HORMONE

Transcription

1 EFFECT OF HYPOTHALAMIC LESIO ON PITUITARY MELANOCYTE-STIMULATING HORMONE S. TALEISNIK, J. DE OLMOS, R. OR\l=I'\ASand MAR\l=I'\AE. TOMATIS Instituto de Investigación Médica, Mercedes y Martín Ferreyra*, Córdoba, Argentina (Received 2 February 1967) SUMMARY The effect on the content of melanocyte-stimulating hormone (MSH) activity in the pituitary ofelectrolytic lesions placed in different regions ofthe hypothalamus was studied in male rats. Lesions in the paraventricular nuclei resulted, after 15 days, in a decrease of pituitary MSH activity to 20\m=.\4\m=+-\ 4\m=.\5%/mg.gland as compared with the controls, without changes in the weight of the hypophyses. In a group of animals in which the lesions failed to destroy the paraventricular nuclei completely the MSH activity in the pituitary was 66\m=.\4\m=+-\7\m=.\5 % of that of controls and the weight of the gland was significantly higher. Hypothalamic lesions in the median eminence of the tuber cinereum produced 24 hr. later a decrease of pituitary MSH activity to 6\m=.\6\m=+-\0\m=.\8 %, but 15 days later the values/mg. gland were almost normal. Lesions placed in the mammillary bodies or in the nucleus caudatus did not affect pituitary MSH content. Extracts of stalk-median eminence or posterior lobe from animals with lesions in the paraventricular nuclei, failed to show MSH-releasing factor as it is found in intact animals, nor did they contain MSH-release-inhibiting factor. The results support the concept that the paraventricular nuclei are involved in the control of pituitary MSH secretion and suggest that the MSH content of the disconnected hypophysis is to some degree regulated autonomously. INTRODUCTION It is a generally accepted fact that in amphibians the central nervous system exerts an inhibitory action on the release of intermediate lobe hormone. Experiments in which the connexions between the pituitary and the hypothalamus were inter rupted by section of the stalk (Etkin, 1962) or transplantation of the gland (Etkin, 1941, 1943) were followed by darkening of the skin as if the secretion of melanocytestimulating hormone (MSH) was increased. Lesions of the hypothalamus produced the same effect (Voitkevich, 1962; Kastin & Ross, 1965). Furthermore, it has been shown (Taleisnik & Tomatis, 1967 a) that toad hypothalamic extracts contain an agent which inhibits the release of MSH. * Supported by the Consejo Nacional de Investigaciones Científicas y Técnicas of Argentina.

2 In mammals evidence is accumulating that the central nervous system is important in the regulation of MSH secretion in spite of the ignorance about the physiological role of the hormone in this vertebrate class. In rats, transplantation of the pituitary to the renal capsule resulted in a decrease in its content of MSH (Kastin & Ross, 1964), and suckling stimuli have been shown to induce an acute drop in the MSH content of the pituitary (Taleisnik & Orias, 1966). It has also been shown that the hypothalamus contains agents which stimulate or inhibit the release of MSH (Taleisnik & Orias, 1965; Taleisnik & Tomatis, 19676). Destruction of restricted areas in the hypothalamus is a valuable method for studying the control by the hypothalamus of the release of various pituitary hor mones. In the present study, this technique was applied to obtain further informa tion concerning the influence of the central nervous system on MSH release. MATERIAL AND METHODS Adult male rats, weighing g., were used. By means of a stereotaxic instrument, electrolytic lesions were placed bilaterally either in the median eminence of the tuber cinereum (ME), in the region of the paraventricular nuclei (PVN) or in other regions of the brain. A direct current of 3 ma for 25 sec. was passed through a nichrome electrode. After the lesions had been made the animals were kept in indivi dual cages and were given free access to food and water. One or 15 days after the operation the animals were killed with ether and the pituitary was removed immedi ately, weighed, suspended in an appropriate volume of distilled water and kept frozen until the MSH content was assayed. In a group of animals with lesions in the PVN, the stalk-median eminence (SME) was removed and suspended in an acid solution to study its content of MSH-releasing factor (MSH-RF). The procedure for preparing these extracts has been described previously (Taleisnik & Orias, 1965). Localization and site of the lesions were determined by brain reconstruction from unstained sections serially cut at 30 p. Assay of MSH. MSH was assayed in vitro using toad skin (Taleisnik & Orias, 1965). The hypophysis of each animal was assayed at two dose levels and the activity/mg. gland was compared with that of a control extract of two pooled glands from normal animals. The MSH content of the control pituitaries was also tested at two dose levels and taken as 100 %. A control extract was prepared each time that operated or normal animals were killed. Five to seven pieces of skin were assigned to each point and the increase between successive doses was twofold. The results are expressed as % of the controls/mg. pituitary and whole gland. Estimates of potency were calculated by standard statistical methods for parallel line assay (Bliss, 1952). Student's (t) test was used to assess the significance of differences between groups. Hypothalamic extracts. The effect of PVN lesions on the content of MSH-RF and MSH-release-inhibiting factor (MSH-R-IF) in the stalk-median eminence was studied. The extracts were injected intravenously into g. male rats, anaesthetized with ether and the animals killed 20 min. later. The SME of two animals were pooled and injected into two rats whose hypophyses were then pooled, suspended in a measured volume of distilled water and kept frozen until the MSH activity was assayed. The decrease of MSH activity in the pituitary induced by the injection of

3 SME extracts was taken as an index of MSH-RF content. In order to find out whether the SME extract contained MSH-release inhibiting factor, the capacity of these extracts to block the depletion of pituitary MSH induced by SME extract from normal animals was tested. RESULTS Histological observations The lesions in the PVN were located at both sides of the third ventricle producing a complete destruction of these nuclei in some animals. The lesions extended from the anterior commissure to a level beyond the caudal end of the PVN. Laterally they were limited by the fornix without affecting it. The lower end of the lesions did not involve the arcuate nucleus and there was no visible injury to the hypophysial portal vessels (Plate, fig. 1). Table 1. Effect of hypothalamic lesions on the melanocyte-stimulating hormone activity content of the pituitary of rats (means ± s.e.) Condition of animals Normal rats Lesions of paraventricular nuclei A. Made with nichrome electrode B. Nichrome electrode (incomplete lesions) C. Made with platinum electrode Lesions of the median eminence A. 1 day after lesion B. 15 days after lesion (large lesion) C. 15 days after lesion (small lesion) Control lesions No. of animals Body weight (g-) Weight of pituitary MSH content of pituitary* Whole gland mg./100 g. Per mg. Whole (mg.) body wt. gland gland 240( ) 7-26 ± ± ( ) 6-85± ( ) ± ( ) 8-57± ± ± ( ) ( ) ( ) ( ) 4-94 ± ± ( ) 6-90± * Percent of control pituitaries. The rats with lesions in the median eminence could be separated into two groups according to the extension of the injury. In the first group there was extensive injury extending from the caudal part of the optic chiasma to the hypophysial stalk with complete or almost complete destruction of the arcuate nuclei. In the second group the lesions were smaller and were located more basally and included the ventral wall of the third ventricle and the hypophysial stalk. In both groups the portal vessels were damaged (Plate, fig. 2). Control lesions were made in the region of the mammillary bodies or the nucleus caudatus.

4 MSH activity of pituitary glands of intact rats Single pituitaries from normal rats were assayed to provide control data and information about the variation in individual glands. Table 1 shows that in eight normal animals the MSH activity/mg. pituitary was % if the activity of the controls (a pool of two hypophyses of intact rats) was taken as 100. The values varied from 90-5 to 117-5%. MSH activity/gland has been calculated by the formula : MSH activity/mg. gland x pituitary/100 g. body weight control pituitary/100 g. body weight The control pituitary/100 g. body weight was obtained from a group of 18 normal rats. In the eight normal animals in which the pituitaries were assayed individually the MSH activity/gland was calculated to be % (Table 1). Effect of lesions in the paraventricular nuclei The results are summarized in Table 1. Animals with complete destruction of the PVN had a mean pituitary weight of 7-31 ± 0-50 (s.e.) mg. The weight/100 g. body weight was These figures were not significantly different from those in the controls. The MSH activity/mg. gland was however % of that of the control group. The hormone content/gland was % of the controls. In ten animals in which the lesions were placed in the region of the PVN but where these nuclei were only destroyed partially or unilaterally, there was a significant increase in pituitary weight considered both in terms of the whole gland or/100 g. body weight. Related to the control group MSH activity/mg. gland was % and that of the whole gland was %. These figures are significantly lower (P < 0-01) than those for intact animals but significantly higher (P < 0-001) than those in animals with complete destruction of the PVN. In order to discard the possibility that the effects of PVN lesions were due to electrolytic deposits from the nichrome electrodes (Everett & Radford, 1961) a group of animals was studied in which the lesions were made with platinum electrodes. Table 1 shows that in four such animals with complete destruction of the PVN, MSH activity was %/mg. gland and %/gland of that of the control group. There were no changes in the weight of the pituitaries. The values obtained were not different from those in which the lesions had been made with nichrome electrodes. Effect of lesions in the median eminence In six animals killed 24 hr. after the lesions in the ME the weight of the hypophyses was not significantly different from that in the control group. However, MSH activity in the pituitary decreased markedly ( %/gland and %/mg. gland). Animals with ME lesions, killed 15 days after the operation, were combined in two groups, one with a small lesion and the other with a large lesion. In spite of the differences in the extent of the lesions no difference in the degree of diabetes insipidus, nor in the weight of the pituitary gland nor in the content of MSH was

5 found. There was a significant decrease in pituitary weight. MSH activity/mg. pituitary was % and %, respectively. These values are not statistically different from those of the group of animals with control lesions. Since glandular weight was reduced the MSH content/gland was about 50 % of those of the controls. Effect of control lesions In three animals with lesions in the mammillary bodies and in three with lesions in the nucleus caudatus there was no change in the pituitary weight and MSH activity/ mg. gland was % of that of the control group. Effect of P VN lesions on the MSH-RF activity of and posterior lobe extracts stalk-median eminence Extracts prepared from SME or the posterior lobe obtained from rats 15 days after lesion of the PVN, were injected into recipient rats to test their effect on pituitary MSH activity. Table 2 shows that in three groups injected with three different pooled extracts there was no significant difference in pituitary MSH activity as compared with controls injected with cerebral cortex extract (in an amount equal to the weight of one SME) and taken as 100% of activity. SME extract from normal animals were also tested (Table 2) although previous work had repeatedly indicated that such extracts produce a depletion in pituitary MSH ; MSH activity dropped to 58-6 %, of that in the control group. Table 2. Effect of lesions in the paraventricular nuclei (PVN) on the content of MSHreleasing factor (MSH-RF) in stalk-median eminence (SME) or posterior lobe (PL) extracts MSH Treatment SME extract from PVN SME extract from intact rats PL extract from PVN Experi- activity in ment pituitary* no. (%) 95% limits A Pf Mean Mean ( ) ( ) ( ) ( ) ( ) ( ) ( ) PL extract from intact rats ( ) < 001 * Referred to that per mg. pituitary of controls (a pool of two glands taken from animals each injected with an extract of rat cerebral cortex in an amount equal to the weight of one SME). t Probability of difference compared with controls. not significant. = < 001 Similarly, posterior lobe extract, prepared from animals with lesions in the PVN, did not affect pituitary MSH activity, whereas the extract prepared from glands of intact animals produced a significant depletion. In order to test whether the lack of activity in the extracts from to deplete pituitary MSH was due to the presence of an MSH-R-IF the capacity of such

6 extracts to inhibit the drop in MSH produced by SME extracts from intact animals was studied. One group of animals was therefore injected with SME extract from intact rats only and two other groups with the same dose of SME extract plus SME or posterior lobe extracts from animals with PVN lesions. No significant difference between the two groups was found (Table 3). Table 3. Effect of lesions in the paraventricular nuclei (PVN) on the content of MSHrelease-inhibiting factor in stalk-median eminence (SME) or posterior lobe (PL) extracts MSH Treatment SME extract from intact rats (A) A plus extract of two SME from A plus extract of two PL from SME extract from intact rats (A) A plus extract of two SME from A plus extract of two PL from Experi- ment 10 activity in pituitary* (%) % limits ( ) ( ) ( ) ( ) ( ) ( ) * Referred to that per mg. pituitary of controls (a pool of two glands taken from animals each injected with an extract of rat cerebral cortex in an amount equal to the weight of one SME). f Compared with rats injected with SME extract of intact rats. not significant. = it DISCUSSION The results of the present study show that lesions of the PVN and complete dis connexion of the pituitary gland by lesions in the median eminence determine two dif ferent responses of the pars intermedia. Destruction of the PVN caused a decrease in the MSH content of the pituitary and this effect seemed to be specific for the ablation of this nucleus since lesions in its vicinity or in other regions did not reproduce it. Animals with lesions in the median eminence had, on the contrary, an almost normal pituitary MSH concentration. It is interesting to notice that Olivecrona (1957) has shown in rats with lesions in the PVN that the volume of the intermediate lobe is reduced by 30%. The effect of the lesions in the PVN on the MSH content of the pituitary cannot be attributed to changes in the secretion of neurohypophysial hormones, since it has been shown that neither oxytocin nor vasopressin affect the release of MSH (Taleisnik & Orias 1965; Taleisnik, Orias & de Olmos, 1966; Taleisnik & Tomatis, 1967 a). We believe that the paraventricular nuclei are concerned with specific neurosecretory agents which regulate the release of MSH. In studying the topographic distribution of MSH-RF in the hypothalamus the maximum activity for this principle was found in the PVN (Taleisnik et al. 1966). It was postulated therefore that MSH-RF could be produced by the neurones of this nucleus whose axons end in the median eminence and the posterior lobe. The results of the present study showing that after lesions in the PVN stalk-median eminence extracts and posterior lobe extracts fail to deplete the pituitary of MSH gives further support to this hypo-

7 thesis. However, from the results obtained with SME and posterior lobe crude extracts it is not possible to say that the destruction of the PVN produced complete loss of MSH-RF. Since the effect of each extract on the MSH content of the pituitary depends on a balance between the factor which produces depletion of MSH and that which counteracts this effect (Taleisnik & Tomatis, 19676), only separation of both principles would resolve this difficulty. With regard to the effect of lesions in the ME on the pituitary concentration of MSH a striking difference was observed between the animals killed 1 and 15 days after the operation. In the former the MSH concentration of the pituitary was very low, but in the groups studied 14 days later most of the activity had been restored. These results contrast with the low values of adrenocorticotrophic (Fortier & de Groot, 1959; McCann & Haberland, 1960) and luteinizing hormone (Taleisnik & McCann, 1961) found in the hypophysis after similar lesions in the hypothalamus. This might be taken as evidence that the pituitary gland can manufacture MSH despite disconnexion from the central nervous system. The almost normal pituitary MSH activity/mg. gland in the animals with ME lesions contrasts with the low value reported in glands transplanted under the renal capsule (Kastin & Ross, 1964). This difference could be due to the release into the systemic circulation of hypothalamic agents acting on the transplanted pituitary. Lesions in the ME would suppress such a mechanism because of the destruction of the areas from which such agents are released. If it is assumed that more MSH is released from the mammalian pars intermedia after disconnexion from the hypothalamus, as has been shown in amphibians (Etkin, 1941, 1943), then the higher pituitary MSH content in the animals killed 15 days after ME lesions (as compared to those killed at 24 hr.), tends to the conclusion that an increase in synthesis also occurs so as to maintain a near normal concentration of MSH an despite increased secretion. Extracts of the supra-optic nuclei have been shown to possess an agent which inhibits the release of MSH (Taleisnik & Tomatis, 19676). Since the PVN is rich in MSH-RF, both the paraventricular and supraoptic nuclei may be involved in the control of MSH secretion and exert antagonistic actions. In the light of the present results it will be of interest to examine the effects of lesions of the supraoptic nuclei on the secretion of MSH. This investigation was supported by (Grant NB ). the United States Public Health Service REFERENCES Bliss, C. T. (1952). The statistics of bioassay. New York: Academic Press. Etkin, W. (1941). On the control of growth and activity of the pars intermedia of the pituitary by the hypothalamus in the tadpole. J. exp. Zool. 86, Etkin, W. (1943). The developmental control of pars intermedia by brain. J. exp. Zool. 92, Etkin, W. (1962). Hypothalamic inhibition of pars intermedia activity in the frog. Gen. cornp. Endocr. Suppl. 1, Everett, J. W. & Radford, H. M. (1961). Irritative deposits from stainless steel electrodes in the preoptic rat brain causing release of pituitary gonadotropin. Proe Soc. exp. Biol. Med. 108, Fortier, C. & de Groot, J. (1959). Release and synthesis of ACTH following electrolytic destruction of the median eminence in the rat. In Abstr. XXI Congr. Int. Physiol. Sei., p. 96. Buenos Aires.

8 Kastin, A. J. & Ross, G. T. (1964). Melanocyte-stimulating hormone (MSH) and ACTH activities of pituitary homografts in albino rats. Endocrinology 75, Kastin, A. J. & Ross, G. T. (1965). Melanocyte-stimulating hormone activity in pituitaries of frogs with hypothalamic lesions. Endocrinology 77, McCann, S. M. & Haberland, P. (1960). Further studies on the regulation of pituitary ACTH in rats with hypothalamic lesions. Endocrinology 66, Olivecrona, H. (1957). Paraventricular nucleus and pituitary gland. Ada physiol. scand. 40, Suppl. 136, Taleisnik, S. & McCann, S. M. (1961). Effect of hypothalamic lesions on the secretion and storage of hypophysial luteinizing hormone. Endocrinology 68, Taleisnik, S. & Orias, R. (1965). A melanocyte-stimulating hormone-releasing factor in hypothalamic extracts. Am. J. Physiol. 208, Taleisnik, S. & Orias, R. (1966). Pituitary melanocyte-stimulating hormone (MSH) after suckling stimulus. Endocrinology 78, Taleisnik, S., Orias, R. & de Olmos, J. (1966). Topographic distribution of the melanocyte-stimulating hormone-releasing factor in rat hypothalamus. Proe Soc. exp. Biol. Med. 122, Taleisnik, S. & Tomatis, M. E. (1967a). Antagonistic effect on melanocyte-stimulating hormone release of two neural tissue extracts. Am. J. Physiol. 212, Taleisnik, S. & Tomatis, M. E. (19676). Melanocyte-stimulating hormone releasing and inhibiting factor in two hypothalamic extracts. Endocrinology (in the Press). Voitkevich, A. A. (1962). Neurosecretory control of the amphibian metamorphosis. Gen. comp. Endocr. Suppl. 1, DESCRIPTION OF PLATE Fig. 1. (a) Serial transverse sections of the hypothalamus showing the region common to all lesions which involved destruction of the paraventricular nuclei, (b) Sagittal reconstruction of the region. Fig. 2. (a) Serial transverse sections of the hypothalamus showing the region common to all lesions in the median eminence of which (6) is a sagittal reconstruction. The two stippled areas correspond to the two groups into which these animals were separated.

9 Journal of Endocrinology, S. TALEISNIK and others Vol. 39, No. 4 Plate (Facing p. 492)