59
J. Physiol. (1979), 292, pp. 59-67 With 3 text-figures Printed in Great Britain
THE EFFECT OF HYPOTHALAMIC LESIONS ON IMMUNO-REACTIVE a-MELANOCYTE STIMULATING HORMONE SECRETION IN THE RAT
BY R. J. PENNY*, F. J. H. TILDERS AND A. J. THODY* From the Department of Pharmacology, Free University, Amsterdam, the Netherlands
(Received 21 November 1978) SUMMARY
We have examined the hypothesis that MSH secretion in the rat is under a tonic inhibitory control by the central nervous system. Electrothermic lesions were made in the medio-basal hypothalamus (m.b.h.) which destroyed the hypothalamo-hypophyseal connexions. Plasma and pituitary a-MSH were measured using a sensitive and specific radioimmunoassay technique. Pituitary x-MSH content decreased to 17% of control levels 24 hr after the lesion and returned to normal by 7 days. Plasma a-MSH was maximally elevated 15 min after the lesion, and returned to normal by 2 hr. No further change over control levels was found for the remainder of the experiment (98 days). The similarity of the plasma ac-MSH response after m.b.h. lesions to that found previously after administration of long acting dopamine receptor blocking drugs suggests that these lesions may interrupt impulse flow in dopaminergic tubero hypophyseal neurones which are thought to be important in the inhibitory control of MSH secretion in the rat. From our present results it seems that the rat pituitary, unlike that of lower vertebrates, is able to re-establish basal MSH secretion very quickly after disconnexion from central control. INTRODUCTION
For many years it has been thought that the secretion of melanocyte-stimulating hormone (MSH) from the pars intermedia of the pituitary is under a tonic inhibitory control by the central nervous system. This hypothesis is largely based on observations in non-mammalian vertebrates where changes in skin colour provide direct evidence for changes in the release of MSH. For instance the prolonged darkening of the skin that occurs in elasmobranchs, teleosts, amphibians and reptiles after pituitary stalk section, hypothalamic lesions, or ectopic transplantation of the pituitary suggests that isolation of the pituitary from central control results in a chronic hyper-secretion of MSH (for review see Goos & Terlou, 1977). Observations of a similar nature in mammals have been difficult due to the lack of satisfactory assays for MSH in the blood. Therefore, changes in the histological appearance of the pars intermedia, or in pituitary MSH content have been taken to reflect changes in MSH secretion. Hypertrophy of the pars intermedia and its cells has been described after pituitary stalk section or hypothalamic lesions in the rat *
Present address: University Department of Dermatology, Newcastle-upon-Tyne, England.
0022-3751/79/4020-0862 $01.50 © 1979 The Physiological Society
R. J. PENNY, F. J. H. TILDERS AND A. J. THODY (Barrnett & Greep, 1951; Bogdanove & Halmi, 1953; Howe & Thody, 1969; Ooki, Kotsu, Kinutari & Daikoku, 1973), goat (Adams, Daniel & Prichard, 1966), rabbit (Brooks, 1938), and dog (Stutinsky, Bonvallet & Dell, 1950), which implied an increased activity of the pars intermedia. Other authors have stated that pituitary stalk section produced no change in the histology of the pars intermedia in the rabbit (Campbell & Harris, 1957) and rat (Daniel, Duchen & Prichard, 1964), and observations of Tilders & Smelik (1978) have shown that the apparent hypertrophy of the pars intermedia which occurs after stalk section is a misinterpretation resulting from the corresponding atrophy of the pars nervosa. Changes in pituitary MSH content after electrical or mechanical lesions which destroyed the median eminence are not only difficult to interpret but are also contradictory. Whilst a decrease in pituitary MSH content has been found one day after removal of the hypothalamic influence (Taleisnik et al. 1967; Tilders & Smelik, 1978) normal pituitary MSH levels have been reported 4 days after the lesion and at various intervals afterwards. (Bal & Smelik, 1967; Carrillo, Kastin, Dunn and Schally 1973; Tilders & Smelik, 1978). Although pituitary decentralization has been reported to induce a chronic increase in plasma MSH as measured by bio-assay (Carrillo et al. 1973; Celis, Macagno & Taleisnik, 1973), we were unable to confirm this in preliminary experiments. It was therefore decided to reinvestigate the effects of hypothalamic lesions on plasma a-MSH in the rat using a radioimmunoassay for a-MSH which allows the routine measurement of circulating hormone (Penny & Thody, 1978). A preliminary report of some of these results has already been published (Penny, Thody & Tilders, 1978). 60
METHODS
Animate Female Wistar rats of approximately 150 g body weight were housed at. a constant temperature (21-23 00) under controlled lighting (08-00-20-00) and received food and water ad libitum. The stage of oestrous was not determined. Rats were anaesthetized with ether and placed in a stereotaxic instrument. A unipolar electrode constructed from 0-5 mm diameter stainless-steel wire insulated with epoxy resin was introduced into the brain at an angle of 7° from vertical and advanced towards the medianeminence. A single electrothermic lesion was produced using a Grass Instruments LM3 Lesion Maker (100 kHz, 10 V). Sham operated animals received the same treatment, but no current was passed. In scme experiments ether anaesthetized and untreated controls were also included. All surgical operations were conducted between 09-00 and 11.30. In experiments where the post-lesioning interval exceeded 24 hr, lesioned animals were placed individually in metabolism cages directly following surgery and urine collected for 24 hr. Subsequently animals were housed in groups under controlled conditions as described previously. At various time intervals -after lesioning animals were transferred to a separate room and decapitated within 2 min. Trunk blood was taken into ice cold heparinized tubes and plasma stored at -30 °C until assayed for a-MSH. Pituitaries were removed and placed into 1 rnl. 0-1 m-HCl and homogenized. Homogenates were centrifuged at 4 0C for 20 min at 2500 g and the supernatant stored at -30 °C until assayed for a-MSH. Brains from lesioned animals were fixed in 10% formal-saline for macroscopic and, in some cases, microscopic examination of the size and extent of the lesion.
CONTROL OF a-MSH SECRETION
61
A88ay of a-MSH Plasma and pituitary a-MSH was measured by radioimnumoassay (Penny & Thody, 1978). Synthetic a-MSH (Ciba-Geigy Ltd) was used as standard and for the production of 125I-labelled preparations.
Statsittcal med8 All results are expressed as mean+ S.E. of mean. Results were tested for homogeneity of variance using the F-ratio test. Student's t test (two tailed) was used as a test of significance between groups. Where the variance of groups was significantly different a modified t test was used (Documenta Geigy, p. 172, 6th ed., Geigy Pharmaceutical Co. Ltd, Manchester).
P
J. Physiol. (1979), 292, pp. 59-67 With 3 text-figures Printed in Great Britain
THE EFFECT OF HYPOTHALAMIC LESIONS ON IMMUNO-REACTIVE a-MELANOCYTE STIMULATING HORMONE SECRETION IN THE RAT
BY R. J. PENNY*, F. J. H. TILDERS AND A. J. THODY* From the Department of Pharmacology, Free University, Amsterdam, the Netherlands
(Received 21 November 1978) SUMMARY
We have examined the hypothesis that MSH secretion in the rat is under a tonic inhibitory control by the central nervous system. Electrothermic lesions were made in the medio-basal hypothalamus (m.b.h.) which destroyed the hypothalamo-hypophyseal connexions. Plasma and pituitary a-MSH were measured using a sensitive and specific radioimmunoassay technique. Pituitary x-MSH content decreased to 17% of control levels 24 hr after the lesion and returned to normal by 7 days. Plasma a-MSH was maximally elevated 15 min after the lesion, and returned to normal by 2 hr. No further change over control levels was found for the remainder of the experiment (98 days). The similarity of the plasma ac-MSH response after m.b.h. lesions to that found previously after administration of long acting dopamine receptor blocking drugs suggests that these lesions may interrupt impulse flow in dopaminergic tubero hypophyseal neurones which are thought to be important in the inhibitory control of MSH secretion in the rat. From our present results it seems that the rat pituitary, unlike that of lower vertebrates, is able to re-establish basal MSH secretion very quickly after disconnexion from central control. INTRODUCTION
For many years it has been thought that the secretion of melanocyte-stimulating hormone (MSH) from the pars intermedia of the pituitary is under a tonic inhibitory control by the central nervous system. This hypothesis is largely based on observations in non-mammalian vertebrates where changes in skin colour provide direct evidence for changes in the release of MSH. For instance the prolonged darkening of the skin that occurs in elasmobranchs, teleosts, amphibians and reptiles after pituitary stalk section, hypothalamic lesions, or ectopic transplantation of the pituitary suggests that isolation of the pituitary from central control results in a chronic hyper-secretion of MSH (for review see Goos & Terlou, 1977). Observations of a similar nature in mammals have been difficult due to the lack of satisfactory assays for MSH in the blood. Therefore, changes in the histological appearance of the pars intermedia, or in pituitary MSH content have been taken to reflect changes in MSH secretion. Hypertrophy of the pars intermedia and its cells has been described after pituitary stalk section or hypothalamic lesions in the rat *
Present address: University Department of Dermatology, Newcastle-upon-Tyne, England.
0022-3751/79/4020-0862 $01.50 © 1979 The Physiological Society
R. J. PENNY, F. J. H. TILDERS AND A. J. THODY (Barrnett & Greep, 1951; Bogdanove & Halmi, 1953; Howe & Thody, 1969; Ooki, Kotsu, Kinutari & Daikoku, 1973), goat (Adams, Daniel & Prichard, 1966), rabbit (Brooks, 1938), and dog (Stutinsky, Bonvallet & Dell, 1950), which implied an increased activity of the pars intermedia. Other authors have stated that pituitary stalk section produced no change in the histology of the pars intermedia in the rabbit (Campbell & Harris, 1957) and rat (Daniel, Duchen & Prichard, 1964), and observations of Tilders & Smelik (1978) have shown that the apparent hypertrophy of the pars intermedia which occurs after stalk section is a misinterpretation resulting from the corresponding atrophy of the pars nervosa. Changes in pituitary MSH content after electrical or mechanical lesions which destroyed the median eminence are not only difficult to interpret but are also contradictory. Whilst a decrease in pituitary MSH content has been found one day after removal of the hypothalamic influence (Taleisnik et al. 1967; Tilders & Smelik, 1978) normal pituitary MSH levels have been reported 4 days after the lesion and at various intervals afterwards. (Bal & Smelik, 1967; Carrillo, Kastin, Dunn and Schally 1973; Tilders & Smelik, 1978). Although pituitary decentralization has been reported to induce a chronic increase in plasma MSH as measured by bio-assay (Carrillo et al. 1973; Celis, Macagno & Taleisnik, 1973), we were unable to confirm this in preliminary experiments. It was therefore decided to reinvestigate the effects of hypothalamic lesions on plasma a-MSH in the rat using a radioimmunoassay for a-MSH which allows the routine measurement of circulating hormone (Penny & Thody, 1978). A preliminary report of some of these results has already been published (Penny, Thody & Tilders, 1978). 60
METHODS
Animate Female Wistar rats of approximately 150 g body weight were housed at. a constant temperature (21-23 00) under controlled lighting (08-00-20-00) and received food and water ad libitum. The stage of oestrous was not determined. Rats were anaesthetized with ether and placed in a stereotaxic instrument. A unipolar electrode constructed from 0-5 mm diameter stainless-steel wire insulated with epoxy resin was introduced into the brain at an angle of 7° from vertical and advanced towards the medianeminence. A single electrothermic lesion was produced using a Grass Instruments LM3 Lesion Maker (100 kHz, 10 V). Sham operated animals received the same treatment, but no current was passed. In scme experiments ether anaesthetized and untreated controls were also included. All surgical operations were conducted between 09-00 and 11.30. In experiments where the post-lesioning interval exceeded 24 hr, lesioned animals were placed individually in metabolism cages directly following surgery and urine collected for 24 hr. Subsequently animals were housed in groups under controlled conditions as described previously. At various time intervals -after lesioning animals were transferred to a separate room and decapitated within 2 min. Trunk blood was taken into ice cold heparinized tubes and plasma stored at -30 °C until assayed for a-MSH. Pituitaries were removed and placed into 1 rnl. 0-1 m-HCl and homogenized. Homogenates were centrifuged at 4 0C for 20 min at 2500 g and the supernatant stored at -30 °C until assayed for a-MSH. Brains from lesioned animals were fixed in 10% formal-saline for macroscopic and, in some cases, microscopic examination of the size and extent of the lesion.
CONTROL OF a-MSH SECRETION
61
A88ay of a-MSH Plasma and pituitary a-MSH was measured by radioimnumoassay (Penny & Thody, 1978). Synthetic a-MSH (Ciba-Geigy Ltd) was used as standard and for the production of 125I-labelled preparations.
Statsittcal med8 All results are expressed as mean+ S.E. of mean. Results were tested for homogeneity of variance using the F-ratio test. Student's t test (two tailed) was used as a test of significance between groups. Where the variance of groups was significantly different a modified t test was used (Documenta Geigy, p. 172, 6th ed., Geigy Pharmaceutical Co. Ltd, Manchester).
P
