Presence of corticotropin in limbic system of normal and hypophysectomized rats

Brain Research, 128 (1977) 575-579 © Elsevier/North-Holland Biomedical Press

575

Presence of corticotropin in limbic system of normal and hypophysectomized rats

DOROTHY T. KRIEGER, ANTHONY LIOTTA and MICHAEL J. BROWNSTEIN

( D.T.K. and A.L.) Division of Endocrinology and Metabolism, Department of Medicine, Mount Sinai School of Medicine, New York, N.Y. 10029 and (M.J.B.) Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Md. 20014 (U.S.A.) (Accepted March 3rd, 1977)

We have recently reported a the presence of similar concentrations of immunoreactive (I-) ACTH (ca. 1100 pg/100 ~g protein) in median eminence and remaining medial basal hypothalamus of both normal and hypophysectomized male rats. The total amount of bioreactive (B-) ACTH found in these areas was approximately 1/100th of that present in pituitary. Much lesser amounts (24-46 pg/100 #g protein, I-ACTH) were found to be present in other brain areas (cerebellum, cortex and thalamus), whereas the concentration in hippocampus was 116 pg/100 #g protein. In view of this latter finding, the present study extends our observations to other brain areas, with special emphasis on the limbic system. Adult male Sprague-Dawley rats were used. All animals were sacrificed between 09.00 and 10.00 h. Studies on hypophysectomized animals were performed l0 days postoperatively. Completeness of hypophysectomy was confirmed by visual inspection of the sellar area at time of sacrifice. Additionally plasma I-ACTH concentrations in all of these animals were below the lower limits of detection (~< l0 pg/ml) in the assay system employeds. Brain dissection was performed as previously described 1. Four pools of a given area were assayed in quadruplicate, each pool containing tissue from 3 animals. Immunoreactive and bioreactive ACTH content was determined and Sephadex G-50 gel chromatography performed as previously describeds, save that immunoreactive characterization was performed utilizing only one antibody. (This was a "midportion" antibody supplied by the NIAMDD Hormone Distribution program, National Pituitary Agency, which reacts with ACTHI-a9 and ACTHI1-24 on an equimolar basis, but not with a-MSH, fl-MSH ACTHI-lo or ACTHlT-a9.) The concentrations of I-ACTH observed in the present study (Fig. 1) were similar to those previously reported 8 for hypothalamus, cerebellum, cortex, thalamus and hippocampus. It should be noted that in the present study the entire hypothalamus, rather than just median eminence and residual medial basal hypothalamus, was analyzed, accounting for the somewhat lower concentrations noted in the present study. (In the previous study B-ACTH concentrations were not determined for cerebellum, cortex, thalamus and hippocampus. The data in the present study indicate

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Fig. 1. Distribution of immunoreactive and bioreactive ACTH-like activity in selected areas of rat brain. In addition to areas depicted, analysis of midbrain, medulla, caudate nucleus and globus pallidus revealed no detectable ACTH-Iike activity, while that of pons was approximately equivalent to that of cerebellum (see text).

that I/B-ACTH ratios in these areas were similar to those previously reported for median eminence and medial basal hypothalamus.) In the present study, other portions of the limbic system (amygdala, preoptic area and septum) had even higher I- and B-ACTH concentrations than those noted for hippocampus. As indicated in Fig. 1, concentrations in olfactory bulb were not different from those observed in cortex or cerebellum; this was also true for stliatum. No detectable I-ACTH ( < 20 pg/100 #g protein) or B-ACTH ( < l0 pg/100 /~g protein) concentrations were present in midbrain, medulla, caudate nucleus or globus pallidus. Concentrations in pons were 23.0 ± 6.0 pg/100 #g protein (I-ACTH) and 23.3 :k 5.7 pg/100 #g protein (B-ACTH). Hypophysectomy was not associated with any significant decrease in !- and B-ACTH concentrations save in hypothalamus. We had previously reported s a similar decrease in median eminence I-ACTH concentrations following hypophysectomy. I/B ratios in areas with significant ACTH concentrations were: hippocampus 1.32, amygdala 1.55, septum 1.31, preoptic area 1.98 and hypothalamus 1.58. We have noted an I/B ratio of 1.5 for anterior pituitary concentrations. Hypophysectomy was not associated with any significant differences in these ratios. Sephadex G-50 gel filtration patterns of preoptic area and amygdalar pools derived from control and hypophysectomized animals are depicted in Fig. 2. Similar elution patterns were obtained for control and hypophysectomized animals, save in the fractions occurring after the major peak. In contrast to chromatograms of anterior pituitary, where the central peak elutes just before the ACTH1-39 marker, the central

577 peak of the chromatograms of these brain areas elutes just after the ACTHI-a9 marker, similar to the pattern we have reported for medial basal hypothalamus. The present study extends our previous observations of brain localization of ACTH-like activity and the persistence of such activity in the hypophysectomized animal. Brain localization of pituitary and pituitary-like hormones has also been recently demonstrated in hypophysectomized animals. These include growth hop mone 16, a-MSHI~, 2°, bioassayable melanotropic and lipolytic peptides 18 and endorphin 2. It is unlikely that the present finding of ACTH-like activity in brain reflects uptake from the systemic vascular system, since systemic injection of radioactively labeled ah-ACTH1-39 results in insignificant amounts of label appearing in brain 14 (Puett, D. personal communication). The persistence of similar concentrations of ACTH-like activity in hypophysectomized animals, in areas remote from the pituitary as well as immunocytochemical demonstration of ACTH in hypothalamus 5 weeks posthypophysectomy (Guillemin, R. and Bloom, F., personal communication), mitigates against a pituitary source, whethei by contamination with hypophyseal tissue or by possible retrograde transport 17 via the pituitary portal system. The possibility 600 500

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Fig. 2. Sephadex G-50 gel filtration of pooled pre-optic area and amygdalar extracts (n = 12). Area of void volume (V0) is marked by HSA-bromphenol blue, that of salt peak by 125I. The per cent of total activity of extract from control brains appearing in void volume (fractions 8-11), elution volume ofp-ACTHx_89 (fractions 12-16), and in the peak eluting after p-ACTHx_39 (fractions 17-23), respectively were 19, 46 and 33 % (preoptic area) and 22, 42 and 35 %) (amygdalar area). Percentages from brains of hypophysectomized animals were 12, 51, 36 (pre-optic area) and 14, 53 and 31 (amygdalar area).

578 therefore remains that such ACTH-Iike activity represents neural synthesis, perhaps of a form somewhat different (although biologically active) from that of pituitary ACTH, as suggested by the slightly different Sephadex G-50 gel filtration patterns of brain and pituitary ACTH. Incubation studies to demonstrate in vitro synthesis of ACTH and ACTH-like peptides by brain tissue are now in progress. The observation that concentrations of I-ACTH- and B-ACTH-Iike activity are, with the exception of those in hypothalamus, highest in limbic system areas, raises several questions with regard to central nervous system (CNS) pituitary-adrenal interrelationships. Different areas of the limbic system have been implicated with regard to the regulation of feedback 12,23 and stress mediated 6 release of A C T H ; involvement of these areas in the regulation of circadian release is more problematical7,1a,2L Additionally, limbic system uptake of tritiated corticosterone has been demonstrated by both biochemical studies ~1 and radioautography4,19. The biological function of such CNS ACTH-like activity is unknown. A C T H and A C T H fragments (especially 4-10) have been reported to play an important role in motivation, learning and memory, although the area reported to be most sensitive to such behavioral effects, as determined from CNS lesion and implantation experiments, appears to be the posterior thalamic area 3, in which ACTH-like activity was of an extremely low order of magnitude in the present study. Other ACTH fragments have been reported to induce excessive grooming, stretching and yawning when administered intraventricularly ~. The neurochemical basis for such peptide hormone effects on behavior is unknown. It may represent a direct neuronal action of ACTH, ACTHinduced changes in neurotransmitter levels9,1°, or in some instances, ACTH-induced changes in adrenal secretion 21. The possible relationship of the demonstration in the limbic system of A C T H like activity (in both normal and hypophysectomized animals) to the role of this system in regulation of A C T H release and in the mediation of some of its behavioral effects is purely conjectural at present. This study was supported by the Lita Annenberg Hazen Charitable Trust.

1 Brownstein, M., Arimura, A., Sato, H., Schally, A. V. and Kizer, J. S., The regional distribution of somatostatin in the rat brain, Endocrinology, 96 (1975) 1456-1461. 2 Cheung, A. L. and Goldstein, A., Failure of hypophysectomy to alter brain content of opioid peptides (Endorphins), Ltlfe Sci., 19 (1976) 1005-1008. 3 De Wied, D., Hormonal influences on motivation, learning and memory processes, Hosp. Practice, ll (1976) 123-131. 4 Gerlach, J. L., McEwen, B. S., Pfaff, D. W., Moskovitz,S., Ferin, M., Carmel, P. W. and Zimmerman, E. A., Cells in regions of rhesus monkey brain and pituitary retain radioactive estradiol, corticosterone and cortisol differentially, Brain Research, 103 (1976) 603-612. 5 Gispen, W. H., Wiegant, V. M., Greven, H. M. and De Wied, D., The induction of excessive grooming in the rat by intraventricular application of peptides derived from ACTH: structureactivity studies, Life Sci., 17 (1976) 645-652. 6 Knigge, K. M., Adrenocortical response to stress in rats with lesions in hippocampus and amygdala, Proc. Soc. exp. Biol. ( N. Y. ) , 108 (1961) 18-21. 7 Krieger, D. T. and Krieger, H. P., The circadian variation of the plasma 17-OHCS in central nervous system disease, J. clin. Endocr., 26 (1966) 929-940.

579 8 Krieger, D. T., Liotta, A. and Brownstein, M. J., Presence of adrenocorticotropin in brain of normal and hypophysectomized rats, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 648-652. 9 Leonard, B. E., Ramaekers, R. and Rigter, H., Effects of adrenorticotrophin-(4-10)-heptapeptide on changes in brain monoamine metabolism associated with retrograde amnesia in the rat, Biochem. Soc. Trans., 3 (1975) 113-115. 10 Leonard, B. E. and Rigter, H., Changes in brain monoamine metabolism and carbon dioxide induced amnesia in the rat, Pharmacol. Biochem. Behav., 3 (1975) 775-781. 11 McEwen, B. S., Interactions between hormones and nerve tissue, Scient. Amer., 235 (1976) 48-59. 12 McHugh, P. R. and Smith, G. P., Negative feedback in adrenocortical response to limbic stimulation in Macaca rnulatta, Amer. J. Physiol., 213 (1967) 1445-1450. 13 Moberg, G. P., Scapagnini, U., De Groot, J. and Ganong, W. F., Effect of sectioning the fornix on diurnal fluctuation in plasma corticosterone levels in the rat, Neuroendocrinology, 7 (1971) 11-15. 14 Nicholson, W. E., Liddle, R. A. and Puett, D., Corticotropin: plasma clearance, catabolism and biotransformation, Prog. 58th Meeting Endocr. Soc., 59 (1976) 976 (Abstr.). 15 Oliver, C., Eskay, R. L. and Porter, J. C., Distribution in the rat brain of a-MSH and its concentration in hypophysial portal blood, Proc. 5th int. Congr. Endocr. Soc., (1976) 244 (Abstr.). 16 Pacold, S. T., Lawrence, A. M. and Kirsteins, L., CNS growth hormone: secretion of GH-like immunoreactivity from monolayer tissue cultures of the amygdala, Clin. Res., 24 (1976) 561 (Abstr.). 17 Page, R. B., Monger, B. L., and Bergland, R. M., Scanning microscopy of pituitary vascular casts, Amer. J. Anat., 146 (1976) 273-301. 18 Rudman, D., Del Rio, A. E., Hollins, B. M., Houser, D. H., Keeling, M. D., Sutin, J., Scott, J. W., Sears, R. A. and Rosenberg, M. Z., Melanotropic-lipolytic peptides in various regions of bovine, simian and human brains and in simian and human cerebrospinal fluids, Endocrinology, 92 (1973) 372-379. 19 Stumpf, W. E. and Sar, M., Topography of extrahypothalamic glucocorticosteroid "feedback" sites in the rat brain. In Excerpta Medica Int. Congr. Ser. 256, Excerpta Medica, Amsterdam, 1972, pp. 120-121. 20 Vaudry, H., Oliver, D., Vaillant, R. and Kraicer, J., Bioactive and immunoreactive a-MSH in the rat brain, Proc. 5th int. Congr. Endocr. Soc., (1976) 274 (Abstr.). 21 Versteeg, F. H. G. and Wurtman, R. J., Effect of ACTH4-10 on the rate of synthesis of [aH]catecholamines in the brains of intact, hypophysectomized and adrenalectomized rats, Brain Research, 93 (1975) 552-557. 22 Wilson, M. and Critchlow, V., Effect of fornix transection or hippocampectomy on rhythmic pituitary-adrenal function in the rat, Neuroendocrinology, 13 (1973/74) 29-40. 23 Wilson, M., Effect of hippocampectomy on dexamethasone suppression of corticosteroid sensitive stress responses, Anat. Rec., 181 (1975)511 (Abstr.).