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Topography of the ACTH-immunoreactive neurons in the basal hypothalamus of the rat brain
Brain Research, 216 (1981) 333-341 © Elsevier/North-Holland Biomedical Press
333
T O P O G R A P H Y OF T H E A C T H - I M M U N O R E A C T I V E N E U R O N S IN T H E BASAL H Y P O T H A L A M U S OF T H E RAT BRAIN
KARL M. KNIGGE, SHIRLEY A. JOSEPH and JAY NOCTON The Neuroendocrine Unit, University of Rochester, School of Medicine and Dentistry, Rochester, N. Y. 14642 (U.S.A.)
(Accepted November 27th, 1980) Key words: hypothalamus -- ACTH -- opiocortin -- immunocytochemistry
SUMMARY Cell bodies of the opiocortin neurons were stained immunocytochemically with ACTH antiserum and their location in the basal hypothalamus of the rat brain was mapped. They are present throughout the entire extent of the hypothalamus, from retrochiasmatic area to mammillary body. In the retrochiasmatic area they are a single, midline group horizontally oriented and lying close to the ventral surface. Throughout the extent of the median eminence, ACTH-ir perikarya are located in the arcuate nucleus, adjacent periventricular stratrum and internuclear space between arcuate and ventromedial nuclei; as a group, they are oriented in a dorso-lateral plane. In the mammillary region, the nucleus assumes a more horizontal orientation again, lying close to the ventral surface. No regional differences were noted in cell density except in the retrochiasmatic area and terminal portion of the mammillary region where they were approximately 30 % fewer in number.
INTRODUCTION Data based upon radioimmunoassay13,26,~s, 30, immunocytochemistryl,7,1s, 19, and in vitro culture methods zs indicate that the mediobasal hypothalamus is a site of high concentration and possible neuronal synthesis of the pro-ACTH/endorphin (opiocortin) peptides (31K, 16K, ACTH, a-MSH, fl-LPH, fl-MSH, and a- and flendorphin). Chemical12, 27 and electrolytic lesionZ0, 36 and deafferentation 31 studies have provided additional evidence that the opiocortin fiber system may originate from perikarya in the mediobasal hypothalamus. Neuronal cell bodies in this region have been shown to be simultaneously immunoreactive with antisera against known opiocortin peptides 6,9,29,a5. Joseph 19 has provided a detailed neuroanatomica map-
334 ping of the fiber distribution within the hypothalamus, to the limblc system, and to the brain stem. Details of the relationship of opiocortin fibers to the m agnocellular nuclei, median eminence, and the pituitary gland have been described by Knigge and Joseph 22,23. This neuropeptide system is distinct from networks containing Met- and Leu-enkephalinS,14,33, 36. In this report we present a more comprehensive map of the ACTH-immunoreactive perikarya in the mediobasal hypothalamus. MATERIALS AND METHODS Male Sprague-Dawley rats, 200-250 g b.wt. were anesthetized and the brains fixed by cardiac perfusion with 100 ml saline followed by 200 ml of Bouin's fixative. Brains were removed and fixed an additional 24 h in fresh fixative at 4 °C. They were sectioned serially in the coronal plane without further treatment on an Oxford Vibratome at 50 #m. Sections from the anterior commissure to the interpeduncular fossa were collected in phosphate buffer-saline (PBS, pH 7.2) and rinsed repeatedly with PBS until colorless. All staining procedures were carried out in 15 x 60 mm plastic dishes with 20-25 sections per dish in 4 ml volumes of staining reagents. In all cases, sections were incubated in the primary antiserum at 4 °C for 48 h in PBS containing 0.02 % Triton-X and 1% bovine serum albumin; sections were agitated during this incubation with the use of an orbital shaker oscillating at 50 cpm. The unlabeled antibody peroxidase-antiperoxidase (PAP) was used. Sections were stained for ACTH with antiserum to ACTH 1-39 (K-531) generated in rabbit using synthetic ACTH 1-39 conjugated to BSA with carbodiamide 19. The initial injection consisted of 4 mg of the immunogen administered intradermally in multiple injection sites along the back. Two booster injections, each consisting of 2 mg immunogen, followed at two-week intervals. The antiserum is normally used at a dilution of 1:128,000 for pituitary immunocytochemistry and a 1:16,000 for staining in brain. For the purpose of intensifying the staining of immunoreactive perikarya, the primary antiserum was used in this study at a dilution of 1:4000. Specificity tests reveal no cross-reactivity with this antiserum when absorption tests were performed with LHRH, somatostatin, fl-endorphin, aand fl-MSH, as well as ACTH 1-10. Absorption studies also indicate cross-reactivity with partially purified preparations of the large molecular weight 31K precursor. Radioimmunoassay specificity tests (at 1 : 16,000 dilution) revealed the following: 13 % cross-reactivity with ACTH1-24; 46 % cross-reactivity with ACTH18-24 ; and no crossreactivity with equimotar concentrations of a-MSH, fl-lipotropin,/%endorphin and aendorphin ag. Mapping of the opiocortin perikarya was performed in the following manner. Initial drawings of the region of the basal hypothalamus were prepared from projections of each slide at an enlargement of >~20. Major nuclear groups and structures were included in these drawings. An antero-posterior coordinate was assigned to each drawing according to the atlas of Konig and Klippel~L Each slide was then examined under a microscope ( × 160), and the location of each profile recognized as an ACTH-immunoreactive perikaryon was plotted in these drawings. The number
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Fig. 1. Frontal 50 pm Vibratome sections of rat hypothalamus. ACTH-ir perikarya (-~) are located in the arcuate nucleus (ARC), adjacent periventricular stratum (ps) and internuclear space laterally between arcuate and ventromedial (VMH) nuclei• At this antero-posterior plane, ACTH-ir fibers are dense in both dorsal and ventral divisions of the dorsomedial nucleus (DMH) perfornical region and lateral hypothalamus (LH). F, fornix; MD median eminence; V, 3rd ventricle; Vr, ventricular recess. x 20. o f profiles were recorded. The opiocortin bed nucleus was mapped in this way on each 50/~m section o f 3 brains. On 4 other brains, maps were prepared at A P coordinates 5340, 4890, 3750 and 3100. Composite final topographical maps were prepared from these working drawings. RESULTS The A CTH-imrnunoreactive neurons
Fig. 1 illustrates the general anatomical organization o f the central opiocortin system in the hypothalamus o f the rat brain. Perikarya o f the opiocortin neurons are located in portions of the arcuate nucleus, extending from the retrochiasmatic area to
336 the m a m m i l l a r y region. F i b e r s originating f r o m these cell bodies distribute extensively t h r o u g h o u t the h y p o t h a l a m u s , limbic system a n d b r a i n stem. The p e r i k a r y a are generally oval, 15-20/zm in d i a m e t e r with i m m u n o r e a c t i v e staining p r o d u c t u n i f o r m l y distributed t h r o u g h o u t the c y t o p l a s m a n d an initial 5-10/~m segment o~'a fairly larged i a m e t e r a x o n (Fig. 2). I n a large n u m b e r o f instances, small i m m u n o r e a c t i v e profiles were seen in contact with either the a x o n or cell body. N o processes which could be identified as dendrites were observed.
Fig. 2. Details of ACTH-ir neurons and fiber profiles in 50/tin Vibratome sections. In 2a, note the granular reaction product present uniformly throughout the cytoplasm and in the first segment of a presumed axon. Profiles of immunoreactive fibers (-~) are present and one (1~) appears to be in contact with the axon. In 2b, another fiber profile (~') appears to be in contact with an ACTH-ir cell body. In 2c, an ACTH-ir neuron is located in the ependymal layer of the median eminence, in apparent contact with CSF of the ventricular recess (V). x 1000.
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3d Fig. 3. Schematic diagrams summarizing the distribution of ACTH-ir neurons in basal hypothalamus of rat brain. The representative sections a-d are assigned an antero-posterior coordinate from the atlas of KSnig and Klippel 2'~. The location of individual perikarya is represented by a single dot. Abbreviations: arc, arcuate nucleus; F, fornix; ha, anterior hypothalamic nucleus; hdd, dorsomediai nucleus, pars dorsdalis; hdr, dorsomedial nucleus, pars ventralis; hp, posterior hypothalamic nucleus; hpv, periventricular stratum; hvma, ventro-medial nucleus, pars anterior; MT, mammillothalamic tract; mml, lateral mammillary nucleus; mmm, medial mammillary nucleus; pr. premammillary nucleus.
338
Topography of the bed nucleus Fig. 3 presents a mapping of the topography of perikarya in the opiocortin bed nucleus. Cell bodies were located throughout the entire antero-posterior extent of the hypothalamus. They appeared first in the retrochiasmatic area, immediately behind the suprachiasmatic nucleus. For a distance of approximately 0.2 mm, they were located as a flat plate of cells along the ventral surface, contiguous across the mid-line and extending 1-1.5 mm laterally (Fig. 3a). Descent of the third ventricle and formation of the median eminence separated the bed nucleus into bilateral groups. Throughout the extent of the median eminence, cell bodies were located in the lateral half of the arcuate nucleus and the internuclear space between the arcuate and ventromedial nuclei laterally and dorsally into the periventricular stratum (Fig. 3b). Posteriorly, the general topography of the bed nucleus shifted to a more vertical orientation with only scattered cells laterally (Fig. 3c). Throughout the entire extent of the median eminence, significant numbers of cells extended into the subependymal zone of the median eminence. Beyond the region of median eminence-stalk separation, the nucleus oriented in a horizontal plane again and continued into the mammillary region in close proximity to the ventriculax recess (Fig. 3d). No cell bodies were located in the mammillary nuclei and only rarely were they seen more than 1 mm from the midline. Approximately equal numbers of cell bodies were present in the bed nucleus in every section of the brain except for its retrochiasmatic portion and the terminal portion in the mammillary region. In the retrochiasmatic region, the total number of cell bodies counted averaged two-thirds the number counted in subsequent sections where the bed nucleus was divided bilaterally. The last 0.3 mm of the bed nucleus in the mammillary region contained one-fifth as many cell bodies. In any given section of the bed nucleus, throughout its entire extent, the density of cell bodies appeared completely uniform. In the main portions of the opiocortin bed nucleus (Fig. 3a, b), the total number of profiles of ACTH-containing cell bodies counted per section averaged 170. We do not know, with the thickness of the sections used in this study (50 /~m), whether all cell bodies were stained. For this reason, no stereological correction factors were applied to correct for the fact that some cells are counted twice in adjacent sections. DISCUSSION The opiocortin perikarya visualized in this study with ACTH antiserum represent a new and complex neuronal system of the brain. Neuropeptides associated with these neurons may be derived by a series of enzymatic cleavages of a large 31 kDalton precursor molecule TM. Among the peptides derived from this precursor are a 16K fragment, ACTH, a-MSH, fl-lipotropin and the endorphins. In our studies (unpublished observations) and those of others 9,29,3~, antisera to each of these peptides immunocytochemically demonstrates the same perikarya and fibers. Zakararian and Smyth 37 have demonstrated that the amounts of biologically active and inactive forms of the endorphins vary in different projection regions of the opiocortin
339 system. Evidence of this type points to the likelihood of distinct functional suborganization in the opiocortin system. In addition to the opiocortin system examined in this study, a number of other new putative transmitters and neurohormones have been identified in the brain and their perikarya and fibers mapped. Some pattern may be emerging with respect to the locations of their perikarya relative to the classically defined nuclear cell groups of the brain. In the hypothalamus, cell bodies containing Substance P, Met- and Leuenkephalin, and angiotensin II appear to be located in defined nuclear groups; Substance P in the dorsomedial, ventromedial and premammillary nuclei, Metenkephalin in the arcuate, ventromedial, paraventricular and premammillary nuclei, and angiotensin in the paraventricular nucleus16. Perikarya containing other neurohormones appear not to be restricted to the boundaries of classical nuclear groups. Although LHRH perikarya have been difficult to demonstrate immunocytochemically, present data indicates a broad distribution in the septal region, preoptic, anterior hypothalamic and arcuate areasa,4,21,24,a4. Somatostatin-containing perikarya are present in the periventricular stratum of the preoptic and anterior hypothalamic regions2,5,11,15. TRH-positive cell bodies are found mainly in the dorsomedial nucleus16. Compared to the information available on these new neurotransmitter and neurohormone systems, the opiocortin bed nucleus appeais to be considerably larger in this hypothalamic distribution and apparent number and size of its cell population. Extending from the retrochiasmatic area to the end of the mammillary region in a continuous field of cell bodies, this group is clearly the longest nuclear structure of the hypothalamus. Its size may be a reflection of its massive fiber distribution in the hypothalamus, preoptic-septal regions, limbic system, and brain stem. The opiocortin bed nucleus is additionally unique in having some of its cell bodies located in the ependymal and subependymal layers of the median eminence. Neurons located in the arcuate region of the hypothalamus have traditionally been viewed as a major source of the tuberoinfundibular projections to the median eminence. Holzwarth-McBride et al. 17 observed little change in the architecture of the median eminence following destruction of 80-90 ~ of arcuate neurons with monosodium glutamate; they suggested that the majority of neurons in this region must project to other areas of the brain. Renaud a2 later found that only 15 ~o of the neurons in the arcuate region could be antidromically activated by stimulation of the median eminence. The opiocortin bed neurons have a small fiber projection into the infundibulum and neural lobe, but none to the external zone of the median eminenceZL These observations, together with the present study, are evolving a clearer understanding of the connections of the arcuate nucleus. Many nuclear groups in the brain have morphologically apparent subdivisions based upon differences in cell size, regional density, or spatial separation of portions of the nucleus. In spite of its multiple distribution of fibers within the hypothalamus, to the limbic system and brain stem, no apparent sub-organization was evident in the opiocortin bed nucleus. Additional studies using other methods may demonstrate regional sub-groups within this nucleus.
340 ACKNOWLEDGEMENTS S. A. J. is a recipient of R C D A HD-00230. This work was supported by N I H G r a n t s AM-22029, HD-07926 a n d NS-15345.
REFERENCES 1 Akil, H., Watson, S., Berger, P. and Barchas, J., Endorphins, fl-LPH and ACTH: biochemical pharmacological, and anatomical studies, Advanc. Biochem. Psychopharm, 18 (1978) 125-137. 2 Alpert, L. C., Brawer, J. R., Patel, Y. C. and Reichlin, S., Somatostatinergic neurons in anterior hypothalamus: immunohistochemical localization, Endocrinology, 98 (1976) 255-258. 3 Bennett-Clarke, C., Immunocytochemical localization of LHRH and SRIF in the rat CNS, Anat. Rec., 187 (1977) 536. 4 Bennett-Clarke, C. and Joseph, S. A., Immunocytochemical distribution of LHRH neurons and processes in the rat: hypothalamic and extrahypothalamic locations, Cell Tiss. Res., submitted. 5 Bennett-Clarke, C., Romagnano, M. A. and Joseph, S. A., Distribution of somatostatin in the rat brain: telencephalon and diencephalon, Brain Research, 188 (1980) 473486. 6 Bloch, B., Bugnon, C., Fellman, D. and Lenys, D., Infundibular neurons of the human hypothalamus simultaneously reactive with antisera against ACTH, MSH, and fl-LPH, Neurosci. Lett. 10 (1978) 147-152. 7 Bloom, F., Rossier, J., Battenberg, R., Bayon, A., French, E., Henriksen, S., Siggins, G., Segal, D., Browne, R., Ling, N. and Guillemin, R., fl-Endorphin cellular localization electrophysiological and behavioral effects, Advanc. Biochem. Psychopharm., 18 (1978) 89-109. 8 Bloom, F., Battenberg, E., Rossier, J., Ling, N. and Guillemin, R., Neurons containing fl-endorphin in rat brain exist separately from those containing enkephalin: immunocytochemical studies, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 1591. 9 Bugnon, C., Bloch, B., Lenys, D. and Fellman, D., Infundibular neurons of the human hypothalamus simultaneously reactive with antisera against endorphins, ACTH, MSH, and fl-LPH, Cell Tiss. Res., 199 (1979) 177-196. 10 Eipper, B. A. and Mains, R. E., Structure and biosynthesis of pro-ACTH/endorphin and related peptides, Endocr. Rev. 1 (1980) 1-27. 11 Elde, R. P. and Parsons, J. A., Immunocytochemical localization of somatostatin in cell bodies of the rat hypothalamus, Amer. J. Anat., 144 (1975) 541-548. 12 Eskay, R. L., Brownstein, M. J. and Long, R. T., a-melanocyte-stimulating hormone: reduction in adult rat brain after monosodium glutamate: treatment of neonates. Science, 205 (1979) 827-829. 13 Fratta, W., Yang, Y., Majane, B. and Costa, E., Distribution of fl-endorphin and related peptides in the hypothalamus, Neuroscience, 4 (1979) 1903-1908. 14 Gramsch, C., Hollt, B., Mehraein, P., Pasi, A. and Herz, A., Regional distribution of methionineenkephalin and beta-endorphin-like immunoreactivity in human brain and pituitary, Brain Research, 171 (1979)261-270. 15 Hokfelt, T., Efendic, S., Hellestrom, C., Johansson, O., Luft, R. and Arimura, A., Cellular localization of somatostatin in endocrine-like cells and neurons of the rat with special reference to the Al-cells of the pancreatic islets and to the hypothalamus, Acta endocr. (Kbh.), 80, Suppl. 200 (1975) 541. 16 Hokfelt, T., Elde, R., Fuxe, K., Johansson, O., Ljungdahl, H., Goldstein, M., Luft, R., Efendic, S., Nilsson, G., Terenius, L., Ganten, D., Jeffcoate, S. L., Rehfeld, J., Said, S., Perez de la Mora, M., Possani, L., Tapia, R., Teran, L. and Palacios, R., Aminergic and peptidergic pathways in the nervous system with special reference to the hypothalamus. In S. Reichlin, R. J. Baldessarini and J. B. Martin (Eds.), The Hypothalarnus, Raven Press, New York, 1978, pp. 69-136. 17 Holzwarth-McBride, M. A., Hurst, E. M. and Knigge, K. M., Monosodium glutamate-induced lesion of the arcuate nucleus. I. Endocrine deficiency and ultrastructure of the median eminence, Anat. Rec., 186 (1976) 185-196. 18 Jacobowitz, D. M. and O'Donohue, T. L., a-melanocyte-stimulating hormone: immunohistochemical identification and mapping in neurons of rat brain, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 6300-6304.
341 19 Joseph, S. A., Immunoreactive adrenocorticotropin in rat brain: a neuroanatomical study using antiserum generated against synthetic ACTH 1-3°. Amer. J. Anat., 158 (1980) 533-548. 20 Joseph, S. A. and Knigge, K. M., Cells of origin of the ACTH-immunoreactiveopiocortin system in the rat brain, Anat. Rec., in press. 21 King, J. C., Gerall, A. A., Fishback, J. B., Elkind, K. E. and Arimura, A., Growth hormonerelease inhibitinghormone (GR-RIH) pathway of the rat hypothalamus revealed by the unlabeled antibody peroxidase-antiperoxidase method, Cell Tiss. Res., 160 (1975) 423430. 22 Knigge, K. M. and Joseph, S. A., Relationship of the central ACTH-immunoreactiveopiocortin system to the supraoptic and paraventricular nuclei, Amer. J. Anat., in press. 23 Knigge, K. M. and Joseph, S., Relationship of the central ACTH-immunoreactive opiocortin system to the supraoptic and paraventricular nuclei, Amer. J. Anat., submitted. 24 Knigge, K. M., Joseph, S. A., Hoffman, G. E., Arimura, A. and Sternberger, L. A., Organization of LRF- and SRIF-neurons in the endocrine hypothalamus. In S. Reichlin, R. J. Baldessarini and J. B. Martin (Eds.), The Hypothalamus, Raven Press, New York, 1978, pp. 49-67. 25 Konig, J. F. R. and Klippel, R. A., The Rat Brain, Williams and Wilkins, Baltimore, Md., 1963. 26 Krieger, D. T., Liotta, A. S. and Brownstein, M.J., Presence of corticotropin in brain of normal and hypophysectomized rats, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 648-652. 27 Krieger, D. T., Liotta, A. S., Nicholsen, G. and Kizer, J. S., Brain ACTH and endorphin reduced in rats with monosodium glutamate-induced arcuate nuclear lesions, Nature (Lond.), 278 (1979) 562-563. 28 Liotta, A. S., Gildersleeve, D., Brownstein, M. J. and Krieger, D. T., Biosynthesis in vitro of immunoreactive 31,000-Dalton corticotropin/fl-endorphin-like material by bovine hypothalamus, Proc. nat. Acad. Sci. (Wash.), 76 (1979) 1448-1452. 29 Nilaver, G., Zimmerman, E. A., Defendini, R., Liotta, A. S., Krieger, D. T. and Brownstein, M. J., Adrenocorticotropin and fl-lipotropin in the hypothalamus, J. Cell BioL 81 (1979) 50-58. 30 O'Donohue, T. L., Miller, R. L., Pendleton, R. C. and Jacobowitz, D. M., A diurnal rhythm of immunoreactive a-melanocyte-stimulatinghormone in discrete regions of the rat brain, Neuroendocrinology, 29 (1979) 281-287. 31 Pelletier, G., Leclerc, R., Saavedra, J. M., Brownstein, M. J., Vaudry, H., Ferland, L. and Labrie, F., Distribution of fl-lipotropin (fl-LPH), adrenocorticotropin (ACTH), and a-melanocyte-stimulating hormone (a-MSH) in the rat brain. I. Origin of the extrahypothalamic fibers, Brain Research, 192 (2) (1980) 433~140. 32 Renaud, L. P., Tuberoinfundibularneurons in the basomedial hypothalamus of the rat: electrophysiological evidence for axon collaterals to hypothalamic and extrahypothalamic areas, Brain Research, 105 (1976) 59-72. 33 Rossiuer, H., Vargo, T. M., Minick, S., Ling, N., Bloom, F. E. and Guillemin, R., Regional dissociation of beta-endorphin and enkephalin contents in rat brain and pituitary, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 5162-5165. 34 Setalo, G., Vigh, S., Schally, A. V., Arimura, A. and Flerko, B., GH-RIH-containingneural elements in the rat hypothalamus, Brahz Research, 90 (1975) 352-356. 35 Sofroniew, M., ACTH and fl-lipotropin in the hypothalamus. Localization in the same arcuate neurons by sequential immunocytochemical procedures, J. Anat. (Lond.), 154 (1979) 283-289. 36 Watson, E., Voigt, K. H. and Martin, R., Concomitant storage of ACTH- and endorphin-like immunoreactivity in the secretory granules of anterior pituitary corticotrophs, Brain Research, 157 (1978) 385-390. 37 Zakararian, S. and Smyth, D., Distribution of active and inactive forms of endorphins in rat pituitary and brain, Proc. nat. Acad. Sci. (Wash.), 76 (1979) 5972-5976.
333
T O P O G R A P H Y OF T H E A C T H - I M M U N O R E A C T I V E N E U R O N S IN T H E BASAL H Y P O T H A L A M U S OF T H E RAT BRAIN
KARL M. KNIGGE, SHIRLEY A. JOSEPH and JAY NOCTON The Neuroendocrine Unit, University of Rochester, School of Medicine and Dentistry, Rochester, N. Y. 14642 (U.S.A.)
(Accepted November 27th, 1980) Key words: hypothalamus -- ACTH -- opiocortin -- immunocytochemistry
SUMMARY Cell bodies of the opiocortin neurons were stained immunocytochemically with ACTH antiserum and their location in the basal hypothalamus of the rat brain was mapped. They are present throughout the entire extent of the hypothalamus, from retrochiasmatic area to mammillary body. In the retrochiasmatic area they are a single, midline group horizontally oriented and lying close to the ventral surface. Throughout the extent of the median eminence, ACTH-ir perikarya are located in the arcuate nucleus, adjacent periventricular stratrum and internuclear space between arcuate and ventromedial nuclei; as a group, they are oriented in a dorso-lateral plane. In the mammillary region, the nucleus assumes a more horizontal orientation again, lying close to the ventral surface. No regional differences were noted in cell density except in the retrochiasmatic area and terminal portion of the mammillary region where they were approximately 30 % fewer in number.
INTRODUCTION Data based upon radioimmunoassay13,26,~s, 30, immunocytochemistryl,7,1s, 19, and in vitro culture methods zs indicate that the mediobasal hypothalamus is a site of high concentration and possible neuronal synthesis of the pro-ACTH/endorphin (opiocortin) peptides (31K, 16K, ACTH, a-MSH, fl-LPH, fl-MSH, and a- and flendorphin). Chemical12, 27 and electrolytic lesionZ0, 36 and deafferentation 31 studies have provided additional evidence that the opiocortin fiber system may originate from perikarya in the mediobasal hypothalamus. Neuronal cell bodies in this region have been shown to be simultaneously immunoreactive with antisera against known opiocortin peptides 6,9,29,a5. Joseph 19 has provided a detailed neuroanatomica map-
334 ping of the fiber distribution within the hypothalamus, to the limblc system, and to the brain stem. Details of the relationship of opiocortin fibers to the m agnocellular nuclei, median eminence, and the pituitary gland have been described by Knigge and Joseph 22,23. This neuropeptide system is distinct from networks containing Met- and Leu-enkephalinS,14,33, 36. In this report we present a more comprehensive map of the ACTH-immunoreactive perikarya in the mediobasal hypothalamus. MATERIALS AND METHODS Male Sprague-Dawley rats, 200-250 g b.wt. were anesthetized and the brains fixed by cardiac perfusion with 100 ml saline followed by 200 ml of Bouin's fixative. Brains were removed and fixed an additional 24 h in fresh fixative at 4 °C. They were sectioned serially in the coronal plane without further treatment on an Oxford Vibratome at 50 #m. Sections from the anterior commissure to the interpeduncular fossa were collected in phosphate buffer-saline (PBS, pH 7.2) and rinsed repeatedly with PBS until colorless. All staining procedures were carried out in 15 x 60 mm plastic dishes with 20-25 sections per dish in 4 ml volumes of staining reagents. In all cases, sections were incubated in the primary antiserum at 4 °C for 48 h in PBS containing 0.02 % Triton-X and 1% bovine serum albumin; sections were agitated during this incubation with the use of an orbital shaker oscillating at 50 cpm. The unlabeled antibody peroxidase-antiperoxidase (PAP) was used. Sections were stained for ACTH with antiserum to ACTH 1-39 (K-531) generated in rabbit using synthetic ACTH 1-39 conjugated to BSA with carbodiamide 19. The initial injection consisted of 4 mg of the immunogen administered intradermally in multiple injection sites along the back. Two booster injections, each consisting of 2 mg immunogen, followed at two-week intervals. The antiserum is normally used at a dilution of 1:128,000 for pituitary immunocytochemistry and a 1:16,000 for staining in brain. For the purpose of intensifying the staining of immunoreactive perikarya, the primary antiserum was used in this study at a dilution of 1:4000. Specificity tests reveal no cross-reactivity with this antiserum when absorption tests were performed with LHRH, somatostatin, fl-endorphin, aand fl-MSH, as well as ACTH 1-10. Absorption studies also indicate cross-reactivity with partially purified preparations of the large molecular weight 31K precursor. Radioimmunoassay specificity tests (at 1 : 16,000 dilution) revealed the following: 13 % cross-reactivity with ACTH1-24; 46 % cross-reactivity with ACTH18-24 ; and no crossreactivity with equimotar concentrations of a-MSH, fl-lipotropin,/%endorphin and aendorphin ag. Mapping of the opiocortin perikarya was performed in the following manner. Initial drawings of the region of the basal hypothalamus were prepared from projections of each slide at an enlargement of >~20. Major nuclear groups and structures were included in these drawings. An antero-posterior coordinate was assigned to each drawing according to the atlas of Konig and Klippel~L Each slide was then examined under a microscope ( × 160), and the location of each profile recognized as an ACTH-immunoreactive perikaryon was plotted in these drawings. The number
335
=
.
~i li i ~¸~¸ ii ....
V
:
;? ? 7
Fig. 1. Frontal 50 pm Vibratome sections of rat hypothalamus. ACTH-ir perikarya (-~) are located in the arcuate nucleus (ARC), adjacent periventricular stratum (ps) and internuclear space laterally between arcuate and ventromedial (VMH) nuclei• At this antero-posterior plane, ACTH-ir fibers are dense in both dorsal and ventral divisions of the dorsomedial nucleus (DMH) perfornical region and lateral hypothalamus (LH). F, fornix; MD median eminence; V, 3rd ventricle; Vr, ventricular recess. x 20. o f profiles were recorded. The opiocortin bed nucleus was mapped in this way on each 50/~m section o f 3 brains. On 4 other brains, maps were prepared at A P coordinates 5340, 4890, 3750 and 3100. Composite final topographical maps were prepared from these working drawings. RESULTS The A CTH-imrnunoreactive neurons
Fig. 1 illustrates the general anatomical organization o f the central opiocortin system in the hypothalamus o f the rat brain. Perikarya o f the opiocortin neurons are located in portions of the arcuate nucleus, extending from the retrochiasmatic area to
336 the m a m m i l l a r y region. F i b e r s originating f r o m these cell bodies distribute extensively t h r o u g h o u t the h y p o t h a l a m u s , limbic system a n d b r a i n stem. The p e r i k a r y a are generally oval, 15-20/zm in d i a m e t e r with i m m u n o r e a c t i v e staining p r o d u c t u n i f o r m l y distributed t h r o u g h o u t the c y t o p l a s m a n d an initial 5-10/~m segment o~'a fairly larged i a m e t e r a x o n (Fig. 2). I n a large n u m b e r o f instances, small i m m u n o r e a c t i v e profiles were seen in contact with either the a x o n or cell body. N o processes which could be identified as dendrites were observed.
Fig. 2. Details of ACTH-ir neurons and fiber profiles in 50/tin Vibratome sections. In 2a, note the granular reaction product present uniformly throughout the cytoplasm and in the first segment of a presumed axon. Profiles of immunoreactive fibers (-~) are present and one (1~) appears to be in contact with the axon. In 2b, another fiber profile (~') appears to be in contact with an ACTH-ir cell body. In 2c, an ACTH-ir neuron is located in the ependymal layer of the median eminence, in apparent contact with CSF of the ventricular recess (V). x 1000.
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3d Fig. 3. Schematic diagrams summarizing the distribution of ACTH-ir neurons in basal hypothalamus of rat brain. The representative sections a-d are assigned an antero-posterior coordinate from the atlas of KSnig and Klippel 2'~. The location of individual perikarya is represented by a single dot. Abbreviations: arc, arcuate nucleus; F, fornix; ha, anterior hypothalamic nucleus; hdd, dorsomediai nucleus, pars dorsdalis; hdr, dorsomedial nucleus, pars ventralis; hp, posterior hypothalamic nucleus; hpv, periventricular stratum; hvma, ventro-medial nucleus, pars anterior; MT, mammillothalamic tract; mml, lateral mammillary nucleus; mmm, medial mammillary nucleus; pr. premammillary nucleus.
338
Topography of the bed nucleus Fig. 3 presents a mapping of the topography of perikarya in the opiocortin bed nucleus. Cell bodies were located throughout the entire antero-posterior extent of the hypothalamus. They appeared first in the retrochiasmatic area, immediately behind the suprachiasmatic nucleus. For a distance of approximately 0.2 mm, they were located as a flat plate of cells along the ventral surface, contiguous across the mid-line and extending 1-1.5 mm laterally (Fig. 3a). Descent of the third ventricle and formation of the median eminence separated the bed nucleus into bilateral groups. Throughout the extent of the median eminence, cell bodies were located in the lateral half of the arcuate nucleus and the internuclear space between the arcuate and ventromedial nuclei laterally and dorsally into the periventricular stratum (Fig. 3b). Posteriorly, the general topography of the bed nucleus shifted to a more vertical orientation with only scattered cells laterally (Fig. 3c). Throughout the entire extent of the median eminence, significant numbers of cells extended into the subependymal zone of the median eminence. Beyond the region of median eminence-stalk separation, the nucleus oriented in a horizontal plane again and continued into the mammillary region in close proximity to the ventriculax recess (Fig. 3d). No cell bodies were located in the mammillary nuclei and only rarely were they seen more than 1 mm from the midline. Approximately equal numbers of cell bodies were present in the bed nucleus in every section of the brain except for its retrochiasmatic portion and the terminal portion in the mammillary region. In the retrochiasmatic region, the total number of cell bodies counted averaged two-thirds the number counted in subsequent sections where the bed nucleus was divided bilaterally. The last 0.3 mm of the bed nucleus in the mammillary region contained one-fifth as many cell bodies. In any given section of the bed nucleus, throughout its entire extent, the density of cell bodies appeared completely uniform. In the main portions of the opiocortin bed nucleus (Fig. 3a, b), the total number of profiles of ACTH-containing cell bodies counted per section averaged 170. We do not know, with the thickness of the sections used in this study (50 /~m), whether all cell bodies were stained. For this reason, no stereological correction factors were applied to correct for the fact that some cells are counted twice in adjacent sections. DISCUSSION The opiocortin perikarya visualized in this study with ACTH antiserum represent a new and complex neuronal system of the brain. Neuropeptides associated with these neurons may be derived by a series of enzymatic cleavages of a large 31 kDalton precursor molecule TM. Among the peptides derived from this precursor are a 16K fragment, ACTH, a-MSH, fl-lipotropin and the endorphins. In our studies (unpublished observations) and those of others 9,29,3~, antisera to each of these peptides immunocytochemically demonstrates the same perikarya and fibers. Zakararian and Smyth 37 have demonstrated that the amounts of biologically active and inactive forms of the endorphins vary in different projection regions of the opiocortin
339 system. Evidence of this type points to the likelihood of distinct functional suborganization in the opiocortin system. In addition to the opiocortin system examined in this study, a number of other new putative transmitters and neurohormones have been identified in the brain and their perikarya and fibers mapped. Some pattern may be emerging with respect to the locations of their perikarya relative to the classically defined nuclear cell groups of the brain. In the hypothalamus, cell bodies containing Substance P, Met- and Leuenkephalin, and angiotensin II appear to be located in defined nuclear groups; Substance P in the dorsomedial, ventromedial and premammillary nuclei, Metenkephalin in the arcuate, ventromedial, paraventricular and premammillary nuclei, and angiotensin in the paraventricular nucleus16. Perikarya containing other neurohormones appear not to be restricted to the boundaries of classical nuclear groups. Although LHRH perikarya have been difficult to demonstrate immunocytochemically, present data indicates a broad distribution in the septal region, preoptic, anterior hypothalamic and arcuate areasa,4,21,24,a4. Somatostatin-containing perikarya are present in the periventricular stratum of the preoptic and anterior hypothalamic regions2,5,11,15. TRH-positive cell bodies are found mainly in the dorsomedial nucleus16. Compared to the information available on these new neurotransmitter and neurohormone systems, the opiocortin bed nucleus appeais to be considerably larger in this hypothalamic distribution and apparent number and size of its cell population. Extending from the retrochiasmatic area to the end of the mammillary region in a continuous field of cell bodies, this group is clearly the longest nuclear structure of the hypothalamus. Its size may be a reflection of its massive fiber distribution in the hypothalamus, preoptic-septal regions, limbic system, and brain stem. The opiocortin bed nucleus is additionally unique in having some of its cell bodies located in the ependymal and subependymal layers of the median eminence. Neurons located in the arcuate region of the hypothalamus have traditionally been viewed as a major source of the tuberoinfundibular projections to the median eminence. Holzwarth-McBride et al. 17 observed little change in the architecture of the median eminence following destruction of 80-90 ~ of arcuate neurons with monosodium glutamate; they suggested that the majority of neurons in this region must project to other areas of the brain. Renaud a2 later found that only 15 ~o of the neurons in the arcuate region could be antidromically activated by stimulation of the median eminence. The opiocortin bed neurons have a small fiber projection into the infundibulum and neural lobe, but none to the external zone of the median eminenceZL These observations, together with the present study, are evolving a clearer understanding of the connections of the arcuate nucleus. Many nuclear groups in the brain have morphologically apparent subdivisions based upon differences in cell size, regional density, or spatial separation of portions of the nucleus. In spite of its multiple distribution of fibers within the hypothalamus, to the limbic system and brain stem, no apparent sub-organization was evident in the opiocortin bed nucleus. Additional studies using other methods may demonstrate regional sub-groups within this nucleus.
340 ACKNOWLEDGEMENTS S. A. J. is a recipient of R C D A HD-00230. This work was supported by N I H G r a n t s AM-22029, HD-07926 a n d NS-15345.
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