Radioautographic study of the rat brain after injection of [1,2-3H]corticosterone

Brain Research, 89 (1975) 61-70

61

,~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

R A D I O A U T O G R A P H I C STUDY OF T H E RAT BRAIN TION OF [1,2-3H]CORTICOSTERONE

AFTER

INJEC-

MARYVONNE WAREMBOURG

Laboratoire d'Histologie, Facultd de Mddecine, Place de Verdun, 59045 Lille Cddex (France) (Accepted December 15th, 1974)

SUMMARY

Cryostat sections of the central nervous system of adrenalectomized male rats injected with [aH]corticosterone were examined by radioautography. One hour after the injection, radioactivity was found to be selectively concentrated in specific neurons of the septum, the hippocampal complex (precommissural hippocampus, cornu Ammonis, gyrus dentatus, subiculum), the indusium griseum, the amygdala and in certain areas of the cortex. In the hippocampus, the pyramidal neurons in fields CA! and CA2 of the cornu Ammonis and the granule neurons of the gyrus dentatus contained more radioactivity than did other regions of the brain. Most of the silver grains were localized in the nuclei of labeled cells. The topographic distribution of corticosterone-concentrating neurons shows that the hormonal target sites in the central nervous system are mainly extrahypothalamic.

INYRODUCTION

Biochemical studies have shown that [3H]corticosterone is selectively accumulated by the hippocampus and the septum in the brain of adrenalectomized rat and have also demonstrated a limited capacity for hormonal uptake 11,ls,19. Macromolecules which bind [3H]corticosterone have been isolated from brain cytosols and evidence presented that these macromolecules have many of the characteristics of a receptor proteinS, 15-17. Radioautography has revealed that [3H]corticosterone is accumulated in neuronal cell bodies of the hippocampus 7 and certain other areas of the brain 27-a°. In this paper, we describe radioautographic studies of the central nervous system after injection of [aH]corticosterone. The work presented here confirms that various regions of the brain have the ability to retain corticosterone, the major glucocorticoid secreted by the rat adrenal 3, and that the hormone is mainly concentrated in the neuronal nuclei of these regions.

62 MATERIALS AND METHODS Eight male S p r a g u e - D a w l e y rats, 30 days old, were adrenalectomized 48 h to 6 days before the experiment and maintained on 0.9 ~ sodium chloride solution. The rats were injected subcutaneously with 100 #Ci o f [l,2-3H]corticosterone (C.E.A. France, spec. act. 26 Ci/mmole) dissolved in 0.15 ml isotonic saline and 0.05 ml ethanol. The animals were decapitated 1 h, 1 h 40 rain or 2 h after the injection. The brains were quickly removed and frozen at - - 2 5 ~C in a W.K. 1150 cryostat (W.K.F. Brandau, Germany). They were cut into 10/~m frontal sections, f r o m the olfactory bulb to the mesencephalon (nuclei pontis), in the cryostat at - - 2 0 °C. The r a d i o a u t o g r a p h i c technique used in order to prevent diffusion artifacts has been previously described 3a,32. After radioautographic exposure for 112-128 days at - - 1 5 °C, the slides were fixed 5 min with methanol, developed 4 rain with the D-19 B K o d a k developer and fixed 6 min in 30 ~ sodium thiosulfate. They were then stained with m e t h y l g r e e n - p y r o n i n . Frontal sections were selected after studying r a d i o a u t o g r a m s and schematic drawings were traced. The identification o f the structures was made according to the rat brain atlas o f K6nig and KlippellL RESULTS

Distribution of radioactivity in the brain 1 h after the injection of l' 3HJ corticosterone In the central nervous system o f all animals studied, the labeled neurons showed similar topographic distribution. One hour after the injection, the t i m e a t which binding reaches its peak in cell nuclei isolated from the hippocampuslS, la, the radioactivity was concentrated in nuclei o f certain cells. These cells are located in distinct and anatomically defined areas (Figs. 1 4 ) w h e r e a s cells o f other regions remain unlabeled.

Figs. 1-4. Selected schematic drawings prepared after frontal section radioautograms from the parolfactory region to the nuclei pontis of male rat brain 1 h after the injection of I00 uCi of [1,2-3H] corticosterone. Areas with accumulation of corticosterone-concentrating neurons are indicated by stipples. Abbreviations: AT, area transitionalis cortico-amygdaloidea; a, n. aecumbens: ab. n. amygdaloideus basalis; aco, n. amygdaloideus corticalis; al, n. amygdaloideus lateralis: am, n. amygdaloideus medialis; ar, n. arcuatus; C, cingulum; CA, commissura anterior; CA1, CA2, CAz and CAa, fields of the pyramidal neuron layer in the cornu Ammonis; CC, crus cerebri; CE, cortex entorhinalis; CFD, commissura fornicis dorsalis; CL, claustrum: CO, chiasma opticum; CPF, cortex piriformis; cp, n. caudatus putamen; FH~ fimbria hippocampi; FLC, fissura longitudinalis cerebri; FOP, fasciculus opticus; FOR, formatio reticularis; GCC, genu corporis callosi; GD, gyrus dentatus: H, habenula; HIA, hippocampus, pars anterior: hp, n. posterior hypothalami; IC, insulae Calleja; IG, indusium griseum; ip, n. interpeduncularis; LM, lemniscus medialis; p, n. pontis; pill, n. principalis n. oculomotorii; pv, n. premamillaris ventralis; RCC, radiato corporis callosi; r, n. tuber: S, subiculum; SGC, substantia grisea centralis; SR, sulcus rhinalis; sl, n. septi lateralis: sm. n. septi medialis: TCC, truncus corporis caHosi; TOL, tractus olfactorius lateralis; TUL, tuberculum olfactorium; TD, tractus diagonalis (Broca); td, n. tractus diagonalis (Broca); VL, vent riculus lateralis: Z1. zona incerta.

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O/factor), and parolJactor)' region.s In the nucleus (n.) olfactorius anterior, labeled neurons existed singly, dispersed or in groups of only a few cells mainly in the lateral and dorsal parts. In these cells, the labeling was relatively low. In the parolfactory region, extensive labeling of neurons was observed in the precommissural hippocampus, pars anterior (Fig. 1). The ktbeled neurons were continuous with the labeled neurons in the indusium griseum (Figs. 2 and 3), that is the supracallosal hippocampus, as well as with the heavily labeled neurons in the preseptal hippocampus anterior. The n. accumbens, the insulae Calleja and the n. tractus diagonatis did not contain labeled neurons.

Septum The uptake of [3H]corticosterone in the septum was not uniform (Fig. 2). Most of the heavily labeled cells were found in its anterior portion: in the n. septi lateralis close to the lateral ventricle and under the corpus callosum (Fig. 5). In m a n y labeled neurons, silver grains were observed over the cell nuclei. The neurons of the n. interstitialis striae terminalis and the n. triangularis septi remained unlabeled. More caudally the organon subfornicale retained some radioactive material.

Hippocampal cornph,x Gyrus hippocampi (cornu Ammonis, gyrus dentatus, subiculum). Many heavily labeled cells were found in the pyramidal layer of the cornu Ammonis. On the basis of characteristic differences in the organization of this layer, Lorente de N6 lz,14 has distinguished among the pyramidal cells 4 zones referred to as fields CA1, CA2, CA3 and CA4. An heterogeneity of labeling among these fields appeared (Fig. 3). Our radioautograms showed that fields CA1 (anterior and posterior portions) and CA2 contained more labeled neurons than fields CAz and CA4. In addition the average intensity of labelingin CA1 (Fig. 6) and CA~ (Fig. 7) was higher than in CA8 (Fig. 8) and CA~. The most weakly labeled area was the field CA4. However, differences in the degree of labeling existed among cells within field CA1: concentration of radioactivity was slightly greater in the anterior neurons than in the posterior neurons. The subiculum also contained radioactively labeled neurons, including the cells of the prosubiculum. Most neurons of the granule layer of the gyrus dentatus incorporated relatively high amounts of radioactivity (Fig. 9). In labeled neurons of the hippocampal complex. the strongest concentration of radioactivity appeared in the nuclei. This nuclear binding was specially marked in the pyramidal neurons (Fig. 10).

Figs. 5-I 1, Radioautograms of the central nervous system, prepared 1 h after subcutaneous injection of [3Hlcorticosterone into adrenalectomized male rats. Frontal plane. Stained with methyl greenpyronin. Exposure time: 120 days. Fig, 5. Radioautogram of the nucleus septi lateralis close to the lateral ventricle, x 660. Figs. 6-8. Radioautograms of the hippocampus showing the labeling of the pyramidal neurons of fields CA1 (Fig. 6, × 535), CA~ (Fig. 7, × 670), CA3 (Fig. 8, × 635). Silver grains are Concentrated over cell nuclei.

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Fig. 9. Radioautogram of the gyrus dentatus. ;. 660. Fig. 10. Radioautogram of neurons of the field CAt illustrating that the radioactive material is concentrated in the nuclei. 1340. Fig. 11. Radioautogram of neurons of the nucleus arnygdaloideus corticalis. × 1340. These neurons are not labeled as heavily as hippocampal neurons (compare Fig. 10 with Fig. 1 I).

Gyrus parahippocampi. O n l y a few labeled n e u r o n s were f o u n d in the p r e s u b i c u l u m a n d in the p a r a s u b i c u l u m . The silver g r a i n density was w e a k e r in these cells than in the n e u r o n s o f gyrus h i p p o c a m p i . Amygdala A few single labeled n e u r o n s were f o u n d in the n. centralis a n d the n. medialis while the n. lateralis a n d the a r e a a m y g d a l a a n t e r i o r were u n l a b e l e d (Fig. 3). The m o s t intensive n e u r o n a l labeling with the highest labeling index was o b s e r v e d in the n. basalis p a r s medialis, in the n. corticalis a n d in the area t r a n s i t i o n a l i s c o r t i c o a m y g daloidae. But these n e u r o n s showed only a c o m p a r a t i v e l y w e a k labeling i f c o m p a r e d with labeled n e u r o n s o f the h i p p o c a m p a l c o m p l e x as can be seen in Figs. 10 a n d 11. The cells o f the m a s s a i n t e r c a l a t a were unlabeled.

Cortex L a b e l e d n e u r o n s were o b s e r v e d in the p e r i a m y g d a l o i d cortex, in the b o r d e r i n g

67 fissura amygdala and in the cortex piriformis, mainly near the sulcus rhinalis. Cells in the area 28 of the cortex entorhinalis were also intensively labeled (Fig. 4). A few scattered labeled neurons, with weak radioactivity, were found in layers of the cortex of the dorsal hemisphere.

Thalamus and hypothalamus There was no accumulation of radioactivity in the thalamic and hypothalamic areas comparable to that found in other regions of the brain referred to above. In these regions a few silver grains could be seen over the cell nuclei and over the cytoplasm of widely dispersed neurons. The n. habenulae retained some radioactivity. Pituita o, A large number of anterior lobe cells showed radioactive labeling. The different types of cells could not be identified by methyl green-pyronin stain. Nevertheless, these labeled cells were found scattered in the whole anterior lobe. Generally, heavily labeled cells were observed adjacent to scantily labeled or non-labeled cells. The radioactive material appeared to be distributed over both the cytoplasm and over the cell nuclei. However, a small number of cells showed retention of radioactivity with silver grains being more heavily concentrated over cell nuclei. In all the structures of the intermediate and posterior lobes, radioactivity was distributed diffusely, with less accumulation in the intermediate lobe compared to two other lobes. The neurohypophysis revealed widely dispersed silver grains without clear cellular or intracellular localization. Distribution of radioactivity 1 h 40 rain and 2 h after the injection of [3H /corticosterone The intensity of labeling clearly decreased in olfactory region, cortex, amygdala and fields CA3 and CA4 of the cornu Ammonis 1 h 40 min and later after administration of the isotope. In contrast, many labeled neurons were still present at these times in septum, indusium griseum, fields CA1 and CA2 and gyrus dentatus. The silver grains were more heavily concentrated over cell nuclei. DISCUSSION

Radioautography has shown that [aH]corticosterone is localized in neuron cell bodies of the central nervous system. Much of the labeling appears to be associated with the cell nuclei. The chemical nature of the radioactivity retained in neurons of the brain has not been studied in our experiments. It has been demonstrated previously 15,1s,19 that at 1 h after the injection of [aH]corticosterone unchanged hormone accounts for 70-80 ~o of radioactivity in the hippocampus and more than 90 ~ of radioactivity in cell nuclei. Thus, the concentrated radioactivity, particularly in the hippocampal neurons, may probably be attributed to unmetabolized [aH]corticosterone. Certain rhinencephalic structures take up and retain greater concentrations of radioactive material than do the hypothalamic structures.

68 The hippocampus and the septum area demonstrate an ability to concentrate radioactive corticosterone in comparison to other nervous tissue sites and the intensity of labeling varied within differents parts of these regions. Other areas, such as the amygdala with overlying cerebral cortex and certain regions of the cortex, show lower but still substantial accumulations of labeled neurons. These observations largely agree with the biochemical uptake results 11,ts,la. The present radioautographic studies are in accord with the localization of [3H]corticosterone in the hippocampus reported by Gerlach and McEwenT; but, we have also found specific concentrations of this hormone in other areas of the brain. Earlier reports 'zv-a0 have described glucocorticoid neurons in the hippocampus, the gyrus dentatus, the indusium griseum, the septum and the amygdala. After a precise regional analysis of the rat brain from the olfactory bulb to the mesencephalon, we confirm this same distribution and give (Figs. 1-4) a survey of the topographic localization of corticosterone-concentrating neurons. The latter is different from the sex steroids -°6. A few dispersed labeled neurons existed in the hippocampus and in the gyrus dentatus after injection of [3H]estradiot. Our observations show that these same cellular groups have a strong capacity to concentrate high quantities of [3H]corticosterone. This is suggestive that these neurons which respond to the hormone in a specific manner are target cells for corticosterone and may be involved with mechanisms controlling ACTH secretion. Using electrophysiological techniques and implantation of corticosteroids, various authors have reported that the hippocampus, the septum and the amygdala are implicated in the control of ACTH releaseZ,9,10,~3. This conclusion contrasts with that obtained by investigators who have shown that the hypothalamus responds functionally and electrically to implantation of both synthetic and natural steroids 4,6, za. Experimental evidence supports the concept of the hypothalamus as the primary neural substrate for neuroendocrine regulation of hypophyseal hormone secretion. The pituitary gland tends to contain more radioactivity than the hypothalamus. Some data in the literature indicate a direct effect of glucocorticoids on the gland 2'),24. But the results are also controversial and additional studies will be necessary before one can conclude whether or not the anterior pituitary is a primary target site for corticosterone. It can thus be supposed that there are several sites of action of corticosteroids within the brain, including the pituitary gland, and some of them may be involved in the regulation mechanism of ACTH secretion. The presence of corticosteroids in the brain produces neurophysiological effects on single-unit activity20, 21 and induces biochemical changes in nervous tissue, for instance, changes in the level of certain enzymes 1,~. These results suggest that glucocorticoids exert effects on brain tissue other than those related to corticotropin secretion. ACKNOWLEDGEMENTS

This work was supported by the I.N.S.E.R.M. and the D.G.R:S.T.

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