Brain Research, 97 (1975) 167-170 (Q Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

167

Increased incorporation of pH]uridine into RNA in the brain subfornical organ of ovariectomized mice

JACK M. GEORGE

and

MARGARET PENROSE

Department of Medicine, The Ohio State University, Columbus, Ohio 43210 (U.S.A.)

(Accepted June 25th, 1975)

The subfornical organ is a tiny hemispheric projection into the third ventricle of the brain at the site where this ventricle connects with the two lateral ventricles. Its function is unknown but it is ideally located to sample the contents of the cerebrospinal fluid and/or to secrete substances into the ventricular system for wide dissemination throughout the brain. It is one of a group of circumventricular structures with an incomplete blood-brain barrier that also includes the pineal gland and median eminence of the hypothalamus 16. Pachomov 8 reported increased specifically stainable neurosecretory material in the subfornical organ after estrogen injection, and Stumpf 15 found uptake of [3H]estradiol by this structure using an autoradiographic technique. Here we present evidence that the subfornical organ responds to ovariectomy by increasing [3H]uridine incorporation into RNA. Eight 25 g mice were ovariectomized and kept for 8 weeks to allow for development of the chronically ovariectomized state. At the end of 8 weeks these mice and a control group of 8 age-matched female mice were injected intraperitoneally with 200 tzCi [aH]uridine (25 Ci//~mole) that had been shown by us to be chromatographically pure. After 45 rain the mice were killed and their brains removed, fixed in icecold modified Carnoy's solution (absolute alcohol-glacial acetic acid, 3:1, v/v), trimmed to a block containing hypothalamus and adjacent structures and embedded in paraffin. Serial frontal sections were cut, mounted on glass slides, and thoroughly washed to remove loosely bound 3H. The slides were then dipped in Kodak NTB-2 photographic emulsion and exposed in the dark at 4 °C for 4 weeks to make autoradiographs before being photographically developed and stained. Developed silver halide grains in photographic emulsion overlying specific brain areas were counted at × 1000 magnification using an ocular grid, and the number of grains was taken as an index of 3H content of specific brain areas. In previous experiments using this technique we have found that pretreatment with ribonuclease of slides containing tissue sections before dipping in photographic emulsion prevented the appearance of > 90~o of silver halide grains when compared with control slides 4. We thus consider that > 90~o of [3H]uridine in washed tissue sections has been incorporated into RNA. Slides were coded before counting to insure objectivity and

168 only silver grains over nuclei in n e u r o n a l cell bodies counted. Non-specific b a c k g r o u n d grains in p h o t o g r a p h i c emulsion over an area o f glass slide away from tissue consistently averaged 0.1/sq.#m. The net c o u n t for each area after s u b t r a c t i o n of b a c k g r o u n d is listed in Table I. O f the 14 areas counted only in the subfornical o r g a n was there a significant difference in activity between control a n d ovariectomized groups of mice. This difference was f o u n d whether the grain c o u n t was expressed per area or per neuron. A n o t h e r way of a n a l y z i n g grain c o u n t / n e u r o n is to determine how m a n y n e u r o n a l cell bodies c o n t a i n a p a r t i c u l a r n u m b e r of grains. When this was d o n e for 5 or m o r e and 10 or m o r e g r a i n s / n e u r o n there were significant differences between the two groups of animals. When the brains were t r i m m e d after initial fixation, the t r i m m i n g s (consisting o f cerebrum, cerebellum a n d brain stem) from each brain were saved individually a n d analyzed for [aHjRNAT. The tissue was homogenized in water at 4 :'C, extracted with ice cold 105',; trichloracetic acid >, 4, and s u p e r n a t e n t discarded. After drying over P2Oa the precipitate was weighed a n d digested with N C S (Nuclear Chicago). a q u a r t e r n a r y a m m o n i u m base, a n d the digest c o u n t e d for tritium. There was no significant difference in [ a H ] R N A c o n t e n t o f b r a i n t r i m m i n g s from ovariectomized mice, 22.9 ::t: 5.6 (S.D.) c o u n t s / m i n / m g , c o m p a r e d with the control group, 25.7 i : 8.0. The pituitary glands o f the two groups of a n i m a l s were similarly exlracted for [!~H]-

TABLE l SILVER HALIDE (}RAINS IN A U T O R A D I O G R A P H S OF HYPOTHALAMI(2 AREAS

Nuclear area

Subfornical organ Anterior hypothalamic nuclei Nucleus preopticus medialis Nucleus triangularis septi Nucleus interstitialis striae terminalis Nucleus preopticus suprachiasmaticus Ependymal cells adjacent to above Nucleus suprachiasmaticus Nucleus arcuatus Ependymal cells adjacent to above Nucleus supraopticus Nucleus paraventricularis Nucleus dorsomedialis Nucleus ventromedialis Subcommissural organ

Control*

2.3 j: 0.84 1.3 i l.l 1.4 0.6 1.0 i 0.3 1.1 I 0.3 I. I t: 0.2 1.2 L 0.6 4.8 ! 1.5 2.2 !: 1.0 1.5 :[ 0.4 1.0 L 0.2 0.6 i: 0.1 1.0 i: 0.2 1.1 f 0.2

Ovariectooty*

P < 0.025

3.9 i 1.6 0.9 :i 0.5 1.6 ::( 0.6 1.3 i: 0.5 1.3 :i 0.6 I,l :t- 0:6 1.2 :} 0.6 4.0? 1.2 1.8 " 0.2 t.8 ::i 0.2 0.8 :~ 0.2 0.6 - 0.2 0.8 ~ 0.4 1.2 ~ 0.6

grains~neuron

Subfornical organ

* grains/sq./tm; mean ~ S.D.

6. l :L 0.20 P < 0.02 9.6 j: 3.1 % neurons with 10 or more grains 13.45_ 16.8 P < 0.02 43.7:!: 27.9 % neurons with 5 or more grains 63.4 :L 24.5 P < 0.05 85.6 :! i 1.2

169 RNA and again no significant difference between the groups was found : ovariectomy, 392 :~- 129 counts/min; control, 300 ~- 164 counts/rain. We conclude that there was no effect of ovariectomy on [aH]uridine incorporation into R N A in the whole brain or pituitary. The subfornical organ appears designed to sample substances in the blood as its capillaries are arranged in bends and coils with fenestra in the capillary walls opening into perivascular spaces to. There are also signs of neurosecretion in this structure in that neuronal cell bodies, axons and axon terminals contain neurosecretory granules 100-200 nm in diameter 11. Dehydration in rats has been reported to cause increased enzymic activity in the subfornical organ ~2 and at the ultrastructural level there was increased rough endoplasmic reticulum 2. Simpson and Routtenberg injected carbachol t0 and angiotensin 1I 13 directly into the subfornical organ of rats with resultant increased drinking, and this drinking response was prevented by ablation of the structure before injection of either substance. Using the autoradiographic method described here we demonstrated increased incorporation of [aH]cytidine into RNA in the subfornical organ in dehydrated mice 5. The sites of estrogen feedback that influence pituitary gonadotropin secretion are unknown but are presumed to be in the hypothalamus rather than the pituitary as estrogen implants in the medial basal hypothalamus are more effective in inhibiting pituitary gonadotropin secretion than implants in the pituitary 9. More definitive evidence supporting hypothalamic location of feedback sites has been reported recently 1. Ovarian implants in the anterior hypothalamus a and estrogen implants into the habenular region of the brain 6 have also been reported to inhibit pituitary gonadotropin secretion but interpretation of such attempts at localization within the brain is difficult because of demonstrated diffusion of [aH]estradiol from the site of implantation 14. The subfornical organ is one of the sites within the brain that selectively takes up estradiol ta and as documented here it responds to ovariectomy with increased incorporation of [aH]uridine into RNA. We examined a number of other hypothalamic nuclear areas that have been found to concentrate estradiol such as nucleus preopticus medialis, nucleus triangularis septi, nucleus interstitialis striae terminalis and nucleus arcuatus 15 and did not find any significant increase in [3H]uridine incorporation into RNA. In these areas the estradiol signal may be transduced to an electrical signal that does not require a new protein synthesis. The increased incorporation of [aH]uridine into RNA which we have found in the subfornical organ following ovariectomy could represent increased'RNA synthesis but could also be due to increased permeability of cells or nuclei to uridine, decreased synthesis of endogenous RNA precursor, or decreased destruction of labeled RNA. In any case the increased cellular activity is evidence that this structure is involved in the response of the brain to ovariectomy. b

This work was supported by Grant RR-34, General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health, Bethesda, Md., U.S.A.

170 I DEBELJUK, L., VILCHI~Z-MARTINEZ,J, A., ARIMURA, A., AND SCHALLY, A. V., Effect ot gonadal steroids on the response lo LH-RF in intact and castrated male rats, Endocrino]og3' , 94 (1974! 1519-1524. 2 DELLMAN, H. D., Ultrastructural changes in the neurons of the rat's subfornical organ dining progressive dehydration, Anat, Rec., 166 (1970) 298 (Abstract). 3 FLERKO, B., AND SZENT,~GOTHAI, J., Oestrogen sensitive nervous structures in the hypothalamub, Acta endocr. (Kbh.), 26 (1957) 121 --I 27. 4 GEORC;e, J. M., Localization in hypothalamus of increased incorporation of [~H]cytidine into RNA in response to oral hypertonic saline, Endocrinology, 92 (1973) 1550-1555. 5 GEORGE, J. M., Hypothalamic sites of incorporation of [3H]cytidine into RNA in response to oral hypertonic saline, Brahl Research, 73 (1974) 184-187. 6 MOTTA, M., FRASCHIN1, F,, GOILIANI, G., AND MARTINI, L., The central nervous system, estrogen and puberty, Endocrinology, 83 (1968) 1101-1107. 7 MUNRO, H. N., In D. GLlCK, (Ed.), Methods' of Biochemical Analysis, 1/ol. 14, Interscience Publ., New York, 1966, p. 113. 8 PACHOMOV,N., Morphologische untersuchungen zur frage der funktion des subfornikalen organs der ratte, Dtsch. Z, Nervenheilk., 185 (1963) 13-19. 9 RAMmEZ, D. V., AND MCCANN, S. M., Comparison of the regulation of luteinizing hormone (LH) secretion in immature and adult rats, Endocr#tology, 72 (1963) 452-464. 10 ROUTtENaErG, A., AND SIMPSON, J. B., Carbachol induced drinking at ventricular and subfornical organ sites of application, Lift, Sci., 10 ( 1971 ) 481-490. I 1 RUD~RT, H., Das subfornikalorgan and seine beziehungen zu dem neurosekretorischen system in zwischenhirn des frosches, Z. Zellforsch., 65 (1965) 790--804. 12 SARRAT, R., Enzymhistochemische untersuchungen am subfornikalorgan der ratte, Exper,,ntia (Basel), 24 (1968) 1239. 13 SIMPSON, J. B., AND ROtO'rT~NBER¢~,A., Subfornical organ: site of drinking elicitation by angiotensin I1, Science, 181 (1973) 1172-1174. 14 SMITH, E. R., AND DAVIDSON, J. M., Role of estrogen in the cerebral control of puberty in female rats, Endocrinology, 82 (1968) 100-108. 15 STUMPy, W. E., Estrogen-neurons and estrogen-neuron systems in the periventricular brain, Amer. J. Anat. , 129 (1970) 207-218. 16 WEINDL, A., Neuroendocrine aspects of circumventricular organs. In W. F. GANONG AND L. MARTIM reds.), Frontiers in Neuroemlocrinology, Oxford Univ. Press, New York, 1973, pp. 3--32.

Increased incorporation of [3H]uridine into RNA in the brain subfornical organ of ovariectomized mice.

Brain Research, 97 (1975) 167-170 (Q Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands 167 Increased incorporation of p...
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