Cell and Tissue Research
Cell Tiss. Res. 186, 423-433 (1978)
9 by Springer-Verlag 1978
Intra- and Extrahypothalamic Vasopressin and Oxytocin Pathways in the Rat R.M. Buijs, D.F. Swaab, J. Dogterom, and F.W. van Leeuwen* Netherlands Institute for Brain Research, Amsterdam, The Netherlands
Summary. Perfusion of rat brain followed by immersion fixation with 2.5 % glutaraldehyde-1% paraformaldehyde, purification of the first antisera and application of the unlabelled antibody enzyme method were used to specifically identify vasopressin and oxytocin containing cells and fibres. The conventional sites of production of these hormones were confirmed as follows: supraoptic and paraventricular nuclei, suprachiasmatic nucleus (only vasopressin), and other cells and cell groups of the hypothalamus. Fibres from the suprachiasmatic nucleus spread out in various directions, and probably project to the nucleus praeopticus periventricularis, organum vasculosum laminae terminalis and in the direction of the supraoptic nucleus. Oxytocin and vasopressin containing pathways could be traced from the paraventricular nucleus to the lateral ventricle, the stria terminalis and the stria medullaris. Some of the oxytocin and vasopressin containing tracts appear to continue onto the septum. The possible importance of these morphological findings for the behavioural effects of vasopressin and oxytocin is discussed.
Key words: Vasopressin - Oxytocin - Immunohistochemistry - Hypothalamus - Memory consolidation. Introduction Arginine-8-vasopressin and related hormones affect memory processes in rats (De Wied et al., 1976). A physiological function for endogenous vasopressin in these processes is suggested because memory deficits were found in Brattleboro rats that are homozygous for hereditary hypothalamic diabetes insipidus (Ho-DI) based on a complete lack of vasopressin (Bohus et al., 1975) and in Wistar rats that were R.M. Buijs, Netherlands Institute for Brain Research, IJdijk 28, Amsterdam, The Netherlands * The authors wish to thank Dr. L. Sternbergerfor his generous gift of peroxidase-antiperoxidase complex, and Miss M.M. Smidt, Mr. A. Potjer and Mr. P. Wolters for their assistance. This work was supported in part by the Foundation for Medical Research FUNGO Send offprint requests to:
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injected i n t r a v e n t r i c u l a r l y with vasopressin a n t i s e r u m (Van W i m e r s m a G r e i d a n u s et al., 1975). T h e r e are v a r i o u s theoretical possibilities for vasopressin to reach those b r a i n a r e a s where it a p p a r e n t l y affects behavior. A route via the p e r i p h e r a l circulation w o u l d seem to be e x c l u d e d by the absence o f a n y c o r r e l a t i o n between p e r i p h e r a l b l o o d levels o f v a s o p r e s s i n a n d passive a v o i d a n c e b e h a v i o r ( D o g t e r o m , 1977). M o r e o v e r , i n t r a v e n o u s l y injected vasopressin a n t i s e r u m was n o t f o u n d to p r o v o k e m e m o r y defects (Van W i m e r s m a G r e i d a n u s et al., 1975). A second possibility for n e u r o h y p o p h y s i a l h o r m o n e s to reach o t h e r b r a i n areas is via the c e r e b r o s p i n a l fluid (CSF). Such releasing sites are t h o u g h t to exist in the third ventricle W i t t k o w s k i , 1968; G o l d s m i t h a n d Z i m m e r m a n , 1975; Z i m m e r m a n et al., 1975) a n d in the lateral ventricle (Brownfield a n d K o z l o w s k i , 1977). This is still a subject o f investigation in o u r o w n a n d in o t h e r l a b o r a t o r i e s . A third possibility is direct t r a n s p o r t o f n e u r o h y p o p h y s i a l h o r m o n e s f r o m the s u p r a o p t i c a n d p a r a v e n t r i c u l a r nuclei (SON, P V N ) a n d the s u p r a c h i a s m a t i c nucleus ( S C N ) to o t h e r b r a i n areas via n e u r o s e c r e t o r y fibres. T h e localization o f such e x t r a h y p o t h a l a m i c p a t h w a y s , a n d the d e t e r m i n a t i o n o f their h o r m o n a l content, w o u l d be o f great value in d e t e r m i n i n g the possible b r a i n areas where vasopressin o r o x y t o c i n m a y influence b e h a v i o r . V a r i o u s studies with u n p u r i f i e d antisera a g a i n s t vasopressin (Burlet et al., 1976) o r cross reacting n e u r o p h y s i n antisera ( W e i n d l et al., 1976; Brownfield a n d K o z l o w s k i , 1977) suggest the existence o f such e x t r a h y p o t h a l a m i c p a t h w a y s . The p r e s e n t s t u d y d e m o n s t r a t e s the specific i m m u n o c y t o c h e m i c a l l o c a l i z a t i o n o f those and other vasopressin and oxytocin pathways.
Materials and Methods Fixation and Staining o f the Tissue
Eight male Wistar rats and four male rats of the Brattleboro strain, homozygous for diabetes insipidus (Ho-DI), were used. Their daily urine production was measured in metabolism cages. The animals, weighing 200-250 g (obtained from TNO, Zeist, The Netherlands) were anesthetized with Nembutal, 0.1 ml/100 g body weight i.p., perfused intracardiallywith 0.9 % saline followed by 2.5 % glutaraldehyde1% paraformaldehyde in 0.1 M cacodylate sucrose buffer pH 7.35 (all reagents Merck) (Sabatini et al., 1963). Brains were immersed in the same fixative for 20 h at 4~C, dehydrated and embedded in paraffin. Sections (6 g) were cut transversally or sagitally, mounted on albuminized slides and brought to phosphate buffered saline (PBS) via xylene and graded ethanol series. Immunoperoxidase staining was accomplished by the sequential application of: a) 10% swine serum for 10 min; b) rabbit vasopressin antiserum (No 125) or oxytoein antiserum (No 0-2-C) (Swaab and Pool, 1975) in several dilutions for 60 min; c) washing in PBS; d) 10% swine serum for 10 rain; e) swine anti-rabbit IgG (Fc fragment) serum 1 : 20 (Nordic) for 60 rain; f) washing in PBS; g) 10 % swine serum for 10 min; h) peroxidase-antiperoxidase (PAP) 1 : 100 for 60 min; i) washing in PBS; j) rinsing in 0.05 M TRIS-HC1 (Merck) pH 7.6. Subsequently a solution of 0.5 mg/ml 3,3'-diamino benzidine (Sigma) in 0.05 M TRIS-HC1 pH 7.6. 0.01% H202 (Merck) Graham and Karnovsky, 1966) was applied on the sections. Staining lasted 10-20 min in the dark, depending upon the dilution of the first antibody. After staining, the sections were washed in HzO, dehydrated and embedded in malinol. Controls included: (1) replacement of the first antiserum by normal rabbit serum; (2) vasopressin antiserum (1 : 200) and oxytocin antiserum (1 : 200) adsorbed to respectively vasopressin or oxytocin containing agarose beads; (3) Ho-DI rat brain sections as a control for the specificity of the purified antivasopressin serum; (4) sections of the suprachiasmatic nucleus of the Wistar rat in order to control for the specificity of the purified oxytocin antiserum. The controls were performed on sections adjacent to those used for the identification of cells and fibres containing oxytocin and vasopressin. All procedures were carried out at room temperature unless otherwise stated.
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Production and Purification of Antisera Vasopressin and oxytocin antisera were purified and tested by means of immunofluorescence in a dilution of 1 : 80 according to Swaab and Pool (1975) by incubation with agarose beads to which oxytocin or vasopressin were coupled, respectively. The purified antiserum was diluted 1 : 200 for the PAP-procedure.
Results
Specificity o f Immunolocalization U n p u r i f i e d v a s o p r e s s i n a n t i s e r u m i n a d i l u t i o n o f 1 : 800 o r less s t a i n e d cells a n d fibres in the hypothalamo-neurohypophysial system (HNS) of Ho-DI rats because
Fig. 1 A and B. Adjacent transverse sections of paraventricular nucleus (PVN) of Wistar rat ( x 300). 3 V third ventricle. A Stained with purified vasopressin antiserum (1 : 200); note staining of cell group (arrow). B Stained with purified oxytocin antiserum (1 : 200); note absence of staining in cell group (arrow) and different staining pattern of PVN cells as compared to Figure 1A
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Fig. 2A-C. Sections of suprachiasmatic nucleus (SCN) of Wistar rat stained with unpufified vasopressin antiserum (1 : 800). 3 Vthird ventricle. A Transverse section of SCN; note thin vasopressin positive fibres running along third ventricle • 275. B Immunopositive vasopressin fibres along lateral ventricle (LV) in nucleus periventricularis in same section, x 500. C Sagittal section of SCN with vasopressin positive fibres running rostrally under ependyma of third ventricle (arrow); OC optic chiasm, x 300 o f cross reaction, while u n p u r i f i e d o x y t o c i n a n t i s e r u m in a dilution o f 1 : 4 0 0 stained the S C N in W i s t a r rats. Sections i n c u b a t e d with n o r m a l r a b b i t serum s h o w e d n o staining at all in the brain. A p a r t f r o m the i m m u n o f l u o r e s c e n c e m e a s u r e m e n t s on a g a r o s e b e a d s a n d the negative staining results on W i s t a r tissue after r e p l a c e m e n t o f the first a n t i b o d y by c o n t r o l p l a s m a a n d after a b s o r p t i o n to the h o m o l o g o u s antigen, the specificity o f the staining with purified antisera on sections was c o n c l u d e d f r o m the following d a t a : no i m m u n o s t a i n i n g o f the H N S o f
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Fig. 3A and B. Transverse sections of Wistar rat brain stained with vasopressin antiserum (1 : 800). A Fibres in lateral septum; note the pericellnlar punctate profiles (right arrow) and branching diversion of other fibre (left arrow) x 1150. B Very thin immunopositive fibres in organum vaseulosum laminae terminalis, just above recessus opticus (RO) x 2200
H o - D I rats was o b s e r v e d after p u r i f i c a t i o n o f v a s o p r e s s i n a n t i s e r u m , while cells a n d fibres in the H N S o f W i s t a r s r e m a i n e d stainable. In a d d i t i o n , With purified o x y t o c i n antiserum, we c o u l d n o t detect a n y i m m u n o s t a i n i n g in S C N o f W i s t a r rats. Ceils t h a t r e a c t e d with the purified v a s o p r e s s i n a n t i s e r u m in n o r m a l W i s t a r rats d i d n o t react in a l t e r n a t i n g serial sections with purified o x y t o c i n a n t i s e r u m , a n d vice versa (Fig. 1 A, B).
Fig. 4 A - D . Transverse sections o f Wistar rat brain showing some extrahypothalamic tracts. A Tract from paraventricular nucleus (PVN) to stria medullaris (SM) stained with purified oxytocin antiserum (1 : 200) x 90. B Same tract in another animal; oxytocin positive cells around capillary (cap) with fibres towards PVN and SM x 650. C Insert of Figure 4B in adjacent section stained with purified vasopressin antiserum (1 : 200); note complete absence of staining x 500. D Tract to stria terminalis (ST) stained with purified vasopressin antiserum; note single vasopressin containing cell in SM; PC plexus chorioideus; LV lateral ventricle x 300
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Intra- and Extrahypothalamic Pathways With purified specific antisera against oxytocin and vasopressin immunoreaction product was found in cells of the PVN and SON, and in the paraventriculosupraoptico-neurohypophysial tract. Moreover, vasopressin and oxytocin containing cells were localized throughout the hypothalamus, immediately below the lining of the third ventricle, and a large cell group containing both hormones was observed ventral from the PVN (cf. Palkovits et al., 1974). Only vasopressin containing cells were found in the SCN. Intra- and extrahypothalamic pathways were localized first by means of nonpurified vasopressin antiserum in a dilution of 1 : 800 staining vasopressin as well as 9xytocin. We found very thin fibres fanning out from the SCN in several directions, dorsally along the lining of the third ventricle (Fig. 2A), rostrally in the direction of the organum vasculosum of the lamina terminalis (OVLT) (Fig. 2 C), laterally in the direction of the SON and caudally in the direction of the median eminence. These fibres could be visualized with purified vasopressin antiserum but not with purified oxytocin antiserum. In addition, these fibres could not be stained with unpurified oxytocin or vasopressin antisera in Ho-DI rats. Thin fibres were located near the lateral ventricle in the nucleus praeopticus periventricularis (Fig. 2B), in the OVLT (Fig. 3 B) and in the lateral and medial septum (Fig. 3A). Most of these fibres were too thin to be identified with purified antisera, although with purified vasopressin antiserum some fibres stained in the nucleus praeopticus periventricularis, while with both purified antisera some fibres could be indicated in the medial septum. In addition, with the use of unpurified oxytocin antiserum in Ho-DI rats, these very thin fibres could be demonstrated in all of the areas mentioned, although in small numbers as compared to the results with unpurified antisera in Wistar rats (especially in the lateral septum where just a few fibres could be indicated). An extrahypothalamic pathway was found running from the rostral part of the PVN in the direction of the stria medullaris. This pathway consisted mainly of oxytocin cells and fibres (Fig. 4A-C). Other fibres proceeded from the PVN towards the stria terminalis. Two tracts were observed, one via the stria pars hypothalamica (Fig. 4D) and one along the capsula interna. The former contained fibres and cells, whereas the latter contained only fibres. Both structures contained oxytocin and vasopressin. Several vasopressin or oxytocin reacting bipolar cells were observed directing their fibres towards the PVN and the lateral ventricle (Fig. 4B). In the region of the stria medullaris and the stria terminalis, oxytocin and vasopressin cells were frequently observed immediately below the ependyma of the lateral ventricle. Sometimes an immunopositive fibre was found in the region of the stria terminalis at the site of attachment of the chorioid plexus and in the mammillary body.
Discussion
The unpurified antisera against oxytocin and vasopressin cross-reacted in the present procedure with vasopressin and oxytocin respectively. However, purifi-
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cation of these antisera according to Swaab and Pool (1975) provided specificity also in this unlabelled antibody technique. A test model to check the purification of the antisera quantitatively, such as that used in immunofluorescence microscopy (Swaab and Pool, 1975), is not yet available for the light microscopical immunolocalization procedure. The best way to check in a tissue section for the absence of cross reaction of a vasopressin antibody with oxytocin is the total absence of staining in the HNS of a Ho-DI rat which contains only oxytocin (Miller and Moses, 1969). Conversely, the SCN which contains only vasopressin (Vandesande et al., 1975; Swaab et al., 1975) should not stain with oxytocin antiserum. Since in alternating sections of the HNS of normal Wistar rats, the respective cells stained either with purified vasopressin antiserum or with purified oxytocin antiserum, we can be quite certain that cross reaction in our procedure is not present. It is not yet known precisely where the fibres from the SCN terminate (Zimmerman, 1976). Electrophysiologically, non-synaptic connections were shown to run from the SCN to the median eminence (Makara et al., 1972). In addition, on the basis of an experiment where 3H-proline was injected into the SCN of one single rat, Swanson and Cowan (1975) reported termination of fibres in the nucleus periventricularis, arcuate nucleus and median eminence. With both of the above mentioned techniques, however, it was impossible to tell whether or not the fibres involved contained indeed vasopressin. The thin vasopressin containing fibres, directed towards the median eminence, support the idea that axons of the SCN do terminate in the external zone of the median eminence (Vandesande et al., 1974). In addition, our observations make it probable that AVP containing fibres of the SCN run not only to the median eminence, but also along the third ventricle to the nucleus praeopticus periventricularis, and to the organum vasculosum of the lamina terminalis. Whether the fibres observed in these areas indeed originate from the SCN cannot be said from the present data, because most fibres were too thin to be followed over long distances. However, our observations on the Ho-DI rat showed that at least some of these fibres contained oxytocin and in consequence must originate from the PVN or SON. Extrahypothalamic neurosecretory pathways, originating from the PVN and SON already described by Legait et Legait (1957), Barry (1961), Sterba (~974), Weindl et al. (1976), Burlet et al. (1976) and Brownfield and Kozlowski (1977) were confirmed in the present study. In addition, information was provided concerning the hormonal content of these pathways. All tracts contained both oxytocin and vasopressin fibres, although the proportions differed. The present observation that the most rostral tract of the PVN to the stria medullaris consists mainly of oxytocin containing cells and fibres agrees with the finding of Swaab et al. (1975), that the rostral part of the PVN contains more oxytocin than vasopressin cells. Although, on several occasions, oxytocin and vasopressin positive cells and fibres were found near capillaries in the region of the stria terminalis, of the stria medullaris and also around cells in the lateral septum, it was not clear whether these fibres actually end in these areas. An answer to this question might be obtained by immunoelectronmicroscopic methods (Van Leeuwen and Swaab, 1977), by which both, the hormonal content and the ultrastructural properties of the endings, can be studied. The punctate perineuronal profiles in the lateral septum indicate, however, that this
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is one of the areas where vasopressin might be released to exert an influence on brain function. Another argument for this hypothesis is that lesions in the septal area caused an impairment of memory consolidation that was impervious to vasopressin treatment (Van Wimersma Greidanus et al., 1976). In their search to PVN and SON afferents in the rabbit, Aulsebrook and Holland (1969) were able to induce oxytocin release by electrically stimulating the septum, stria terminalis and periventricular areas. Tindal et al. (1969) reported the same effect for the mamillary body. Since we have found oxytocin and vasopressin containing fibres in these areas, the release of oxytocin need not necessarily be brought about via afferents, but, rather by direct stimulation of neurosecretory fibres. These data, and the observed bipolarity of many cells in the extrahypothalamic pathways and in the PVN, make it plausible that it is the same cell that sends its fibres to the neurohypophysis and to extrahypothalamic areas. This possibility could, in addition, explain the simultaneous elevation of vasopressin and oxytocin levels in the CSF and in the blood (Heller et al., 1968; Vorherr et al., 1968) following vagal stimulation or hemorrhage. Another route by which vasopressin or oxytocin could influence brain function might be via the CSF. Injected vasopressin or vasopressin antisera into the cerebroventricular system respectively improved or impaired memory consolidation (De Wied, 1976; Van Wimersma Greidanus et al., 1975). The extrahypothalamic fibres might be of importance also for this second route. In the regions of the stria terminalis and stria medullaris, both vasopressin and oxytocin containing cells and fibres were frequently noted directly under the ependyma of the lateral ventricle. If neurohypophysial hormones are released in this area they would presumably be transported into the CSF via perivascular channels (Cserr and Ostrach, 1974). The view that vasopressin and oxytocin have neurotransmitter function (e.g., Barker, 1976) is supported by the finding of various areas in which fibres were present containing these hormones. Even more so the punctate pericellular profiles in the lateral septum argue for a release. Such a peptide transmitter function might be the basis for the influence of these and related peptides on memory consolidation.
References
Aulsebrook, L.H., Holland, R.C.: Central regulation of oxytocin release with and without vasopressin release. Amer. J. Physiol. 216, 818-829 (1969) Barker, J.L. Peptides: roles in neuronal excitability. Physiol. Rev. 56, 438452 (1976) Barry, J.: Recherches morphologiques et exp6rimentales sur la glande dienc6phalique et l'appareil hypothalamo-hypophysaire. Ann. Sci. Univ. Besangon, Zool. Physiol., Ser. 2, 3-133 (1961) Bohus, B., Van Wimersma Greidanus, Tj.B., De Wied, D.: Behavioral and endocrine responses of rats with hereditary hypothalamic diabetes insipidus (Brattleboro strain). Physiol. Behav. 14, 609-615 (1975) Brownfield, M.S., Kozlowski, G.P.: The hypothalamo-choroidal tract. I. Immunohistochemical demonstration of neurophysin pathways to telencephalic choroid plexuses and cerebrospinal fluid. Cell Tiss. Res. 178, 111-127 (1977)
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Burlet, A., Chateau, M., Marchetti, J.: Contributions of immunoenzymatic technique to the study of diencephalic localization of vasopressin. First intern. Symp. on Immunoenzymatic Techniques INSERM. Symp. Nr. 2 (G. Feldmann, P. Druet, J. Bignon, S. Avrameas, eds.), pp~ 333-343. Amsterdam: North Holland Publishing Comp. (1976) Cserr, H.F., Ostrach, L.H.: Bulk flow of interstitial fluid after intracranial injection of Blue Dextran 2000. Exp. Neurol. 45, 50-60 (1974) Dogterom, J.: The release and presence of vasopressin in plasma and cerebrospinal fluid as measured by radioimmunoassay; studies on vasopressin as a mediator of memory processes in the rat. Ph.D. Thesis, State Univ. Utrecht, The Netherlands (1977) Goldsmith, P.C., Zimmerman, E.A.: Ultrastructural localization of neurophysin and vasopressin in the rat median eminence. Endocrinology 96, A 239 (1975) Graham, R.C., Karnovsky, M.J.: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291-302 (1966) Heller, H., Hasan, S.A., Saifi, A.Q.: Antidiuretic activity in the cerebrospinal fluid. J. Endocr. 41, 273280 (1968) Leeuwen, F.W. van, Swaab, D.F.: Specific immunoelectronmicroscopic localization of vasopressin and oxytocin in the neurohypophysis of the rat. Cell Tiss. Res. 177, 493-501 (1977) Legait, H., Legait, E.: Les voies extra-hypophysaires des noyaux neuros6cr~toires hypothalamiques chez les batraciens et les reptiles. Acta anat. (Basel) 30, 429-443 (1957) Makara, G.B., Harris, M.C., Spyer, K.M.: Identification and distribution of tuberoinfundibular neurons. Brain Res. 40, 283-290 (1972) Miller, M., Moses, A.M. : Radioimmunoassay of vasopressin with a comparison of immunological and biological activity in the rat posterior pituitary. Endocrinology 84, 557-562 (1969) Palkovits, M., Z~iborski, L., Ambach, G.: Accessory neurosecretory cell groups in the rat hypothalamus. Acta morph. Acad. Sci. hung. 22 (1), 21-33 (1974) Sabatini, D.D., Bensch, K., Barrnett, R2.: Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 19 59 (1963) Sterba, G.: Ascending neurosecretory pathways of the peptidergic type. In: Neurosecretion - The final neuroendocrine pathway (F. Knowles, L. Vollrath, eds.), pp. 38-47. Berlin-Heidelberg-New York: Springer 1974 Swaab, D.F., Pool, C.W. : Specificity of oxytocin and vasopressin immunofluorescence. J. Endocr. 66, 263-272 (1975) Swaab, D.F., Pool, C.W., Nijveldt, F.: Immunofluorescence of vasopressin and oxytocin in the rat hypothalamo-neurohypophyseal system. J. neural Transm. 36, 195-215 (1975) Swanson, L.W., Cowan, W.M.: The efferent connections of the suprachiasmatic nucleus of the hypothalamus. J. comp. Neurol. 160, 1-12 (1975) Tindal, J.S., Knaggs, G.S., Turvey, A.: The afferent path of the milk-ejection reflex in the brain of the rabbit. J. Endocr. 43, 663 671 (1969) Vandesande, F., Dierickx, K., De Mey, J.: Identification of the vasopressin-neurophysin producing neurons of the rat suprachiasmatic nuclei. Cell Tiss. Res. 156, 377-380 (1975) Vandesande, F., De Mey, J.: Dierickx, K.: Identification of neurophysin producing cells. I. The origin of the neurophysin like substance-containing nerve fibres of the external region of the median eminence of the rat. Cell Tiss. Res. 151, 187 200 (1974) Vorherr, H., Bradbury, M.W.B., Hoghoughi, M., Kleeman, C.R.: Antidiuretic hormone in cerebrospinal fluid during endogenous and exogenous changes in its blood level. Endocrinology 83, 246-250 (1968) Weindl, A., Sofroniew, M.V., Schinko, I.: Psychotrope Wirkungen hypothalamischer Hormone: Immunohistochemische Identifikation extrahypophys/irer Verbindungen neuroendokriner Neurone. Arzneimittel-Forsch. 26, 1191 1194 (1976) Wied, D., de: Behavioral effects of intraventricularly administered vasopressin and vasopressin fragments. Life Sci. 19, 685-690 (1976) Wied, D. de, Van Wimersma Greidanus, Tj. B., Bohus, B., Urban, I., Gispen, W.H. : Vasopressin and memory consolidation. In: Perspectives in brain research (M.A. Corner, D.F. Swaab, eds.), pp. 181 194. Progr. Brain Res. 45, Amsterdam: Elsevier 1976 Wimersma Greidanus, Tj. B. van, Bohus, B., Wied, D. de: CNS sites of action ofACTH, MSH and
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vasopressin in relation to avoidance behavior. In: Anatomical neuroendocrinology (W.E. Stumpf and L.D. Grant, eds.), pp. 284-289, Basel: Karger 1976 Wimersma Greidanus, Tj. B. van, Dogterom, J., Wied, D. de. Intraventricular administration of antivasopressin serum inhibits memory consolidation in rats. Life Sci. 16, 637-644 (1975) Wittkowski, W. : Elektronenmikroskopische Studien zur intraventrikul/iren Neurosekretion in den Recessus infundibularis der Maus. Z. Zellforsch. 92, 207-216 (1968) Zimmerman, E.A.: Localization of hypothalamic hormones by immunocytochemical techniques. Front. Neuroendocrin. 4, 25-62 (1976) Zimmerman, E.A., Defendini, R., Sokol, H.W., Robinson, A.G.: The distribution of neurophysinsecreting pathways in the mammalian brain: Light microscopic studies using the immunoperoxidase technique. Ann. N.Y. Acad. Sci. 24, 9~111 (1975)
Accepted July 11, 1977
Cell Tiss. Res. 186, 423-433 (1978)
9 by Springer-Verlag 1978
Intra- and Extrahypothalamic Vasopressin and Oxytocin Pathways in the Rat R.M. Buijs, D.F. Swaab, J. Dogterom, and F.W. van Leeuwen* Netherlands Institute for Brain Research, Amsterdam, The Netherlands
Summary. Perfusion of rat brain followed by immersion fixation with 2.5 % glutaraldehyde-1% paraformaldehyde, purification of the first antisera and application of the unlabelled antibody enzyme method were used to specifically identify vasopressin and oxytocin containing cells and fibres. The conventional sites of production of these hormones were confirmed as follows: supraoptic and paraventricular nuclei, suprachiasmatic nucleus (only vasopressin), and other cells and cell groups of the hypothalamus. Fibres from the suprachiasmatic nucleus spread out in various directions, and probably project to the nucleus praeopticus periventricularis, organum vasculosum laminae terminalis and in the direction of the supraoptic nucleus. Oxytocin and vasopressin containing pathways could be traced from the paraventricular nucleus to the lateral ventricle, the stria terminalis and the stria medullaris. Some of the oxytocin and vasopressin containing tracts appear to continue onto the septum. The possible importance of these morphological findings for the behavioural effects of vasopressin and oxytocin is discussed.
Key words: Vasopressin - Oxytocin - Immunohistochemistry - Hypothalamus - Memory consolidation. Introduction Arginine-8-vasopressin and related hormones affect memory processes in rats (De Wied et al., 1976). A physiological function for endogenous vasopressin in these processes is suggested because memory deficits were found in Brattleboro rats that are homozygous for hereditary hypothalamic diabetes insipidus (Ho-DI) based on a complete lack of vasopressin (Bohus et al., 1975) and in Wistar rats that were R.M. Buijs, Netherlands Institute for Brain Research, IJdijk 28, Amsterdam, The Netherlands * The authors wish to thank Dr. L. Sternbergerfor his generous gift of peroxidase-antiperoxidase complex, and Miss M.M. Smidt, Mr. A. Potjer and Mr. P. Wolters for their assistance. This work was supported in part by the Foundation for Medical Research FUNGO Send offprint requests to:
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injected i n t r a v e n t r i c u l a r l y with vasopressin a n t i s e r u m (Van W i m e r s m a G r e i d a n u s et al., 1975). T h e r e are v a r i o u s theoretical possibilities for vasopressin to reach those b r a i n a r e a s where it a p p a r e n t l y affects behavior. A route via the p e r i p h e r a l circulation w o u l d seem to be e x c l u d e d by the absence o f a n y c o r r e l a t i o n between p e r i p h e r a l b l o o d levels o f v a s o p r e s s i n a n d passive a v o i d a n c e b e h a v i o r ( D o g t e r o m , 1977). M o r e o v e r , i n t r a v e n o u s l y injected vasopressin a n t i s e r u m was n o t f o u n d to p r o v o k e m e m o r y defects (Van W i m e r s m a G r e i d a n u s et al., 1975). A second possibility for n e u r o h y p o p h y s i a l h o r m o n e s to reach o t h e r b r a i n areas is via the c e r e b r o s p i n a l fluid (CSF). Such releasing sites are t h o u g h t to exist in the third ventricle W i t t k o w s k i , 1968; G o l d s m i t h a n d Z i m m e r m a n , 1975; Z i m m e r m a n et al., 1975) a n d in the lateral ventricle (Brownfield a n d K o z l o w s k i , 1977). This is still a subject o f investigation in o u r o w n a n d in o t h e r l a b o r a t o r i e s . A third possibility is direct t r a n s p o r t o f n e u r o h y p o p h y s i a l h o r m o n e s f r o m the s u p r a o p t i c a n d p a r a v e n t r i c u l a r nuclei (SON, P V N ) a n d the s u p r a c h i a s m a t i c nucleus ( S C N ) to o t h e r b r a i n areas via n e u r o s e c r e t o r y fibres. T h e localization o f such e x t r a h y p o t h a l a m i c p a t h w a y s , a n d the d e t e r m i n a t i o n o f their h o r m o n a l content, w o u l d be o f great value in d e t e r m i n i n g the possible b r a i n areas where vasopressin o r o x y t o c i n m a y influence b e h a v i o r . V a r i o u s studies with u n p u r i f i e d antisera a g a i n s t vasopressin (Burlet et al., 1976) o r cross reacting n e u r o p h y s i n antisera ( W e i n d l et al., 1976; Brownfield a n d K o z l o w s k i , 1977) suggest the existence o f such e x t r a h y p o t h a l a m i c p a t h w a y s . The p r e s e n t s t u d y d e m o n s t r a t e s the specific i m m u n o c y t o c h e m i c a l l o c a l i z a t i o n o f those and other vasopressin and oxytocin pathways.
Materials and Methods Fixation and Staining o f the Tissue
Eight male Wistar rats and four male rats of the Brattleboro strain, homozygous for diabetes insipidus (Ho-DI), were used. Their daily urine production was measured in metabolism cages. The animals, weighing 200-250 g (obtained from TNO, Zeist, The Netherlands) were anesthetized with Nembutal, 0.1 ml/100 g body weight i.p., perfused intracardiallywith 0.9 % saline followed by 2.5 % glutaraldehyde1% paraformaldehyde in 0.1 M cacodylate sucrose buffer pH 7.35 (all reagents Merck) (Sabatini et al., 1963). Brains were immersed in the same fixative for 20 h at 4~C, dehydrated and embedded in paraffin. Sections (6 g) were cut transversally or sagitally, mounted on albuminized slides and brought to phosphate buffered saline (PBS) via xylene and graded ethanol series. Immunoperoxidase staining was accomplished by the sequential application of: a) 10% swine serum for 10 min; b) rabbit vasopressin antiserum (No 125) or oxytoein antiserum (No 0-2-C) (Swaab and Pool, 1975) in several dilutions for 60 min; c) washing in PBS; d) 10% swine serum for 10 rain; e) swine anti-rabbit IgG (Fc fragment) serum 1 : 20 (Nordic) for 60 rain; f) washing in PBS; g) 10 % swine serum for 10 min; h) peroxidase-antiperoxidase (PAP) 1 : 100 for 60 min; i) washing in PBS; j) rinsing in 0.05 M TRIS-HC1 (Merck) pH 7.6. Subsequently a solution of 0.5 mg/ml 3,3'-diamino benzidine (Sigma) in 0.05 M TRIS-HC1 pH 7.6. 0.01% H202 (Merck) Graham and Karnovsky, 1966) was applied on the sections. Staining lasted 10-20 min in the dark, depending upon the dilution of the first antibody. After staining, the sections were washed in HzO, dehydrated and embedded in malinol. Controls included: (1) replacement of the first antiserum by normal rabbit serum; (2) vasopressin antiserum (1 : 200) and oxytocin antiserum (1 : 200) adsorbed to respectively vasopressin or oxytocin containing agarose beads; (3) Ho-DI rat brain sections as a control for the specificity of the purified antivasopressin serum; (4) sections of the suprachiasmatic nucleus of the Wistar rat in order to control for the specificity of the purified oxytocin antiserum. The controls were performed on sections adjacent to those used for the identification of cells and fibres containing oxytocin and vasopressin. All procedures were carried out at room temperature unless otherwise stated.
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Production and Purification of Antisera Vasopressin and oxytocin antisera were purified and tested by means of immunofluorescence in a dilution of 1 : 80 according to Swaab and Pool (1975) by incubation with agarose beads to which oxytocin or vasopressin were coupled, respectively. The purified antiserum was diluted 1 : 200 for the PAP-procedure.
Results
Specificity o f Immunolocalization U n p u r i f i e d v a s o p r e s s i n a n t i s e r u m i n a d i l u t i o n o f 1 : 800 o r less s t a i n e d cells a n d fibres in the hypothalamo-neurohypophysial system (HNS) of Ho-DI rats because
Fig. 1 A and B. Adjacent transverse sections of paraventricular nucleus (PVN) of Wistar rat ( x 300). 3 V third ventricle. A Stained with purified vasopressin antiserum (1 : 200); note staining of cell group (arrow). B Stained with purified oxytocin antiserum (1 : 200); note absence of staining in cell group (arrow) and different staining pattern of PVN cells as compared to Figure 1A
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Fig. 2A-C. Sections of suprachiasmatic nucleus (SCN) of Wistar rat stained with unpufified vasopressin antiserum (1 : 800). 3 Vthird ventricle. A Transverse section of SCN; note thin vasopressin positive fibres running along third ventricle • 275. B Immunopositive vasopressin fibres along lateral ventricle (LV) in nucleus periventricularis in same section, x 500. C Sagittal section of SCN with vasopressin positive fibres running rostrally under ependyma of third ventricle (arrow); OC optic chiasm, x 300 o f cross reaction, while u n p u r i f i e d o x y t o c i n a n t i s e r u m in a dilution o f 1 : 4 0 0 stained the S C N in W i s t a r rats. Sections i n c u b a t e d with n o r m a l r a b b i t serum s h o w e d n o staining at all in the brain. A p a r t f r o m the i m m u n o f l u o r e s c e n c e m e a s u r e m e n t s on a g a r o s e b e a d s a n d the negative staining results on W i s t a r tissue after r e p l a c e m e n t o f the first a n t i b o d y by c o n t r o l p l a s m a a n d after a b s o r p t i o n to the h o m o l o g o u s antigen, the specificity o f the staining with purified antisera on sections was c o n c l u d e d f r o m the following d a t a : no i m m u n o s t a i n i n g o f the H N S o f
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Fig. 3A and B. Transverse sections of Wistar rat brain stained with vasopressin antiserum (1 : 800). A Fibres in lateral septum; note the pericellnlar punctate profiles (right arrow) and branching diversion of other fibre (left arrow) x 1150. B Very thin immunopositive fibres in organum vaseulosum laminae terminalis, just above recessus opticus (RO) x 2200
H o - D I rats was o b s e r v e d after p u r i f i c a t i o n o f v a s o p r e s s i n a n t i s e r u m , while cells a n d fibres in the H N S o f W i s t a r s r e m a i n e d stainable. In a d d i t i o n , With purified o x y t o c i n antiserum, we c o u l d n o t detect a n y i m m u n o s t a i n i n g in S C N o f W i s t a r rats. Ceils t h a t r e a c t e d with the purified v a s o p r e s s i n a n t i s e r u m in n o r m a l W i s t a r rats d i d n o t react in a l t e r n a t i n g serial sections with purified o x y t o c i n a n t i s e r u m , a n d vice versa (Fig. 1 A, B).
Fig. 4 A - D . Transverse sections o f Wistar rat brain showing some extrahypothalamic tracts. A Tract from paraventricular nucleus (PVN) to stria medullaris (SM) stained with purified oxytocin antiserum (1 : 200) x 90. B Same tract in another animal; oxytocin positive cells around capillary (cap) with fibres towards PVN and SM x 650. C Insert of Figure 4B in adjacent section stained with purified vasopressin antiserum (1 : 200); note complete absence of staining x 500. D Tract to stria terminalis (ST) stained with purified vasopressin antiserum; note single vasopressin containing cell in SM; PC plexus chorioideus; LV lateral ventricle x 300
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Intra- and Extrahypothalamic Pathways With purified specific antisera against oxytocin and vasopressin immunoreaction product was found in cells of the PVN and SON, and in the paraventriculosupraoptico-neurohypophysial tract. Moreover, vasopressin and oxytocin containing cells were localized throughout the hypothalamus, immediately below the lining of the third ventricle, and a large cell group containing both hormones was observed ventral from the PVN (cf. Palkovits et al., 1974). Only vasopressin containing cells were found in the SCN. Intra- and extrahypothalamic pathways were localized first by means of nonpurified vasopressin antiserum in a dilution of 1 : 800 staining vasopressin as well as 9xytocin. We found very thin fibres fanning out from the SCN in several directions, dorsally along the lining of the third ventricle (Fig. 2A), rostrally in the direction of the organum vasculosum of the lamina terminalis (OVLT) (Fig. 2 C), laterally in the direction of the SON and caudally in the direction of the median eminence. These fibres could be visualized with purified vasopressin antiserum but not with purified oxytocin antiserum. In addition, these fibres could not be stained with unpurified oxytocin or vasopressin antisera in Ho-DI rats. Thin fibres were located near the lateral ventricle in the nucleus praeopticus periventricularis (Fig. 2B), in the OVLT (Fig. 3 B) and in the lateral and medial septum (Fig. 3A). Most of these fibres were too thin to be identified with purified antisera, although with purified vasopressin antiserum some fibres stained in the nucleus praeopticus periventricularis, while with both purified antisera some fibres could be indicated in the medial septum. In addition, with the use of unpurified oxytocin antiserum in Ho-DI rats, these very thin fibres could be demonstrated in all of the areas mentioned, although in small numbers as compared to the results with unpurified antisera in Wistar rats (especially in the lateral septum where just a few fibres could be indicated). An extrahypothalamic pathway was found running from the rostral part of the PVN in the direction of the stria medullaris. This pathway consisted mainly of oxytocin cells and fibres (Fig. 4A-C). Other fibres proceeded from the PVN towards the stria terminalis. Two tracts were observed, one via the stria pars hypothalamica (Fig. 4D) and one along the capsula interna. The former contained fibres and cells, whereas the latter contained only fibres. Both structures contained oxytocin and vasopressin. Several vasopressin or oxytocin reacting bipolar cells were observed directing their fibres towards the PVN and the lateral ventricle (Fig. 4B). In the region of the stria medullaris and the stria terminalis, oxytocin and vasopressin cells were frequently observed immediately below the ependyma of the lateral ventricle. Sometimes an immunopositive fibre was found in the region of the stria terminalis at the site of attachment of the chorioid plexus and in the mammillary body.
Discussion
The unpurified antisera against oxytocin and vasopressin cross-reacted in the present procedure with vasopressin and oxytocin respectively. However, purifi-
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cation of these antisera according to Swaab and Pool (1975) provided specificity also in this unlabelled antibody technique. A test model to check the purification of the antisera quantitatively, such as that used in immunofluorescence microscopy (Swaab and Pool, 1975), is not yet available for the light microscopical immunolocalization procedure. The best way to check in a tissue section for the absence of cross reaction of a vasopressin antibody with oxytocin is the total absence of staining in the HNS of a Ho-DI rat which contains only oxytocin (Miller and Moses, 1969). Conversely, the SCN which contains only vasopressin (Vandesande et al., 1975; Swaab et al., 1975) should not stain with oxytocin antiserum. Since in alternating sections of the HNS of normal Wistar rats, the respective cells stained either with purified vasopressin antiserum or with purified oxytocin antiserum, we can be quite certain that cross reaction in our procedure is not present. It is not yet known precisely where the fibres from the SCN terminate (Zimmerman, 1976). Electrophysiologically, non-synaptic connections were shown to run from the SCN to the median eminence (Makara et al., 1972). In addition, on the basis of an experiment where 3H-proline was injected into the SCN of one single rat, Swanson and Cowan (1975) reported termination of fibres in the nucleus periventricularis, arcuate nucleus and median eminence. With both of the above mentioned techniques, however, it was impossible to tell whether or not the fibres involved contained indeed vasopressin. The thin vasopressin containing fibres, directed towards the median eminence, support the idea that axons of the SCN do terminate in the external zone of the median eminence (Vandesande et al., 1974). In addition, our observations make it probable that AVP containing fibres of the SCN run not only to the median eminence, but also along the third ventricle to the nucleus praeopticus periventricularis, and to the organum vasculosum of the lamina terminalis. Whether the fibres observed in these areas indeed originate from the SCN cannot be said from the present data, because most fibres were too thin to be followed over long distances. However, our observations on the Ho-DI rat showed that at least some of these fibres contained oxytocin and in consequence must originate from the PVN or SON. Extrahypothalamic neurosecretory pathways, originating from the PVN and SON already described by Legait et Legait (1957), Barry (1961), Sterba (~974), Weindl et al. (1976), Burlet et al. (1976) and Brownfield and Kozlowski (1977) were confirmed in the present study. In addition, information was provided concerning the hormonal content of these pathways. All tracts contained both oxytocin and vasopressin fibres, although the proportions differed. The present observation that the most rostral tract of the PVN to the stria medullaris consists mainly of oxytocin containing cells and fibres agrees with the finding of Swaab et al. (1975), that the rostral part of the PVN contains more oxytocin than vasopressin cells. Although, on several occasions, oxytocin and vasopressin positive cells and fibres were found near capillaries in the region of the stria terminalis, of the stria medullaris and also around cells in the lateral septum, it was not clear whether these fibres actually end in these areas. An answer to this question might be obtained by immunoelectronmicroscopic methods (Van Leeuwen and Swaab, 1977), by which both, the hormonal content and the ultrastructural properties of the endings, can be studied. The punctate perineuronal profiles in the lateral septum indicate, however, that this
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is one of the areas where vasopressin might be released to exert an influence on brain function. Another argument for this hypothesis is that lesions in the septal area caused an impairment of memory consolidation that was impervious to vasopressin treatment (Van Wimersma Greidanus et al., 1976). In their search to PVN and SON afferents in the rabbit, Aulsebrook and Holland (1969) were able to induce oxytocin release by electrically stimulating the septum, stria terminalis and periventricular areas. Tindal et al. (1969) reported the same effect for the mamillary body. Since we have found oxytocin and vasopressin containing fibres in these areas, the release of oxytocin need not necessarily be brought about via afferents, but, rather by direct stimulation of neurosecretory fibres. These data, and the observed bipolarity of many cells in the extrahypothalamic pathways and in the PVN, make it plausible that it is the same cell that sends its fibres to the neurohypophysis and to extrahypothalamic areas. This possibility could, in addition, explain the simultaneous elevation of vasopressin and oxytocin levels in the CSF and in the blood (Heller et al., 1968; Vorherr et al., 1968) following vagal stimulation or hemorrhage. Another route by which vasopressin or oxytocin could influence brain function might be via the CSF. Injected vasopressin or vasopressin antisera into the cerebroventricular system respectively improved or impaired memory consolidation (De Wied, 1976; Van Wimersma Greidanus et al., 1975). The extrahypothalamic fibres might be of importance also for this second route. In the regions of the stria terminalis and stria medullaris, both vasopressin and oxytocin containing cells and fibres were frequently noted directly under the ependyma of the lateral ventricle. If neurohypophysial hormones are released in this area they would presumably be transported into the CSF via perivascular channels (Cserr and Ostrach, 1974). The view that vasopressin and oxytocin have neurotransmitter function (e.g., Barker, 1976) is supported by the finding of various areas in which fibres were present containing these hormones. Even more so the punctate pericellular profiles in the lateral septum argue for a release. Such a peptide transmitter function might be the basis for the influence of these and related peptides on memory consolidation.
References
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vasopressin in relation to avoidance behavior. In: Anatomical neuroendocrinology (W.E. Stumpf and L.D. Grant, eds.), pp. 284-289, Basel: Karger 1976 Wimersma Greidanus, Tj. B. van, Dogterom, J., Wied, D. de. Intraventricular administration of antivasopressin serum inhibits memory consolidation in rats. Life Sci. 16, 637-644 (1975) Wittkowski, W. : Elektronenmikroskopische Studien zur intraventrikul/iren Neurosekretion in den Recessus infundibularis der Maus. Z. Zellforsch. 92, 207-216 (1968) Zimmerman, E.A.: Localization of hypothalamic hormones by immunocytochemical techniques. Front. Neuroendocrin. 4, 25-62 (1976) Zimmerman, E.A., Defendini, R., Sokol, H.W., Robinson, A.G.: The distribution of neurophysinsecreting pathways in the mammalian brain: Light microscopic studies using the immunoperoxidase technique. Ann. N.Y. Acad. Sci. 24, 9~111 (1975)
Accepted July 11, 1977
