170
Brain Research, 597 (I 992) 170-175 @ 1992 Elsevi,:r Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 25450
Magnocellular neurosecretory neurons with ferritin-like immunoreactivity in the hypothalamic supraoptic and paraventricular nuclei of the rat A k i r a T o k u n a g a a, K a t s u h i k o O n o a, T o s h i r o O n o b a n d M a k o t o O g a w a a " Third Department o f A n a t o m y a n d h Department o f Parasitology, O k a y a m a Unicersity Medical School, O k a y a m a (Japan)
(Accepted 8 September 1992)
Kts, words: Ferritin; Transferrin receptor: lmmunohistochemistry; Supraoptic nucleus; Paraventricular nucleus; Arginine vasopressin;
Water deprivation
Immunohistochemistry for rat liver ferritin (FRT) revealed an intensive labeling in some structures of the rat brain, in the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei, almost all neurosecretory neurons with vasopressin (AVP)-Iike immunoreactivity were immunostained with FRT. After water deprivation, a marked enlargement of cell body and an immunoreactivity to transferrin receptors were found in AVP-, FRT- and double (AVP+ FRT)-Iabeled neurons in the SON and PVN.
Since iron was first detected histochemically in human brain by Zaleski ~'~ in 1886, many studies have been devoted to the distribution of iron in the central nervous system of various animal species including man (See Youdim~). Chemical quantitative 4.ts." or histochemical studies :'.'~ revealed that iron was mainly distributed in the extrapyramidal motor systems. It is said that 1/3 of the iron in the brain is stored in ferritin (FRT), a family of protein for iron storage and iron detoxification, and the remaining 2/3 are incorporated into enzymes, lipids, and other low molecular weight proteins7'~2. Recently, histochemical and immunohistochemical studies on the regional distribution of iron 5'7
and FRT 5 in the brain demonstrated that they were distributed not only in the extrapyramidal motor systems but also in the various brain structures, including the sensory and limbic systems. in the present study, a polyclonal antibody against rat liver FRT t4 immunohistochemically stained neurosecretory neurons in the hypothalamic supraoptic (SON) and paraventricular nuclei (PVN). This observation may reveal additional functions of iron and/or FRT in the brain I°'lt'ls. Twenty-one adult Wistar rats weighing 300-360 g were used in this study. Five animals were deeply anesthetized with sodium pentobarbital (30 mg/kg,
TABLE ! Competition studies with anti-FRT, anti-A VP a n d a n t i . O X T antisera
Staining intensity of neurosecretory cells in SON and PVN: + , strong, + , very weak, - , absence of staining. 4ntiserunc
anti-FRT
anti-A VP
Anti-OXT
Concentration o f competing substances:
!0 - "~
I0 - ~'
I0 - 7
! 0 - "~
I0 - ~
!0 - z
i 0 - "~
! 0 - t,
!0 - 7
Rat liver FRT AVP OXT
+ +
_ + +
_ + +
+ _ +
+ _ +
+ _ +
+ _+ _
+ _+ _
+ + _
Correspondence: A. Tckunaga, Third Department of Anatomy, Okayama University Medical School, Shikata-Cho 2-5-1, Okayama 700, Japan.
171 i.p.) and perfused intracardially with 150 ml of Ringer's solution and followed by 900 ml of either 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) or Zamboni's fixative. The brains were postfixeddn the same fixative for 5 h at 4°C and then immersed in 20% sucrose PB (pH 7.4, 4°C). Transverse frozen sections were cut serially at a thickness of 30/zm. Every other section was incubated with an anti-rat liver FRT antibody 14 (1 : 4,000 in 0. l M phosphate-buffered saline, PBS) for 24 h at 4°C, and stained with the ABC method. Preadsorption testing was performed as follows: anti-FRT antiserum was preincubated overnight at 4°C with varying concentrations of rat FRT, AVP and O X T (Table I). Using the preadsorbed antiserum, sections through the hypothalamus were processed by the immunohistochemical procedures as described above. Six brains fixed with 4% paraformaldehyde in 0.1 M PB (pH 7.4, 4°C) were embedded into paraffin and the coronal mirror-image sections (3-5 p.m in thickness) through the hypothalamus were immunohistochemically stained either with anti-FRT (1:2,000 in PBS)
and anti-arginine vasopressin (AVP) antibodies (1:2,000 in PBS, Immunonuclear Co., USA) or with anti-FRT and anti-oxytocin (OXT) antibodies (1 : 2,000 in PBS, Immunonuclear Co., USA). Cross-reactivities of anti-AVP with OXT and of anti-OXT with AVP Were studied (Table I). Ten adult Wistar rats (150-200 g) were allowed free access to tap water and standard laboratory chow for at least 3 days prior to the start of the experiment• Five of them were deprived of water for 8 days and the others were given ordinary care as controls. Three animals from each group were perfused with 4% paraformalde~hyde PB solution between 11.00 and 13.00 h to avoid the effect of possible circadian fluctuations in AVP and OXT secretions. The hypothalamic region and the pituitary gland were embedded in paraffin, and, the mirror-image sections were immunohistochemically stained with FRT and AVP or with FRT and OXT as described above, i ' The two ~emaining animals from. each group were perfused with 300 mi of Ringer's solution, and the brain and the pituitary gland were immediately re-
B
A
i
'C?• ~ ,-_ :V":,-',;'
.~,
ii ~
,~.
,,
.
Fig. 1. Photomicrographsof rat brain structures with intensive immunoreactivityfor rat liver ferritin. A" the .medial habenular nucleus (MH). B: the interpeduncular nucleus (INT) and pars reticulata of the substantia nigra (SNR). C: the molecular layer (ML) and the Purkinje cells (P) of the cerebellar cortex. D: the dorsal cochlear nucleus (DCO). Frozen sections with 30-p,m thickness. Bars in A, C and D, and B indicate 100 and 500 ~m, respectively.
172 cleus of the inferior coiliculus, nucleus of the lateral lemniscus, superior olivary nuclei and cerebellar nuclei. Some pyramidal neurons in the cerebral cortex were weakly immunoreactive to FRT. Furthermore, some glial cells with dense FRTi were occasionally scattered in areas of the brain other than the above-mentioned structures. Cells in the area postrema and the ependymal cells were intensely immunostained with anti-FRT antiserum. No specific labeling was found after the preadsorption of anti-FRT antiserum with rat FRT (Fig. 2A, Table I) and no cross-reactivity of anti-FRT with either AVP or OXT was detected (Table I). Hill and Switzer 7 reported localization of iron in the rat brain using a modified Perls' histochemicai reaction intensified with diaminobenzidine. Among 72 iron-containing areas as determined by densitometry 5'7, almost all the FRTi structures in the present study corresponded to iron-rich areas by Hill and his co-workers 5'7. In the present observation, very dense FRTi was detected in the medial habenular nucleus. Hill et al. 5'7 histochemically exhibited moderate amounts of iron in the lateral habenular nucleus but none in the medial habenula. However, a striking enrichment of Tf-R was
moved and kept at -80°C. Cryosections of 7-~m-thickness were prepared and then air-dried for 60 min. They were fixed with acetone for 5 min at room temperature and treated with monoclonal antibody against rat transferrin receptor (Tf-R) (1:5 in PBS, Sera-lab., Sussex, England) for 12 h at 4°C by the ABC method described above, immunohistochemicaily stained sections were counterstained with hematoxyline. In the rat brain, dense FRT-like immunoreactivity (FRTi) was found in the neuropils of the glomerular layer in the olfactory bulb, Calleja's islands, interpeduncular nucleus (Fig. IB), pars reticulata of the substantia nigra (Fig. 1B) and molecular layer of the cerebellar cortex (Fig. 1C), and in the neurons in the supraoptic (SON) (Fig. 2B), paraventricular (PVN) (Fig. 2C) and suprachiasmatic hypothalamic nuclei, medial habenular nucleus (Fig. IA), ventral and dorsal cochlear nuclei (Fig. 1D) and cerebellar Purkinje cells (Fig. 1C). A weak labeling by FRT was observed in neurons a n d / o r neuropils of the putamen and caudate nucleus, pyramidal and molecular layers of the hippocampus, nucleus ruber, superficial layers of the superior colliculus, parabigeminal nucleus, central nu-
.
*
.
,o
. _
iA
D
Q ,D
III •
t.
Fig. 2. A: no immunoreactivityto ferritin (FRT) is seen in the supraoptic nucleus(SON) after preadsorption of FRT-antiserumwith rat FRT. B and C: FRT-like immunoreactivityof neurosecretorycells in the SON and the paraventricularnucleus (PVN). D: enlargementof cell body and intensive FRT-like immunoreactivityin the SON neurosecretoryneurons after water deprivation for 8 days. OT: optic tract. Frozen sectionswith 30/zm thickness.Bars indicate 100 ~m.
173 detected not only in the interpeduncular nucleus but also in the medial habenular nucleus 6']2. In the hypothalamic SON and PVN, almost all the AVP- and some of the OXT-immunoreactive magnocellular neurons (Fig. 2B and C) were intensely immunostained with anti-FRT antiserum. Thick primary dendrites near the cell body and perikarya of the hypothalamic neurosecretory cells were labeled with FRT. Although the labeling was not distinct in axons within either the SON or PVN, weak but discernible immunoreactivity to FRT was observed in axonal terminals of the neurohypophysis (not illustrated). In the SON and PVN of the water-deprived rats, the cell size of the magnocellular neurons with FRT- and AVP-like immunoreactivities increased markedly, as was determined by length of the short and long axes (Figs. 2C and 3A). These findings are consistent with those of Hyodo et al. 8. Most of these magnocellular
cells showed double-labeling with both AVP and FRT (Fig. 3C). Immunoreactivity to AVP in the neurosecretory neurons in both nuclei was slightly weaker in the water-deprived rats than that in the controls, whereas the immunoreactivity to AVP in the posterior pituitary gland was increased in the water-deprived group than that of controls. Immunoreactivity for FRT in the SON neurosecretory cells seemed to be not changed in both groups. On the other hand, only a small number of enlarged neurons were immunopositive to OXT, FRT and a combination of both (Fig. 3B). Since cross-reactivities of anti-AVP with OXT and anti-OXT with AVP were not negligible ]7 (Table I), it is not ruled out a possibility that anti-OXT antiserum may bind to AVP in some neurosecretory neurons in the water-deprived SON and PVN. Autoradiographic hybridization signals indicating the localization of AVP mRNA were significantly in-
o, on
~'o9,%-~O~o ~_" % vo .8... o ooo .oo
oc~ o
13
.
.ogOo~,9Oo PVN ,(3, o°
Long axis
0 FRT positive O OXT positive • Double labeled , Blood vessel
20.
ooo °
oo
B
r,~__ o o'~'o~3~ o soopm
9246-aw00
Jm
4" '' '' ~
°
,.,.g..e.l.e
a - l o
m
us, US, m , In, m ,
•
Ql~qt
.,.,.~ S O N Opt. tr. o FRT positive Control (N:380) Water Oeprivatioa iH:348) %i
A
Shortaxis
9226-1WD8
5 0 0 IJm
AVP positive • Double labeled , Blood vessel
C
Fig. 3. A: cell size distribution of the supraoptic neurosecretory neurons with ferritin (FRT)- and arginine vasopressin (AVP)-like immunoreactivities in the untreated and water-deprived rats. B: distribution of FRT- and oxytocin (OXT)-Iike immunoreactive neurons (open circle and open square, respectively) in the paraventricular nucleus (PVN) of a water-deprived rat (9246-3WD8). A small number of OXT-like immunoreactive neurosecretory cells show FRT-like immunoreactivity (closed circle). C: distribution of FRT- and AVP-like immunoreactive neurons (open circle and open triangle, respectively) in the supraoptic nucleus (SON) of a water-deprived rat (9226-1WD8). Double-labeling with FRT and AVP is observed in a large number of neurosecretory neurons (closed circle). Opt. tr., optic tract, lllrd V., the third ventricle.
174
Fig. 4. Immunocytochemical demonstration of tansferrin receptor (Tf-R) in the supraoptic nucleus in a control (A) and a water-deprived rat (B). Note neurosecretory neurons (asterisks in B) with Tf-R immunoreactivity after water deprivation. Arrows in A and B: Tf-R immunoreactive endothelial cells. Cryosections with 7-/~m thickness. Bars = 50 #m.
creased in the magnoceUular neurosecretory cells of the SON and PVN after the deprivation of water s'~3. The present findings showing a reduction in the neurosccrctory cells and an increase in the neurohypophysis of immunostainability to AVP may indicate that the release of AVP from axonal terminals in the posterior pituitary gland was more active in the water-deprived rats than that in the untreated animals. In the SON of the untreated rats, immunoreactivity to Tf-R was found in the capillary endothelial cells (Fig. 4A), as reported by Jefferies et al.'~ and Fishmann ~,t al. ~, but hardly detected in the neurosecretory neurons. However, in the hypothalamic nucleus of waterdeprived rats, not only capillary endothelium but also the enlarged magnocellular neurosecretory neurons were densely stained with monoclonal antibody against Tf-R (Fig. 4B). Capillary endothelial cells in the neurobypophysis, however, showed very weak immunoreact~vity to Tf-R in both the control and water-deprived groups (not illustrated). Hill et alJ' demonstrated immunohistochemically that Tf-R binding sites occurred nat only over the capillary endothelial cells but also over n'europils and the cell body of neurons and glial cells. The present findings suggest that iron is taken up by neurosecretory neurons in the form of iron-transfertin via the capillary endothelium, and stored in FRT. Yince immunoreactivity to Tf-R was apparently increased in the AVP neurons after water deprivation, 1t is possible that iron or FRT plays some roles in the
synthesis, metabolism or transport of the antidiuretic hormone in the hypothalamic ncurosecretory neurons. Acknowledgemaets. The authors would like to express their appreciation to Dr. Michiyasu Awai, Emeritus professor of Okayama University, for his helpful suggestions and discussions. We are also grateful to Miss. Kazuko Mori for her technical assistance.
REFERENCES I Fishmann, J.B., Rubin, J.B., Handrahan, J.V., Connor. J.R. and Fine, R.E., Receptor-mediated transcytosis of transferrin across the blood-brain barrier, J. Neurosci. Res., 18 (1987) 299-304. 2 Fran£ois, C., Nguyen-Legros, J. and Percheron, G., Topographical and cytological localization of iron in rat and monkey brains, Brain Res., 215 (1981) 317-322. 3 Guizzetti, P., Principali risulati dell'appricazione grossolona a fresco delle reazioni istochimiche del ferro sul sistema nervosa centrale dell'uomo e di aluni mammiferi domestici, Rir. Pathd. Nerc. Ment., 20 (1915) 102-117. Cited by Youdim Is. 4 Hallgren, B. and Sourander, P., The effect of age on the nonhaemin iron in the human brain, J. Neurochem., 3 (1958) 41-51. 5 Hill, J.M., The distribution of iron in the brain. In M.B.H. Youdim (Ed.), Topk's in Neurochemistry and Neuropharmacology. Vol. 2. Brain Iron: Neurochemical and Beharioral Aspects, Taylor & Francis, London, 1988, pp. 1-24. 6 Hill, J.M., Ruff, M.R., Weber, RJ. and Pert, C.B., Transferrin receptors in rat brain: neuropeptide-like pattern and relationship to iron distribution, Proc. Natl. Acad. Sci. USA, 82 (1985) 45534557. 7 Hill, J.M. and Switzer, R.C., The regional distribution and cellular localization of iron in the rat brain, Neuroscience, I I 0984) 595 -603. 8 Hyodo, S., Sato, M. and Urano, A., Molecular- and immunohistochemical study on expressions of vasopressin and oxytocin gene following water deprivation, Zool. Sci., 6 (1989) 335-343.
175 9 Jefferies, W.A., Brandon, MR., Hunt, S.V., Williams, A.F., Gatter, K.C. and Mason, D.Y., Transferrin receptor on endothelium of brain capillaries, Nature, 312 (1984) 162-163. 10 Joshi, J.G. and Clauberg, M., Ferritin: an iron storage protein with diverse functions, BioFactors, 1 (1988) 207-212. 11 Joshi, J.G. and Zimmerman, A., Ferritin: an expanded role in metabolic regulation, Toxicology, 48 (1988) 21-29. 12 Mash, D.C., Pablo, J., Flynn, D.D., Efange, S.M.N., Weiner, W.J., Characterization and distribution of transferrin receptors in the rat brain, J. Neurochem., 55 (1990) 1972-1979. 13 Meeker, R.B., Greenwood, R.S. and Hayward, J.N., Vasopressin mRNA expression in individual magnocellular neuroendocrine cells of the supraoptic and paraventricular nucleus in response to water deprivation, Neuroendocrinology, 54 (1991) 236-247. 14 Ono, T., Tsujii, T. and Seno, S., A morphological study of ferritin synthesis in macrophages with ingested ferric hydroxide-potas-
15
sium polyvinyi sulfate complexes, Cell Struct. Funct., 8 (1983) 267-279. Rajan, K.S., Coiburn, R.W. and Davis, J.M., Distribution of metal ions in the subceilular fractions of several rat brain areas, Life Sci., 18 (1976) 423-432. Spatz, H., Uber den Eisennachweis im Gehirn, besonders in Zentren des extrapyramidal-motorischen Systems, Ztschr. gesam. Neurol. Psychiat., 77 (1922) 261-390. Swaab, D.F. and Pool, C.W., Specificity of oxytocin an3. vasopressin immunofluorescence, J. Endocrinol., 66 (1975) 263-272. Youdim, M.B.H., Brain iron metabolism: biochemical and behavioral aspects in relation to dopaminergic neurotransmission. In A. Lajtha (Ed.), Handbook of Neurochemistry. Vol. 10. Pathological Neurochemistry, Plenum, New York, 1985, pp. 731-755. Zaleski, S., Das Eisen der Organe bei Morbus maculosus Werlhofii, Arch. Exp. Pathol. Pharmakol., 23 (1887) 77-99. ..
16 17 18
19
Brain Research, 597 (I 992) 170-175 @ 1992 Elsevi,:r Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 25450
Magnocellular neurosecretory neurons with ferritin-like immunoreactivity in the hypothalamic supraoptic and paraventricular nuclei of the rat A k i r a T o k u n a g a a, K a t s u h i k o O n o a, T o s h i r o O n o b a n d M a k o t o O g a w a a " Third Department o f A n a t o m y a n d h Department o f Parasitology, O k a y a m a Unicersity Medical School, O k a y a m a (Japan)
(Accepted 8 September 1992)
Kts, words: Ferritin; Transferrin receptor: lmmunohistochemistry; Supraoptic nucleus; Paraventricular nucleus; Arginine vasopressin;
Water deprivation
Immunohistochemistry for rat liver ferritin (FRT) revealed an intensive labeling in some structures of the rat brain, in the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei, almost all neurosecretory neurons with vasopressin (AVP)-Iike immunoreactivity were immunostained with FRT. After water deprivation, a marked enlargement of cell body and an immunoreactivity to transferrin receptors were found in AVP-, FRT- and double (AVP+ FRT)-Iabeled neurons in the SON and PVN.
Since iron was first detected histochemically in human brain by Zaleski ~'~ in 1886, many studies have been devoted to the distribution of iron in the central nervous system of various animal species including man (See Youdim~). Chemical quantitative 4.ts." or histochemical studies :'.'~ revealed that iron was mainly distributed in the extrapyramidal motor systems. It is said that 1/3 of the iron in the brain is stored in ferritin (FRT), a family of protein for iron storage and iron detoxification, and the remaining 2/3 are incorporated into enzymes, lipids, and other low molecular weight proteins7'~2. Recently, histochemical and immunohistochemical studies on the regional distribution of iron 5'7
and FRT 5 in the brain demonstrated that they were distributed not only in the extrapyramidal motor systems but also in the various brain structures, including the sensory and limbic systems. in the present study, a polyclonal antibody against rat liver FRT t4 immunohistochemically stained neurosecretory neurons in the hypothalamic supraoptic (SON) and paraventricular nuclei (PVN). This observation may reveal additional functions of iron and/or FRT in the brain I°'lt'ls. Twenty-one adult Wistar rats weighing 300-360 g were used in this study. Five animals were deeply anesthetized with sodium pentobarbital (30 mg/kg,
TABLE ! Competition studies with anti-FRT, anti-A VP a n d a n t i . O X T antisera
Staining intensity of neurosecretory cells in SON and PVN: + , strong, + , very weak, - , absence of staining. 4ntiserunc
anti-FRT
anti-A VP
Anti-OXT
Concentration o f competing substances:
!0 - "~
I0 - ~'
I0 - 7
! 0 - "~
I0 - ~
!0 - z
i 0 - "~
! 0 - t,
!0 - 7
Rat liver FRT AVP OXT
+ +
_ + +
_ + +
+ _ +
+ _ +
+ _ +
+ _+ _
+ _+ _
+ + _
Correspondence: A. Tckunaga, Third Department of Anatomy, Okayama University Medical School, Shikata-Cho 2-5-1, Okayama 700, Japan.
171 i.p.) and perfused intracardially with 150 ml of Ringer's solution and followed by 900 ml of either 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) or Zamboni's fixative. The brains were postfixeddn the same fixative for 5 h at 4°C and then immersed in 20% sucrose PB (pH 7.4, 4°C). Transverse frozen sections were cut serially at a thickness of 30/zm. Every other section was incubated with an anti-rat liver FRT antibody 14 (1 : 4,000 in 0. l M phosphate-buffered saline, PBS) for 24 h at 4°C, and stained with the ABC method. Preadsorption testing was performed as follows: anti-FRT antiserum was preincubated overnight at 4°C with varying concentrations of rat FRT, AVP and O X T (Table I). Using the preadsorbed antiserum, sections through the hypothalamus were processed by the immunohistochemical procedures as described above. Six brains fixed with 4% paraformaldehyde in 0.1 M PB (pH 7.4, 4°C) were embedded into paraffin and the coronal mirror-image sections (3-5 p.m in thickness) through the hypothalamus were immunohistochemically stained either with anti-FRT (1:2,000 in PBS)
and anti-arginine vasopressin (AVP) antibodies (1:2,000 in PBS, Immunonuclear Co., USA) or with anti-FRT and anti-oxytocin (OXT) antibodies (1 : 2,000 in PBS, Immunonuclear Co., USA). Cross-reactivities of anti-AVP with OXT and of anti-OXT with AVP Were studied (Table I). Ten adult Wistar rats (150-200 g) were allowed free access to tap water and standard laboratory chow for at least 3 days prior to the start of the experiment• Five of them were deprived of water for 8 days and the others were given ordinary care as controls. Three animals from each group were perfused with 4% paraformalde~hyde PB solution between 11.00 and 13.00 h to avoid the effect of possible circadian fluctuations in AVP and OXT secretions. The hypothalamic region and the pituitary gland were embedded in paraffin, and, the mirror-image sections were immunohistochemically stained with FRT and AVP or with FRT and OXT as described above, i ' The two ~emaining animals from. each group were perfused with 300 mi of Ringer's solution, and the brain and the pituitary gland were immediately re-
B
A
i
'C?• ~ ,-_ :V":,-',;'
.~,
ii ~
,~.
,,
.
Fig. 1. Photomicrographsof rat brain structures with intensive immunoreactivityfor rat liver ferritin. A" the .medial habenular nucleus (MH). B: the interpeduncular nucleus (INT) and pars reticulata of the substantia nigra (SNR). C: the molecular layer (ML) and the Purkinje cells (P) of the cerebellar cortex. D: the dorsal cochlear nucleus (DCO). Frozen sections with 30-p,m thickness. Bars in A, C and D, and B indicate 100 and 500 ~m, respectively.
172 cleus of the inferior coiliculus, nucleus of the lateral lemniscus, superior olivary nuclei and cerebellar nuclei. Some pyramidal neurons in the cerebral cortex were weakly immunoreactive to FRT. Furthermore, some glial cells with dense FRTi were occasionally scattered in areas of the brain other than the above-mentioned structures. Cells in the area postrema and the ependymal cells were intensely immunostained with anti-FRT antiserum. No specific labeling was found after the preadsorption of anti-FRT antiserum with rat FRT (Fig. 2A, Table I) and no cross-reactivity of anti-FRT with either AVP or OXT was detected (Table I). Hill and Switzer 7 reported localization of iron in the rat brain using a modified Perls' histochemicai reaction intensified with diaminobenzidine. Among 72 iron-containing areas as determined by densitometry 5'7, almost all the FRTi structures in the present study corresponded to iron-rich areas by Hill and his co-workers 5'7. In the present observation, very dense FRTi was detected in the medial habenular nucleus. Hill et al. 5'7 histochemically exhibited moderate amounts of iron in the lateral habenular nucleus but none in the medial habenula. However, a striking enrichment of Tf-R was
moved and kept at -80°C. Cryosections of 7-~m-thickness were prepared and then air-dried for 60 min. They were fixed with acetone for 5 min at room temperature and treated with monoclonal antibody against rat transferrin receptor (Tf-R) (1:5 in PBS, Sera-lab., Sussex, England) for 12 h at 4°C by the ABC method described above, immunohistochemicaily stained sections were counterstained with hematoxyline. In the rat brain, dense FRT-like immunoreactivity (FRTi) was found in the neuropils of the glomerular layer in the olfactory bulb, Calleja's islands, interpeduncular nucleus (Fig. IB), pars reticulata of the substantia nigra (Fig. 1B) and molecular layer of the cerebellar cortex (Fig. 1C), and in the neurons in the supraoptic (SON) (Fig. 2B), paraventricular (PVN) (Fig. 2C) and suprachiasmatic hypothalamic nuclei, medial habenular nucleus (Fig. IA), ventral and dorsal cochlear nuclei (Fig. 1D) and cerebellar Purkinje cells (Fig. 1C). A weak labeling by FRT was observed in neurons a n d / o r neuropils of the putamen and caudate nucleus, pyramidal and molecular layers of the hippocampus, nucleus ruber, superficial layers of the superior colliculus, parabigeminal nucleus, central nu-
.
*
.
,o
. _
iA
D
Q ,D
III •
t.
Fig. 2. A: no immunoreactivityto ferritin (FRT) is seen in the supraoptic nucleus(SON) after preadsorption of FRT-antiserumwith rat FRT. B and C: FRT-like immunoreactivityof neurosecretorycells in the SON and the paraventricularnucleus (PVN). D: enlargementof cell body and intensive FRT-like immunoreactivityin the SON neurosecretoryneurons after water deprivation for 8 days. OT: optic tract. Frozen sectionswith 30/zm thickness.Bars indicate 100 ~m.
173 detected not only in the interpeduncular nucleus but also in the medial habenular nucleus 6']2. In the hypothalamic SON and PVN, almost all the AVP- and some of the OXT-immunoreactive magnocellular neurons (Fig. 2B and C) were intensely immunostained with anti-FRT antiserum. Thick primary dendrites near the cell body and perikarya of the hypothalamic neurosecretory cells were labeled with FRT. Although the labeling was not distinct in axons within either the SON or PVN, weak but discernible immunoreactivity to FRT was observed in axonal terminals of the neurohypophysis (not illustrated). In the SON and PVN of the water-deprived rats, the cell size of the magnocellular neurons with FRT- and AVP-like immunoreactivities increased markedly, as was determined by length of the short and long axes (Figs. 2C and 3A). These findings are consistent with those of Hyodo et al. 8. Most of these magnocellular
cells showed double-labeling with both AVP and FRT (Fig. 3C). Immunoreactivity to AVP in the neurosecretory neurons in both nuclei was slightly weaker in the water-deprived rats than that in the controls, whereas the immunoreactivity to AVP in the posterior pituitary gland was increased in the water-deprived group than that of controls. Immunoreactivity for FRT in the SON neurosecretory cells seemed to be not changed in both groups. On the other hand, only a small number of enlarged neurons were immunopositive to OXT, FRT and a combination of both (Fig. 3B). Since cross-reactivities of anti-AVP with OXT and anti-OXT with AVP were not negligible ]7 (Table I), it is not ruled out a possibility that anti-OXT antiserum may bind to AVP in some neurosecretory neurons in the water-deprived SON and PVN. Autoradiographic hybridization signals indicating the localization of AVP mRNA were significantly in-
o, on
~'o9,%-~O~o ~_" % vo .8... o ooo .oo
oc~ o
13
.
.ogOo~,9Oo PVN ,(3, o°
Long axis
0 FRT positive O OXT positive • Double labeled , Blood vessel
20.
ooo °
oo
B
r,~__ o o'~'o~3~ o soopm
9246-aw00
Jm
4" '' '' ~
°
,.,.g..e.l.e
a - l o
m
us, US, m , In, m ,
•
Ql~qt
.,.,.~ S O N Opt. tr. o FRT positive Control (N:380) Water Oeprivatioa iH:348) %i
A
Shortaxis
9226-1WD8
5 0 0 IJm
AVP positive • Double labeled , Blood vessel
C
Fig. 3. A: cell size distribution of the supraoptic neurosecretory neurons with ferritin (FRT)- and arginine vasopressin (AVP)-like immunoreactivities in the untreated and water-deprived rats. B: distribution of FRT- and oxytocin (OXT)-Iike immunoreactive neurons (open circle and open square, respectively) in the paraventricular nucleus (PVN) of a water-deprived rat (9246-3WD8). A small number of OXT-like immunoreactive neurosecretory cells show FRT-like immunoreactivity (closed circle). C: distribution of FRT- and AVP-like immunoreactive neurons (open circle and open triangle, respectively) in the supraoptic nucleus (SON) of a water-deprived rat (9226-1WD8). Double-labeling with FRT and AVP is observed in a large number of neurosecretory neurons (closed circle). Opt. tr., optic tract, lllrd V., the third ventricle.
174
Fig. 4. Immunocytochemical demonstration of tansferrin receptor (Tf-R) in the supraoptic nucleus in a control (A) and a water-deprived rat (B). Note neurosecretory neurons (asterisks in B) with Tf-R immunoreactivity after water deprivation. Arrows in A and B: Tf-R immunoreactive endothelial cells. Cryosections with 7-/~m thickness. Bars = 50 #m.
creased in the magnoceUular neurosecretory cells of the SON and PVN after the deprivation of water s'~3. The present findings showing a reduction in the neurosccrctory cells and an increase in the neurohypophysis of immunostainability to AVP may indicate that the release of AVP from axonal terminals in the posterior pituitary gland was more active in the water-deprived rats than that in the untreated animals. In the SON of the untreated rats, immunoreactivity to Tf-R was found in the capillary endothelial cells (Fig. 4A), as reported by Jefferies et al.'~ and Fishmann ~,t al. ~, but hardly detected in the neurosecretory neurons. However, in the hypothalamic nucleus of waterdeprived rats, not only capillary endothelium but also the enlarged magnocellular neurosecretory neurons were densely stained with monoclonal antibody against Tf-R (Fig. 4B). Capillary endothelial cells in the neurobypophysis, however, showed very weak immunoreact~vity to Tf-R in both the control and water-deprived groups (not illustrated). Hill et alJ' demonstrated immunohistochemically that Tf-R binding sites occurred nat only over the capillary endothelial cells but also over n'europils and the cell body of neurons and glial cells. The present findings suggest that iron is taken up by neurosecretory neurons in the form of iron-transfertin via the capillary endothelium, and stored in FRT. Yince immunoreactivity to Tf-R was apparently increased in the AVP neurons after water deprivation, 1t is possible that iron or FRT plays some roles in the
synthesis, metabolism or transport of the antidiuretic hormone in the hypothalamic ncurosecretory neurons. Acknowledgemaets. The authors would like to express their appreciation to Dr. Michiyasu Awai, Emeritus professor of Okayama University, for his helpful suggestions and discussions. We are also grateful to Miss. Kazuko Mori for her technical assistance.
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