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Induction by nerve growth factor (NGF) of NGF receptor-like immunoreactivity in the preoptic area and hypothalamus of neonatal rats
Neuroscience Letters, 138 (1992) 241-244 ~© 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
241
NSL 08567
Induction by nerve growth factor (NGF) of NGF receptor-like immunoreactivity in the preoptic area and hypothalamus of neonatal rats M a s a k o Nishizuka and Yasumasa Arai Department of Anatomy, Juntendo University School o[ Medicine, Tokyo (Japan) (Received 29 October 1991; Revised version received 31 January 1992; Accepted 2 February 1992)
Key words: Nerve growth factor; Nerve growth factor receptor; Preoptic area; Hypothalamus; Neonatal rat Nerve growth factor (NGF) was injected in the brain of neonatal rats. In eleven of thirty-one rats that received 1/tg of mouse NGF (2.5S form) on postnatal day 3, NGF receptor-like immunoreactivity was found in the preoptic area (POA) and hypothalamus until 12 days after birth. However, no immunoreactivity was seen in the region of rats in the same group examined on day 20 or 45 and of uninjected control rats on days 145. These results indicate that NGF induces the transient appearance of the receptor for NGF in the POA and hypothalamus of neonatal rats.
Nerve growth factor (NGF) acts as atrophic factor in certain populations of neurons in the central nervous system of the rat [3, 11]. The distribution of NGF [10, 13, 16], the mRNA for NGF [16], the NGF receptor (NGFR) [14, 18], and the mRNA for NGFR [9] has been clarified in the brain of developing [16] and adult [9, 10, 13, 14, 16, 18] rats. NGFR-specific immunoreactivity and expression o f m R N A for NGFR are increased in the cholinergic neurons in the forebrain of adult rats by intracerebral infusion of NGF [2, 7]. NGFR-like immunoreactive neurons are seen in the preoptic area (POA) and hypothalamus of adult rats [14, 15, 18]. We have suggested that a population of tyrosine hydroxylase (TH)-containing neurons in the hypothalamus of perinatal rats are responsive to the NGF, based on the findings that NGF increased the TH-specific immunoreactivity in the fetal hypothalamus transplanted into the ventricle of adult rats [12]. The aim of the present study was to elucidate whether appearance of NGFR in the hypothalamic neurons is regulated by NGF during the neonatal period. The profile of distribution of NGFR-Iike immunoreactivity and of TH immunoreactivity was examined in the POA and hypothalamus of neonatal rats afer the infusion of NGF into the brain. Wistar rats were used. Rat pups of I (the day of birth), Correspondence: M. Nishizuka, Department of Anatomy, Juntendo University School of Medicine, 2 Hongo 1-1, Bunkyo-ku, Tokyo 113, Japan.
3, 5, 8, 12, 20, and 45 days of age were used as members of an intact control group. Three-day-old rats in another group were injected intracerebrally with 0.1 or 1/lg of 2.5S NGF (an extract from mouse submandibular glands, Biomedical Technologies Inc., Stoughton, MA) and dissolved in 5/tl of 0.1% bovine serum albumin (BSA; Biomedical Technologies Inc.) through a microruler syringe (701-RN; Hamilton Co., Reno, NV). They were killed at 5, 8, 12, 20, or 45 days of age. Another group of rats was given 1/lg of NGF dissolved in 5/A of BSA on day 5 and killed at 8 or 12 days of age. Other rats were injected with the same volume of vehicle on day 3 and killed on day 5 or day 8. The brains of 1- to 5-day-old rats were removed after decapitation and immersed overnight at 4°C in fixative that consisted of 4% paraformaldehyde and 15 ml saturated picric acid in 100 ml phosphate buffer (PB, pH 7.4). The other rats were deeply anesthetized with pentobarbital and perfused with the same fixative. All the brains were cryoprotected in 10% sucrose dissolved in the PB, frozen in isopentane chilled in liquid nitrogen, and cut at 10Hm on a cryostat. The sections were mounted on glass slides and treated with 0.1% hydrogen peroxide in PBS. Every third section was incubated overnight at 4°C in a solution of affinitypurified 192-IgG (2.5 /lg IgG/ml, supplied by E.M. Johnson, Jr. [1, 18]) or the supernatant of a hybridoma cell culture that produced 192-IgG (supplied by H. Hatanaka). The neighboring sections were incubated with a TH-specific monoclonal antibody (PCTH-7, supplied by
242 H. H a t a n a k a [6]). F o r negative control staining, the other set of neighboring sections was incubated without primary antibody or with a preparation of mouse I g G (2.5 /.tg/ml; Chemicon, Temecula, CA). After being washed, all of the sections were incubated with biotinylated anti-mouse I g G and ABC reagent (Vector Laboratories, Burlingame, CA), and then with a solution of diaminobenzidine that contained hydrogen peroxide. The site of injection (Fig. 1) was verified on the immunostained sections and the rats that received injections in the POA or the hypothalamus were examined. Since there are reports that tissue damage induces and/or increases N G F R immunoreactivity in the forebrain of neonatal [17] and adult rats [2, 4], the rats in which the medial region of the POA and hypothalamus was damaged by the infusion were excluded from the experiment. The neurons with N G F R - l i k e immunoreactivity were seen in the basal forebrain of all of the rats that received N G F and of the control rats, irrespective of age. The neighboring sections stained with 192-IgG in the supernatant of the hybridoma culture and with the purified antibody showed similar N G F R - l i k e immunoreactivity in the forebrain region. The immunoreactivity specific for N G F R was seen in the POA and hypothalamus of rats that had received 1 p g of N G F at 3 days of age (Fig. 2). Four rats out of 11 at 5 days of age, 4 rats out of 12 at 8 days of age (Fig. 2a), and 3 of 8 at 12 days of age (Fig. 2b), that is, 11 rats in all out of a total of 31, had N G F R - I i k e immunoreactive neurons in the POA and hypothalamus (Table I). Since, in the control preparations, no immunostaining of any neurons was observed in the same region, the immunoreactivity of these neurons was specific to the antibody against N G F R . In the eleven rats, the immunoreactivity was localized in the neuronal cell bodies and the proximal part of their processes in the periventricular gray of the POA and anterior hypothalamus (Fig. 2a) and the arcuate nucleus of the hypothalamus (Fig. 2b) ipsi- and contralateral to the
site of injection (Fig. 2a,b). The number of neurons with N G F R - l i k e immunoreactivity varied a m o n g these eleven rats, for example, ten neurons in total could be seen in the region stained with 192-IgG in one rat. By contrast, m a n y immunoreactive neurons were seen in a section taken from another rat, as shown in Fig. 2b. Additionally the ependymal cells of the third ventricle of the eleven rats showed specific N G F R - l i k e immunoreactivity at 5 (Fig. 2e) and 8 days of age. The suprachiasmatic nucleus in some of the eleven rats showed specific N G F R immunoreactivity, but the immunoreactivity was localized in the fibers, in good accord with previously reported findings [14, 15]. In contrast to the above results, no immunoreactivity was encountered in the POA or hypothalamus of the rats in the same group when they were killed at 20 or 45 days of age, rats that received 0.1 p g N G F at 3 days of age, or rats that received 1 p g on day 5 (Table I). N o immunoreactivity was found in the POA or hypothalamus of uninjected control 1- to 45-day-old rats (Table I). The immunoreactivity specific for T H was present in the POA (Fig. 2c) and hypothalamus (Fig. 2d) of all of the rats, as has been fully documented by others [8]. In the eleven rats in which the neurons with N G F R - l i k e immunoreactivity were visualized, the area in which these neurons were localized (Fig. 2a,b) corresponded to a part of the area in which the TH-containing neurons were distributed (Fig. 2c,d). The present results indicate that the immunoreactivity specific to 192-IgG was expressed in the POA and hypothalamus during a neonatal period after the infusion of TABLE I APPEARANCE OF NEURONS WITH NGFR-L1KE IMMUNOREACTIVITY IN THE PREOPTIC AREA (POA) AND HYPOTHALAMUS OF NEONATAL RATS Numbers of rats that had immunoreactive neurons in the POA and hypothalamus among rats injected with mouse 2.5S NGF on day 3 or 5, among rats injected with vehicle on day 3 and among uninjected control rats. Number of rats studied are given in parentheses. Postnatal days Group
1
Normal
0(8) 0(5) 0(7) 0(5) 0(5) 0(7) 0(3)
NGF on day 3 1 pg 0.1pg Fig. 1. Sites of injection in the eleven rats that had neurons with NGFR-like immunoreactivity in the POA and hypothalamus after the infusion of the NGF. Injections are superimposed on three representative coronal sections, ac, anterior commissure; arc, arcuate nucleus; f, fornix; pe, periventricular gray; mpo, medial POA: ox, optic chiasma, 3v, third ventricle.
NGF on day 5 l pg Vehicle on day 3
3
5
8
12
20
45
4(11) 4(12) 3(8) 0(5) 0(3) 0(5) 0(5) 0(4)
0(5) 0(6) 0(5) 0(5)
243
Fig. 2. NGFR-Iike immunoreactivity in the POA and hypothalamus of rats given NGF on day 3 and TH-containing neurons. NGFR-Iike immunoreactive neurons are visible in the POA of an 8-day-old rat (a, inset in a higher magnification) and in the arcuate nucleus of the hypothalamus of a 12-day-old rat (b, inset in an higher magnification), and TH-containing neurons are seen in the neighboring sections (c, d, respectively). NGFR-Iike immunoreactivity in the ependymal cells of a 5-day-old rat (e). *, third ventricle. Bar -- 50 pro.
N G F . T h e 192-IgG a n t i b o d y raised a g a i n s t the r a t N G F R [1] recognizes a high- a n d / o r a low-affinity N G F R [5]. Thus, a t r a n s i e n t increase in levels o f i m m u n o r e a c t i v e N G F R seems to have o c c u r r e d in the i m m u n o r e a c t i v e neurons. I n f u s i o n o f m o u s e N G F into the b r a i n o f a d u l t rats is k n o w n to induce the e x p r e s s i o n o f N G F R [2] a n d the m R N A for N G F R [7] in the choli-
nergic n e u r o n s o f the striatum. T h e i n d u c t i o n o f N G F R b y N G F is likely to be c o m m o n to all n e u r o n s t h a t are responsive to N G F , irrespective o f the n e u r o n a l subsets a n d the age o f the animal. Existence o f N G F R - l i k e i m m u n o r e a c t i v i t y in the neurons a n d the e p e n d y m a l cells o f the P O A a n d h y p o t h a l a m u s has been r e p o r t e d in a d u l t rats [14, 18]. L o c a l i z a -
244
tion of the neurons with NGFR-like immunoreactivity in the NGF-infused neonatal rats is in good accord with that in adults [14, 18]. The absence of immunoreactivity in normal neonatal rats consequently indicates that the infusion of NGF on day 3 induced the NGFR immunoreactivity in the neurons that would subsequently produce NGFR molecules. Our previous study [12] in which fetal hypothalamic tissues were transplanted into the adult cerebral ventricle suggested that the TH-containing neurons in the hypothalamus responded to NGF. Since, in the POA and hypothalamus of the neonatal rats, the localization of the NGFR-Iike immunoreactivity coincided with the distribution of TH-containing neurons, the neurons that expressed NGFR-like immunoreactivity were, presumably, a subpopulation of the TH-containing neurons. The alternative possibility, namely, that the NGFR-like immunoreactive neurons and the TH-containing neurons are members of different neuronal subsets but are interconnected cannot be excluded. We thank Drs. H. Hatanaka, E.M. Johnson, Jr. who kindly provided 192-IgG and PCTH-7 and Dr. E.M. Shooter for his kind suggestions.
1 Chandler, C.E., Parsons, L.M., Hosang, M. and Shooter, E.M., A monoclonal antibody modulates the interaction of nerve growth factor with PC12 cells, J. Biol. Chem., 259 (1984) 6882 6889. 2 Gage, F.H., Batchelor, P., Chen, K.S., Chin, D., Higgins, G.A., Koh, S., Deputy, S., Rosenberg, M.B., Fischer, W. and Bj6rklund, A., NGF receptor reexpression and NGF-mediated cholinergic neuronal hypertrophy in the damaged adult neostriatum, Neuron, 2(1989) 1177 1184. 3 Gnahn, H., Hefti, F., Heumann, R., Schwab, M.E. and Thoenen, H., NGF-mediated increase of choline acetyltransferase (CHAT) in the neonatal rat forebrain: evidence for a physiological role of NGF in the brain?, Dev. Brain Res., 9 (1983) 45-52. 4 Gomez-Pinilla, F., Cotman, C.W. and Nieto-Sampedro, M., NGF receptor immunoreactivity in the rat brain: topographic distribution and response to entorhinal ablation, Neurosci. Lett., 82 (1987) 260-266. 5 Green, S.H. and Green, L.A., A single M r ~ 103,000 12~l-fl-nerve growth factor-affinity-labeled species represents both the low and high affinity forms of the nerve growth factor receptor, J. Biol. Chem., 261 (1986) 15316 15326.
6 Hatanaka, H. and Arimatsu, Y., Monoclonal antibodies to tyrosine hydroxylase from rat pheochromocytoma PC12h cells with special reference to nerve growth factor-mediated increase of the immunoprecipitable enzymes, Neurosci. Res., 1 (1984) 253 263. 7 Higgins, G.A., Koh, S., Chen, K.S. and Gage, F.H., NGF induction of NGF receptor gene expression and cholinergic neuronal hypertrophy within the basal forebrain of the adult rat, Neuron, 3 (1989) 247-256. 8 H6kfelt, T., Martensson, R., Bj6rklund, A., Kleinau, S. and Goldstein, M., Distributional maps of tyrosine-hydroxylase-immunoreactive neurons in the rat brain. In A. BjOrklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 2, Classical Transmitters in the CNS, Part 1, Elsevier, Amsterdam, 1984, pp. 277-379. 9 Koh, S., Oyler, G.A. and Higgins, G.A., Localization of nerve growth factor receptor messenger RNA and protein in the adult rat brain, Exp. Neurol., 106 (1989) 397~,04. 10 Korsching, S., Auburger, G., Heumann, R., Scott, J. and Thoenen, H., Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation, EMBO J., 4 (1985) 1389-1393. 11 Mobley, W.C., Rutkowski, J.L., Tennekoon, G.I., Buckannan, K. and Johnstom, M.V., Choline acetyltransferase activity in striatum of neonatal rats increased by nerve growth factor, Science, 229 (1985) 284-287. 12 Nishizuka, M. and Arai, Y., Modification of nerve growth factor and sex steroids in development of hypothalamic neurons. Endocr. Exp., 24 (1990) 77-86. 13 Nishizuka, M., Katoh-Semba, R., Eto, K., Arai, Y., lizuka, R. and Kato, K., Age- and sex-related differences in the nerve growth factor distribution in the rat brain, Brain Res. Bull., 27 ( 1991 ) 685 688. 14 Pioro, E.P. and Cuello, A.C., Distribution of nerve growth factor receptor-like immunoreactivity in the adult rat central nervous system. Effect of colchicine and correlation with the cholinergic system 1. Forebrain, Neuroscience, 34 (1990) 57 87. 15 Sofroniew, M.V., Isacson, O. and O'Brien, T.S., Nerve growth factor receptor immunoreactivity in the rat suprachiasmatic nucleus, Brain Res., 476 (1989) 358 362. 16 Whittemore, S.C., Ebendal. T., L~irkfors, L., Olson, L., Seiger, A,., Str6mberg, I. and Persson, H., Developmental and regional expression offl-nerve growth factor messenger RNA and protein in the rat central nervous system, Proc. Natl. Acad. Sci. USA, 83 (1986) 817 821. 17 Whittemore, S.R., L~rkfors, L., Ebendal, T., Holets. V.R., Ericsson, A. and Persson, H., Increased fl-nerve growth factor messenger RNA and protein levels in neonatal rat hippocampus following specific cholinergic lesions, J. Neurosci., 7 (1987) 244-251. 18 Yan, Q. and Johnson Jr., E.M., Immunohistochemical localization and biochemical characterization of nerve growth factor receptor in adult rat brain, J. Comp. Neurol., 290 (1989) 585 598.
241
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Induction by nerve growth factor (NGF) of NGF receptor-like immunoreactivity in the preoptic area and hypothalamus of neonatal rats M a s a k o Nishizuka and Yasumasa Arai Department of Anatomy, Juntendo University School o[ Medicine, Tokyo (Japan) (Received 29 October 1991; Revised version received 31 January 1992; Accepted 2 February 1992)
Key words: Nerve growth factor; Nerve growth factor receptor; Preoptic area; Hypothalamus; Neonatal rat Nerve growth factor (NGF) was injected in the brain of neonatal rats. In eleven of thirty-one rats that received 1/tg of mouse NGF (2.5S form) on postnatal day 3, NGF receptor-like immunoreactivity was found in the preoptic area (POA) and hypothalamus until 12 days after birth. However, no immunoreactivity was seen in the region of rats in the same group examined on day 20 or 45 and of uninjected control rats on days 145. These results indicate that NGF induces the transient appearance of the receptor for NGF in the POA and hypothalamus of neonatal rats.
Nerve growth factor (NGF) acts as atrophic factor in certain populations of neurons in the central nervous system of the rat [3, 11]. The distribution of NGF [10, 13, 16], the mRNA for NGF [16], the NGF receptor (NGFR) [14, 18], and the mRNA for NGFR [9] has been clarified in the brain of developing [16] and adult [9, 10, 13, 14, 16, 18] rats. NGFR-specific immunoreactivity and expression o f m R N A for NGFR are increased in the cholinergic neurons in the forebrain of adult rats by intracerebral infusion of NGF [2, 7]. NGFR-like immunoreactive neurons are seen in the preoptic area (POA) and hypothalamus of adult rats [14, 15, 18]. We have suggested that a population of tyrosine hydroxylase (TH)-containing neurons in the hypothalamus of perinatal rats are responsive to the NGF, based on the findings that NGF increased the TH-specific immunoreactivity in the fetal hypothalamus transplanted into the ventricle of adult rats [12]. The aim of the present study was to elucidate whether appearance of NGFR in the hypothalamic neurons is regulated by NGF during the neonatal period. The profile of distribution of NGFR-Iike immunoreactivity and of TH immunoreactivity was examined in the POA and hypothalamus of neonatal rats afer the infusion of NGF into the brain. Wistar rats were used. Rat pups of I (the day of birth), Correspondence: M. Nishizuka, Department of Anatomy, Juntendo University School of Medicine, 2 Hongo 1-1, Bunkyo-ku, Tokyo 113, Japan.
3, 5, 8, 12, 20, and 45 days of age were used as members of an intact control group. Three-day-old rats in another group were injected intracerebrally with 0.1 or 1/lg of 2.5S NGF (an extract from mouse submandibular glands, Biomedical Technologies Inc., Stoughton, MA) and dissolved in 5/tl of 0.1% bovine serum albumin (BSA; Biomedical Technologies Inc.) through a microruler syringe (701-RN; Hamilton Co., Reno, NV). They were killed at 5, 8, 12, 20, or 45 days of age. Another group of rats was given 1/lg of NGF dissolved in 5/A of BSA on day 5 and killed at 8 or 12 days of age. Other rats were injected with the same volume of vehicle on day 3 and killed on day 5 or day 8. The brains of 1- to 5-day-old rats were removed after decapitation and immersed overnight at 4°C in fixative that consisted of 4% paraformaldehyde and 15 ml saturated picric acid in 100 ml phosphate buffer (PB, pH 7.4). The other rats were deeply anesthetized with pentobarbital and perfused with the same fixative. All the brains were cryoprotected in 10% sucrose dissolved in the PB, frozen in isopentane chilled in liquid nitrogen, and cut at 10Hm on a cryostat. The sections were mounted on glass slides and treated with 0.1% hydrogen peroxide in PBS. Every third section was incubated overnight at 4°C in a solution of affinitypurified 192-IgG (2.5 /lg IgG/ml, supplied by E.M. Johnson, Jr. [1, 18]) or the supernatant of a hybridoma cell culture that produced 192-IgG (supplied by H. Hatanaka). The neighboring sections were incubated with a TH-specific monoclonal antibody (PCTH-7, supplied by
242 H. H a t a n a k a [6]). F o r negative control staining, the other set of neighboring sections was incubated without primary antibody or with a preparation of mouse I g G (2.5 /.tg/ml; Chemicon, Temecula, CA). After being washed, all of the sections were incubated with biotinylated anti-mouse I g G and ABC reagent (Vector Laboratories, Burlingame, CA), and then with a solution of diaminobenzidine that contained hydrogen peroxide. The site of injection (Fig. 1) was verified on the immunostained sections and the rats that received injections in the POA or the hypothalamus were examined. Since there are reports that tissue damage induces and/or increases N G F R immunoreactivity in the forebrain of neonatal [17] and adult rats [2, 4], the rats in which the medial region of the POA and hypothalamus was damaged by the infusion were excluded from the experiment. The neurons with N G F R - l i k e immunoreactivity were seen in the basal forebrain of all of the rats that received N G F and of the control rats, irrespective of age. The neighboring sections stained with 192-IgG in the supernatant of the hybridoma culture and with the purified antibody showed similar N G F R - l i k e immunoreactivity in the forebrain region. The immunoreactivity specific for N G F R was seen in the POA and hypothalamus of rats that had received 1 p g of N G F at 3 days of age (Fig. 2). Four rats out of 11 at 5 days of age, 4 rats out of 12 at 8 days of age (Fig. 2a), and 3 of 8 at 12 days of age (Fig. 2b), that is, 11 rats in all out of a total of 31, had N G F R - I i k e immunoreactive neurons in the POA and hypothalamus (Table I). Since, in the control preparations, no immunostaining of any neurons was observed in the same region, the immunoreactivity of these neurons was specific to the antibody against N G F R . In the eleven rats, the immunoreactivity was localized in the neuronal cell bodies and the proximal part of their processes in the periventricular gray of the POA and anterior hypothalamus (Fig. 2a) and the arcuate nucleus of the hypothalamus (Fig. 2b) ipsi- and contralateral to the
site of injection (Fig. 2a,b). The number of neurons with N G F R - l i k e immunoreactivity varied a m o n g these eleven rats, for example, ten neurons in total could be seen in the region stained with 192-IgG in one rat. By contrast, m a n y immunoreactive neurons were seen in a section taken from another rat, as shown in Fig. 2b. Additionally the ependymal cells of the third ventricle of the eleven rats showed specific N G F R - l i k e immunoreactivity at 5 (Fig. 2e) and 8 days of age. The suprachiasmatic nucleus in some of the eleven rats showed specific N G F R immunoreactivity, but the immunoreactivity was localized in the fibers, in good accord with previously reported findings [14, 15]. In contrast to the above results, no immunoreactivity was encountered in the POA or hypothalamus of the rats in the same group when they were killed at 20 or 45 days of age, rats that received 0.1 p g N G F at 3 days of age, or rats that received 1 p g on day 5 (Table I). N o immunoreactivity was found in the POA or hypothalamus of uninjected control 1- to 45-day-old rats (Table I). The immunoreactivity specific for T H was present in the POA (Fig. 2c) and hypothalamus (Fig. 2d) of all of the rats, as has been fully documented by others [8]. In the eleven rats in which the neurons with N G F R - l i k e immunoreactivity were visualized, the area in which these neurons were localized (Fig. 2a,b) corresponded to a part of the area in which the TH-containing neurons were distributed (Fig. 2c,d). The present results indicate that the immunoreactivity specific to 192-IgG was expressed in the POA and hypothalamus during a neonatal period after the infusion of TABLE I APPEARANCE OF NEURONS WITH NGFR-L1KE IMMUNOREACTIVITY IN THE PREOPTIC AREA (POA) AND HYPOTHALAMUS OF NEONATAL RATS Numbers of rats that had immunoreactive neurons in the POA and hypothalamus among rats injected with mouse 2.5S NGF on day 3 or 5, among rats injected with vehicle on day 3 and among uninjected control rats. Number of rats studied are given in parentheses. Postnatal days Group
1
Normal
0(8) 0(5) 0(7) 0(5) 0(5) 0(7) 0(3)
NGF on day 3 1 pg 0.1pg Fig. 1. Sites of injection in the eleven rats that had neurons with NGFR-like immunoreactivity in the POA and hypothalamus after the infusion of the NGF. Injections are superimposed on three representative coronal sections, ac, anterior commissure; arc, arcuate nucleus; f, fornix; pe, periventricular gray; mpo, medial POA: ox, optic chiasma, 3v, third ventricle.
NGF on day 5 l pg Vehicle on day 3
3
5
8
12
20
45
4(11) 4(12) 3(8) 0(5) 0(3) 0(5) 0(5) 0(4)
0(5) 0(6) 0(5) 0(5)
243
Fig. 2. NGFR-Iike immunoreactivity in the POA and hypothalamus of rats given NGF on day 3 and TH-containing neurons. NGFR-Iike immunoreactive neurons are visible in the POA of an 8-day-old rat (a, inset in a higher magnification) and in the arcuate nucleus of the hypothalamus of a 12-day-old rat (b, inset in an higher magnification), and TH-containing neurons are seen in the neighboring sections (c, d, respectively). NGFR-Iike immunoreactivity in the ependymal cells of a 5-day-old rat (e). *, third ventricle. Bar -- 50 pro.
N G F . T h e 192-IgG a n t i b o d y raised a g a i n s t the r a t N G F R [1] recognizes a high- a n d / o r a low-affinity N G F R [5]. Thus, a t r a n s i e n t increase in levels o f i m m u n o r e a c t i v e N G F R seems to have o c c u r r e d in the i m m u n o r e a c t i v e neurons. I n f u s i o n o f m o u s e N G F into the b r a i n o f a d u l t rats is k n o w n to induce the e x p r e s s i o n o f N G F R [2] a n d the m R N A for N G F R [7] in the choli-
nergic n e u r o n s o f the striatum. T h e i n d u c t i o n o f N G F R b y N G F is likely to be c o m m o n to all n e u r o n s t h a t are responsive to N G F , irrespective o f the n e u r o n a l subsets a n d the age o f the animal. Existence o f N G F R - l i k e i m m u n o r e a c t i v i t y in the neurons a n d the e p e n d y m a l cells o f the P O A a n d h y p o t h a l a m u s has been r e p o r t e d in a d u l t rats [14, 18]. L o c a l i z a -
244
tion of the neurons with NGFR-like immunoreactivity in the NGF-infused neonatal rats is in good accord with that in adults [14, 18]. The absence of immunoreactivity in normal neonatal rats consequently indicates that the infusion of NGF on day 3 induced the NGFR immunoreactivity in the neurons that would subsequently produce NGFR molecules. Our previous study [12] in which fetal hypothalamic tissues were transplanted into the adult cerebral ventricle suggested that the TH-containing neurons in the hypothalamus responded to NGF. Since, in the POA and hypothalamus of the neonatal rats, the localization of the NGFR-Iike immunoreactivity coincided with the distribution of TH-containing neurons, the neurons that expressed NGFR-like immunoreactivity were, presumably, a subpopulation of the TH-containing neurons. The alternative possibility, namely, that the NGFR-like immunoreactive neurons and the TH-containing neurons are members of different neuronal subsets but are interconnected cannot be excluded. We thank Drs. H. Hatanaka, E.M. Johnson, Jr. who kindly provided 192-IgG and PCTH-7 and Dr. E.M. Shooter for his kind suggestions.
1 Chandler, C.E., Parsons, L.M., Hosang, M. and Shooter, E.M., A monoclonal antibody modulates the interaction of nerve growth factor with PC12 cells, J. Biol. Chem., 259 (1984) 6882 6889. 2 Gage, F.H., Batchelor, P., Chen, K.S., Chin, D., Higgins, G.A., Koh, S., Deputy, S., Rosenberg, M.B., Fischer, W. and Bj6rklund, A., NGF receptor reexpression and NGF-mediated cholinergic neuronal hypertrophy in the damaged adult neostriatum, Neuron, 2(1989) 1177 1184. 3 Gnahn, H., Hefti, F., Heumann, R., Schwab, M.E. and Thoenen, H., NGF-mediated increase of choline acetyltransferase (CHAT) in the neonatal rat forebrain: evidence for a physiological role of NGF in the brain?, Dev. Brain Res., 9 (1983) 45-52. 4 Gomez-Pinilla, F., Cotman, C.W. and Nieto-Sampedro, M., NGF receptor immunoreactivity in the rat brain: topographic distribution and response to entorhinal ablation, Neurosci. Lett., 82 (1987) 260-266. 5 Green, S.H. and Green, L.A., A single M r ~ 103,000 12~l-fl-nerve growth factor-affinity-labeled species represents both the low and high affinity forms of the nerve growth factor receptor, J. Biol. Chem., 261 (1986) 15316 15326.
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