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p75 Neurotrophin receptor immunoreactivity in the aged human hypothalamus
Neuroscience Letters 231 (1997) 9–12
p75 Neurotrophin receptor immunoreactivity in the aged human hypothalamus Margaret M. Moga*, Taihung Duong Department of Anatomy, Indiana University School of Medicine, Terre Haute, IN 47809, USA Received 11 February 1997; received in revised form 27 June 1997; accepted 3 July 1997
Abstract Low-affinity p75 neurotrophin receptor (p75NTR) immunoreactivity in the aged human hypothalamus was examined in autopsied material. Numerous p75NTR-immunoreactive cells were found in the paraventricular and supraoptic hypothalamic nuclei. The suprachiasmatic nucleus was devoid Of p75NTR-immunostaining. Many p75NTR-immunoreactive fibers extended laterally and ventrally from the paraventricular and supraoptic nuclei into the pituitary stalk and median eminence. Our results suggest that neurotrophins may be present within the human hypothalamo-hypophyseal system. 1997 Elsevier Science Ireland Ltd. Keywords: Paraventricular hypothalamic nucleus; Supraoptic nucleus; Suprachiasmatic nucleus; Nerve growth factor
The low-affinity nerve growth factor receptor (p75) is now termed the p75 neurotrophin receptor (p75NTR) based on its affinity and binding to all known neurotrophins, not just to nerve growth factor (NGF) [5,7]. Current evidence indicates several potential roles for p75NTR, including the retrograde transport of neurotrophins, the modulation of Trk receptor activity and specificity, and the regulation of programmed cell death [6–8]. In primates and rodents, p75NTR-immunoreactivity is prominent in the cholinergic basal forebrain, cerebellum and many brainstem nuclei [14,26,29,30,34,39]. In the rat, an additional locus of p75NTR-immunoreactivity has been identified in the hypothalamus, specifically in the suprachiasmatic, paraventricular and supraoptic nuclei [29,35,37,39]. The p75NTR-immunoreactivity in the suprachiasmatic nucleus (SCN) is one of the most heavily stained areas in the rat forebrain[29,35,37,39]. This finding has led to a number of recent studies examining the role of neurotrophins in the SCN and in the regulation of circadian timing, the major function of the SCN [3,21,22]. In the present study, we describe the distribution of p75NTR-immunoreactivity in the aged human hypothalamus.
* Corresponding author. 135 Holmstedt Hall, ISU, Terre Haute, IN 47809-9989, USA. Tel.: +1 812 2373420; fax: +1 812 2377646; e-mail: [email protected]
This study (#EX9606-04) was approved by the Indiana University-Purdue University at Indianapolis Institutional Review Board, USA. Brains from three female patients (mean age, 84 years; range, 81–87) with no clinical signs of neurologic or psychiatric illness were obtained at autopsy, 2-3 h postmortem. The brains were hemisected; the right half was sliced transversely into 2 cm slabs; and the slabs were immersion-fixed in phosphate buffered 4% paraformaldehyde. Blocks containing the hypothalamus were cryoprotected in 30% sucrose, and then cut into 30 mm sections on a freezing microtome. To assess the cytoarchitectural integrity of the regions under study, one series of sections (1-of-6) was Nissl-stained with cresyl violet. To detect the potential presence of neurofibrillary tangles and/or amyloid deposits, one series of sections was immunolabeled with a monoclonal antibody to paired helical filaments (PHF1) [13], and another series, with a monoclonal antibody to amyloid b protein (4G8) [18], according to a previously described peroxidase-antiperoxidase method [10]. The remaining three series of sections were processed as follows: one series with a monoclonal antibody to human p75NTR (clone 8211; Boehringer Mannheim) [14,31], the second series with a polyclonal antibody to vasopressin (VP; Incstar), and the third series with a polyclonal antibody to vasoactive intestinal polypeptide (VIP; Incstar). VP-/ VIP-immunohistochemistry was used as an aid in determining the nuclear boundaries of the human SCN [15]. The
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specificity of the p75NTR antibody was tested by (1) incubation of representative sections in 1 ml of the diluted antibody preincubated with 50 mg of p75NTR peptide, (2) omission of the primary antibody, and (3) substitution of the primary antibody with non-specific IgG (i.e. normal mouse serum; Jackson ImmunoResearch). No specific immunohistochemical staining was observed in any of these controls. However, the possibility that the p75NTR antibody may react with structurally related proteins in the human hypothalamus cannot be excluded. Therefore, the term p75NTR-immunoreactivity in the present investigation refers to p75NTR ‘like’ immunoreactivity. The immunohistochemical procedure for p75NTR, VP and VIP is as follows. Sections are pretreated with 1% sodium borohydride for 5–10 min, rinsed in 0.01 M phosphate-buffered saline (PBS; pH 7.4) treated with 3% hydrogen peroxide–10% methanol in PBS for 5 min, and preincubated for 1 h in a PBS solution containing 2% normal donkey serum (NDS), 0.3% Triton X-100 (TX), and 2% bovine serum albumin (BSA). The sections are then incubated in
the primary antibody (anti-p75NTR, diluted 1:50 in PBSNDS-TX-BSA; anti-VP and anti-VIP, diluted 1:20 000 in PBS-NDS-TX) with gentle agitation for 40–48 h at 4°C. Next, the sections are rinsed in PBS, incubated in the secondary antibody (biotin-SP-conjugated donkey anti-mouse IgG or biotin-SP-conjugated donkey anti-rabbit IgG; Jackson ImmunoResearch; diluted 1:200 in PBS-NDS-TX) for 1–2 h, rinsed again, and reacted with ABC reagent (Elite ABC kit, Vector) for 1–2 h. After several rinses in PBS, the sections are reacted in a solution of 0.05% diaminobenzidine (DAB)–0.025% nickel chloride–0.01% H2O2 in 0.1 M Tris buffer (pH 7.4) for 10 min. The sections are rinsed in PBS, mounted on gel-coated slides, dehydrated through alcohol and xylene, and then coverslipped with Permount (Fisher). In each of the three cases, occasional PHF1-immunoreactive (-ir) neurofibrillary tangles as well as sparse, diffuse 4G8-ir amyloid deposits are found within the hypothalamus; these lesions are concentrated within the lateral hypothalamic and preoptic areas. The supraoptic, paraventricular and
Fig. 1. Photomicrographs of coronal sections through the human hypothalamus illustrate the distribution of p75NTR-ir cells and fibers. p75NTR-ir neurons are present in (A) the paraventricular nucleus (PVH), and (B) the supraoptic nucleus (SON). In (A), a few p75NTR-ir fibers coursing from the PVH are indicated by arrowheads. (C) A dense bundle of p75NTR-ir fibers is found in the infundibulum (two fibers indicated by arrowheads). (D) The median eminence (ME) shows intense immunostaining (p75NTR-ir neurons in the adjacent infundibulum are indicated by arrowheads). fx, fornix; opt, optic tract; 3V, third ventricle. Bar, 200 mm.
M.M. Moga, T. Duong / Neuroscience Letters 231 (1997) 9–12
suprachiasmatic nuclei show no immunostaining for either of these lesion types. The human p75NTR monoclonal antibody labels numerous neurons in the supraoptic, paraventricular, and accessory magnocellular hypothalamic nuclei (Fig. 1A,B). Many of the p75NTR-ir neurons are similar in morphology (i.e. large soma, multipolar) and distribution to VP-ir neurons observed in adjacent sections of the hypothalamus, potentially identifying these cells as magnocellular neurons. Some p75NTR-ir cells in the paraventricular nucleus are relatively small and may represent parvocellular neurons. Many p75NTR-ir fibers course ventro-laterally from the paraventricular nucleus to join similar fibers from the supraoptic nucleus; together, these fibers form a dense bundle within the infundibular stalk (Fig. 1C). Scattered p75NTR-ir cells, possibly supraoptic neurons, are present in the infundibular stalk (Fig. 1D). The median eminence shows a high concentration of p75NTR-immunoreactivity, consisting of both cells and fibers (Fig. 1D). The suprachiasmatic nucleus and the remainder of the hypothalamus are devoid of p75NTR-immunostaining. Outside the hypothalamus, p75NTR-ir neurons are numerous in the basal forebrain. Our results show that p75NTR-ir neurons are present in the paraventricular, supraoptic and accessory magnocellular nuclei of the aged human hypothalamus, and that p75NTRir fibers extend from these nuclei into the infundibular stalk and median eminence. Talamini and Aloe [37] observed an identical pattern of labeling in the rat hypothalamus. Furthermore, they noted p75NTR-ir neurons in both magnocellular and parvocellular divisions of the paraventricular nucleus, and p75NTR-ir fibers in both the internal and the external layers of the median eminence, indicating that the paraventricular nucleus-derived p75NTR-ir fibers terminate in both the neurohypophysis and the median eminence. Recently, Borson et al. [4] detected p75NTR-ir fibers in the neurohypophysis of a non-human primate; these fibers were in close proximity to neurotrophin-ir cells in the adjacent pars tuberalis. Based on these findings, an examination of p75NTR-immunoreactivity in the human neurohypophysis appears warranted. A role for neurotrophins in the hypothalamic–pituitary axis is supported by a number of other studies. Supraoptic and paraventricular hypothalamic neurons express mRNAs for a variety of neurotrophins and neurotrophin receptors, including mRNA for the low-affinity neurotrophin receptor, p75NTR [25], the high-affinity neurotrophin receptors, trkB and trkC [1,24,25], and the neurotrophins, NGF and brainderived neurotrophic factor (BDNF) [25]. TrkB- and trkC-ir neurons have been observed in the supraoptic nucleus [32], and BDNF-ir neurons, in the paraventricular nucleus [40]. Neurotrophins (i.e. NGF and BDNF) and their receptors (p75NTR, trkA, trkB, and trkC) have been localized in the pituitary [4,19,28]. The hypothalamic–pituitary axis is activated by NGF; this effect is mediated by VP neurons in the hypothalamus [27,33,36]. In a recent study of the fetal human brain, Chen et al. [9]
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found no p75NTR-immunoreactivity in the supraoptic or paraventricular hypothalamic nuclei. This negative result may be due to a number of factors: (1) the different monoclonal antibodies (8211 versus NGFR-5) used in the two studies, (2) the difference in the monoclonal antibody dilutions, 1:80 000 in Chen et al. versus 1:50 in the present study, or (3) a possible difference in p75NTR protein production, storage or release between the fetal and aged hypothalamus. Regarding the first possibility, one study has shown that different p75NTR antibodies recognize different epitopes [38]. Based on the presence of multiple isoforms of trkB and trkC in the brain [2], we speculate that the 8211 antibody may recognize a truncated form of p75NTR in the human hypothalamus. Alternatively, there is some support for the third possibility, namely a difference in hypothalamic function with age. Magnocellular neurons in the supraoptic and paraventricular nuclei show evidence of increased activation in the elderly human, as indicated by an increase in the size of the Golgi apparatus [23], the cell soma [11], and the nucleolus [17]. Blood levels of neurophysins, which are produced by magnocellular hypothalamic neurons, increase gradually with age [12]. Thus, p75NTR may be present in the fetal hypothalamus at levels not detectable by immunohistochemistry. We observed no p75NTR-immunoreactivity within the SCN. This lack of staining is most likely due to a species difference as hamsters [21] and two different non-human primates [20,34] show a similar lack of p75NTR-immunoreactivity in the SCN. In support of this finding, Lehman et al. [21] recently detected p75NTR and trkC mRNA in the rat SCN, but only TrkA and TrkC mRNA in the hamster SCN. A number of neuronal cell populations express Trk neurotrophin receptors but not p75NTR [6,8,16], highlighting the fact that the low-affinity p75 neurotrophin receptor is not essential for Trk receptor function or for neurotrophin responsiveness [6]. Thus, neurons in the human SCN may express high-affinity Trk neurotrophin receptors in the absence of p75NTR. Future experiments will examine this possibility. This study was supported by funds from the Indiana University School of Medicine to M.M.M. and by a NIH grant (NS 31524) to T.D. The authors gratefully acknowledge the technical assistance of Eric Chaney and Paul Acton, and the generous gift of PHF1 antibody from Dr. Peter Davies. [1] Altar, C.A., Siuciak, J.A., Wright, P., Ip, N.Y., Lindsay, R.M. and Wiegand, S.J., In situ hybridization of trkB and trkC receptor mRNA in rat forebrain and association with high-affinity binding of [125I]BDNF, [125I]NT-4/5 and [125I]NT-3, Eur. J. Neurosci., 6 (1994) 1389–1405. [2] Barbacid, M., Structural and functional properties of the TRK family of neurotrophin receptors, Ann. N. Y. Acad. Sci., 766 (1995) 442– 458. [3] Bina, K.G. and Rusak, B., Nerve growth factor phase shifts circadian activity rhythms in Syrian hamsters, Neurosci. Lett., 206 (1996) 97– 100.
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[4] Borson, S., Schatteman, G., Claude, P. and Bothwell, M., Neurotrophins in the developing and adult primate adenohypophysis: a new pituitary hormone system?, Neuroendocrinology, 59 (1994) 466– 476. [5] Bothwell, M., Keeping track of neurotrophin receptors, Cell, 65 (1991) 915–918. [6] Bothwell, M., Functional interactions of neurotrophins and neurotrophin receptors, Annu. Rev. Neurosci., 18 (1995) 223–253. [7] Chao, M.V., The p75 neurotrophin receptor, J. Neurobiol., 25 (1994) 1373–1385. [8] Chao, M.V. and Hempstead, B.L., p75 and Trk: a two-receptor system, Trends Neurosci., 18 (1995) 321–326. [9] Chen, E.-Y., Mufson, E.J. and Kordower, J.H., TRK and p75 neurotrophin receptor systems in the developing human brain, J. Comp. Neurol., 369 (1996) 591–618. [10] Duong, T., Duocette, T., Zidenberg, N.A., Jacobs, R.W. and Scheibel, A.S., Microtubule-associated proteins tau and amyloid P component in Alzheimer’s disease, Brain Res., 603 (1993) 74– 86. [11] Fliers, E., Swaab, D.F., Pool, C.W. and Verwer, R.W.H., The vasopressin and oxytocin neurons in the human supraoptic and paraventricular nucleus; changes with aging and in senile dementia, Brain Res., 342 (1985) 45–53. [12] Frolkis, V., Golovchenko, S.F., Medved, V. and Frolkis, R.A., Vasopressin and cardiovascular system in aging, Gerontology, 28 (1982) 290–302. [13] Greenberg, S.G. and Schein, J., The isolation of a relatively specific paired helical filament antibody, Soc. Neurosci. Abstr., 15 (1989) 1038. [14] Hefti, F., Hartikka, J., Salvatierra, A., Weiner, W.J. and Mash, D.C., Localization of nerve growth factor receptors in cholinergic neurons of the human basal forebrain, Neurosci. Lett., 69 (1986) 37–41. [15] Hofman, M.A., Zhou, J.-N. and Swaab, D.F., Suprachiasmatic nucleus of the human brain: an immunocytochemical and morphometric analysis, Anat. Rec., 244 (1996) 552–562. [16] Holtzman, D.M., Kilbridge, J., Li, Y., Cunningham, E.T., Lenn, N.J., Clary, D.O., Reichardt, L.F. and Mobley, W.C., TrkA expression in the CNS: evidence for the existence of several novel NGF-responsive CNS neurons, J. Neurosci., 15 (1995) 1567–1576. [17] Hoogendijk, J.E., Fliers, E., Swaab, D.F. and Verwer, R.W.H., Activation of vasopressin neurons in the human supraoptic and paraventricular nucleus in senescence and senile dementia, J. Neurol. Sci., 69 (1985) 291–299. [18] Kim, K.S., Miller, D.L., Sapienza, V.J., Chen, C.M.J., Baii, C., Grundke-Iqbal, I., Currie, J.R. and Wisniewski, H.M., Production and characterization of monoclonal antibodies reactive to synthetic cerebrovascular amyloid peptide, Neurosci. Res. Commun., 2 (1988) 121–130. [19] Kononen, J., Soinila, S., Persson, H., Honkaniemi, J., Ho¨kfelt, T. and Pelto-Huikko, M., Neurotrophins and their receptors in the rat pituitary gland: regulation of BDNF and trkB mRNA levels by adrenal hormones, Mol. Brain Res., 27 (1994) 347–354. [20] Kordower, J.H., Bartus, R.T., Bothwell, M., Schatteman, G. and Gash, D.M., Nerve growth factor receptor immunoreactivity in the nonhuman primate (Cebus apella): distribution, morphology, and colocalization with cholinergic enzymes, J. Comp. Neurol., 277 (1988) 465–486. [21] Lehman, M.N., Jansen, H.T., Wortman, M., Stevens, P., Kim, C., Norgren, R.B. and Zeitler, P., Neurotrophin and neurotrophin receptor expression in the suprachiasmatic nucleus (SCN) of rats and hamsters, Soc. Neurosci. Abstr., 22 (1996) 1141. [22] Liang, F.-O., Miranda, R., Sohrabji, F., Brooks, C. and Earnest, D., Brain-derived neurotrophic factor and trkB receptors in the rat SCN: distribution and functional implications, Soc. Neurosci. Abstr., 22 (1996) 1141. [23] Lucassen, P.J., Ravid, R., Gonatas, N.K. and Swaab, D.F., Activation of the human supraoptic and paraventricular nucleus neurons with
[24]
[25]
[26]
[27]
[28] [29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
aging and in Alzheimer’s disease as judged from increasing size of the Golgi apparatus, Brain Res., 632 (1993) 105–113. Merlio, J.-P., Ernfors, P., Jaber, M. and Persson, H., Molecular cloning of rat trkC and distribution of cells expressing messenger RNAs for members of the trk family in the rat central nervous system, Neuroscience, 51 (1992) 513–532. Miranda, R.C., Sohrabji, F. and Toran-Allerand, C.D., Neuronal colocalization of mRNAs for neurotrophins and their receptors in the developing central nervous system suggests a potential for autocrine interactions, Proc. Natl. Acad. Sci. USA, 90 (1993) 6439– 6443. Mufson, E.J., Brashers-Krug, T. and Kordower, J.H., p75 nerve growth factor receptor immunoreactivity in the human brainstem and spinal cord, Brain Res., 589 (1992) 115–123. Otten, U., Baumann, J.B. and Girard, J., Stimulation of the pituitaryadrenocortical axis by nerve growth factor, Nature, 282 (1979) 413– 414. Patterson, J.C. and Childs, G.V., Nerve growth factor and its receptor in the anterior pituitary, Endocrinology, 135 (1994) 1689–1696. 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 – I. Forebrain, Neuroscience, 34 (1990) 57–87. 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 – II. Brainstem, cerebellum and spinal cord, Neuroscience, 34 (1990) 89–110. Ross, A., Grob, P., Bothwell, M., Elder, D.E., Ernst, C.S., Marano, N., Ghrist, B.F.D., Slemp, C.C., Herlyn, M., Atkinson, B. and Koprowski, H., Characterization of nerve growth factor receptor in neural crest tumors using monoclonal antibodies, Proc. Natl. Acad. Sci. USA, 81 (1984) 6681–6685. Salehi, A., Verhaagen, J., Dijkhuizen, P.A. and Swaab, D.F., Colocalization of high-affinity neurotrophin receptors in nucleus basalis of Meynert neurons and their differential reduction in Alzheimer’s disease, Neuroscience, 75 (1996) 373–387. Scaccianoce, S., Cigliana, G., Nicolai, R., Muscolo, L.A., Porcu, A., Navarra, D., Perez-Polo, J.R. and Angelucci, L., Hypothalamic involvement in the activation of the pituitary-adrenocortical axis by nerve growth factor, Neuroendocrinology, 58 (1993) 202–209. Schatteman, G.C., Gibbs, L., Lanahan, A.A., Claude, P. and Bothwell, M., Expression of NGF receptor in the developing and adult primate central nervous system, J. Neurosci., 8 (1988) 860– 873. 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. Taglialatela, G., Angelucci, L., Scaccianoce, S., Foreman, P.J. and Perez-Polo, J.R., Nerve growth factor modulates the activation of the hypothalamo-pituitary-adrenocortical axis during the stress response, Endocrinology, 129 (1991) 2212–2218. Talamini, L.M. and Aloe, L., Immunohistochemical localization of nerve growth factor (NGF) and NGF-receptor in the hypothalamus of adult rats, Arch. Ital. Biol., 131 (1993) 255–266. Vissavajjhala, P., Leszyk, J.D., Lin-Goerke, J. and Ross, A.H., Structural domains of the extracellular domain of human nerve growth factor receptor detected by partial proteolysis, Arch. Biochem. Biophys., 294 (1992) 244–252. Yan, Q. and Johnson, E.M. Jr., Immunohistochemical localization and biochemical characterization of nerve growth factor receptor in adult rat brain, J. Comp. Neurol., 290 (1989) 585–598. Yan, Q., Rosenfeld, R.D., Matheson, C.R., Hawkins, N., Lopez, O.T., Bennett, L. and Welcher, A.A., Expression of brain-derived neurotrophic factor protein in the adult rat central nervous system, Neuroscience, 78 (1997) 431–448.
p75 Neurotrophin receptor immunoreactivity in the aged human hypothalamus Margaret M. Moga*, Taihung Duong Department of Anatomy, Indiana University School of Medicine, Terre Haute, IN 47809, USA Received 11 February 1997; received in revised form 27 June 1997; accepted 3 July 1997
Abstract Low-affinity p75 neurotrophin receptor (p75NTR) immunoreactivity in the aged human hypothalamus was examined in autopsied material. Numerous p75NTR-immunoreactive cells were found in the paraventricular and supraoptic hypothalamic nuclei. The suprachiasmatic nucleus was devoid Of p75NTR-immunostaining. Many p75NTR-immunoreactive fibers extended laterally and ventrally from the paraventricular and supraoptic nuclei into the pituitary stalk and median eminence. Our results suggest that neurotrophins may be present within the human hypothalamo-hypophyseal system. 1997 Elsevier Science Ireland Ltd. Keywords: Paraventricular hypothalamic nucleus; Supraoptic nucleus; Suprachiasmatic nucleus; Nerve growth factor
The low-affinity nerve growth factor receptor (p75) is now termed the p75 neurotrophin receptor (p75NTR) based on its affinity and binding to all known neurotrophins, not just to nerve growth factor (NGF) [5,7]. Current evidence indicates several potential roles for p75NTR, including the retrograde transport of neurotrophins, the modulation of Trk receptor activity and specificity, and the regulation of programmed cell death [6–8]. In primates and rodents, p75NTR-immunoreactivity is prominent in the cholinergic basal forebrain, cerebellum and many brainstem nuclei [14,26,29,30,34,39]. In the rat, an additional locus of p75NTR-immunoreactivity has been identified in the hypothalamus, specifically in the suprachiasmatic, paraventricular and supraoptic nuclei [29,35,37,39]. The p75NTR-immunoreactivity in the suprachiasmatic nucleus (SCN) is one of the most heavily stained areas in the rat forebrain[29,35,37,39]. This finding has led to a number of recent studies examining the role of neurotrophins in the SCN and in the regulation of circadian timing, the major function of the SCN [3,21,22]. In the present study, we describe the distribution of p75NTR-immunoreactivity in the aged human hypothalamus.
* Corresponding author. 135 Holmstedt Hall, ISU, Terre Haute, IN 47809-9989, USA. Tel.: +1 812 2373420; fax: +1 812 2377646; e-mail: [email protected]
This study (#EX9606-04) was approved by the Indiana University-Purdue University at Indianapolis Institutional Review Board, USA. Brains from three female patients (mean age, 84 years; range, 81–87) with no clinical signs of neurologic or psychiatric illness were obtained at autopsy, 2-3 h postmortem. The brains were hemisected; the right half was sliced transversely into 2 cm slabs; and the slabs were immersion-fixed in phosphate buffered 4% paraformaldehyde. Blocks containing the hypothalamus were cryoprotected in 30% sucrose, and then cut into 30 mm sections on a freezing microtome. To assess the cytoarchitectural integrity of the regions under study, one series of sections (1-of-6) was Nissl-stained with cresyl violet. To detect the potential presence of neurofibrillary tangles and/or amyloid deposits, one series of sections was immunolabeled with a monoclonal antibody to paired helical filaments (PHF1) [13], and another series, with a monoclonal antibody to amyloid b protein (4G8) [18], according to a previously described peroxidase-antiperoxidase method [10]. The remaining three series of sections were processed as follows: one series with a monoclonal antibody to human p75NTR (clone 8211; Boehringer Mannheim) [14,31], the second series with a polyclonal antibody to vasopressin (VP; Incstar), and the third series with a polyclonal antibody to vasoactive intestinal polypeptide (VIP; Incstar). VP-/ VIP-immunohistochemistry was used as an aid in determining the nuclear boundaries of the human SCN [15]. The
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specificity of the p75NTR antibody was tested by (1) incubation of representative sections in 1 ml of the diluted antibody preincubated with 50 mg of p75NTR peptide, (2) omission of the primary antibody, and (3) substitution of the primary antibody with non-specific IgG (i.e. normal mouse serum; Jackson ImmunoResearch). No specific immunohistochemical staining was observed in any of these controls. However, the possibility that the p75NTR antibody may react with structurally related proteins in the human hypothalamus cannot be excluded. Therefore, the term p75NTR-immunoreactivity in the present investigation refers to p75NTR ‘like’ immunoreactivity. The immunohistochemical procedure for p75NTR, VP and VIP is as follows. Sections are pretreated with 1% sodium borohydride for 5–10 min, rinsed in 0.01 M phosphate-buffered saline (PBS; pH 7.4) treated with 3% hydrogen peroxide–10% methanol in PBS for 5 min, and preincubated for 1 h in a PBS solution containing 2% normal donkey serum (NDS), 0.3% Triton X-100 (TX), and 2% bovine serum albumin (BSA). The sections are then incubated in
the primary antibody (anti-p75NTR, diluted 1:50 in PBSNDS-TX-BSA; anti-VP and anti-VIP, diluted 1:20 000 in PBS-NDS-TX) with gentle agitation for 40–48 h at 4°C. Next, the sections are rinsed in PBS, incubated in the secondary antibody (biotin-SP-conjugated donkey anti-mouse IgG or biotin-SP-conjugated donkey anti-rabbit IgG; Jackson ImmunoResearch; diluted 1:200 in PBS-NDS-TX) for 1–2 h, rinsed again, and reacted with ABC reagent (Elite ABC kit, Vector) for 1–2 h. After several rinses in PBS, the sections are reacted in a solution of 0.05% diaminobenzidine (DAB)–0.025% nickel chloride–0.01% H2O2 in 0.1 M Tris buffer (pH 7.4) for 10 min. The sections are rinsed in PBS, mounted on gel-coated slides, dehydrated through alcohol and xylene, and then coverslipped with Permount (Fisher). In each of the three cases, occasional PHF1-immunoreactive (-ir) neurofibrillary tangles as well as sparse, diffuse 4G8-ir amyloid deposits are found within the hypothalamus; these lesions are concentrated within the lateral hypothalamic and preoptic areas. The supraoptic, paraventricular and
Fig. 1. Photomicrographs of coronal sections through the human hypothalamus illustrate the distribution of p75NTR-ir cells and fibers. p75NTR-ir neurons are present in (A) the paraventricular nucleus (PVH), and (B) the supraoptic nucleus (SON). In (A), a few p75NTR-ir fibers coursing from the PVH are indicated by arrowheads. (C) A dense bundle of p75NTR-ir fibers is found in the infundibulum (two fibers indicated by arrowheads). (D) The median eminence (ME) shows intense immunostaining (p75NTR-ir neurons in the adjacent infundibulum are indicated by arrowheads). fx, fornix; opt, optic tract; 3V, third ventricle. Bar, 200 mm.
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suprachiasmatic nuclei show no immunostaining for either of these lesion types. The human p75NTR monoclonal antibody labels numerous neurons in the supraoptic, paraventricular, and accessory magnocellular hypothalamic nuclei (Fig. 1A,B). Many of the p75NTR-ir neurons are similar in morphology (i.e. large soma, multipolar) and distribution to VP-ir neurons observed in adjacent sections of the hypothalamus, potentially identifying these cells as magnocellular neurons. Some p75NTR-ir cells in the paraventricular nucleus are relatively small and may represent parvocellular neurons. Many p75NTR-ir fibers course ventro-laterally from the paraventricular nucleus to join similar fibers from the supraoptic nucleus; together, these fibers form a dense bundle within the infundibular stalk (Fig. 1C). Scattered p75NTR-ir cells, possibly supraoptic neurons, are present in the infundibular stalk (Fig. 1D). The median eminence shows a high concentration of p75NTR-immunoreactivity, consisting of both cells and fibers (Fig. 1D). The suprachiasmatic nucleus and the remainder of the hypothalamus are devoid of p75NTR-immunostaining. Outside the hypothalamus, p75NTR-ir neurons are numerous in the basal forebrain. Our results show that p75NTR-ir neurons are present in the paraventricular, supraoptic and accessory magnocellular nuclei of the aged human hypothalamus, and that p75NTRir fibers extend from these nuclei into the infundibular stalk and median eminence. Talamini and Aloe [37] observed an identical pattern of labeling in the rat hypothalamus. Furthermore, they noted p75NTR-ir neurons in both magnocellular and parvocellular divisions of the paraventricular nucleus, and p75NTR-ir fibers in both the internal and the external layers of the median eminence, indicating that the paraventricular nucleus-derived p75NTR-ir fibers terminate in both the neurohypophysis and the median eminence. Recently, Borson et al. [4] detected p75NTR-ir fibers in the neurohypophysis of a non-human primate; these fibers were in close proximity to neurotrophin-ir cells in the adjacent pars tuberalis. Based on these findings, an examination of p75NTR-immunoreactivity in the human neurohypophysis appears warranted. A role for neurotrophins in the hypothalamic–pituitary axis is supported by a number of other studies. Supraoptic and paraventricular hypothalamic neurons express mRNAs for a variety of neurotrophins and neurotrophin receptors, including mRNA for the low-affinity neurotrophin receptor, p75NTR [25], the high-affinity neurotrophin receptors, trkB and trkC [1,24,25], and the neurotrophins, NGF and brainderived neurotrophic factor (BDNF) [25]. TrkB- and trkC-ir neurons have been observed in the supraoptic nucleus [32], and BDNF-ir neurons, in the paraventricular nucleus [40]. Neurotrophins (i.e. NGF and BDNF) and their receptors (p75NTR, trkA, trkB, and trkC) have been localized in the pituitary [4,19,28]. The hypothalamic–pituitary axis is activated by NGF; this effect is mediated by VP neurons in the hypothalamus [27,33,36]. In a recent study of the fetal human brain, Chen et al. [9]
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found no p75NTR-immunoreactivity in the supraoptic or paraventricular hypothalamic nuclei. This negative result may be due to a number of factors: (1) the different monoclonal antibodies (8211 versus NGFR-5) used in the two studies, (2) the difference in the monoclonal antibody dilutions, 1:80 000 in Chen et al. versus 1:50 in the present study, or (3) a possible difference in p75NTR protein production, storage or release between the fetal and aged hypothalamus. Regarding the first possibility, one study has shown that different p75NTR antibodies recognize different epitopes [38]. Based on the presence of multiple isoforms of trkB and trkC in the brain [2], we speculate that the 8211 antibody may recognize a truncated form of p75NTR in the human hypothalamus. Alternatively, there is some support for the third possibility, namely a difference in hypothalamic function with age. Magnocellular neurons in the supraoptic and paraventricular nuclei show evidence of increased activation in the elderly human, as indicated by an increase in the size of the Golgi apparatus [23], the cell soma [11], and the nucleolus [17]. Blood levels of neurophysins, which are produced by magnocellular hypothalamic neurons, increase gradually with age [12]. Thus, p75NTR may be present in the fetal hypothalamus at levels not detectable by immunohistochemistry. We observed no p75NTR-immunoreactivity within the SCN. This lack of staining is most likely due to a species difference as hamsters [21] and two different non-human primates [20,34] show a similar lack of p75NTR-immunoreactivity in the SCN. In support of this finding, Lehman et al. [21] recently detected p75NTR and trkC mRNA in the rat SCN, but only TrkA and TrkC mRNA in the hamster SCN. A number of neuronal cell populations express Trk neurotrophin receptors but not p75NTR [6,8,16], highlighting the fact that the low-affinity p75 neurotrophin receptor is not essential for Trk receptor function or for neurotrophin responsiveness [6]. Thus, neurons in the human SCN may express high-affinity Trk neurotrophin receptors in the absence of p75NTR. Future experiments will examine this possibility. This study was supported by funds from the Indiana University School of Medicine to M.M.M. and by a NIH grant (NS 31524) to T.D. The authors gratefully acknowledge the technical assistance of Eric Chaney and Paul Acton, and the generous gift of PHF1 antibody from Dr. Peter Davies. [1] Altar, C.A., Siuciak, J.A., Wright, P., Ip, N.Y., Lindsay, R.M. and Wiegand, S.J., In situ hybridization of trkB and trkC receptor mRNA in rat forebrain and association with high-affinity binding of [125I]BDNF, [125I]NT-4/5 and [125I]NT-3, Eur. J. Neurosci., 6 (1994) 1389–1405. [2] Barbacid, M., Structural and functional properties of the TRK family of neurotrophin receptors, Ann. N. Y. Acad. Sci., 766 (1995) 442– 458. [3] Bina, K.G. and Rusak, B., Nerve growth factor phase shifts circadian activity rhythms in Syrian hamsters, Neurosci. Lett., 206 (1996) 97– 100.
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[4] Borson, S., Schatteman, G., Claude, P. and Bothwell, M., Neurotrophins in the developing and adult primate adenohypophysis: a new pituitary hormone system?, Neuroendocrinology, 59 (1994) 466– 476. [5] Bothwell, M., Keeping track of neurotrophin receptors, Cell, 65 (1991) 915–918. [6] Bothwell, M., Functional interactions of neurotrophins and neurotrophin receptors, Annu. Rev. Neurosci., 18 (1995) 223–253. [7] Chao, M.V., The p75 neurotrophin receptor, J. Neurobiol., 25 (1994) 1373–1385. [8] Chao, M.V. and Hempstead, B.L., p75 and Trk: a two-receptor system, Trends Neurosci., 18 (1995) 321–326. [9] Chen, E.-Y., Mufson, E.J. and Kordower, J.H., TRK and p75 neurotrophin receptor systems in the developing human brain, J. Comp. Neurol., 369 (1996) 591–618. [10] Duong, T., Duocette, T., Zidenberg, N.A., Jacobs, R.W. and Scheibel, A.S., Microtubule-associated proteins tau and amyloid P component in Alzheimer’s disease, Brain Res., 603 (1993) 74– 86. [11] Fliers, E., Swaab, D.F., Pool, C.W. and Verwer, R.W.H., The vasopressin and oxytocin neurons in the human supraoptic and paraventricular nucleus; changes with aging and in senile dementia, Brain Res., 342 (1985) 45–53. [12] Frolkis, V., Golovchenko, S.F., Medved, V. and Frolkis, R.A., Vasopressin and cardiovascular system in aging, Gerontology, 28 (1982) 290–302. [13] Greenberg, S.G. and Schein, J., The isolation of a relatively specific paired helical filament antibody, Soc. Neurosci. Abstr., 15 (1989) 1038. [14] Hefti, F., Hartikka, J., Salvatierra, A., Weiner, W.J. and Mash, D.C., Localization of nerve growth factor receptors in cholinergic neurons of the human basal forebrain, Neurosci. Lett., 69 (1986) 37–41. [15] Hofman, M.A., Zhou, J.-N. and Swaab, D.F., Suprachiasmatic nucleus of the human brain: an immunocytochemical and morphometric analysis, Anat. Rec., 244 (1996) 552–562. [16] Holtzman, D.M., Kilbridge, J., Li, Y., Cunningham, E.T., Lenn, N.J., Clary, D.O., Reichardt, L.F. and Mobley, W.C., TrkA expression in the CNS: evidence for the existence of several novel NGF-responsive CNS neurons, J. Neurosci., 15 (1995) 1567–1576. [17] Hoogendijk, J.E., Fliers, E., Swaab, D.F. and Verwer, R.W.H., Activation of vasopressin neurons in the human supraoptic and paraventricular nucleus in senescence and senile dementia, J. Neurol. Sci., 69 (1985) 291–299. [18] Kim, K.S., Miller, D.L., Sapienza, V.J., Chen, C.M.J., Baii, C., Grundke-Iqbal, I., Currie, J.R. and Wisniewski, H.M., Production and characterization of monoclonal antibodies reactive to synthetic cerebrovascular amyloid peptide, Neurosci. Res. Commun., 2 (1988) 121–130. [19] Kononen, J., Soinila, S., Persson, H., Honkaniemi, J., Ho¨kfelt, T. and Pelto-Huikko, M., Neurotrophins and their receptors in the rat pituitary gland: regulation of BDNF and trkB mRNA levels by adrenal hormones, Mol. Brain Res., 27 (1994) 347–354. [20] Kordower, J.H., Bartus, R.T., Bothwell, M., Schatteman, G. and Gash, D.M., Nerve growth factor receptor immunoreactivity in the nonhuman primate (Cebus apella): distribution, morphology, and colocalization with cholinergic enzymes, J. Comp. Neurol., 277 (1988) 465–486. [21] Lehman, M.N., Jansen, H.T., Wortman, M., Stevens, P., Kim, C., Norgren, R.B. and Zeitler, P., Neurotrophin and neurotrophin receptor expression in the suprachiasmatic nucleus (SCN) of rats and hamsters, Soc. Neurosci. Abstr., 22 (1996) 1141. [22] Liang, F.-O., Miranda, R., Sohrabji, F., Brooks, C. and Earnest, D., Brain-derived neurotrophic factor and trkB receptors in the rat SCN: distribution and functional implications, Soc. Neurosci. Abstr., 22 (1996) 1141. [23] Lucassen, P.J., Ravid, R., Gonatas, N.K. and Swaab, D.F., Activation of the human supraoptic and paraventricular nucleus neurons with
[24]
[25]
[26]
[27]
[28] [29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
aging and in Alzheimer’s disease as judged from increasing size of the Golgi apparatus, Brain Res., 632 (1993) 105–113. Merlio, J.-P., Ernfors, P., Jaber, M. and Persson, H., Molecular cloning of rat trkC and distribution of cells expressing messenger RNAs for members of the trk family in the rat central nervous system, Neuroscience, 51 (1992) 513–532. Miranda, R.C., Sohrabji, F. and Toran-Allerand, C.D., Neuronal colocalization of mRNAs for neurotrophins and their receptors in the developing central nervous system suggests a potential for autocrine interactions, Proc. Natl. Acad. Sci. USA, 90 (1993) 6439– 6443. Mufson, E.J., Brashers-Krug, T. and Kordower, J.H., p75 nerve growth factor receptor immunoreactivity in the human brainstem and spinal cord, Brain Res., 589 (1992) 115–123. Otten, U., Baumann, J.B. and Girard, J., Stimulation of the pituitaryadrenocortical axis by nerve growth factor, Nature, 282 (1979) 413– 414. Patterson, J.C. and Childs, G.V., Nerve growth factor and its receptor in the anterior pituitary, Endocrinology, 135 (1994) 1689–1696. 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 – I. Forebrain, Neuroscience, 34 (1990) 57–87. 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 – II. Brainstem, cerebellum and spinal cord, Neuroscience, 34 (1990) 89–110. Ross, A., Grob, P., Bothwell, M., Elder, D.E., Ernst, C.S., Marano, N., Ghrist, B.F.D., Slemp, C.C., Herlyn, M., Atkinson, B. and Koprowski, H., Characterization of nerve growth factor receptor in neural crest tumors using monoclonal antibodies, Proc. Natl. Acad. Sci. USA, 81 (1984) 6681–6685. Salehi, A., Verhaagen, J., Dijkhuizen, P.A. and Swaab, D.F., Colocalization of high-affinity neurotrophin receptors in nucleus basalis of Meynert neurons and their differential reduction in Alzheimer’s disease, Neuroscience, 75 (1996) 373–387. Scaccianoce, S., Cigliana, G., Nicolai, R., Muscolo, L.A., Porcu, A., Navarra, D., Perez-Polo, J.R. and Angelucci, L., Hypothalamic involvement in the activation of the pituitary-adrenocortical axis by nerve growth factor, Neuroendocrinology, 58 (1993) 202–209. Schatteman, G.C., Gibbs, L., Lanahan, A.A., Claude, P. and Bothwell, M., Expression of NGF receptor in the developing and adult primate central nervous system, J. Neurosci., 8 (1988) 860– 873. 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. Taglialatela, G., Angelucci, L., Scaccianoce, S., Foreman, P.J. and Perez-Polo, J.R., Nerve growth factor modulates the activation of the hypothalamo-pituitary-adrenocortical axis during the stress response, Endocrinology, 129 (1991) 2212–2218. Talamini, L.M. and Aloe, L., Immunohistochemical localization of nerve growth factor (NGF) and NGF-receptor in the hypothalamus of adult rats, Arch. Ital. Biol., 131 (1993) 255–266. Vissavajjhala, P., Leszyk, J.D., Lin-Goerke, J. and Ross, A.H., Structural domains of the extracellular domain of human nerve growth factor receptor detected by partial proteolysis, Arch. Biochem. Biophys., 294 (1992) 244–252. Yan, Q. and Johnson, E.M. Jr., Immunohistochemical localization and biochemical characterization of nerve growth factor receptor in adult rat brain, J. Comp. Neurol., 290 (1989) 585–598. Yan, Q., Rosenfeld, R.D., Matheson, C.R., Hawkins, N., Lopez, O.T., Bennett, L. and Welcher, A.A., Expression of brain-derived neurotrophic factor protein in the adult rat central nervous system, Neuroscience, 78 (1997) 431–448.