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Localization of nerve growth factor receptors in cholinergic neurons of the human basal forebrain
Neuroscience Letters', 69 (1986) 37 41 Elsevier Scientific Publishers Ireland Ltd.
37
N S L 04094
LOCALIZATION OF NERVE G R O W T H FACTOR RECEPTORS IN CHOLINERGIC N E U R O N S OF THE H U M A N BASAL FOREBRAIN
F R A N Z HEFTP'*. J U K K A H A R T I K K A I, A N A SALVATIERRA', W I L L I A M J. W E I N E R ' and DEBORAH C. MASH 2
:Department o/Neurology, University o1" Miami School of Medicine, P.O. Box 016960, Miami. FL 33101 and :Divisi~n qf Neur~science and Behavi~ra~ Neur~h~gy' Harvard University' B~st~n' MA ~22~5 U.S.A.: (Received April 28th, 1986; Accepted May 12th, 1986)
Key wordw cholinergic neuron - nerve growth factor - nucleus basalis
Alzheimer's disease
Nerve growth factor ( N G F ) receptors were visualized in the basal human forebrain using an lmmunohistochemial procedure with a monoclonal antibody previously shown to recognize human melanoma cell N G F receptors. The receptors were found to be exclusively located in the medial septal nucleus, the diagonal band of Broca, and the nucleus basalis. This location coincided with that of cell bodies of ascending cholinergic neurons of the basal forebrain. In addition, N G F receptor-positive cells were costained for acetylcholinesterase. These findings indicate that cholinergic neurons of the basal forebrain but none of the other neurons located in this area express receptors for N G F . Results suggest that N G F acts as a lrophic factor for cholinergic neurons in the human brain in a similar way as has been established in recent years ['or the rat brain.
Nerve growth factor (NGF) is a specific neurotrophic factor for peripheral sympathetic neurons and sensory neurons which require N G F for development and maintenance of function [12, 21, 31, 32]. Other neurons of the peripheral mammalian nervous system and central catecholaminergic neurons are not affected by N G F [7, 18, 32]. In recent years evidence has accumulated that N G F acts as neurotrophic factor for ascending cholinergic neurons of the basal forebrain. Cell bodies of these cholinergic neurons are located in medial septal nucleus, diagonal band of Broca, substantia innominata, nucleus basalis and they project to hippocampus, cortex and amygdala [22, 34]. The evidence suggesting a role of N G F in the function of these cholinergic cells includes the following points. First, N G F as well as the m R N A coding for N G F are present in the rat brain and their levels correlate with the anatomical distribution of cholinergic neurons [19, 29]. Second, application of N G F to rat cholinergic neurons in vivo and in vitro stimulates the expression of choline acetyltransferase (CHAT) [11, 14, 15, 17, 23, 33] and promotes survival of forebrain cholinergic neurons in vivo [16]. Third, forebrain cholinergic neurons have been shown to internalize NGF, suggesting that they contain receptors for N G F [27, 28]. N G F receptors have been demonstrated by immunoprecipitation in the rat brain, and their concentration was highest in the medial septum [30]. Finally, the notion that N G F *Author for correspondence.
0304-3940/86/$ 03.50 O 1986 Elsevier Scientific Publishers Ireland Ltd.
38
acts as a neurotrophic factor for cholinergic neurons of the basal forebrain is also supported by results from transplantation studies [4]. These findings have important implications for Alzheimer's disease, since it is associated with a selective loss of these cholinergic neurons in the human brain [1, 3, 5, 24]. Even though other neuronal systems are also affected in Alzheimer's disease, the Joss of cholinergic neurons seems to be a principal factor responsible for the characteristic memory loss [2, 5, 6, 26]. The previously summarized findings indicating that N G F trophically affects forebrain cholinergic neurons suggest that providing N G F to these neurons might retard or even prevent their degeneration. However, all studies describing N G F ' s effect on cholinergic neurons have been carried out in rodents. In order to provide evidence that N G F is a neurotrophic factor for chotinergic neurons of the human brain, we attempted to demonstrate the existence of N G F receptors on these neurons in human tissue. Brain tissue was obtained from a 7-year-old girl who died of heart failure and a 35-year-old woman who died of a pulmonary infection. Tissue blocks were cut from the area of the ventral basal forebrain after postmortem delays of 5 and 9 h, respectively. The tissue was immersed in 4% formaldehyde in phosphate-buffered saline (PBS) for 24 h and was then transferred to PBS containing 30% sucrose. Tissue blocks were frozen in liquid nitrogen and sections of 40/am thickness were cut on a cryostat. For immunohistochemical demonstration of N G F receptors, free floating sections were incubated in PBS containing a monoclonal antibody to human N G F receptors (ME82.11, ref. 25, ascites fluid 1:50), normal horse serum (1:100), 5% bovine serum albumin, and 5% sucrose. The specificity of ME82.11 was earlier directly demonstrated by immunoprecipitation of N G F receptors which were affinity labeled with iodinated N G F [25]. Sections were then incubated in PBS containing a biotinylated anti-mouse antibody, followed by a biotin-avidin conjugate of peroxidase (Vectastain). The peroxidase was developed with diaminobenzidine. In control staining, the specific monoclonal antibody was replaced by unspecific mouse IgG (10/tg/ml). AChE histochemistry performed according to Geneser-Jenden and Blackstad [9] was used to visualize cholinergic neurons because no anti-ChAT antibody was available for immunohistochemical visualization of CHAT. In the rat brain, AChE has been established as a reliable marker for cholinergic neurons in the basal forebrain [8, 20]. The view that this is also true for the human brain is supported by a very recent study demonstrating that immunohistochemistry with an antibody to human ChAT visualized the same neurons in the basal human forebrain as AChE histochemistry [10]. In the areas of the basal forebrain, the monoclonal antibody ME82.11 visualized cells in the nucleus basalis, the diagonal band of Broca and the medial septal nucleus (Fig. 1). The areas studied included the caudate nucleus, internal and external segments of the globus pallidus, putamen, preoptic area, anterior nuclei of thalamus and hypothatamus, parts of the amygdala, anterior commissure, internal capsule, column of the fornix, and the olfactory tubercle [22, 34]. None of these additional structures contained positively stained cells for N G F receptors. The localization of N G F receptor-positive cells corresponds very closely to the location of cholinergic neurons of the basal forebrain in the human brain and animal brain [22, 34]. This suggests that
39
Fig. I. Localization of NGF receptors in the human brain. Receptors were visualized using a monoclonal antibody against human melanoma cell NGF receptors. The antibody was visualized using an avidin biotin peroxidase procedure. A: section of the basal forebrain stained for ACHE. B: adjacent section stained for NGF receptors. C: control section in which the specific monoclonal antibody was replaced by unspecitic mouse lgGs (outlined anatomical structures are also valid for panels A and B. D, E: nucleus basalis neurons visualized by NGF receptor immunocytochemistry. F: localization of NGF receptors in perinuclear area and outer membranes of nucleus basalis neuron. G: double-staining for NGF-receptors and ACHE. The brown product of the peroxidase reaction on cell body and processes is overlaid by black crystals of the product of AChE histochemistry. Two clearly visible crystals are indicated by white arrows). Anatomical structures: ac, anterior commissure: gpe, external segment of globus pallidus: gpi, internal segment of globus pallidus: ic, internal capsule: nb, nucleus basalis; rt, reticular nucleus of thalamus. Bars represent 30 ~tm in panels D, E, F, and 5 ]am in panel G.
N G F r e c e p t o r s are e x p r e s s e d by these c h o l i n e r g i c n e u r o n s b u t n o t by o t h e r n e u r o n s in the s a m e area. I n d e e d , n u m b e r , m o r p h o l o g y a n d l o c a l i z a t i o n o f N G F
receptor-
p o s i t i v e cells m a t c h e d t h a t o f A C h E - p o s i t i v e cells v i s u a l i z e d in a d j a c e n t s e c t i o n s (Fig. 1). F u r t h e r m o r e , c o - s t a i n i n g for N G F
receptors and AChE directly confirmed that
N G F r e c e p t o r s are e x c l u s i v e l y l o c a t e d in the c h o l i n e r g i c n e u r o n s o f the b a s a l foreb r a i n (Fig. 1). In the n u c l e u s basalis, all A C h E - p o s i t i v e cells also w e r e l a b e l l e d for NGF-receptors.
In c o n t r a s t , A C h E - p o s i t i v e cell b o d i e s in the o t h e r a r e a s ( c a u d a t e
n u c l e u s , p u t a m e n , h y p o t h a l a m u s ) did n o t c o n t a i n the b r o w n p r o d u c t o f the p e r o x i dase reaction. T h e r e a c t i o n p r o d u c t v i s u a l i z i n g N G F r e c e p t o r s was l o c a t e d o n the o u t e r m e m b r a n e o f the cell b o d y a n d p r o x i m a l p r o c e s s e s , a n d it was h e a v i l y c o n c e n t r a t e d in
the p e r i n u c l e a r a r e a o f the cell b o d y (Fig. 1). Such a d i s t r i b u t i o n was a n t i c i p a t e d since p r e v i o u s r e p o r t s c o n c e r n i n g n e u r o n s o f the r a t p e r i p h e r a l n e r v o u s system have established t h a t N G F r e c e p t o r s are synthesized in cell bodies, t r a n s p o r t e d to a x o n a l t e r m i n a l s and, after h a v i n g b o u n d N G F , t r a n s p o r t e d r e t r o g r a d e l y to the cell b o d y [31, 32]. The fact t h a t N G F r e c e p t o r s were l o c a t e d on the o u t e r m e m b r a n e o f cell b o d i e s a d d s s u p p o r t to the n o t i o n t h a t e x o g e n o u s N G F is able to influence cholinergic neurons after loss o f their a x o n s [16]. W e failed to observe positively stained fibers or t e r m i n a l s in the a m y g d a l a , i.e. in one o f the target a r e a s o f f o r e b r a i n cholinergic neurons, suggesting t h a t the h i s t o c h e m i c a l p r o c e d u r e is n o t sensitive e n o u g h to locate N G F r e c e p t o r s on these structures. T h e findings f r o m the present s t u d y s t r o n g l y suggest the c o n c e p t that N G F is a specific n e u r o t r o p h i c f a c t o r for cholinergic n e u r o n s o f the h u m a n basal forebrain. W e have earlier h y p o t h e s i z e d t h a t the lack o f N G F m a y be involved in the p a t h o g e n esis o f A l z h e i m e r ' s disease a n d t h a t c o m p o u n d s m i m i c k i n g its a c t i o n might be benificial in the t r e a t m e n t o f this disease [13]. Beneficial effects o f N G F m i g h t also be a n t i c i p a t e d even if c a u s a t i v e factors o t h e r t h a n those i n v o l v i n g N G F are responsible for the d e g e n e r a t i o n o f cholinergic neurons. I n c r e a s i n g the a v a i l a b i l i t y o f N G F to cholinergic n e u r o n s is likely to s t i m u l a t e m a n y synthetic processes in these cells a n d to m a k e t h e m less v u l n e r a b l e to a p r i m a r y disease process. These studies were s u p p o r t e d b y g r a n t s f r o m the A l z h e i m e r ' s Disease a n d R e l a t e d D i s o r d e r s A s s o c i a t i o n , C h i c a g o IL, a n d the N a t i o n a l P a r k i n s o n F o u n d a t i o n , M i a m i F L . The a u t h o r s gratefully a c k n o w l e d g e the gift o f the m o n o c l o n a l a n t i b o d y from Dr. M. Herlyn, W i s t a r Institute, P h i l a d e l p h i a . 1 Bartus, R.T., Dean, R.L., Beer, B. and Lippa, A.S., The cholinergic hypothesis of geriatric memory dysfunction, Science, 217 (1982) 408-417. 2 Biedermann, G.B., The search for the chemistry of memory; recent trends and the logic of investigation in the role of cholinergic and adrenergic transmitters. In G.A. Kerkut and J.W. Phyllis (Eds.), Progress in Neurobiology, Vol. 2, Pergamon, New York, 1975. 3 Coyle, J.T., Price, D.L. and DeLong, M.R., Alzheimer's disease: a disease of cortical eholinergic innervation, Science, 219 (1983) 1184-1190. 4 Crutcher, K.A. and Davis. J.N., Sympathetic noradrenergic sprouting in response to central cholinergic denervations, Trends Neurosci., 4 (1981) 70 72. 5 Davies, P., Is it possible to design rational treatment for the symptoms of Alzheimer's disease? Drug Develop. Res., 5 (1985) 69-75. 6 Deutsch, J.A., The cholinergic synapse and the site of memory, Science, 174 (1971) 788-794. 7 Dreyfus, C.F., Peterson, E.R. and Crain, S.M., Failure of nerve growth factor to affect fetal mouse brain catecholaminergic neurons in culture, Brain Res., 194 (1980) 540-551. 8 Eckenstein, F. and Sofroniew, M.W., Identification of central cholinergic neurons containing both choline acetyltransferase and acetylcholinesterase and of central neurons containing only acetylcholinesterase, J. Neurosci., 3 (1983) 2286-2291. 9 Geneser-Jenden, F.A. and Blackstad, T.E., Distribution of acetylcholinesterase in the hippocampal region of the guinea pig. 1. Entorhinal area, parasubiculum, and pre-subiculum, Z. Zellforsch., 114 (1971) 460-481. 10 German, D.C., Bruce, G. and Hersh, L.B., lmmunohistochemical staining of cholinergic neurons in the human brain using a polyclonal antibody to human choline acetyltransferase, Neurosci. Len., 61 (1985) 1-5.
11 Gnahn, H., Hefti, F., Heumann, R., Schwab, M. and Thoenen, H., NGF-mediated increase of choline acetyltransferase (CHAT) in the neonatal forebrain; evidence for a physiological role of NGF in the brain? Dev. Brain Res., 9 (1981) 45-52. 12 Greene, L.A. and Shooter, E.M., The nerve growth factor: biochemistry, synthesis, and mechanism of action, Annu. Rev. Neurosci., 3 (1980) 353-402. 13 t tefti, F., Alzheimer's disease caused by a lack of nerve growth factor? Ann. Neurol., 13 (1983) 109 110.
14 Hcfti, F., Dravid, A. and Hartikka, J., Chronic intraventricular injections of nerve growth factor elevate hippocampal choline acetyltransferase activity in adult rats with partial septo-hippocampal lesions, Brain Res., 293 (1984) 305-309. 15 Hefti, F., Hartikka, J., Eckenstein, F., Gnahn, H., Heumann, R. and Schwab, M., Nerve growth factor (NG F) increases choline acetyltransferase but not survival or fiber growth of cultured septal cholinergic neurons, Neuroscience, 14 (1985) 55-68. 16 Hefti, F., Nerve growth factor (NGF) promotes survival of septal cholinergic neurons after fimbrial transsections, J. Neurosci., 6 (1986) 2155 2162. 17 Honegger, P. and Lenoir, D., Nerve growth factor (NGF) stimulation of cholinergic telencephalic neurons in aggregating cell cultures, Dev. Brain Res., 3 (1982) 229 238. 18 Konkol, R.J., Mailman, R.B., Bendeich, E.G., Garrison, A.M., Mueller, R.A. and Breese, G.R., Evaluation of the effects of nerve growth factor and anti-nerve growth factor on the development of central catecholaminergic neurons, Brain Res., 144 (1978) 277-285. 19 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. 20 Lcvey, A.1., Wainer, B.H., Mufson, E.J. and Mesulam, M.M., Co-localization of acetylcholinesterase and choline acetyltransferase in the rat cerebrum, Neuroscience, 9 (1983) 9-22. 21 Levi-Montalcini, R., Developmental neurobiology and the natural history of nerve growth factor, Annu. Rev. Neurosci., 5 (1982) 341 362. 22 Mesulam, M.M., Mufson, E.J., Wainer, B.H. and Levey, A.I., Central cholinergic pathways in the rat: all overview based on an alternative nomenclature (Chl-Ch6), Neuroscience, 10 (1983) 1185 1202; 1158: 1211-1218. 23 Mobley, W.C., Rutkowski, J.L., Tennekoon, G.I., Buchanan, K. and Johnston, M.V., Choline acetyltransferase activity in striatum of neonatal rats increased by nerve growth factor, Science, 229 (1985) 284 286. 24 Perry, E.H., and Perry, R.H., The cholinergic system in Alzheimer's disease. In Roberts P.J. (Ed.), Biochemistry of Dementia, Wiley, London, 1984, pp. 135-183. 25 Ross, A.H., Grob, P., Bothwell, M., Elder, D.E., Ernst, C.S., Marano, N., Ghrist, B.F., 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. U.S.A., 81 (1984) 6681-6685. 26 Russell, R.W., Cholinergic system in behavior: the search for mechanisms of action, Annu. Rev. Pharmacol. Toxicol., 22 (1982) 435-463. 27 Schwab, M., Otten, U., Agid, Y. and Thoenen, H., Nerve growth factor (NGF) in the rat CNS: absence of specific retrograde axonal transport and tyrosine hydroxylase induction in locus coeruleus and substantia nigra, Brain Res., 168 (1979) 473-483. 28 Seiler, M. and Schwab, M.E., Specific retrograde transport of nerve growth factor (NGF) from neocortex to nucleus basalis in the rat, Brain Res., 300 (1984) 33 36. 29 Shelton, D i . and Reichardt, L.F., Expression of the nerve growth factor gene correlates with the density of sympathetic innervation in effector organs, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 7951 7955. 30 Taniuchi, M., Schweitzer, J.B. and Johnson, E.M,, Nerve growth factor receptor molecules in rat brain, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 1950-1954. 31 Thoenen, H. and Barde, Y.A., Physiology of nerve growth factor, Physiol. Rev., 60 (1980) 1284-1335. 32 Thoenen, H. and Edgar, D., Neurotrophic factors, Science, 229 (1985) 238-242. 33 Toniolo, G., Dunnett, S.B., Hefti, F. and Will, B., Acetylcholine-rich transplants in the hippocampus: influence of intrinsic growth factors and application of NGF on choline acetyttransferase activity, Brain Res., 345 (1986) 141 146. 34 Wainer, B.H., Levey, A.I., Mufson, E.F. and Mesulam, M.M., Cholinergic systems in mammalian brain identified with antibodies against choline acetyltransferase, Neurosci. Int., 6 (1984) 163 182.
37
N S L 04094
LOCALIZATION OF NERVE G R O W T H FACTOR RECEPTORS IN CHOLINERGIC N E U R O N S OF THE H U M A N BASAL FOREBRAIN
F R A N Z HEFTP'*. J U K K A H A R T I K K A I, A N A SALVATIERRA', W I L L I A M J. W E I N E R ' and DEBORAH C. MASH 2
:Department o/Neurology, University o1" Miami School of Medicine, P.O. Box 016960, Miami. FL 33101 and :Divisi~n qf Neur~science and Behavi~ra~ Neur~h~gy' Harvard University' B~st~n' MA ~22~5 U.S.A.: (Received April 28th, 1986; Accepted May 12th, 1986)
Key wordw cholinergic neuron - nerve growth factor - nucleus basalis
Alzheimer's disease
Nerve growth factor ( N G F ) receptors were visualized in the basal human forebrain using an lmmunohistochemial procedure with a monoclonal antibody previously shown to recognize human melanoma cell N G F receptors. The receptors were found to be exclusively located in the medial septal nucleus, the diagonal band of Broca, and the nucleus basalis. This location coincided with that of cell bodies of ascending cholinergic neurons of the basal forebrain. In addition, N G F receptor-positive cells were costained for acetylcholinesterase. These findings indicate that cholinergic neurons of the basal forebrain but none of the other neurons located in this area express receptors for N G F . Results suggest that N G F acts as a lrophic factor for cholinergic neurons in the human brain in a similar way as has been established in recent years ['or the rat brain.
Nerve growth factor (NGF) is a specific neurotrophic factor for peripheral sympathetic neurons and sensory neurons which require N G F for development and maintenance of function [12, 21, 31, 32]. Other neurons of the peripheral mammalian nervous system and central catecholaminergic neurons are not affected by N G F [7, 18, 32]. In recent years evidence has accumulated that N G F acts as neurotrophic factor for ascending cholinergic neurons of the basal forebrain. Cell bodies of these cholinergic neurons are located in medial septal nucleus, diagonal band of Broca, substantia innominata, nucleus basalis and they project to hippocampus, cortex and amygdala [22, 34]. The evidence suggesting a role of N G F in the function of these cholinergic cells includes the following points. First, N G F as well as the m R N A coding for N G F are present in the rat brain and their levels correlate with the anatomical distribution of cholinergic neurons [19, 29]. Second, application of N G F to rat cholinergic neurons in vivo and in vitro stimulates the expression of choline acetyltransferase (CHAT) [11, 14, 15, 17, 23, 33] and promotes survival of forebrain cholinergic neurons in vivo [16]. Third, forebrain cholinergic neurons have been shown to internalize NGF, suggesting that they contain receptors for N G F [27, 28]. N G F receptors have been demonstrated by immunoprecipitation in the rat brain, and their concentration was highest in the medial septum [30]. Finally, the notion that N G F *Author for correspondence.
0304-3940/86/$ 03.50 O 1986 Elsevier Scientific Publishers Ireland Ltd.
38
acts as a neurotrophic factor for cholinergic neurons of the basal forebrain is also supported by results from transplantation studies [4]. These findings have important implications for Alzheimer's disease, since it is associated with a selective loss of these cholinergic neurons in the human brain [1, 3, 5, 24]. Even though other neuronal systems are also affected in Alzheimer's disease, the Joss of cholinergic neurons seems to be a principal factor responsible for the characteristic memory loss [2, 5, 6, 26]. The previously summarized findings indicating that N G F trophically affects forebrain cholinergic neurons suggest that providing N G F to these neurons might retard or even prevent their degeneration. However, all studies describing N G F ' s effect on cholinergic neurons have been carried out in rodents. In order to provide evidence that N G F is a neurotrophic factor for chotinergic neurons of the human brain, we attempted to demonstrate the existence of N G F receptors on these neurons in human tissue. Brain tissue was obtained from a 7-year-old girl who died of heart failure and a 35-year-old woman who died of a pulmonary infection. Tissue blocks were cut from the area of the ventral basal forebrain after postmortem delays of 5 and 9 h, respectively. The tissue was immersed in 4% formaldehyde in phosphate-buffered saline (PBS) for 24 h and was then transferred to PBS containing 30% sucrose. Tissue blocks were frozen in liquid nitrogen and sections of 40/am thickness were cut on a cryostat. For immunohistochemical demonstration of N G F receptors, free floating sections were incubated in PBS containing a monoclonal antibody to human N G F receptors (ME82.11, ref. 25, ascites fluid 1:50), normal horse serum (1:100), 5% bovine serum albumin, and 5% sucrose. The specificity of ME82.11 was earlier directly demonstrated by immunoprecipitation of N G F receptors which were affinity labeled with iodinated N G F [25]. Sections were then incubated in PBS containing a biotinylated anti-mouse antibody, followed by a biotin-avidin conjugate of peroxidase (Vectastain). The peroxidase was developed with diaminobenzidine. In control staining, the specific monoclonal antibody was replaced by unspecific mouse IgG (10/tg/ml). AChE histochemistry performed according to Geneser-Jenden and Blackstad [9] was used to visualize cholinergic neurons because no anti-ChAT antibody was available for immunohistochemical visualization of CHAT. In the rat brain, AChE has been established as a reliable marker for cholinergic neurons in the basal forebrain [8, 20]. The view that this is also true for the human brain is supported by a very recent study demonstrating that immunohistochemistry with an antibody to human ChAT visualized the same neurons in the basal human forebrain as AChE histochemistry [10]. In the areas of the basal forebrain, the monoclonal antibody ME82.11 visualized cells in the nucleus basalis, the diagonal band of Broca and the medial septal nucleus (Fig. 1). The areas studied included the caudate nucleus, internal and external segments of the globus pallidus, putamen, preoptic area, anterior nuclei of thalamus and hypothatamus, parts of the amygdala, anterior commissure, internal capsule, column of the fornix, and the olfactory tubercle [22, 34]. None of these additional structures contained positively stained cells for N G F receptors. The localization of N G F receptor-positive cells corresponds very closely to the location of cholinergic neurons of the basal forebrain in the human brain and animal brain [22, 34]. This suggests that
39
Fig. I. Localization of NGF receptors in the human brain. Receptors were visualized using a monoclonal antibody against human melanoma cell NGF receptors. The antibody was visualized using an avidin biotin peroxidase procedure. A: section of the basal forebrain stained for ACHE. B: adjacent section stained for NGF receptors. C: control section in which the specific monoclonal antibody was replaced by unspecitic mouse lgGs (outlined anatomical structures are also valid for panels A and B. D, E: nucleus basalis neurons visualized by NGF receptor immunocytochemistry. F: localization of NGF receptors in perinuclear area and outer membranes of nucleus basalis neuron. G: double-staining for NGF-receptors and ACHE. The brown product of the peroxidase reaction on cell body and processes is overlaid by black crystals of the product of AChE histochemistry. Two clearly visible crystals are indicated by white arrows). Anatomical structures: ac, anterior commissure: gpe, external segment of globus pallidus: gpi, internal segment of globus pallidus: ic, internal capsule: nb, nucleus basalis; rt, reticular nucleus of thalamus. Bars represent 30 ~tm in panels D, E, F, and 5 ]am in panel G.
N G F r e c e p t o r s are e x p r e s s e d by these c h o l i n e r g i c n e u r o n s b u t n o t by o t h e r n e u r o n s in the s a m e area. I n d e e d , n u m b e r , m o r p h o l o g y a n d l o c a l i z a t i o n o f N G F
receptor-
p o s i t i v e cells m a t c h e d t h a t o f A C h E - p o s i t i v e cells v i s u a l i z e d in a d j a c e n t s e c t i o n s (Fig. 1). F u r t h e r m o r e , c o - s t a i n i n g for N G F
receptors and AChE directly confirmed that
N G F r e c e p t o r s are e x c l u s i v e l y l o c a t e d in the c h o l i n e r g i c n e u r o n s o f the b a s a l foreb r a i n (Fig. 1). In the n u c l e u s basalis, all A C h E - p o s i t i v e cells also w e r e l a b e l l e d for NGF-receptors.
In c o n t r a s t , A C h E - p o s i t i v e cell b o d i e s in the o t h e r a r e a s ( c a u d a t e
n u c l e u s , p u t a m e n , h y p o t h a l a m u s ) did n o t c o n t a i n the b r o w n p r o d u c t o f the p e r o x i dase reaction. T h e r e a c t i o n p r o d u c t v i s u a l i z i n g N G F r e c e p t o r s was l o c a t e d o n the o u t e r m e m b r a n e o f the cell b o d y a n d p r o x i m a l p r o c e s s e s , a n d it was h e a v i l y c o n c e n t r a t e d in
the p e r i n u c l e a r a r e a o f the cell b o d y (Fig. 1). Such a d i s t r i b u t i o n was a n t i c i p a t e d since p r e v i o u s r e p o r t s c o n c e r n i n g n e u r o n s o f the r a t p e r i p h e r a l n e r v o u s system have established t h a t N G F r e c e p t o r s are synthesized in cell bodies, t r a n s p o r t e d to a x o n a l t e r m i n a l s and, after h a v i n g b o u n d N G F , t r a n s p o r t e d r e t r o g r a d e l y to the cell b o d y [31, 32]. The fact t h a t N G F r e c e p t o r s were l o c a t e d on the o u t e r m e m b r a n e o f cell b o d i e s a d d s s u p p o r t to the n o t i o n t h a t e x o g e n o u s N G F is able to influence cholinergic neurons after loss o f their a x o n s [16]. W e failed to observe positively stained fibers or t e r m i n a l s in the a m y g d a l a , i.e. in one o f the target a r e a s o f f o r e b r a i n cholinergic neurons, suggesting t h a t the h i s t o c h e m i c a l p r o c e d u r e is n o t sensitive e n o u g h to locate N G F r e c e p t o r s on these structures. T h e findings f r o m the present s t u d y s t r o n g l y suggest the c o n c e p t that N G F is a specific n e u r o t r o p h i c f a c t o r for cholinergic n e u r o n s o f the h u m a n basal forebrain. W e have earlier h y p o t h e s i z e d t h a t the lack o f N G F m a y be involved in the p a t h o g e n esis o f A l z h e i m e r ' s disease a n d t h a t c o m p o u n d s m i m i c k i n g its a c t i o n might be benificial in the t r e a t m e n t o f this disease [13]. Beneficial effects o f N G F m i g h t also be a n t i c i p a t e d even if c a u s a t i v e factors o t h e r t h a n those i n v o l v i n g N G F are responsible for the d e g e n e r a t i o n o f cholinergic neurons. I n c r e a s i n g the a v a i l a b i l i t y o f N G F to cholinergic n e u r o n s is likely to s t i m u l a t e m a n y synthetic processes in these cells a n d to m a k e t h e m less v u l n e r a b l e to a p r i m a r y disease process. These studies were s u p p o r t e d b y g r a n t s f r o m the A l z h e i m e r ' s Disease a n d R e l a t e d D i s o r d e r s A s s o c i a t i o n , C h i c a g o IL, a n d the N a t i o n a l P a r k i n s o n F o u n d a t i o n , M i a m i F L . The a u t h o r s gratefully a c k n o w l e d g e the gift o f the m o n o c l o n a l a n t i b o d y from Dr. M. Herlyn, W i s t a r Institute, P h i l a d e l p h i a . 1 Bartus, R.T., Dean, R.L., Beer, B. and Lippa, A.S., The cholinergic hypothesis of geriatric memory dysfunction, Science, 217 (1982) 408-417. 2 Biedermann, G.B., The search for the chemistry of memory; recent trends and the logic of investigation in the role of cholinergic and adrenergic transmitters. In G.A. Kerkut and J.W. Phyllis (Eds.), Progress in Neurobiology, Vol. 2, Pergamon, New York, 1975. 3 Coyle, J.T., Price, D.L. and DeLong, M.R., Alzheimer's disease: a disease of cortical eholinergic innervation, Science, 219 (1983) 1184-1190. 4 Crutcher, K.A. and Davis. J.N., Sympathetic noradrenergic sprouting in response to central cholinergic denervations, Trends Neurosci., 4 (1981) 70 72. 5 Davies, P., Is it possible to design rational treatment for the symptoms of Alzheimer's disease? Drug Develop. Res., 5 (1985) 69-75. 6 Deutsch, J.A., The cholinergic synapse and the site of memory, Science, 174 (1971) 788-794. 7 Dreyfus, C.F., Peterson, E.R. and Crain, S.M., Failure of nerve growth factor to affect fetal mouse brain catecholaminergic neurons in culture, Brain Res., 194 (1980) 540-551. 8 Eckenstein, F. and Sofroniew, M.W., Identification of central cholinergic neurons containing both choline acetyltransferase and acetylcholinesterase and of central neurons containing only acetylcholinesterase, J. Neurosci., 3 (1983) 2286-2291. 9 Geneser-Jenden, F.A. and Blackstad, T.E., Distribution of acetylcholinesterase in the hippocampal region of the guinea pig. 1. Entorhinal area, parasubiculum, and pre-subiculum, Z. Zellforsch., 114 (1971) 460-481. 10 German, D.C., Bruce, G. and Hersh, L.B., lmmunohistochemical staining of cholinergic neurons in the human brain using a polyclonal antibody to human choline acetyltransferase, Neurosci. Len., 61 (1985) 1-5.
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