Effects of brain-derived neurotrophic factor and nerve growth factor on remaining neurons in the lesioned nucleus basalis magnocellularis

BRAIN RESEARCH ELSEVIER

Brain Research 639 (1994) 149-155

Research Report

Effects of brain-derived neurotrophic factor and nerve growth factor on remaining neurons in the lesioned nucleus basalis magnocellularis Ad J. Dekker a,b,,, Anne M. Fagan a, Fred H. Gage a, Leon J. Thai a,b a Department of Neurosciences, University of California, San Diego, CA, USA b Neurology Service, VA Medical Center, San Diego, CA 92161, USA (Accepted 2 November 1993)

Abstract

Rats received a unilateral lesion of the nucleus basalis magnocellularis (NBM) by infusion of ibotenic acid. Starting 2 weeks after the lesion, the animals were treated with nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) by intraparenchymal infusion of 3/zg per day for 4 weeks. Lesioned control animals received a similar amount of cytochrome c. The activity of choline acetyltransferase (CHAT) in the frontal neocortex was significantly reduced by the lesion (-39%). However, the intraparenchymal treatment with NGF or BDNF did not affect cortical ChAT activity. The number of p75 NGF receptor-immunoreactive neurons in the NBM was significantly decreased ( - 49%) by the lesion and was not affected by NGF or BDNF. The size of the remaining neurons was significantly increased by NGF (+ 32%), but not by BDNF (+ 12%). Similarly, in situ hybridization showed enhanced expression of the p75 NGF receptor following treatment with NGF, but not with BDNF. These results suggest that although BDNF occurs in the target area of cholinergic NBM neurons, its effects on these neurons are less pronounced than those of NGF.

Key words: Nucleus basalis magnocellularis; Nerve growth factor; Brain-derived neurotrophic factor; p75 NGF receptor; In situ hybridization; Choline acetyl transferase

I. Introduction

The projection from nucleus basalis magnocellularis (NBM) to neocortex, part of the basal forebrain cholinergic system, plays an important role in learning and memory. Lesions of the N B M result in an impaired performance in tasks for learning and memory and decreased cholinergic innervation in the neocortex (for review, see ref. 11). These lesions serve as a model for extensive (up to 50%) loss of cholinergic neurons, as well as other neurons in the NBM, and can be used to study the effects of treatment on remaining cholinergic neurons under such conditions. T r e a t m e n t with nerve growth factor (NGF), starting 2 weeks after a lesion of the NBM, increases choline acetyl transferase (CHAT) activity, ChAT-positive fiber staining and acetylcholine release in the neocortex [8,9,10,12,13,15,20,21], and increases the size of remaining neurons in the N B M

* Corresponding author. Groenhovenweg 161, 2803 DD Gouda, The Netherlands. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 5 0 1 - S

[9]. These results suggest that N G F stimulates the functioning of remaining cholinergic neurons in the NBM. In addition, N G F rescues N B M neurons under conditions of retrograde degeneration following cortical devascularizing lesions [38]. The role of trophic factors other than N G F on basal forebrain cholinergic neurons in vivo is less well known. Brain-derived neurotrophic factor (BDNF), originally purified from pig brain [4], shows structural similarities to N G F [35] and binds to the same low affinity receptor [45,47]. A possible effect of B D N F on basal forebrain cholinergic neurons was suggested by the fact that the highest levels of its m R N A are found in the target areas of these neurons, neocortex and hippocampus [16,25,37, 42], areas that also contain the highest amount of N G F [32]. In addition, B D N F increases C h A T activity of septal cholinergic neurons in vitro [1,30,40] and rescues medial septum neurons following transection of the fimbria-fornix [31,33,39], suggesting that B D N F has a trophic effect on cholinergic neurons in vivo. Finally, there is a lower level of the m R N A for B D N F in the hippocampus of patients with Alzheimer's disease [43],

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A.J. Dekker et al./ Brain Research 639 (1994) 149-155

whereas the m R N A for N G F is not affected [19]. Since it has been proposed that the loss of cholinergic neurons in Alzheimer's disease is due to a lack of trophic support [3,22], this would suggest an important role of B D N F in the maintenance of cholinergic neurons. It was therefore of interest to compare the effects of B D N F with those of N G F on remaining cholinergic neurons following a lesion of the NBM.

2. Materials and methods 2.1. Surgery Male Fischer-344 albino rats (Harlan Sprague Dawley, Indianapolis, IN) weighing 280-300 g were used. Animals, were anesthetized by an i.m. injection consisting of 62.5 mg/kg ketamine (Ketaset, 100 mg/ml, Bristol Laboratories, Syracuse, NY), 3.175 mg/kg xylazine (Rompun, 20 mg/ml, Miles Laboratories, Shawnee, KS) and 0.625 mg/kg of acepromazine maleate (10 mg/ml, TechAmerica Group Inc., Elwood, KS) dissolved in 0.9% sterile saline. After anesthesia the rats were mounted in a small animal stereotaxic apparatus (David Kopf, Tujunga, CA) with bregma and lambda in the same horizontal plane. Ibotenic acid (4.5 /zg//zl in sterile 0.9% saline, adjusted to pH = 7.4, Sigma, St Louis, MO) was infused at a rate of 0.1 /xl/min through a Hamilton 10 /zl syringe (model 701SN, Reno, NV) connected to an infusion pump (model BSP-99, Braintree Inc., Braintree, MA). Infusion took place for 6 min at AP + 8.1, LAT + 2.7, DV + 2.2 from the interaural line and for 6 min at AP + 7.2, LAT + 2.9, DV + 3.4 (coordinates according to the atlas by Paxinos and Watson [41]). After each infusion the needle was left in place for another 5 min to ensure proper diffusion of the solution. In total, 21 animals received NBM lesions and 7 animals served as unlesioned controls. Two weeks after the lesion, osmotic minipumps (model 2002, Alza Co. Palo Alto, CA) were implanted subcutaneously. The pumps were connected by 3.5 cm vinyl tubing (size V/4, Bolab Inc., Lake Havasu City, AZ) to a stainless-steel cannula (28 gauge, 5 mm length, Plastic Products, Roanoke, VA). The cannula was placed dorsal to the site of the lesion (AP + 7.7, LAT + 2.8) in the parenchyma rather than in the lateral ventricle, since intraventricular infusion of BDNF is less effective [2], presumably because the ventricle wall has a high density of trk-B receptors [29], which bind BDNF [47]. The pumps contained either human recombinant NGF (500 ~ g / ml, Syntex, Palo Alto, CA), BDNF (500 /zg/ml, Regeneron, Tarrytown, NY) or cytochrome c (500 ~g/ml, Sigma, St. Louis, MO) in artificial CSF (148 mM NaCt, 3 mM KCI, 1.4 mM CaCI 2, 0.8 mM MgCI2, 1.5 mM Na2HPO 4 0.2 mM NaH2PO 4, pH = 7.4) containing 100 /zg/ml rat serum albumin (Sigma) and 50 p,g/ml gentamycin (Sigma). The pumps were paraffin coated for 13 mm, resulting in a flow rate of 0.25/xl/h and a delivery of 3 ~g per day. Pumps were changed after 3 weeks. At a concentration of 500/~g/ml the recovery of NGF and BDNF after 3 weeks is approximately 100% (ref. 9 and R. Lindsay, personal communication). The 21 animals that received an NBM lesion were randomly assigned to 3 treatment groups (NGF, BDNF, cytochrome c) of 7 animals each.

2.2. ChAT activity After 4 weeks of treatment, the animals were sacrificed by decapitation. The brains were rapidly removed and dissected on ice. The frontal and parietal cortex and hippocampus were stored at -70"C. A 3 mm slice (approximately 6.5-9.5 AP) was saved for histology and in situ hybridization. The tissue was sonicated in 400

tzl of 50 mM phosphate buffer (pH = 7.4). ChAT activity was determined by the method of Fonnum [17]. This method measures the conversion of [14C]acetyl-coenzyme A to [14C]ACh. The protein content of the samples was determined by the method of Lowry et al. [36].

2.3. Histology The 3-mm slice collected during dissection was fixed in 10% formalin for 2 days and stored in 0.32 M sucrose. Forty-tzm sections were cut, using a sliding microtome, mounted on slides and stored at -20°C for in situ hybridization. The remaining sections were stored in a cryoprotectant solution (glycerol, ethyleneglycol and 0.1 M phosphate buffer, pH = 7.4, 3:3:4 by vol). Sections were immunostained for the low affinity NGF receptor (p75) using the procedure described by Batchelor et al. [6]. Briefly, endogenous peroxidase enzyme activity was blocked with 0.6% H20 2 for 15-30 min, after which sections were rinsed with 1% horse serum and 0.25% Triton X-100 in 0.1 M Tris-buffered saline, pH = 7.4. Subsequently, sections were incubated overnight with a monoclonal antibody against p75 NGF receptor (1 mg/125 ml, 192 lgG, from mouse hybridoma [48], rinsed twice for 10 min with Tris-buffered saline, incubated for 1 h with the secondary antibody (horse anti-mouse, biotinylated, 1:160, Vector Laboratories Inc., Burlingame, CA), rinsed twice for 10 min with Tris-buffered saline and incubated for 1 h with avidin and biotinylated peroxidase (1 : 100, Vector). Staining was visualized with 0.025% diaminobenzidine, 0.01% H20 2 and 0.6% nickel chloride in Tris-buffered saline for 6-12 min. For each animal the number of neurons and their size was measured in the left and right NBM (in two independently selected sections, approximately 8 mm AP). Quantification was performed using an IBAS 2000 image analysis system (Kontron, Munich, Germany). Data from at least 6 animals per group were used for quantification.

2.4. In situ hybridization In situ hybridization was performed as described by Higgins et al. [23,24]. A full-length 1679 bp cDNA, complementary to rat p75 NGF receptor mRNA [44], subcloned into the BglII-EcoRI site of the pGEM3 plasmid vector (Promega Biotec, Madison, Wl), was used for production of 35S-labeled anti-sense or sense strand RNA probes. Transcription was performed in a volume of 20/.d, containing 40 mM Tris-HCl, pH = 7.5, 6 mM MgCI 2, 40 units of ribonuclease inhibitor (Promega), 10 mM dithiothreitol (DTT), 1 mM rATP, 1 mM rGTP, 1 mM rUTP, 125 /~M [35S]rCTP (800 C i / mmol, Amersham), 2 p,g linearized DNA template and 20 units of RNA polymerase (SP6 for the anti-sense probe, T7 for the sense probe, Stratagene Cloning Systems, La Jolla, CA). Transcription proceeded for 4 h at 37°C, after which the DNA template was removed by digestion with RNase-free DNase (15 min at 37°C with 30 U of DNase I, Boehringer-Mannheim, Indianapolis, IN). The unincorporated nucleotides were removed by Sephadex filtration (Sephadex G50, Pharmacia), using denatured salmon sperm DNA as a carrier. RNA probes were size-reduced to an average length of 100 nucleotides by alkaline hydrolysis (0.1 M NaOH, 4°C for 12 min), prior to phenol/ chloroform extraction and ethanol precipitation. Tissue pretreatment was performed on slides placed in glass racks in glass containers. All glassware and solutions were treated with diethylpyrocarbonate (1:1000 by vol, Sigma) to avoid RNA degradation by contaminating RNase. Tissue was fixed for 1 min in 4% formaldehyde in phosphate buffered saline (0.28 M, pH ~ 7.4). Subsequently, the tissue was treated with proteinase K (50/~g/ml in 50 mM Tris, 5 mM EDTA, pH=8.0) for 5 rain at 37°C and post-fixed as described above. Following acetylation (10 min in 0.25% acetic anhydride in 0.9% NaCl, containing 0.1 M tri-

A.J. Dekker et al. / Brain Research 639 (1994) 149-155 ethanolamine, p H = 8.0), the tissue was dehydrated through a graded ethanol series and air dried. The prehybridization buffer consisted of 50% formamide, 0.5 M NaCI, 10 m M Pipes buffer (pH = 8.0), 10 m M E D T A , 250 m M DTT, 5 × D e n h a r d t ' s solution (0.02% BSA, 0.02% Ficoll, 0.02% polyvinyipyrrolidone), 0.2% SDS, 10% dextran sulfate and denatured yeast t R N A (200 p . g / m l ) and salmon sperm D N A (500 /zg/ml). Tissue prehybridization was performed at 56°C in a humidified chamber for 1 - 3 h. Hybridization was carried out in a humidified chamber at 56°C for 17-18 h, with 8 0 / z l of hybridization buffer, containing 1.5 × 106 cpm of the probe per slide. Following hybridization, slides were rinsed for 15 min in 4 × SSC (0.6 M NaCl, 50 m M sodium citrate), containing 25 m M D T T and for 15 min in 4 × SSC. Excess probe was removed by R N a s e digestion for 30 min at 37°C ( 7 5 / z g / m l R N a s e A (Sigma) in 500 m M NaCl, 50 m M Tris, p H = 8.0), followed by futher washing steps: 30 min at 37°C in 500 m M NaCi, 50 m M Tris, pH = 8.0; 15 min at 42°C in 2 × S S C and 1 min at 56°C in 0.1×SSC. Next, the tissue was dehydrated through graded ethanol series and air dried. Hybridized slides were dipped in liquid emulsion (NTB-2, Kodak, diluted 1:1 in H 2 0 ) , exposed for 10 days at - 2 0 ° C and developed for autoradiography in Kodak D19. Tissue sections were lightly counterstained with thionin, dehydrated to xylene, coverslipped. Grain clusters overlying the N B M were counted u n d e r dark-field optics. For each animal, four counts on each side of the brain were averaged.

2. 5. Statistical analysis T r e a t m e n t effects were analyzed with one-way analysis of variance ( A N O V A ) followed by a N e w m a n - K e u l s post hoc test. In view of the variability of the in situ hybridization, these data were analyzed with a Kruskal-WaUis test, followed by a M a n n - W h i t n e y U-test.

3. Results

3.1. ChAT activity The ChAT activity in the frontal and parietal cortex on both the intact and the lesioned side are given in Table 1. The ChAT activity in the frontal cortex on the

151

Table 2 Effects of lesions of the N B M and treatment with N G F or B D N F on the n u m b e r of p75 N G F receptor-immunoreactive neurons in the NBM

Control Cytochrome c BDNF NGF

Lesioned side

Intact side

87.7+ 46.0 + 38.6 + 57.4+

84.8+8.1 73.3 + 5.8 95.6 + 5.8 101.3+8.9

3.3 11.1 * * 3.8 * * 6.1 * *

Data are expressed as m e a n + S.E.M. * * P < 0.01 vs. control (Newman-Keuls).

lesioned side showed significant differences between groups (F3,24 = 36.97, P < 0.0001). This was due to a decrease in all the lesioned groups ( P < 0.01 vs. control, Newman-Keuls) without any differences between the three lesioned groups themselves ( P > 0.05, Newman-Keuls). In the parietal cortex, the lesioned side showed significant differences between groups (F3,24 = 3.43, P < 0.03). All lesioned groups showed lower ChAT activities, but the reduction reached statistical significance only in the BDNF-treated group ( P < 0.05, Newman-Keuls). ChAT activity on the intact side was not affected by the contralateral lesion or the treatment with trophic factors (frontal cortex: F3,24 = 0.29, P > 0.05; parietal cortex: F3,z4 = 0.49, P > 0.05).

3.2. Histological results The number and size of p75 N G F receptor immunoreactive neurons in the NBM are given in Table 2 and Fig. 1. The number of neurons on the lesioned side was significantly different between groups (F3,22 = 10.26, P < 0.0002). This was due to a reduction in all lesioned groups ( P < 0.01, Newman-Keuls), without any difference between these groups themselves ( P > 0.05, Newman-Keuls). The size of the neurons was also different between groups (F3,22 = 10.63, P <

350

Table 1 Effects of lesions of the N B M and treatment with N G F or B D N F on C h A T activity in the neocortex

Frontal cortex Control Cytochrome c BDNF NGF Parietal cortex Control Cytochrome c BDNF NGF

Lesioned side

Intact side

43.4 + 27.8 + 27.2 + 27.1 +

46.2 + 45.7 + 44.9 + 44.2 +

37.7 + 33.1 + 30.0+ 33.0+

0.9 1.7 * * 1.3 * * 1.3 * * 1.6 2.1 1.4 * 1.8

38.1 + 36.5 + 37.4+ 37.4+

2.2 1.5 0.5 1.9 0.7 0.7 1.3 1.1

C h A T activity is expressed as nmol of A C h formed per h per mg protein ( m e a n + S . E . M . , n = 7). * , * * Different from control, P < 0.05, 0.01 ( N e w m a n - K e u l s ) .

500

E .5

20o

•

150

N

•~

lo0 50 0

|

i

t

Control

Cyt. C

BDNF

w

NGF

group Fig. 1. Effect of treatment with N G F or B D N F on the size of remaining neurons following lesions of the NBM. Animals were treated for 4 weeks with NGF, B D N F or cytochrome c (3 /zg per day) by intraparenchymal infusion, starting 2 weeks after a unilateral lesion of the NBM. Data ( m e a n + S.E.M.) from at least 6 animals were included. * P < 0.05 vs. cytochrome c, N e w m a n - K e u l s ) .

A.J. Dekker et al. /Brain Research 639 (1994) 149-155

152

Fig. 2. In situ hybridization using the p75 NGF receptor probe. Dark-field photomicrograph showing a 1 × 1.5 mm region of the intact NBM adjacent to the ventral striatum; 50 grain clusters can be counted in the photographed field.

0.0002). This effect was d u e to an i n c r e a s e in size in t h e N G F - t r e a t e d g r o u p ( P < 0.01 vs. e a c h o f the o t h e r groups, N e w m a n - K e u l s ) . T h e n u m b e r o f n e u r o n s in t h e u n l e s i o n e d N B M was n o t a f f e c t e d (F3,23 = 2.83, P > 0.05). T h e size o f n e u r o n s on t h e intact side was as follows: control: 224.0 + 8.0, c y t o c h r o m e c: 238.4 + 6.9, B D N F : 243.1 + 7.8, N G F : 235.4 + 4.7; m e a n + s.e.m.,

90

!. o control

cyt. c BDNF group

NGF

Fig. 3. Effect of treatment with NGF, BDNF or cytochrome c on p75 NGF receptor mRNA expression in the lesioned NBM. Animals were treated for 4 weeks with NGF, BDNF or cytochrome c (3 /zg per day) by intraparenchymal infusion, starting 2 weeks after a unilateral lesion of the NBM. The number of grain clusters was counted on two sides in 4 brain sections. Data (mean + s.e.m.) from at least 6 animals were included. Cross-hatched bars, intact side; dark bars, lesioned side. * P < 0.05 vs. cytochrome c.

/xm2). T h e size was n o t significantly d i f f e r e n t b e t w e e n g r o u p s (F3,23 = 1.31, P > 0.05).

3.3. In situ hybridization In situ h y b r i d i z a t i o n o f t h e a n t i - s e n s e p r o b e for mRNAN~FR(p75 ) was visible as clusters of grains in t h e r e g i o n o f the N B M (Fig. 2, cf. ref. 24). T h e n u m b e r o f g r a i n clusters in t h e N B M , c o u n t e d u n d e r d a r k f i e l d microscopy, is given in Fig. 3. O n the l e s i o n e d side, t h e r e was a significant d i f f e r e n c e b e t w e e n g r o u p s ( H = 10.97, P = 0.01). This was d u e to r e d u c t i o n s in all t h e l e s i o n e d g r o u p s ( P < 0.05, M a n n - W h i t n e y U). H o w ever, t h e r e w e r e significantly m o r e g r a i n clusters in the N G F - t r e a t e d group, b u t n o t the B D N F - t r e a t e d group, c o m p a r e d to t h e c y t o c h r o m e - t r e a t e d g r o u p ( P < 0.05, M a n n - W h i t n e y U). O n t h e intact side t h e r e was no significant d i f f e r e n c e b e t w e e n g r o u p s (Fig. 3, H = 4.27, P > 0.05). T h e sense p r o b e s h o w e d no d i s c e r n i b l e hyb r i d i z a t i o n signal ( d a t a not shown).

4. Discussion 4.1. Local administration o f trophic factors A f t e r i.c.v, a d m i n i s t r a t i o n , B D N F is less effective t h a n a f t e r i n t r a p a r e n c h y m a l infusion [2], possibly b e -

A.J. Dekker et al. / Brain Research 639 (1994) 149-155

cause B D N F binds to the ventricle wall, which has a high concentration of its high affinity receptors, trk-B [29,47]. In contrast, the high affinity receptor for NGF, trk-A, is concentrated in basal forebrain neurons and N G F reaches the brain tissue even after a single i.c.v. injection [34]. Therefore, in this study, to avoid a confounded comparison between B D N F and NGF, trophic factors were administered directly into the parenchyma. The results for N G F can be compared with those of previous studies in which N G F was administered i.c.v., and where this treatment increased ChAT activity in the neocortex [9,13]. In the present study, cortical ChAT activity was not affected by NGF. Similarly, implantation of NGF-producing fibroblasts dorsal to the lesioned NBM does not affect cortical ChAT actvity [14]. On the other hand, both i.c.v, and intraparenchymal administration of N G F increases the size of remaining neurons in the NBM, and NGF-producing fibroblasts as well as i.c.v, administered N G F increase the ChAT-positive fiber staining in the ventral areas of the neocortex (refs. 9, 13, 14). This suggests that reinnervation of the target area is influenced qualitatively by the route of administration of the trophic factor. This effect could be due to obstruction of fiber growth by the graft or the cannula. However, Kawaja et al. [28] found that NGF-producing grafts, placed in the cavity of a fimbria-fornix aspiration lesion, allowed regrowing axons to pass and reinnervate the hippocampus. It is therefore more likely that local administration of N G F influences reinnervation from the NBM by tropic effects [27], as shown in vitro by Campenot [7]. The grafts in the experiment by Kawaja et al. [28] were placed along the reinnervation trajectory. It is not known if a different graft placement would also change the reinnervation in the case of the septo-hippocampal pathway. 4.2. Expression o f mRNANGFR(p75 )

B D N F had only a marginal effect on the m R N A for the p75 N G F receptor in NBM neurons, whereas N G F significantly increased expression of this mRNA. Since immunoreactive p75 N G F receptor in the NBM is only found in neurons [6,13], of which the number does not increase following treatment with N G F [9,13], the increased expression probably reflects an upregulation in neurons that previously had m R N A levels below the detection threshold. The upregulation of p75 by N G F has been shown in a variety of cell systems, both in vivo and in vitro (for review, see ref. 5). Since p75 binds N G F and B D N F with equal affinity [45,47], these resuits suggest that binding to this receptor is not sufficient for its own upregulation and that the extent of p75 upregulation is determined by high affinity receptors (trk-A, trk-B) or their interaction with p75 (cf. ref.

153

5). Such a mechanism may also differ across cell types, since B D N F does lead to p75 upregulation in C6-glioma cells [46]. 4.3. Histological results

In the present study, local infusion of 3/zg N G F per day resulted in a 32% increase in the size of remaining neurons in the NBM. This is in agreement with the results from a previous study, where i.c.v, treatment with N G F (10 /xg/day) increased neuronal size by 45% [9]. It can be estimated that a 4 ~g i.c.v, dose would have a similar effect as local infusion of 3 /xg, suggesting that local infusion is only slightly more effective in delivering N G F to the NBM. B D N F had no significant effect on neuronal size. This is in agreement with results by Kniisel et al.. [31] and Lapchak and Hefti [33], showing less pronounced effects of BDNF compared to N G F on cholinergic reinnervation of the hippocampus following partial fimbrial transections. However, these authors administered BDNF by i.c.v, injection, so that the smaller effects of BDNF might be the result of a lower availability. Morse et al. [39] showed that septal infusion of BDNF reduced the loss of medial septum neurons after a fimbria-fornix transection by 50%. However, the dose used (12 ~ g / day) was 40 times higher than the dose of NGF, needed to rescue 90% of the neurons [18]. Taken together, the findings from several studies suggest that B D N F is less effective than N G F as a neurotrophic factor for basal forebrain cholinergic neurons. Therefore, the role of BDNF in a region such as the neocortex is not clear. It is possible that trk-B receptors are concentrated on NBM neuron terminals, rather than the cell bodies. In this case, BDNF would be more effective when administered in the neocortex. Alternatively, the effect of B D N F on cholinergic neurons could be more pronounced in the presence of exogenous NGF. Finally, the main target of cortical B D N F might be a different neuronal population projecting to the neocortex, such as the dopaminergic neurons [26]. Acknowledgements. This study was supported by the VA Medical research Service,NSF (9010252),NIH (AG06088 and PO1AG10435), the Broad Foundation and the Margaret and Herbert Hoover Foundation. BDNF was provided by Dr. R.M. Lindsay, Regeneron Pharmaceuticals, Tarrytown,NY. The authors gratefullyacknowledgethe excellent technical assistance of Andy Chen (CHAT activity) and Thuy-Ann Vu (immunostaining).

5. References

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