Synaptic loss in cognitively impaired aged rats is ameliorated by chronic human nerve growth factor infusion

Neuroscience Vol. 68, No. 1, pp. 19 27, 1995

~

Pergamon

Elsevier ScienceLid

IBRO

0306-4522(95)00099-2

Printed in Great Britain

S Y N A P T I C LOSS IN C O G N I T I V E L Y I M P A I R E D A G E D R A T S IS A M E L I O R A T E D BY C H R O N I C H U M A N N E R V E GROWTH FACTOR INFUSION K. S. C H E N , E. M A S L I A H , * M. M A L L O R Y and F. H. G A G E University of California, San Diego, School of Medicine, Department of Neurosciences, La Jolla, CA 92093-0624, U.S.A.

Abstract--lnthe present study, we assessed the synaptic changes in aged impaired and unimpaired rats, and the effect of exogenous human nerve growth factor administration on behavioral activity and synaptic density. Human nerve growth factor was administered into the rat ventricles with a cannula connected to an osmotic pump in adult, aged impaired and unimpaired rats. Behavioral performance was evaluated in the Morris water maze. Aged impaired rats had an 18 + 4% decrease in the number of synaptophysinimmunoreactive presynaptic terminals as compared to aged unimpaired rats. After a continuous four-week human nerve growth factor, the aged impaired rats displayed a significant 16+ 3% increase in the number of synaptophysin-immunoreactive presynaptic terminals in the frontal cortex, as compared to aged impaired rats treated with vehicle. This increase correlated with an improvement in water maze performance (r = - 0 . 7 4 , P <0.001). Measurements of synaptophysin-immunoreactive presynaptic terminals in other cortical and subcortical regions did not show any statistically significant difference or correlations among the various groups. These results support the possibility that nerve growth factor mediates the induction of other trophic factors which, in turn, might potentially produce a sprouting response of non-cholinergic fibers that ameliorate the cognitive deficits in impaired, aged rats.

d e f i c i t s . 2"833'17'28"38 These studies were focused on the

The density o f synaptic p o p u l a t i o n s in the brain p r o b a b l y depends on the balance between synaptic elimination and plasticityJ During the aging process, synaptic contacts are lost and the mechanisms responsible for restructuring the dysfunctional microcircuitries are less effective. In the rat, approximately 30% o f synaptic input into the cortex is lost during agingJ Cognitive deficits emerge in some aged rats, and to date these deficits have been correlated with a decrease in cellular markers o f cholinergic neurons in the basal forebrain. 9 F u r t h e r m o r e , the cognitive deficits and cellular changes can be partially reversed by exogenous nerve growth factor ( N G F ) infusion. 8 Recent reports have s h o w n that exogenous N G F administration into the rat brain increased the spine density in the aged rat frontal cortex, 2~ as well as the n u m b e r o f cholinergic synaptic terminals in the decorticated rat. ~5 Previous studies have also shown that exogenous administration o f NGF" rescues forebrain neurons after f i m b r i a - f o r n i x lesionJ ~'39 Thus, the exogenous administration o f growth factors might partially reverse the age-associated synapse and neuronal loss, as well as ameliorate m e m o r y

use o f murine N G F 2.5s. F o r the present study we assessed the effect o f exogenous administration o f h u m a n N G F ( h N G F ) on behavioral activity and the numbers o f s y n a p t o p h y s i n - i m m u n o r e a c t i v e ( S Y N - I R ) presynaptic terminals in aged impaired and unimpaired rats.

EXPERIMENTALPROCEDURES Water maze testing

The female Fisher 344 rats (Harlan Sprague-Dawley) used in this study were divided into four groups: (i) aged (26 months old), behaviorally impaired rats that received hNGF infusion, (ii) aged impaired rats that received vehicle only, (iii) aged unimpaired rats and (iv) three-month-old adult rats. Briefly, as described previously, s each rat was given four consecutive trials a day. Collection of data was automated by an on-line video tracking system. The following variables were analysed: total distance a rat swam, the swim distance in each quadrant and the latency to find the platform. All animals were first pretested for two weeks on a visible platform version of the water maze. This screening procedure eliminated about 30% of the aged rats. Aged animals were designated "impaired" if they were able to learn the visible platform task but, on the last three days of testing, had a mean swim distance that was greater than two standard deviations above the mean swim distance of the adult rats. If the aged rats had a mean swim distance that was less than two standard deviations above the mean swim distance of the adult rats, they were designated as "unimpaired". The aged impaired animals were assigned equally to groups that would receive infusions of either recombinant hNGF (aged NGF) or vehicle alone (aged

*To whom correspondence should be addressed. Abbreviations: AD, Alzheimer's disease; CHAT, choline

acetyltransferase; DB, diagonal band; hNGF, human nerve growth factor; MS, medial septum; NBM, nucleus basalis magnocellularis; NGF, nerve growth factor; PBS, phosphate-buffered saline; P75-R nerve growth factor receptor; SYN-IR, synaptophysin-immunoreactive. 19

20

K. S. Chen et al.

vehicle). The infusion vehicle was a phosphate-buffered artificial cerebrospinal fluid containing 150ram NaC1, 1.8 m M CaCI2, 1.2 m M MgSO4, 2.0 m M K2HPO 4, 10.0 m M glucose and 0.01% autologous rat serum, pH7.4. The h N G F (courtesy of Syntex-Synergen Research) was solubilized in the vehicle and administered at a concentration of 50#g/ml. The rats received a total of 10.51tg of h N G F during the five weeks of infusion. Ai'ter two weeks of chronic infusions, all the animals were retested on the hidden platform water maze for five days (retest 1). At that point, new filled p u m p s were implanted and the animals were infused for two more weeks followed by five days of testing (retest 2).

Surgical procedures and tissue preparation Aged impaired rats were deeply anesthetized and a 28-gauge stainless steel cannula (3280P osmotic p u m p connector cannula, Plastic Products, Inc.), embedded in a dental acrylic stabilization platform, was implanted into the right lateral ventricle (coordinates: AP - 0 . 5 ram, ML 4 . 0 m m and DV - 4 . 5 r a m relative to bregma). The minipump was placed subcutaneously in the neck/shoulder area and changed every two weeks. The animals were perfused transcardially with 50 ml of 0.1 M phosphatebuffered saline (PBS; pH 7.4) followed by 250 ml of 10% buffered formalin for 10 15 min. The brains were removed, postfixed overnight in 10% buffered formalin and cryoprotected in 30% sucrose for two days. Forty-micrometerthick sections were cut on a sliding microtome and stored in cryoprotectant at 20'C until immunohistochemical processing.

Nerve growth ['actor receptor immunocytochemistry image analysis

and

In order to verify that the h N G F administered was biologically active in the rat, every fourth tissue section was immunostained with a mouse anti-rat monoclonal antibody to N G F receptor (P75-R) obtained from cell culture supernatant (gift of E. M. Johnson). Tissue sections were processed using the avidin biotin-peroxidase method with nickel sulfate intensification, as described previously, t2 A computerized image analysis technique (Cue-2 Image Analysis System, Olympus) was employed for quantification of the number and size of P75-R-immunolabeled cells using a magnification of x 33. Cells were sized and counted if their cross-sectional area was greater than 70/~m ~'. The number of cells in sections that were immunohistochemically processed for the P75-R was s u m m e d to obtain the total number of cells per side in the medial septum (MS), diagonal band (DB) and nucleus basalis magnocellularis (NBM) for each animal. The size of each counted P75-R-positive cell was averaged to obtain the mean cell size for each of the three regions.

Synaptophysin immunocytoehemistry In order to determine the effect of aging and h N G F on presynaptic terminals in the neocortex, hippocampal, septal region and basal ganglia of the animals which were tested behaviorally and in which we performed the P75-R immunocytochemistry, sections were immunolabeled with the monoclonal antibody against the synaptic protein marker synaptophysin (SY38, Boehringer-Mannheim, Indianapolis, IN)J TM Previous studies in denervated rodent models, 24'35as well as immunoelectron microscopic studies] 3 support the validity of this approach to evaluate the presynaptic terminal density. Briefly, as described previously, 24"27 blind-coded 40-/~m-thick coronal sections (Plate 13, Paxinos Atlas) from all four groups were washed in PBS, pH 7.4, followed by Triton X-100, blocked with normal horse serum and incubated overnight at 4 C with synaptophysin (1 #g/ml). The next day, the sections were washed in PBS and incubated in fluorescein-conjugated anti-mouse

immunoglobulin St Louis, MO). taneously under from each group results.

G made in horse (Vector Laboratories, All the sections were processed simulthe same conditions. Sets of three slides were used to assess the reproducibility of

Laser scanning conjocal microscopy and image analysis Blind-coded immunolabeled sections were viewed with a Zeiss x 63 (N.A. 1.4) objective on a Zeiss Axiovert 35 microscope with attached laser scanning confocal microscope M R C 600 (Bio-Rad, Watford, U.K.). 27 From each case serial optical sections were obtained from the frontoparietal region, hippocampus, caudate p u t a m e m ventral pallidum, intermediate lateral septal nucleus, ventral lateral septal nucleus and primary olfactory cortex. All series of sections from each case were digitized under standardized conditions, maintaining the same levels of gain, aperture and contrast, Quantitative analysis of the merged serial optical sections displaying the SYN-IR presynaptic terminals was carried out as described previously, 24'2627 with the aid of the Image 1.43 software program running on a Macintosh IIci. Measurements were made of the number, size and percentage of the area occupied by presynaptic terminals in a selected field. These values were expressed relative to the area of the region. For each case, four fields from each region were recorded and averaged. Each field represented 5800/~m 2 and the results were expressed as the number of SYN-IR presynaptic terminals per 100 #m2,

Statistical analysis After all data were collected, the blind code was broken and statistical analyses of the results were performed using the STAT VIEW II software package running on a Macintosh IIci personal computer. Comparisons a m o n g the different groups were done with a two-tailed, unpaired Student's t-test and with the one-way ANOVA. Values were expressed as mean _+ S.E.M. Pearson Product-Moment correlations and the correlation coefficient (r) 29 were calculated with simple linear regression analyses.

RESULTS

Behavioral alterations during aging and nerve growth factor infusion

qfter

human

In t h e p r e s e n t s t u d y , we a s s e s s e d t h e c h a n g e s in p r e s y n a p t i c t e r m i n a l s in a g e d i m p a i r e d a n d u n i m p a i r e d r a t s a n d t h e effect o f e x o g e n o u s h N G F a d m i n i s t r a t i o n o n b e h a v i o r a l activity a n d the numbers of SYN-IR presynaptic terminals, hNGF w a s a d m i n i s t e r e d i n t o t h e ventricles o f a d u l t , a g e d i m p a i r e d a n d a g e d u n i m p a i r e d rats. B e h a v i o r a l perf o r m a n c e w a s e v a l u a t e d in t h e M o r r i s w a t e r m a z e . All a d u l t r a t s were able to l e a r n to e s c a p e o n t o t h e h i d d e n p l a t f o r m in t h e w a t e r m a z e w i t h i n t h r e e d a y s o f testing. S e v e n o f 16 a g e d r a t s h a d a m e a n s w i m d i s t a n c e o n t h e last t h r e e d a y s o f t e s t i n g o n t h e h i d d e n p l a t f o r m t a s k t h a t w a s less t h a n t w o s t a n d a r d deviations above the mean swim distance of the a d u l t rats a n d were d e s i g n a t e d as a g e d u n i m p a i r e d . T h e r e w a s n o s i g n i f i c a n t difference b e t w e e n t h e a g e d u n i m p a i r e d a n d t h e a d u l t r a t s o n a n y p r e i n f u s i o n test d a y ( d a y s 1-10). T h e r e m a i n i n g n i n e a g e d rats h a d mean swim distances greater than two standard deviations above the mean swim distance of the adult rats, a n d were a s s i g n e d to t h e a g e d i m p a i r e d g r o u p .

Human NGF ameliorates synapse loss

Summary

of water

maze

testing

z~ = • o

2250 2000 1750

E

21

Aged NGF Aged VEH Aged UNIMP Adult

1500

i-

1250 0 u rm

~3

1000 750

500 250 0

Visible test

H;dden' test

Retest 1" Retest 2'

Fig. 1. Mean swim distance on postinfusion retest l (days 24 and 25) and retest 2 (days 33 and 35) in the water maze. AG NGF, aged impaired group that received NGF infusions; AG VEH, aged impaired group that received infusions of vehicle alone; AG UNIMP, aged behaviorally unimpaired group; ADULT, adult group. In the visible test no statistically significant differences were observed. In the hidden test, aged NGF and aged vehicle were different from the aged unimpaired and adult (*P < 0.002). In Retest l, no statistically significant differences were observed, while in Retest 2 aged NGF, aged unimpaired and adult rats were different from aged vehicle (**P < 0.001, one-way ANOVA).

These aged impaired rats were subsequently divided into an h N G F - i n f u s e d group (aged N G F ) and a vehicle-infused group (aged vehicle). There was no significant difference between the aged N G F and aged vehicle groups on any preinfusion test day. There was a significant difference between both groups of aged impaired animals (aged N G F and aged vehicle) and the aged unimpaired and adult animals (P < 0.05; Fig. 1). Following two weeks of chronic infusions there was still no significant difference between the aged N G F and aged vehicle rats on postinfusion retest 1, either on day 24 or on day 25 (Fig. 1). However, on the first day of postinfusion retest 2 (day 33), there was a significant difference between the aged N G F and aged vehicle rats (P < 0.05; Fig. 1). There was no significant difference between either the adult or the aged unimpaired rats and aged N G F rats on day 33. In contrast, a significant difference remained between the mean swim distance of aged vehicle rats and adult or aged unimpaired rats. On the next two days of retest 2 (days 34 and 35), the aged vehicle animals

were able to relearn the spatial location of the hidden platform and were no longer significantly different from the aged N G F animals. Therefore, h N G F can ameliorate the performance of aged impaired animals on the Morris water maze by facilitating retention of information learned during chronic infusion (Fig. 1).

Nerve growth factor receptor immunoreactivity after human nerve growth factor infusion in the aged rat In order to confirm that the administered h N G F had a biological effect in the rat, sections immunostained with anti-P75R were evaluated with an Image analysis system. Aged impaired rats infused with N G F displayed a trend toward an increase in the mean size of P75-R-positive cells in the MS, DB and the N B M (Table 1). In the MS, a statistically significant difference was observed between aged N G F and adult rats (P < 0.05). In the DB, aged N G F rats were statistically different compared to aged unimpaired and adult rats (P < 0.05). In the N B M , aged N G F rats were statistically different compared to aged unimpaired rats (P < 0.05; Table 1). There was no

Table 1. Mean nerve growth factor receptor-positive cell size Cell size (/~m ~) Group Aged NGF Aged vehicle Aged unimpaired Adult *P < 0.05.

MS 179.9+ 13.1" 168.0 _+ 20.4 158.5 + 7.8 146.4 + 5.7

DB 187.5__+7.0" 159.8 ± 16.9 157.8 _ 4.9 165.7 _ 7.8

NBM 192.6+ 10.1" 167.1 + 23.2 156.0 _+ 13.0 178.1 + 7.9

K. S. Chen et al.

22

Table 2. Mean nerve growth factor receptor-positive cell number Total number of cells per side Group Aged NGF Aged vehicle Aged unimpaired Adult

MS

DB

NBM

639-+ 127.7 635 ± 85.2 718±35.9 857 _+ 64.8

1677_+103.3 1655 ± 216.0 2076_+97.0* 2059 _+ 80.0

1256 ± 190.7 1255 + 64.2 1454_+140.0 1503 ± 136.9

*P < 0.05.

significant difference between cells on the left a n d right sides. Thus, h N G F can increase the size of P75-R-positive cells in the basal forebrain but has no effect u p o n the total n u m b e r of cells, which is reduced in aged rats t h a t exhibit behavioral deficits on the water maze. Tables 1 a n d 2 show the size and m e a n n u m b e r , respectively, of P75-R-positive cells for the adult, aged u n i m p a i r e d a n d aged impaired ( b o t h N G F and vehicle) animals in the MS, D B and N B M . There were no significant differences in P75-R-positive cell n u m b e r or size between aged u n i m p a i r e d a n d adult rats in any of the three areas examined (Table 2). The n u m b e r of P75-R-positive cells tended to be greater

in the adult and aged u n i m p a i r e d animals in all three regions, a l t h o u g h significant differences ( P < 0.05) were observed only in the DB (Table 2). There were no significant differences between the n u m b e r of cells c o u n t e d in aged N G F a n d aged vehicle rats (Table 2). Additionally, there was no significant difference between the n u m b e r of cells on the left and right sides. No t u m o r s were observed grossly in the brains of any of the aged animals.

Human nerve growth factor administration induces sprouting that ameliorates cognitit, e deficits In the frontoparietal region a n d primary olfactory cortex, a n t i - s y n a p t o p h y s i n i m m u n o l a b e l e d

Fig. 2. Laser scanning confocal imaging of SYN-IR in aged impaired rats treated with NGF (A) and aged impaired rats treated with vehicle (B). (C) Synaptophysin-immunolabeled distended neuritic processes were observed in the periventricular region in aged impaired rats treated with NGF. (D) A threedimensional reconstruction of the synaptophysin-immunolabeled abnormal neuritic and synaptic elements shows thal many of these distended boutons were derived from the same neuritic process.

Human N G F ameliorates synapse loss

a homogeneous population of presynaptic terminals evenly distributed throughout the neuropil (Fig. 2A, B). Quantification of the SYN-IR presynaptic terminals showed that the frontoparietal region of aged impaired rats treated with vehicle (aged vehicle) displayed an 1 8 + 4 % decrease in the numbers of SYN-IR presynaptic terminals, as compared to aged impaired rats treated with N G F and aged unimpaired rats (Table 3, Fig. 2A, B). The number of SYN-IR presynaptic terminals in the frontoparietal region of aged rats that were initially impaired and were treated with N G F for four weeks (aged N G F ) was similar to that of aged unimpaired rats (Table 3). Aged unimpaired rats and aged N G F rats showed a slight decrease in the number of SYN-IR presynaptic terminals in the frontoparietal region compared to adult rats (Table 3). SYN-IR in the molecular layer of the hippocampal dentate gyrus was characterized by a fine granular pattern distributed in layers. In the primary olfactory cortex and in the inner and outer molecular layer of the hippocampus, no statistically significant differences in SYN-IR presynaptic terminals was observed among the four groups (Table 3). In the caudate-putamen complex and ventral lateral septal nucleus, anti-synaptophysin immunolabeled an evenly distributed dense synaptic population. In contrast, in the ventral globus pallidus and midseptal nucleus the SYN-IR presynaptic terminals were aligned along the dendrites and around neuronal soma. The mid-septal nucleus of the aged NGF-treated rats showed a trend toward an increase in the number of SYN-IR presynaptic terminals; however, this difference was not statistically significant (Table 3). The SYN-IR presynaptic terminals in other subcortical areas analysed were not significantly different among the four groups (Table 3). In the periventricular region adjacent to the septal nuclei, the aged N G F rats displayed clusters of tortuous, abnormal, SYN-IR presynaptic boutons (Fig. 2C, D). Serial section analysis and reconstruction showed that several of these clusters of dilated terminals were continuous, with distorted SYN-IR neurites (Fig. 2C, D). Linear regression analysis between the water maze performance and the number of SYN-IR presynaptic terminals in cortical and subcortical areas showed a strong correlation between the frontoparietal region and improved performance in aged rats (r = - 0.74, P < 0.001) and four weeks (r = - 0.76, P < 0.001) after N G F infusion (Fig. 3). Other cortical and subcortical regions did not show significant correlations with the water maze performance at any point of testing. Among regions analysed, only SYN-IR presynaptic terminals in the ventral lateral septal nucleus correlated with the density in the mid-septal nucleus (r = 0.83, P < 0.001). The measurements of SYN-IR presynaptic terminals among other cortical and subcortical regions analysed were not significantly correlated.

23

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DISCUSSION

tion, 26'27"34 and is a good indicator of the dynamic

The present study showed that in aged impaired rats treated with vehicle the number of SYN-IR presynaptic terminals in the frontoparietal region was lower compared to aged unimpaired and adult rats, while N G F increased the numbers of SYN-IR presynaptic terminals, and that these changes correlated with water maze performance. Consistent with these results, a recent study has shown that age-dependent decrease in SYN-IR in the rat neocortex could be prevented by rearing under an enriched environment. 12 Alterations in SYN-IR due to aging and/or N G F administration could be interpreted as the result of: (i) overall changes in synaptic density populations, (ii) changes in the numbers of synaptic vesicles per terminal and (iii) changes in the amount of synaptophysin per synaptic vesicle. For the present study, we showed that the overall numbers of SYN-IR presynaptic terminals were modified during aging and by NGF, but that the levels of SYN-IR per terminal were preserved, supporting the contention that changes in SYN-IR indicate overall changes in synaptic numbers. Furthermore, previous studies have shown that SYN-IR per terminal is preserved during aging, 25 denervation24'35 and neurodegenera-

changes in synaptic populations. During normal aging, in humans and other animals, there is a variable decrease in synaptic content in the cortex. L32It has been hypothesized that the decline in cognitive abilities during aging might be related to synaptic loss and loss of cholinergic projections to the neocortex. Previous studies have shown a decrease in choline acetyltransferase (CHAT) activity in aged rats, 22 as well as a loss of neurons in the NBM. l° In Alzheimer's disease (AD), normal aging accounts for 5 10% of the 45% average synaptic loss observed. 34 Furthermore, the degree of synaptic loss was strongly correlated with the severity of the dementia, as assessed by tests of global cognition. There has been extensive speculation as to whether N G F infusion might have a therapeutic effect in AD, both by enhancing cholinergic system survival and by inducing a sprouting response in the neocortex. 2~ Nevertheless, it is not clear if N G F administration in AD might induce more damage that repair. 21 It is possible that N G F administered in normal aged individuals with small behavioral deficits might have a beneficial effect in improving cognition.

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Human NGF ameliorates synapse loss

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Fig. 4. Schematic representation of the hypothetical mechanisms by which NGF could induce an amplification of the sprouting response in the aged rat neocortex. In the adult unimpaired animals (normal), the basal nucleus neurons (BNN) project to the neocortical neuronal targets (NCN), which in turn project to other cortical targets. During aging, some basal nucleus neurons die (solid ball) and others are atrophied (hatched ball), resulting in a decrease of cholinergic input into the neocortex which, in turn, results in a trans-synaptic degeneration of the neocortical neuronal targets. NGF cannot recover dead basal nucleus neurons, but it is capable of rescuing partially impaired neurons, which in turn results in an increase in the number of cholinergic terminals in the neocortex. The trans-synaptic effect of this cholinergic sprouting is the recovery and, consequently, the sprouting of presynaptic terminals of the neocortical neuronal targets.

Exogenous administration of N G F in aged rats improves behavioral performance on the water maze and correlates with an increase in the numbers of S Y N - I R presynaptic terminals in the frontoparietal region. These results are consistent with previous reports showing that a four week infusion of N G F into aged rat ventricles reverses changes in neocortical spines, characterized by an increase in spine number similar to the density seen in young controls. 28 Other studies have reported that, in the decorticated adult rat, intraventricular administration of N G F for seven days induced an increase in C h A T - i m m u n o r e a c t i v e varicosities and an increase in size of cholinergic boutons and in the number of synaptic contacts. 15 Furthermore, N G F administration into aged rats has been shown to affect their behavioral performance, 8'j° stimulating cholinergic transmission. 39 N G F infusion also improves spatial m e m o r y in aged rats and hypertrophy of P75-R- and ChAT-positive cells in the basal forebrain, s In most of these studies it has been suggested that improved performance is related to increased cholinergic activity in the cortex, j6~37In the present study, as well as in other studies, N G F induced at least a 16 _+ 3% or greater increase in the number of S Y N - I R presynaptic terminals in the neocortex. In contrast, lesion to the N B M in the aged rat induced a greater synaptic loss in the neocortex 5 than would be predicted from the known percentage of terminals that are cholinergic in the neocortex. 33 Furthermore, lesions of the basal forebrain in neonatal mouse not only result in severe retardation of cholinergic markers in the neocortex, but also in damage to other neuronal populations. 18 Therefore, a sprouting response in the neocortex greater than 5 - 8 % 33 might suggest that: (i) N G F induces sprouting of non-

cholinergic fibers; (ii) N G F mediates induction of other trophic factors which in turn induce a sprouting response of non-cholinergic fibers; (iii) a partial cholinergic reinnervation induced by N G F promotes sprouting of adjacent neurons (Fig. 4); or (iv) N G F might induce cholinergic hyperinnervation. Previous studies have shown that N G F increases the ratio of amyloid precursor protein-695 to amyloid precursor protein-751 m R N A in the basal forebrain. 17 In young adult rats, chronic N G F infusion produces an increase in amyloid precursor protein m R N A , P75-R m R N A , P75-R immunoreactivity and hypertrophy of ChAT-immunoreactive neurons/4 In PC-12 cells, N G F treatment stimulates the secretion of high levels of two Kunitz protease inhibitor containing amyloid precursor protein subtypes (120,000 and 125,000 molecular weights). 3~ N G F has also been found to induce neuritic outgrowth and sprouting via growthassociated protein-43 production. 6"7"2°The N-terminal region of amyloid precursor protein at low doses induces trophic activity characterized by increased neuritic branching and increased neuronal survival. 3'4° This indicates that N G F might exert an indirect trophic effect in non-cholinergic neurons by stimulating amyloid precursor protein biosynthesis 3° or other putative trophic factors. CONCLUSIONS

The present results reveal a selective decrease in the number of S Y N - I R presynaptic terminals in the frontoparietal cortex of aged rats, which correlated with a spatial learning deficit. Furthermore, chronic N G F infusion improved spatial learning in the cognitively impaired rats, increased cholinergic cells and induced synaptic sprouting selectively in the frontal

26

K.S. Chen et al.

cortex. The causal relationship between these correlations is suggested by related research, and the clarification o f these relationships will likely reveal much a b o u t the basic mechanisms o f age-related function, degeneration and regeneration in the CNS.

authors would like to thank M. L. Gage for her help in preparing the manuscript. We thank the members of Syntex-Synergen Research for the human recombinant NGF and for technical assistance. This work was supported by grants from the National Institute on Aging (AG10435, AG06088) and the Broad Family Foundation to F. H. G. and AG10689, AG05131 to E. M. Acknowledgements--The

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