Brain Research, 293 (1984) 305-311 305 Elsevier

Chronic Intraventricular Injections of Nerve Growth Factor Elevate Hippocampal Choline Acetyltransferase Activity in Adult Rats with partial Septo-Hippocampal

Lesions

F. HEFTI, A. DRAVID and J. HARTIKKA

Sandoz Ltd., Preclinical Research, 4002 Basel (Switzerland)

(Accepted June 21st, 1983)

Key words: choline acetyltransferase - - cholinergic neurons - - hippocampus - - septum - - nerve growth factor - - Alzheimer's disease

Nerve growth factor (NGF) was injected intraventricularly during 4 weeks into adult rats with unilateral partial lesions of the cholin- ergic septo-hippocampal pathway. On the lesioned side, NGF treatment elevated choline acetyltransferase (CHAT) activity up to 60% above the activity measured on the lesioned side of cytochrome c-treated controls. On the unlesioned side, NGF treatment in- creased ChAT activity only to an insignificant degree. ChAT activity in the septum of NGF-treated animals was increased by 60% as compared to controls. The NGF-induced increases on the lesioned side and in the septum were not accompanied by elevations in ace- tylcholinesterase (ACHE) activity. Furthermore, histochemical analysis revealed no difference in AChE staining pattern or intensity between NGF-treated and control animals. The lack of effect on AChE strongly suggests that the increases in ChAT activity in hippo- campus and septum are due to an elevation of ChAT activity within cholinergic neurons surviving the lesion rather than to a promotion of sprouting of cholinergic fibers.

INTRODUCTION

Nerve growth factor (NGF) is a protein essential

for development and maintenance of function of pe- ripheral sympathetic and sensory neurons (for review see ref. 32). Administration of N G F to rodents pro-

duces a hypertrophy of sympathetic neurons, where- as treatment with antibodies to NGF (anti-NGF) re-

sults in atrophy. Furthermore, N G F selectively in- duces the synthesis of tyrosine hydroxylase (TH) and

dopamine fl-hydroxylase in sympathetic ganglia. In contrast to these pronounced effects on peripheral sympathetic neurons, central catecholaminergic neu- rons are not affected by N G F or anti-NGF8,19, 26.

However, the following observations have suggested that NGF might play a role in the function of central

cholinergic neurons. First, N G F injected into the hip- pocampus of rats is taken up by nerve terminals and transported retrogradely to the cell bodies of cholin- ergic neurons in the medial septal nucleus and the nu- cleus of the diagonal band of Broca 26. Since recep- tor-mediated uptake is a prerequisite for specific ret- rograde transport 27, the above finding suggests the

existence of NGF-receptors on cholinergic terminals

in the hippocampus. Second, cells from the forebrain of fetal rats grown in aggregating cultures respond to

NGF with an increase in choline acetyltransferase (CHAT) activity 16. Third, destruction of the choliner-

gic input to the hippocampus results in an ingrowth of peripheral sympathetic fibers which matches the pre-

vious distribution of cholinergic terminals in the hip- pocampus 6,21,29. As peripheral sympathetic neurons

are known to react with fiber outgrowth to NGF, a similarity between the putative hippocampal signal and NGF has been postulated 7.

A recent study 11, designed to further investigate the role of NGF in the function of central cholinergic

neurons, confirmed that these cells react to exoge-

nous NGF. Repeated intraventricular administration of NGF to rats during their first postnatal week el- evated C h A T activity in the forebrain. Furthermore, NGF was shown to increase C h A T activity in cultures of dissociated neurons from the septal region. How- ever, this study failed to confirm the view that NGF is identical with an endogenous neurotrophic factor for central cholinergic neurons. Intraventricular injec-

0006-8993/84/$03.00 (~) 1984 Elsevier Science Publishers B.V.

306

tions of anti-NGF to neonatal rats did not reduce ChAT activity in the forebrain. Furthermore, the off-

spring of rats autoimmunized against N G F showed

no reduction in C h A T activity in forebrain areas. Despite the lack of evidence for a physiological

role of NGF in the function of central cholinergic

neurons, their ability to react to exogenous N G F is

worth further investigation in light of the fact that

these neurons seem to be specifically affected in Alz- heimer's disease. Post-mortem analysis of brains from such patients revealed a significant reduction in

ChAT activity in cortex and hippocampus (for review

see ref. 31). We therefore decided to study the ef-

fects of intraventricular administration of NG F in

adult rats with partial lesions of the cholinergic septo- hippocampal pathway. The septo-hippocampal pro-

jection was chosen because its anatomy is clearly de-

scribed and because the cholinergic fibers coursing through the fimbria are easily accessible for ie- sioning22,23, 28. We found that repeated intraventricu-

lar administration of N G F significantly elevated hip-

pocampai ChAT activity in animals with partial sep- to-hippocampal lesions. The increases in C h A T ac-

tivity were not accompanied by an increase in ace- tylcholinesterase (ACHE) activity, indicating that

NGF treatment elevated ChAT activity within re-

maining terminals but failed to promote sprouting of cholinergic fibers.

METHODS

Unilateral, partial transection of the fimbria was

performed using a slight modification of a method to be presented in detail elsewhere (Dravid and van Deusen, in preparation). Briefly, female rats (Wis-

tar, 200 g) were anesthetized with hexobarbital (100 mg/kg i.p.) and placed in a stereotaxic device. A

specially designed knife (8 mm long, 0.7 mm wide) was lowered into the brain at the antero-posterior level A6100 (according to the atlas of Koenig and KlippepS) and at a position of 1.0 mm lateral to the midline. The knife was then moved laterally to 5.0 mm lateral of the midline and retrieved at this po- sition. After performing the lesion, a cannula (inner diameter 0.3 mm) was inserted on the lesioned side of the brain into the lateral ventricle and permanent- ly fixed with dental cement. For intraventricular in- jections, a hypodermic needle attached to a Hamii-

ton syringe was inserted through the cannula into the ventricle. Control injections with cresyl violet re-

vealed rapid spreading of the dye into both lateral and the third ventricle.

Half of the animals were injected twice weekly during 4-10 weeks with 10 ~g of mouse submaxillary

gland NGF, control animals received equal amounts

of cytochrome c (a protein with similar molecular

weight and isoelectric point as NGF). Both proteins were dissolved in 5 gl of isotonic saline; the solutions

were injected intraventricularly at 1/~l/min, the nee-

dle was left in place for approximately 2 min after ter- mination of the injection.

Rats were decapitated 3 days after the last injec-

tion. The brains were removed and manually dis-

sected into septum, hippocampus, cortex, and 'rest of

PARTIAL TRANSSECTION OF FIMRRIA

INTRAVENTRICULAR INJECTION OF NGF DURING 4 WEEKS

=~

ChAT pmol/min/ pg protein

11.5

]] ILl 1 2 3 4

~ SEPTAL H I P P O C A M P U S TEMPORAl.

Fig. 1. Effect of intraventricular administration of NGF (10/~g twice weekly during 4 weeks) on ChAT activity in hippocampus of rats with unilateral partial lesions of the cholinergic septo- hippocampal pathway. Lesions were made by cutting the lat- eral two-thirds of the fimbria. Hippocampus was dissected into 4 pieces along its septo-temporal axis. Lightly stippled and open bars represent unlesioned and lesioned sides of cyto- chrome c-treated controls, respectively. The lesions resulted in a gradual reduction of ChAT activity from the septal to the tem- poral hippocampal pole (significantly different in pieces 2, 3, 4, P < 0.01). Heavily stippled and black bars represent unle- sioned and lesioned sides of NGF-treated animals, respec- tively. NGF treatment only slightly increased ChAT activity on the unlesioned side, wheres pronounced increases were seen on the lesioned side. Bars represent mean + S.E.M. (n = 14; significantly different from corresponding control by P < 0.01).

brain'. Hippocampus was further dissected into 4

pieces of equal size along its septo-temporal axis. Tis- sue pieces were immediately frozen on dry ice and

later homogenized in 50 mM Tris-HCl buffer, pH 6.0, containing 0.1% Triton X-100. Aliquots of the ho-

mogenate were used for the assay of C h A T activity

according to Fonnum ~0, A C h E activity according to Wilson et al. 34, TH activity by a modification of the method of Levitt et al. 20 as described by Edgar et al. 9,

and protein content according to Bradford 5. Some

brains were frozen intact and taken for histochemical

demonstration of A C h E by a modification of the method of Karnovsky 17. Non-specific cholinesterases

were inhibited by the inclusion of 30 g M tetra-isopro- pylpyrophosphoramide ( iso-OMPA) in the incuba-

tion medium.

Chemicals were of analytical grade and purchased

from Merck (Darmstadt, F .R.G.) , Serva (Heidel- berg, F .R.G.) , or Sigma (St. Louis, MO). Mouse NGF was purified from adult mouse submandibular

glands according to the method of Bocchini and An- geletti 3, with the modifications described by Suda et al.30.

RESULTS

Unilateral partial fimbrial transection (affecting

307

the lateral part of the fimbria) reduced C h A T and

A C h E activities in the 3 most temporal regions of the

hippocampus analyzed (Fig. 1, Table I). The reduc-

tion in C h A T activity progressively increased from

27% in region 2 to 77% in region 4. This finding is in

agreement with results from anatomical studies showing that the cholinergic fibers innervating the

temporal hippocampus course through the lateral part of the fimbria22, 28.

Repeated intraventricular injections of N G F dur-

ing 4 weeks to animals with partial fimbrial transec-

tion resulted in increases of hippocampal C h A T ac- tivities on the lesioned side of 20--60% above the ac-

tivities measured on the lesioned side of control ani-

mals (Fig. 1). The relative increases were largest in

the temporal parts of the hippocampus. On the unle- sioned side, only small increases (approximately

10%) were observed which did not reach statistical

significance. ChAT activity in the septum of NGF- treated rats was increased by 60% as compared to

control animals (Table II). C h A T activity in cortex and 'rest of brain' was not affected by N G F treat-

ment.

Contrary to the marked elevations in C h A T activ- ity, the activity of A C h E was not affected in any of the brain areas investigated. As illustrated by similar

reductions in C h A T and A C h E activity after fimbrial

TABLE I

Effect of NGF treatment (10 I~g intraventricularly twice weekly during 4 weeks) on AChE activity in the brain of rats with partial transec- tions of the fimbria

Values are in pmol/min/~g protein; mean + S.E.M., n = 10-14.

Controls N G F

Hippocampus 1 (septal pole)* 15.9 + 0.6 14.8 + 0.5 unlesioned side 3 17.5 + 0.7 15.8 + 0.5

3 20.4 + 0.8 18.2 + 0.9 4 (temporal pole) 24.1 + 1.0 22.3 + 0.9

Hippocampus 1 (septal pole) 15.7 + 1.1 14.5 + 1.2 lesioned side 2 11.6 __+ 2.6** 12.2 + 0.7**

3 7.9 + 1.6"* 8.7 + 1.0"* 4 (temporal pole) 5.1 + 0.7** 5.6 + 0.8**

Septum 23.4 + 4.4 20.0 ___ 2.3

Cortex 14.5 + 2.8 14.6 + 1.5

'Rest of brain' 46.5 + 5.0 49.2 + 5.4

* Numbers of hippocampal pieces correspond to those used in Fig. 1. ** Significant reductions as compared to the corresponding tissue piece on the unlesioned side, P < 0.01.

308

Fig. 2. AChE-stained coronal sections through the lateral hippocampus of rats with partial septo-hippocampal Lesions. A: unlesioned side. B: lesioned side of an animal treated with NGF (10/~g twice weekly into the lateral ventricle) during 10 weeks. C: unlesioned side. D: lesioned side of a cytochrome c-treated control. The lesions largely reduce AChE staining, the reduction being most pro- nounced in the CA3 field. The sections reveal no difference between NGF-treated and control animals.

TABLE II

Effect of NGF treatment (10 pg intraventricularly twice weekly during 4 weeks) on ChAT activity in the brain of rats with partial transections of the firnbria

Values are pmol/min/pg protein; mean + S.E.M. ; n = 10.

Controls NGF

Septum 0.343 + 0.028 0.550 + 0.037** Cortex 0.101 + 0.008 0.104 + 0.005 'Rest of brain'* 0.464 + 0.028 0.514 + 0.042

* Brain tissue remaining after dissection of hippocampus, cortex and septum;

** Significantly different from controls, P < 0.001.

lesions (cf. Fig. 1, Tab le II, and ref. 4) and s imilar

d e v e l o p m e n t a l pa t te rns of the two enzymes 23, h ippo-

campal A C h E is to a large ex ten t local ized within

chol inergic te rminals and synapses. A n increase in

C h A T activity wi thou t a c o n c o m i t a n t e leva t ion of

A C h E activity the re fo re speaks against a p r o m o t i o n

of sprout ing by N G F .

A possible de layed increase in A C h E activity was

assessed in a g roup of animals which was t r ea ted with

N G F or cy toch rome c for 10 weeks. B iochemica l ly ,

similar increases in C h A T activity in sep tum and hip-

p o c a m p u s were obse rved as in animals t r ea ted for 4

309

weeks. The activity of AChE again remained un- changed in all brain areas (data not shown). Histo- chemical analysis of such animals confirmed the ef- fectiveness of the fimbrial transections: AChE stain- ing was strongly reduced in the temporal parts of the hippocampus, whereas only slight reductions were observed in the septal parts. In the temporal hippo- campus, staining intensity was most strongly reduced in the CA3 field. No differences in staining pattern or intensity were observed between NGF-treated and control animals (Fig. 2).

Repeated intraventricular injections of NGF dur- ing 4 weeks failed to affect central catecholaminergic neurons, inasmuch as TH activity was not increased in 'rest of brain', which contained locus coeruleus, substantia nigra, hypothalamus and striatum, i.e. areas of origin and termination of dopaminergic and noradrenergic neurons. TH activity in 'rest of brain' of control and NGF-treated animals was 0.013 + 0.001, and 0.013 _+ 0.002 pmol/min//~g protein, re- spectively (m _+ S.E.M., n = 10). TH activity in supe- rior cervical ganglia was 0.142 _+ 0.020 in controls and 0.114 _+ 0.015 pmol/min/pg protein (m _+ S.E.M., n -- 9) in NGF-treated animals, suggesting that NGF injected intraventricularly did not reach the periphery in sufficient amounts to affect the sym- pathetic nervous system. TH activity in hippocampus was below the limit of detection of the method used. This prevented us from confirming biochemically the ingrowth of peripheral sympathetic fibers into the hippocampus, which has been shown to occur histo- chemically after lesions of the cholinergic input to the hippocampus6,21, 29.

DISCUSSION

The results of the present study indicate that NGF is able to elevate ChAT activity in cholinergic septo- hippocampal neurons after partial lesion of their pathway. The increase in ChAT activity most proba- bly is due to an activation or induction of the enzyme within cholinergic terminals rather than to a promo- tion of sprouting of cholinergic fibers. This is sug- gested by the fact that NGF treatment did not elevate AChE activity and failed to increase staining inten- sity of AChE in histochemical sections. The results of the present study are in line with those obtained on neonatal rats, where NGF also increased ChAT ac-

tivity without affecting AChE activity 11. Further-

more, the concept that NGF's action on cholinergic septo-hippocampal neurons is limited to an elevation of ChAT activity is supported by findings obtained on cultures of dissociated septal neurons, where NGF increases ChAT activity without affecting survival or fiber outgrowth from cholinergic neurons (unpub- lished observations).

Mouse NGF, as was used in the present study, is known to be contaminated by small amounts of re- nin2,12. Even though renin-free NGF was not used in this study, there is strong circumstantial evidence suggesting that the observed increases in ChAT ac- tivity are due to NGF rather than to the renin con- tamination: (i) in neonatal rats, increases in ChAT activity were obtained with both mouse-NGF and NGF isolated from bovine seminal plasma, which is free of renin contaminationlaA4; (ii) in cultures of dis- sociated septal neurons, ChAT activity was elevated by both mouse NGF and bovine NGF. Furthermore, the effect of mouse NGF was blocked by specific anti- bodies to NGF 11.

In contrast to its ability to elevate ChAT activity in cholinergic septo-hippocampal neurons during de- velopment and after partial lesions of their pathway, NGF failed to affect these neurons in intact adult ani- mals and on the control sides of rats with unilateral partial fimbrial transection (ref. 11 and Fig. 1). The mechanism is unknown by which a partial septo-hip-

pocampal lesion induces the cholinergic neurons to become responsive to NGF. A partial lesion might in- duce the formation of NGF receptors or might couple existing receptors to a functional process. The fact that specific retrograde transport of NGF has been demonstrated in intact brains of adult rats suggests that the receptors are present also in adult animals. It is also unclear whether the NGF-mediated increases in ChAT activity take place in neurons with com- pletely transected axons or, alternatively, in neurons which lost only part of their terminal network due to lesion.

In developing rats, NGF treatment increases hip- pocampal as well as cortical ChAT activity 11, indicat- ing that cholinergic neurofis of both the septo-hippo- campal projection and the projection from the nucle- us basalis to the cortex can respond to NGF. These projections apparently are homologous to those spe- cifically affected in Alzheimer's disease25.31.33. It is

310

still controversial whether the decreases in hippo-

campal and cortical ChAT activity observed in such

patients reflect degeneration of cholinergic neurons

or a reduction of ChAT activity within these neu-

rons 24.33. In the latter case, NGF or compounds mim-

icking its action might help to restore ChAT to nor-

mal levels. However, since NGF does not elevate

ChAT activity in intact brains of adult animals, it is

not certain that cholinergic neurons in brains of pa-

tients with Alzheimer 's disease respond to NGF.

It has been speculated that Alzheimer 's disease

might be caused by a lack of a specific neurotrophic

factor for cholinergic neurons 1 and, furthermore,

that such a factor might be identical to NGF 15. How-

ever, as was discussed in more detail in a recent pub-

lication 11, it is unlikely that the NGF-media ted in-

creases in ChAT activity reflect a physiological role

of NGF in the central nervous system. (i) Ant i -NGF

does not decrease ChAT activity after intraventricu-

lar administration to neonatal rats. (ii) The offspring

of rats autoimmunized against N G F show no de-

crease in central ChAT activity. (iii) In cultures of

dissociated septal neurons, in which N G F increases

ChAT activity, ant i -NGF fails to reduce basal ChAT

levels. The data are compatible with the view that

NGF mimicks the effect of an endogenous NGF-like

factor, which is immunologically different from

NGF. The lack of such an NGF-like factor might be

responsible for the development of Alzheimer 's dis-

ease. However, until the demonstrat ion of an endo-

genous factor, it cannot be excluded that the N G F re-

ceptors mediating the increases in ChAT activity

might have no physiological role.

ACKNOWLEDGEMENT

We are grateful for the excellent technical assis-

tance of Mrs. B. Burckhardt and Miss C. Messer.

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