NGF-Mediated increase of choline acetyltransferase (ChAT) in the neonatal rat forebrain: Evidence for a physiological role of NGF in the brain?

Developmental Brain Research, 9 (1983) 45-52 Elsevier

45

NGF-Mediated Increase of Choline Acetyltransferase (CHAT) in the Neonatal Rat Forebrain: Evidence for a Physiological Role of NGF in the Brain? H. GNAHN, F. HEFTI 1, R. HEUMANN, M. E. SCHWAB and H. THOENEN Max-Planck-lnstitute for Psychiatry, Department of Neurochemistry, Martinsried/Munich (F. R. G.) and t Sandoz Ltd., Preclinical Research, Basel (Switzerland) (Accepted January 18th, 1983) Key words: nerve growth factor - - forebrain cholinergic neurons - - neonatal rat - - choline acetyltransferase

Neonatal rats received intraventricular injections of mouse submandibular gland nerve growth factor (NGF) on days 1, 3, 5 and 7 postpartum. After killing the animals at day 8, activities of choline acetyltransferase (CHAT), acetylcholine esterase (ACHE) and tyrosine hydroxylase (TH) were measured in different brain areas. NGF treatment increased ChAT activity in the septal area by 78%, in the hippocampus by 30%, and in the cortex by 73% relative to control animals. No increase was observed in other brain areas. The elevation of ChAT activity was not accompanied by an increased activity of ACHE, and the concomitant 30-40% increase of TH activity observed in the cortex and brainstem was abolished after immunosympathectomy, reflecting the ingrowth of peripheral sympathetic peripheral fibers into the central nervous system (CNS) in response to centrally administrated NGF23. In adult rats, repeated injections of NGF over 4 weeks caused a small but statisticallysignificant increase of ChAT activity (15%) in the forebrain. In contrast, repeated intraventricular or intracortical injections into neonatal rats of large amounts of purified antibodies against mouse NGF (anit-NGF) failed to reduce ChAT activity in the same forebrain areas. Moreover, the offspring of rats autoimmunized against mouse NGF showed no reduction of ChAT activity in the brain, even though the TH activity was reduced by 76% in the superior cervical ganglia (SCG) of these animals. Antibodies against mouse NGF were also without effect on ChAT activity in cultures of dissociated septal neurons, though these cells also responded to NGF with an increase in ChAT activity. Anti-NGF blocked the effect of exogenous NGF but failed to reduce basal ChAT activity in these cultures. It is concluded that exogenous NGF can affect forebrain cholinergic neurons during their development. NGF does not seem to be identical with an endogenous neurotrophic factor produced by hippocampus or neocortex acting on cholinergic neurons of the forebrain. INTRODUCTION

to N G F do not affect central dopaminergic and noradrenergic neurons11.22.31. However, the following ob-

Nerve growth factor ( N G F ) is a protein that is essential for the d e v e l o p m e n t and m a i n t e n a n c e of function of sympathetic and sensory neurons in the peripheral nervous system (for review see T h o e n e n and Barde38). In s y m p a t h e t i c ganglia of rats and mice N G F causes h y p e r t r o p h y , and administration of antibodies against N G F results in atrophy. In addition to these general effects, N G F in these ganglia specifically increases the synthesis of tyrosine hydoxylase (TH) and d o p a m i n e fl-hydroxylase ( D B H ) , the key enzymes in the synthesis of n o r e p i n e p h r i n e , the transmitter in s y m p a t h e t i c neurons. The physiological significance of N G F is d e m o n s t r a t e d by the fact that antibodies against N G F lead to a drastic decrease in T H and D B H levels. In contrast to these p r o n o u n c e d effects on catecholaminergic neurons in the p e r i p h e r a l nervous system, N G F and antibodies

servations suggest that N G F might play a role in forebrain cholinergic neurons similar to that seen in peripheral adrenergic neurons. Schwab et ai.31 found that N G F injected into the rat h i p p o c a m p u s is specifically taken up by nerve terminals, and t r a n s p o r t e d retrogradely to the cholinergic neurons of the medial septal nucleus and the nucleus of the diagonal band of Broca. This strongly suggests the presence of N G F - r e c e p t o r s on the cell m e m b r a n e s of these cholinergic neurons. R e c e p t o r - m e d i a t e d uptake is the prerequisite for a specific r e t r o g r a d e transport, and indeed, all N G F - r e s p o n s i v e neurons have been shown to internalize and transport N G F (for review see Schwab and Thoenen32). A direct biochemical response to N G F was in fact d e m o n s t r a t e d by Honegger and Lenoirl9: cells from the forebrain of foetal rats grown in aggregation cultures r e s p o n d to low

0165-3806/83/$03.00 © 1983 Elsevier Science Publishers B.V.

46 doses of N G F by an increase in ChAT activity. Finally, destruction of the cholinergic input to the hippocampus results in an ingrowth of peripheral sympathetic neurons which matches the previous distribution of cholinergic terminals in the hippocampUS6,27,34. The results of these lesion experiments indicate that peripheral sympathetic neurons and central cholinergic neurons might respond to the same neuronotrophic signal produced by the hippocampus. As peripheral sympathetic neurons are known to react with fiber outgrowth to NGF, a similarity of the putative hippocampal signal to N G F has been postulated 8. To obtain further information on the role of N G F in the central nervous system, and to test the hypothesis that N G F might act as a neurotrophic factor for central cholinergic neurons, we have examined the effects of intraventricular administrations of N G F and antibodies against N G F on ChAT activity in the rat brain. Our results indicate that N G F is able to increase ChAT levels in forebrain cholinergic neurons. They, however, do not support the concept that N G F is identical to an endogenous neurotrophic factor produced in the hippocampus and neocortex and acting on cholinergic neurons of the forebrain. MATERIALS AND METHODS Mouse N G F was purified from adult mouse submandibular glands according to the method of Bocchini and Angeletti 3, with the modifications described by Suda et al. 37. Bovine N G F was purified from seminal plasma as described by Harper et al. ~7. Sheep antibodies against mouse N G F 37 were purified by affinity chromatography as described by St6ckel et al. 36. Of these antibodies, 200/ag inhibited the biological activity of 10 yg of mouse N G F in vitro. Chemicals used were of analytical grade and, if not otherwise stated, were purchased from Merck (Darmstadt, F.R.G.) or Sigma (St. Louis, MO, U.S.A.).

frontal cortex. The animals were immobilized by cooling them on ice and were then placed in a specially fitted form made of dental moulding material2¢x Stereotaxic coordinates were derived from the atlas of Sherwood and Timiras 33. The location of the injected material was checked by making injections of cresyl violet into age- and weight-matched animals: the dye was found to be evenly distributed in the lateral and third ventricles after intraventricular injections. N G F (30/~g in 3 ,ul of Dulbecco's phosphatebuffered saline, PBS) and purified antibodies against NGF (200 pg in 3 pi) were injected on postnatal days 1, 3, 5 and 7. Injections were made at 1/~l/min. [,ittermate control animals received equal amounts and volumes of cytochrome C or sheep IgG. Adequate diffusion of antibodies to N G F throughout the brain was controlled by making injections of fluorescein isothiocyanate-labeled antibodies, and the subsequent examination of frozen sections under the fluorescence microscope. The litter size was always ~< 10 pups. On day 8 postpartum, the pups were killed by decapitation. Their brains were removed and dissected on ice into the 'septal area', hippocampus, cortex, and 'rest of the brain'. The 'septal area' contained the septum and the medioventral part of the telencephalon, including the nucleus of the diagonal band of Broca, and the cell group equivalent to the nucleus basalis Meynert of higher mammals, i.e. the areas containing the cell bodies of the cholinergic neurons projecting to the hippocampus and the cortex 5~7,9-28.29. In some litters, the superior cervical ganglia (SCG) were also dissected. Tissues were frozen on dry-ice and stored at - - 7 0 °C for biochemical analysis. Adult female Wistar rats were chronically implanted with cannulae into the right lateral ventricle. NGF (20~g in 10/A of PBS) was injected twice weekly over 4 weeks; control animals received PBS. Animals were killed by decapitation, and septal area, hippocampus, and cortex were dissected as above.

Immunosympathectomy Intracerebral injections Mouse NGF, bovine NGF, or antibodies against NGF were injected into the third ventricles of neonatal rats (Wistar) using a 5/tl syringe (Hamilton) attached to a stereotaxic apparatus. Antibodies to NGF were in addition injected bilaterally into the

Immunosympathectomy was performed by systemic (subcutaneous) application of 100 mg/kg body weight of antibodies to NGF on the first day postpartum. According to Goedert et al. 1~the effect of antibodies to N G F is reversible on addition of N G F up to 48 h, Therefore we began the intraventricular NGF injections only on day 4 postpartum.

47

Auto-immunization Female rats (Wistar) were immunized against mouse N G F according to Johnson et al. 20. After two boosts at monthly intervals, anti-NGF titers of 1000-1500 were attained as evaluated by the abolition of the fiber outgrowth from PC12 cells in response to 10 ng/ml. NGF. The animals were then mated. The offspring were killed on the first day after birth and their brains and SCGs were dissected as described above• Age- and weight-matched litters from nonimmunized mothers were taken as controls.

05-

•

C"

[~

Enzyme determinations Samples of brain tissues and cultured septal neurons were homogenized in 5 mM Tris-HCl buffer pH 7.5, containing 0.1% Triton X-100. Aliquots of the homogenate were used for the enzyme assays: (i) ChAT according to Fonnum13; (ii) T H by a modification of the method of Levitt et al. 24 as described by Edgar et al.12; (iii) A C h E according to Wilson et al. 39. Proteins were measured by the method of Bradford 4 using bovine y-globulin as standard. RESULTS

Effects of intraventricular injections of NGF on ChA T activity. Mouse N G F injected intraventricularly on days 1, 3, 5 and 7 postpartum led to an increased specific activity of ChAT in the forebrain of rats at day 8 (Fig. 1). The increase was most pronounced in the septal

Mouse - NGF

03-

I--

02

Primary cultures of dissociated septal neurons Brains from foetal rats (embryonic day 17) were removed and the septal area was dissected. Tissue pieces were incubated in 0.5% trypsin (Gibco) and 0•05% DNAase (type I, Sigma) in PBS for 15 min at 37 °C. The pieces were washed twice with L-15 medium (Gibco, supplemented with 9 g/l of glucose). Cells were dissociated by mild trituration in the same medium using a fire-polished Pasteur pipette• Cells were plated at a density of 200,000-500,000 cells per well in polyornithine-coated multiwell plates of 16 mm diameter (Falcon). Cells were cultured for 8 days in a modified L-15 medium (L-15/CO2 growth medium TM omitting Methocel, but containing 5% horse serum, 1% rat serum, and 10 btM cytosine arabinonucleoside in a 5% CO2 incubator.

I-"-] Controls

0.4-

H

;~

-X-

0.1 :/3 Sepia! Area Hippocampus Cortex

Rest of Brain

Fig. 1. Effect of intraventricular injections of mouse NGF on ChAT activity in the brain of neonatal rats; 30 #g of NGF were injected at postnatal days 1, 3, 5, and 7; controls received equal amounts of cytochrome C. The animals were killed at postnatal day 8. Data show 1 out of 4 corresponding experiments. Columns represent means _+ S.E.Ms. (n = 5). Statistical significance: *P < 0.05; **P < 0.001, relative to controls. area (78%) followed by cortex (73%) and hippocampus (30%). No significant increase was observed in the thalamus, brainstem and cerebellum, ('rest of brain') (Fig. 1). Total brain wet weight and protein content were not affected• The preparation of mouse N G F used in this study contains some renin activity, which has been shown to be responsible for the increases in ornithine decarboxylase15 and sodium appetite 2 following intracerebral N G F injections. In order to exclude the possibility that renin was responsible for the elevated ChAT activity, we compared the effect of mouse N G F with that of bovine NGF. Bovine NGF, which is purified from the seminal plasma, contains no renin activity16. The injection of bovine N G F caused increases of ChAT activity in all 3 forebrain areas that were virtually identical to those elicited by mouse N G F (Fig. 2). Injections of mouse N G F into the ventricles of adult rats caused only small, though statistically significant (P < 0.05) increases of ChAT activity in the forebrain (Table I). These increases were only observed after multiple injections of N G F over 4

4~

o51 D Contro,s

0.4-

]

30"

2_

[]

Controls

[]

~ , ~ , ~ - NG~

Bovine- NGF

i! /

20

I

•

!

i !!:

_1_ W ¢J

, t ,o2 g,

'

r7

(31-

)i 10

I

j• )

1

Septal Area Hippocnmpus Cortex

,,J

Septol Area Hippocompus Cortex

R~t of Brain

Fig. 2. Effect of intraventricular injections of bovine N G F on C h A T activity in the brain of neonatal rats. A n i m a l s were treated as in the legend to Fig. 1. C o l u m n s represent m e a n s + S.E.M. (n = 4). Statistical significance: * P < 0.05: * * P < 0.001, relative to controls.

weeks; a single injection failed to elevate ChAT activity (data not shown). Effects of intraventricular injections of N G F on A C h E and TH activities Intraventricular injections of NGF into neonatal rats producing increases in ChAT activity in the forebrain failed to affect the specific activity of AChE (Fig. 3). However, an increase of 30-40% in specific TH activity was observed in the cortex and the TABLE I

Increased ChAT activity in the brain of adult rats after the intraventricular administration of mouse NGF over 4 weeks Animals were implanted with a eannula in the right lateral ventricle; 2 0 ~ g N G F was injected twice weekly. Controls were injected with vehicle (phosphate-buffered saline), nmoi/min/mg protein, m e a n s + S.E.M.

Septum

Hippocampus Cortex

Controls(n=7) 0.617+0.015 0.504+0.030 N G F 8 × 20/~g 4 weeks (n = 8) 0.707+0.033* 0.576+0.039 % increase 15% 14% * Different from controls, P < 0.05 (t-test).

0.344_+0.015 0.393_+0.020* 14%

Rest of Brain

Fig. 3. Effect of intraventricular injection of bovine N G F on the activity of A C h E in the brain of neonatal rats. Results were obtained from the same animals as those shown in Fig. 2.

brainstem after NGF injections concomitant to a 180% increase of specific T H activity in the SCGs (data not shown). Immunosympathectomy prior to the intracerebral injections of NGF completely abolished the increases of TH activity in the CNS. These increases of TH activity therefore reflect the ingrowth of peripheral sympathetic fibers into the CNS, a phenomenon known to occur in neonatal rodents after intracerebral injections of large doses of NGFn3. Effect of administration of antibodies to NGF on ChA T activity in the brain Intraventricular or intracortical injections of equal amounts of affinity-purified antibodies to mouseNGF failed to reduce ChAT activity in all brain areas (Fig. 4A and B). This result cannot be explained by insufficient penetration of the antibodies into the brain tissue, since fluorescein isothiocyanate-conjugated antibodies injected intraventricularly or intracortically could be detected in all brain areas by fluorescence microscopy (data not shown). Moreover, intracerebral injections of antibodies to NGF decreased by 60% the specific TH activity in the SCGs, presumably due to leakage of antibodies from the central injection site into the peripheral circulation (sheep IgG injected: TH activity = 1.021 _+_.0.029:

49 TABLE II

ChAT activity in the brains and TH activity in the superior cervical ganglia (SCG) of newborn rats exposed in utero to antibodies against NGF Control animals were age- and weight-matched offspring from non-immunized mothers. Septum, hippocampus and cortex: CHAT, nmol/min/mg protein, means + S.E.M. SCG: TH, nmol/min/mg protein, means + S.E.M.

Controls (n = 5) Prenatal exposure to antibodies to NGF (n = 5)

Septum

Hippocampus

Cortex

SCG

0.032 + 0.004

0.011 + 0.001

0.020 + 0.001

0.316 + 0.018

0.037 + 0.003

0.011 + 0.001

0.018 + 0.001

0.154 + 0.021"

* Different from controls, P < 0.001.

0.3 A ._

intra ventricular

f~

Controls Anti - Mouse

activity when compared with control litters (Table - NGF

0.2

/,

Lc ' i I-¢(.3

ble II). The lower C h A T activity in 1-day-old as compared to 8-day-old animals (Fig. 1) reflects the devel-

0.1-

o p m e n t of the cholinergic septo-hippocampal projection during the first postnatal week28, 29. As in the pre-

Septal Area Hippocampus 0.3IB

_j_

intra-

cortical _~

,

II). The effectiveness of the antibody to N G F transfer through the placenta in these animals was indicated by the fact that T H activity per SCG was reduced by 76%, and T H specific activity by 51% (Ta-

0.~

Cortex

F~

30

Rest at

Brain

natal rat, the b l o o d - b r a i n barrier for antibodies is not yet establishedl, inadequate penetration of the antibodies can be excluded.

Controls

Anti - Mouse -NGF

J~

IE 0.1

Effects o f N G F and antibodies to N G F on C h A T activity in primary cultures o f dissociated septal neurons N G F treatment of cultured septal n e u r o n s for 7 days caused a more than 2-fold increase in C h A T specific activity (Table III). This increase could be blocked by antibodies to N G F , but these antibodies

i,/i Septal Area Hippocampus

Cortex

Rest of

Brain

Fig. 4. Effects of intraventricular (A) or intracortieal (B) injections of antibodies to mouse NGF on ChAT activity in the brain of neonatal rats; 200/~g of purified antibodies were injected at postnatal days 1, 3, 5, and 7; animals were killed one day after the last injection. Both treatments reduced TH specific activity in SCGs by 60%. Columns represent means _+S.E.M. (n = 5).

antibodies to N G F injected: T H activity = 0.416 + 0.016; nmol D O P A formed/min/mg protein; P < 0.001).

Prenatal exposure to antibodies against N G F The offspring of rats that were a u t o i m m u n i z e d against N G F showed no decrease in central C h A T

TABLE III

Effects of NGF and antibodies against NGF in primary cultures of dissociated rat septal neurons, which were prepared from foetal rats of 17 days gestation The cells were cultured for 8 days at a density of 200,000-500,000 cells per well (16 mm diameter). Antibodies to NGF: 2/zl/ml of a sheep antiserum against NGFsT. Means + S.E.M., n = 6.

Controls NGF 100 ng/ml Antibodies to NGF NGF 100 ng/ml + antibodies to NGF

Protein content (Izg)

ChAT (nmol/min/mg protein)

123 + 25 124 + 15 124 + 14

0.185 + 0.013 0.477 + 0.018" 0.202 + 0.008

130 + 9

0.217 + 0.022

* Different from control, P < 0.001.

50 failed to reduce the basal ChAT activity in cultures not given NGF. DISCUSSION lntraventricular injections of N G F into rats during their first postnatal week elevate ChAT activity in septal area, hippocampus, and cortex, i.e. in the areas of origin and termination of the cholinergic septo-hippocampal pathway, and the projection from the basal nucleus to the n e o c o r t e x 5,9,21,25,2s,29. Following the demonstration of specific retrograde transport of NGF by the septo-hippocampal neurons, which strongly suggested the presence of receptors for NGF or a closely related molecule on these neurons 31, the results of the present study show that these cholinergic CNS neurons are also able to respond to N G F with a well-defined biochemical response in vivo. In contrast, we found no NGF-mediated ChAT increase in the brainstem motoneurons, in agreement with the absence of retrograde transport of NGF in spinal motoneurons 35. Even though large amounts of N G F (30/xg per injection) were given intraventricularly, the effect on ChAT activity is not due to the well-known renin contamination of 2.5 S NGF, since the same response was also obtained with renin-free bovine N G F 16. As the amount of N G F that actually penetrates into the brain after intraventricular administration is unknown, it is not possible to establish an exact doseresponse relationship between N G F concentration and effect on ChAT activity in vivo. However, in aggregate cultures of forebrain neurons 19 and of dissociated septal neurons (Table III), N G F elevated ChAT activity when present at concentrations similar to those eliciting biochemical responses from peripheral sympathetic neurons (10-9-10 -10 M18,38). In the peripheral sympathetic nervous system of neonatal rodents, the administration of NGF not only elevates the activity of the key enzymes for the synthesis of norepinephrine, T H and DBH, but also causes enhanced neuronal survival and increased production of nerve fibers. All these effects are detectable in vivo and in vitro 3s. It remains to be shown whether the increase in ChAT activity observed after intraventricular injections of N G F to newborn rats is accompanied by an increase in the density of the cholinergic fibers innervating hippocampus and cortex.

In this study, the increase of C h A T activity was nol accompanied by an elevation of AChE activity, which, in the hippocampus, is mainly contained in cholinergic axons 2s,29. In the in vitro studies, where ChAT was increased by NGF, there was no increase in survival of AChE-positive cells by NGF (unpublished results). Whereas the presence of NGF-receptors, the specific retrograde axonal transport of NGF~ and the specific increase of ChAT by NGF in forebrain cholinergic neurons clearly indicate a responsiveness of these CNS neurons to NGF, the results of intracerebral injections of antibodies against NGF into newborn rats, of such antibody applications to septal cultures, and of ChAT measurements in newborn rats whose mothers have high titers of circulating antibodies against N G F indicate that this responsiveness does not reflect a physiological role for N G F in these neurons during their normal development and function. Under all these experimental conditions antibodies against NGF failed to show any effect on ChAT levels although, in the same animals, the levels of TH and of total cell protein were severely depressed in the peripheral nervous system (SCG). The pronounced effects of the antibodies in the peripheral nervous system show that these antibodies, directed against mouse submandibular gland NGF, are clearly able to neutralize the peripheral endogenous rat NGF, as has been previously s h o w n 14,2°. Therefore, if an NGF-like molecule exists in the rat CNS, it would have to display the same receptor-binding properties as the 'classical' NGF molecule, but the CNS factor would have to differ completely from NGF in its immunological characteristics. A similar situation (only 10% of antibody cross-reactivity) has been found so far only for NGF from two different sources in very different species: mouse submandibular gland NGF and bovine seminal plasma NGF 1". The presence of NGF-receptors and a well-defined NGF response, as well as the results from lesion experiments (sprouting of peripheral sympathetic fibers into cholinergically denervated hippocampus or cortex) justifies the search for such a brain NGF-like factor. However, it has to be borne in mind that these NGF-receptors possibly have no obvious functional role. Only the demonstration of the ligand itself can resolve the question of the physiological relevance of the response of central cholinergic neurons to NGF.

51 However, up until now, reports on the occurrence of NGF activity in these brain regions have been highly

ACKNOWLEDGEMENTS

controversial10,30

The authors thank Dr. G. H a r p e r for supplying bovine NGF. They are grateful for the excellent technical assistance of Mrs. M. Reitmayer and Ms. C. Messer. H . G . is a fellow of the Deutsche Forschungsgemeinschaft.

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