The effects of nerve growth factor on the development of septal cholinergic neurons in reaggregate cell cultures

Neuroscience Vol. 29, No. Printed in Great Britain

I, pp. 209-223,

1989

0306-4522/89 $3.00 + 0.00 Pergamon Press plc 0 1989 IBRO

THE EFFECTS OF NERVE GROWTH FACTOR ON THE DEVELOPMENT OF SEPTAL CHOLINERGIC NEURONS IN REAGGREGATE CELL CULTURES J. HSIANG,* A. HELLER,? P. C. HOFFMANN, W. C. MOBLEY~ and B.‘H. WAImR*t§ Departments of *Pathology and tPharmacologica1 and Physiological Sciences, The University of Chicago, Chicago, IL 60637, U.S.A., SDepartment of Neurology and the Neuroscience Program, The University of California at San Francisco, San Francisco, CA 94143, U.S.A. Abstract-Recent

studies suggest that nerve growth factor is present within the central nervous system where it may exert selective trophic effects on cholinergic neurons. We have measured the effects of nerve growth factor on septal cholinergic neurons in three-dimensional reaggregating cell cultures, a system which closely simulates the cellular environment in situ. Septal cells obtained from 15day-old mouse embryos were dissociated into a single cell suspension and then allowed to reaggregate in culture in a rotary incubator shaker. After 17 days in culture, half of the reaggregates from a flask were sonicated for measurement of choline acetyltransferase activity, and the remaining reaggregates were processed for acetylcholinesterase histochemistry. Addition of nerve growth factor to medium containing septal reaggregates resulted in greater than a three-fold increase in choline acetyltransferase activity and in the number of acetylcholinesterase-positive cells, as well as an enhancement in the staining of acetylcholinesterase-positive fibers. All of these effects of nerve growth factor could be neutralized by antibodies to nerve growth factor. In order to evaluate the possible role of endogenous hippocampal-derived nerve growth factor, antiserum to nerve growth factor was added to the culture media containing septal-hippocampal coaggregates. After 21 days in culture, the presence of nerve growth factor antibodies did not qualitatively affect the pattern or density of cholinergic fibers observed. Synapse formation between cholinergic axons and hippocampal target cells was still in evidence as revealed by electron microscopy. However, there was o) and cholinergic cell number (30%) when a modest decrease in choline acetyltransferase activity (ZOO/ compared with coaggregates grown in culture medium either without nerve growth factor antiserum or with non-immune serum. The magnitude of these effects was markedly less than the effects observed when exogenous nerve growth factor was added to septal cells grown alone in reaggregate culture. These results suggest that nerve growth factor may play a role during central cholinergic development, but that additional trophic mechanisms are likely to be required.

Nerve growth factor (NGF) is a neurotrophic factor which is required for the development and maintenance of sympathetic and sensory neurons of the peripheral nervous system.30,48 It is released by target tissues and binds to specific receptors on the innervating nerve endings. 22However, the actions of NGF are not limited to the peripheral nervous system. Recent evidence suggests that NGF may also function as a neurotrophic factor for central cholinergic neurons. First, following intraventricular injection in neonatal rats12.36 or addition to cultures of striatal cell~‘~ or reaggregating telencephalic cells,” NGF increases the activity of choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine.

§To whom correspondence should be addressed. Abbreviations: ACh, acetylcholine; AChE, acetylcholinesterase; acetyl-CoA, a&y1 co-enzyme A; ChAT, choline acetyltransferase; DAB, 3.3’-diaminobenzidine: DFP. diisopropylfluorophosphate; DMEM, DulbecceVogt modification of Eagle’s medium; E, embryonic day; EDTA, ethylenediaminetetra-acetic acid; iso-OMPA, tetraisopropylpyrophosphoramide; mRNA, messenger ribonucleic acid; NGF, nerve growth factor; P, postnatal day; PBS, phosphate-buffered saline; S-H, septal-hippocampal; T,, triiodothyronine.

Second, the levels of NGF and its mRNA in the CNS correlate with the location and developmental time course of cholinergic innervation.‘.24.2*,43 Relatively high NGF levels are found both in the septum, which contains the cholinergic cell bodies, and in the hippocampus, which receives heavy cholinergic innervation from the septum. High levels of NGF mRNA are found in the hippocampus relative to the septum,24 suggesting that NGF is synthesized in the target regions of the magnocellular cholinergic neurons, taken up by their terminals, and then transported retrogradely to their cell bodies; a situation analogous to that of the peripheral nervous system. Third, NGF receptors are found to be selectively located in areas occupied by cholinergic neurons of the basal forebrain as demonstrated by immunoprecipitation with a monoclonal antibody to the NGF receptor protein’7.46,47 and by binding of radioiodinated NGF.4’ Finally, intraventricular administration of NGF has been reported to promote survival of septal cholinergic neurons following fornix-fimbrial transection.‘6,27*52 In spite of the evidence supporting a physiological role for NGF as a target cell-derived cholinergic neurotrophic factor, there are certain questions about 209

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the function of NGF in the CNS that remain to be answered. Although existing evidence suggests that NGF prevents retrograde degeneration following axotomy of adult cholinergic neurons,16 what role it plays in supporting the development of central cholinergic neurons is unclear. Previous studies addressing this question in vitro have reported conflicting findings. For example, Hefti et al. reported that although addition of NGF to embryonic day 17 rat septal monolayer cultures resulted in an increase of ChAT activity, there was no apparent effect on either cholinergic cell numbers or fiber proliferation.15 In contrast, recent studies have shown that application of NGF greatly increases the number of septohippocampal projections in organotypic co-cultures from the postnatal day 7 septum and hippocampus.” One possible explanation for this discrepancy is that the monolayer culture experimentsi employed less mature septal cell populations which have not as yet interacted with their hippocampal target cells. In the organotypic co-culture experimentslo the tissues were derived from postnatal animals at a time when septal neurites had migrated into the hippocampus. It is therefore possible that NGF “responsiveness” might require cues resulting from a septal-hippocampal cellkell interaction. It is noteworthy that the NGF effects on central cholinergic neurons recorded to date have largely involved administration of exogeneous NGF. However, the crucial role of endogeneous NGF in the peripheral nervous system has been most clearly demonstrated by the effect of NGF antiserum administered to newborn mammals, which resulted in a massive destruction of sympathetic nerve cells.3’ Unfortunately, similar deleterious effects were not elicited in the central system with either intraventricular or intracortical administration of anti-NGF antibodies.” As a result it was concluded that NGF has only limited physiological function in the development of this system. Contrary to these findings, by injecting antibodies to NGF in embryonic animals it was possible to demonstrate a reduction of ChAT activity.40 In addition, administration of NGF antibodies to septal-hippocampal explant cultures decreases the number of septal cholinergic fibers projecting to the hippocampus.” The latter experiments thus argue for a physiological role of endogenous NGF. We have previously studied the effects of hippocampal target cells on the developing septal cholinergic neurons in reaggreating tissue culture.” We demonstrated that hippocampal target cells promote the survival as well as the differentiation of the innervating septal cholinergic neurons.“*” Based on the recent hypothesis that NGF is a hippocampalderived cholinergic trophic factor,24*25the present study employed the dissociated cell-reaggregate system to examine the effects of NGF on developing septal cholinergic neurons, especially its effect on cholinergic cell number. The septal cells were derived

from 15day-old mouse embryos, a stage just following neurogenesis in this area.* At this early stage, there is no septal cholinergic projection to the hippocampal target cells,35 and thus the septal cholinergic cells probably have not been subjected to target cell signals. Preliminary results of these studies have been presented earlier.49 In addition, the effect of anti-NGF antibodies on the interaction between septal cholinergic neurons and their target cells was examined. EXPERIMENTAL PROCEDURES

Materials The embryos used in these experiments were from pregnant C57BL/6J mice (Jackson Laboratories). The pregnant mice were housed with a constant light-dark cycle of 12 h and fed a breeding diet of mouse chow containing 10% fat (Teklab). All solutions used in tissue culture were filtered through 0.22nm filters (Millipore Corporation) and stored in sterile bottles at 4°C. Fetal bovine and horse sera, penicillin-streptomycin (5000 units penicillin, 5000 pg streptomycin/ml), trypsin, Basal Medium Eagle’s, DulbeccoVogt modification of Eagle’s Medium (DMEM, catalog number 320-1965) insulin, triiodothyronine (I,), hydrocortisone, transferrin and trace elements mix were all obtained from Grand Island Biological Company. Vitamin A, vitamin E, vitamin B,, , biotin, lipoic acid, linoleic acid, sodium selenite, 3,3’-diaminobenzidine (DAB), tetraisopropylpyrophosphoramide (iso-OMPA), and diisopropylfluorophosphate (DFP) were all obtained from Sigma. DNase was purchased from Cooper Biomedical. 2.5S-NGF antiserum was purchased from Collaborative Research, Inc. (catalog number: 40015). Mouse NGF was purified by ion exchange chromatography and characterized as previously described.37 Dissection and cell culture The dissection of the embryonic mouse brain and the techniques for reaggregating the embryonic cells have been previously described.*O Briefly, septa (source of cholinergic cells) and hippocampi (source of target cells) were removed from the 15day mouse embryos. Like tissues were combined and dissociated into single cells by treatment with 0. I % trypsin followed by gentle flushing through a fine-bore Pasteur pipette. The number of cells was counted with a hemocytometer and either 10 million septal cells, or 5 million septal cells and 5 million hippocampal cells, were placed into 25-ml Erlenmeyer flasks containing 3.5 ml of initial culture medium at pH 7.4. The initial culture medium consisted of Basal Medium Eagle’s plus 10% (v/v) fetal bovine serum, 1% (v/v) of the penicillin-streptomycin solution and 0.0025% (w/v) of the DNase. The flasks were placed in a rotatory incubator shaker at 80 rpm and 37°C. After 24 h, when initial aggregation had taken place, the initial culture medium was replaced with fresh medium supplemented with horse serum in place of fetal bovine serum. The medium was changed every 2 days. At the sixth day in culture, the serum-supplemented medium was replaced with serum-free, chemically-defined medium*O which was changed every 2-3 days until the day of harvest. Treatment of septal reaggregates

with nerve growlh factor

Septal reaggregates were prepared from 15-day-old mouse embryos as described above. On the sixth day in culture, the day on which the medium was changed to defined medium, NGF was added to the media to yield final concentrations of either 10, 50 or 100 ng/ml. The same concentrations of NGF were included in each medium change thereafter. Control cultures received normal medium without the addition of NGF. In some experiments, NGF antiserum

Effects of nerve growth factor on development of cholinergic neurons

211

or non-immune serum (normal rabbit serum) was added (final concentration = 1: 200 dilution in both cases), along with long/ml NGF, to the septal reaggregates. After a total of 17 days in culture, half of the reaggregates from the flask were sonicated for measurement of ChAT activity (see below). The remaining reaggregates were processed for acetylcholinesterase (AChE) histochemistry as described below.

experimental samples), and corrections were made for counting efficiency (approximately 50%) using a computergenerated quench curve. Results are expressed as pmol of [3H]ACh formed/min/mg protein.

Diisopropylfluorophosphate

Preparation

pharmacohistochemistry

In septal reaggregates treated with NGF, it was difficult to discern individual ACHE-positive neurons because of intense fiber staining. In order to facilitate the quantification of AChE-positive neurons, a pharmacohistochemical procedure employing the AChE inhibitor DFP was applied as described in uiuos*29and in uitro.‘j In the present experiments DFP was added to the culture medium (final concentration, 40 PM) of appropriate flasks on the day of harvest. Following a 5-min exposure, the reaggregates were thoroughly washed 5 times with defined medium and then transferred to new flasks containing 3.5ml of defined medium. The reaggregates were cultured for an additional 67 h in order to allow resynthesis of AChE and then harvested and processed for AChE histochemistry as described below. Histochemical staining for these new stores of AChE at this time point enables the visualization of discrete neuronal somata and proximal enzyme-containing processes5 Treatment of septal-hippocampal growth factor antiserum

coaggregates

with nerve

of choline acetyltransferase

Protein was measured by the method of Bradford3 using bovine serum albumin as a standard. of tissue sections

After 21 days in culture (17 days in some experiments), reaggregates from individual flasks were collected, transferred to a Maximov slide and photographed. From these photographs, the number of reaggregates present in each flask could be determined and their long diameters measured. The reaggregates were then washed in phosphatebuffered saline (PBS) (0.01 M nhosnhate. 0.15 M NaCl. DH 7.4) and immersion &ed in 4% paraformaldehyde (w/vi in 0.1 M sodium phosphate buffer (pH 7.4) for 2(r-24 h at 4°C. After fixation, the reaggregates were washed several times in PBS and embedded in 10% (w/v) gelatin. Gelatin blocks were fixed overnight in 4% paraformaldehyde in 0.1 M sodium phosphate buffer @H 7.4). The gelatin blocks were sectioned with a Vibratome at 50 PM and collected in PBS. The sections were then processed for acetylcholinesterase (AChE) histochemistry. Acetylcholinesterase

histochemistry

rationale for employing AChE histochemistry as opposed to ChAT immunohistochemistry has been previously discussed.2’ AChE staining was carried out on the reaggregate sections employing the method described recently by Tago et al. 45 Briefly, the sections were rinsed in 0.1 M maleate buffer (pH 6.0), incubated for 45 min in Kamovsky and Roots medium (0.03mM cupric sulfate, 0.05 mM sodium citrate, 0.1 M maleate buffer, 0.005 mM potassium ferricyanide, 18 PM acetylchiocholine iodide) containing 30 PM iso-OMPA as an inhibitor of non-specific cholinesterase, and then rinsed with 0.05 M Tris-HCl (pH 7.6). After rinsing, the sections were incubated in 0.025 M Tris-HCl @H 7.6) containing 0.02% (w/v) DAB and 0.15% (w/v) nickel ammonium sulfate for 5 min. H,02 was then added to the incubation medium to a final concentration of 0.003% (v/v) and the sections were allowed to react for 30 min. The sections were then washed in 0.05 M Tris-HCl (pH 7.6) and mounted. The

For the purpose of evaluating the possible role of “endogenous NGF” in mediating the observed target-cell effects on the development of septal choline@ neurons, coaggregates of septal and hippocampal cells were prepared and cultured in the presence of anti-NGF antiserum (final concentration = 1: 200 dilution) starting on the second day of culture. Thereafter, fresh anti-NGF was added whenever the medium was changed. This concentration of anti-NGF antiserum is sufficient to neutralize at least long/ml of exogenous NGF as demonstrated by the present study. This concentration of NGF is about 9000 times the concentration of endogenous NGF produced by hippocampus, since it has been shown that hippocampus from an adult rat produces 1300 fg of NGF per mg of tissue,2* and there were about 3 mg of hippocampal tissue per septal-hippocampal (S-H) flask in our studies. Control cultures received normal medium or were treated with non-immune serum (normal rabbit serum, final concentration = 1: 200 dilution). After 21 days in culture, half of the S-H coaggregates from the flask were sonicated for measurement of ChAT activity. The remaining half was processed for AChE histochemistry. Measurement

Protein &termination

activity

The measurement of ChAT activity was similar to that described by Fonnum? Briefly, aliquots (25 ~1) of sonicated cell lysates (15 s with a Branson cell disruptor 200 at output control = 3) in 100 mM NaCl in 0.01 M sodium phosphate buffer (PH = 7.4) were mixed with 25 ~1 of enzyme assay media containing: 0.1 mM acetyl co-enzyme A (acetylCoA), 139,000 dpm [3H]-acetyl-CoA (1.4 Ci/mmol, New England Nuclear), 12.5mM choline bromide, 300mM NaCl, 25 mM EDTA, and 0.2 mM physostigmine in 50 mM sodium phosphate buffer, pH 7.4. Following incubation at 37°C for 40 min, the reactions were stopped by transferring 25 ~1 of the incubation mixture to a scintillation vial containing 2.0 ml of 10 mM sodium phosphate buffer, pH 7.4, 2.5 ml of toluene-based scintillation cocktail, and 0.55 ml of acetonitrile containing 2.8 mg sodium tetraphenylboron and counted. For controls, either cell lysates which had been placed in boiling water for 10 min or buffer blanks (minus lysates) were employed. Counts of radioactivity from the controls (background) were subtracted from the counts of experimental samples (the background is less than 5% of the

Comparison flasks

of acetylcholinesterase

cell

number

between

A method of morphometric analysis, described previously,” was used to estimate the number of AChE neurons in a given flask. Briefly, 30 reaggregate proties obtained from the reaggregates in a given flask were randomly chosen for analysis. The number of AChE-nositive cells with nroximal dendrites was counted and the area of the sections was measured. The volume of the individual reaggregate profiles in which cells were counted was calculated from the summed section areas and the section thickness. This morphometric analysis yields an estimate of the number of AChE neurons per volume of tissue analysed. Total tissue volume per flask was estimated by using the long diameters of each of the reaggregates to determine their volume (assuming the reaggregates approximate spheres) and summing the volumes. With this data it is possible to compare the number of AChE-positive cells per flask from a finite sample of histochemical sections. A statistical test for comparing cell numbers among flasks has been devised.18 This test is based on the difference between the number of cells estimated per flask weighted to account for differences in flask tissue volumes and the area of the reaggregate profiles. This weighting normalizes the statistic to more closely approximate a normal random distribution.

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Electron microscopy After AChE staining, some of the septal-hippocampal coaggregate sections were processed for electronmicroscopic examination according to the method described earlier.” Briefly, these sections were washed with 0.1 M sodium phosphate buffer (pH 7.4), then osmicated in 1% osmium tetroxide in 0.1 M sodium phosphate buffer (pH 7.4) for 30min, dehydrated, embedded in Durcupan resin and mounted onto slides. The resin was then allowed to polymerize on the section-mounted slides in a 60°C oven for 48 h. Coaggregate profiles were then cut from the sections with a fine scalpel under a dissecting microscope and glued to Beem capsules. Serial ultrathin sections were then cut, placed on Formvar-coated slot grids, stained with uranyl acetate and lead citrate, and examined under a Philips 201 electron microscope.

RESULTS

Efect of nerve growth factor on choline acetyltransferase activity of septal cholinergic neurons The trophic effect of NGF in enhancing ChAT activity has been addressed previously using reaggregates from rat telencephahc cells.‘9 Since the latter cultures contained chohnergic neurons from a variety of brain areas, we examined the effects of NGF on reaggregate cultures derived specifically from the septal region. After 17 days in culture, septal reaggregates from media containing different concentrations of NGF were processed for measurement of ChAT activity. The addition of long/ml NGF markedly increased the ChAT activity to almost four times the control value, and the levels of ChAT activity appeared to plateau when higher concentrations of NGF were added (Table 1). The specificity of this NGF effect was evaluated by adding antibodies to NGF (1: 200 dilution) to the culture medium containing 10 ng/ml of NGF. The results indicated that the trophic effect of NGF on ChAT activity could be neutralized by the addition of anti-NGF antibodies (Table 2), i.e. the ChAT activity was not significantly different from the control value. To control for non-specific toxic effects of the anti-NGF antiserum, non-immune serum (normal rabbit serum) was added (1:200 dilution) instead of anti-NGF. As shown in Table 2, the trophic effect of NGF on ChAT activity was not affected by the non-immune serum.

Effect of nerve growth factor on septal cholinergicjber staining The effects of NGF on promoting septohippocampal projections were reported recently by Gahwiler et al.” using organotypic co-cultures from 7-day-old rats. These results were in contrast to the results of a previous report where NGF treatment of septal monolayer cultures did not enhance chohnergic fiber staining.15 Employing the reaggregate system, we studied the effect of NGF on fiber staining of septal cholinergic neurons grown in the absence of their target cells. After 17 days in culture, sections from septal reaggregates treated with NGF revealed a marked increase in the density of AChE-positive

fibers when compared with sections from the control reaggregates (no NGF added) (Fig. 1). Staining was especially dense at the periphery of the reaggregates (Figs 1C and 2B, D). While there was clearly enhanced fiber staining in the presence of NGF, these AChE fibers did not exhibit the well-defined “axonlike” appearance that we have observed in the presence of target cells (Fig. 4).20 Instead, these fibers resembled those observed in septal reaggregates (Fig. lA, B) or septal-cerebellar coaggregates, i.e. they were poorly defined, often discontinuous, and varicosities were seldom observed. By visual inspection, higher concentrations of NGF (Fig. 1C) did not seem to increase the density of AChE fibers compared to that of the septal reaggregates treated with long/ml of NGF (Fig. 2B). This observation correlates well with the effect of NGF on ChAT activity. In order to examine the specificity of the effects of NGF on AChE-positive fiber staining, antibodies to NGF (I:200 dilutions) were added to the culture medium containing 10 ng/ml of NGF. The results indicated that in the presence of anti-NGF antibodies, the effect of NGF on fiber staining was eliminated, i.e. the density of AChE fibers in the reaggregates was similar to that of the control (Fig. 2C). If non-immune rabbit serum was added (1:200 dilution) instead of anti-NGF, the trophic effect of NGF on fiber staining was not affected (Fig. 2D). Efect of nerve growth factor on the estimation acetylcholinesterase-positive cells

of

To date conflicting results have been reported with respect to the effect of NGF on survival (or maintenance) of central cholinergic neurons during their normal development. ‘3~‘5It has been suggested that NGF either doesI or does not increase septal cholinergic cell survival.15 In evaluating the effect of NGF on septal cholinergic cell numbers in the reaggregate system, our initial results were derived from 5 experiments: 11 flasks containing varying concentrations of NGF; and 5 control flasks. In 10 of the experimental flasks, increased numbers of AChE-positive cells were observed relative to controls; however, in only 5 of them were the differences statistically significant (P < 0.001). Although these results may suggest a trophic effect of NGF on AChE-cell number, they were too inconsistent to support a conclusion. The inconsistency was thought to be most likely due to the densely-stained AChE fibers of NGF-treated septal reaggregate sections obscuring the somata and proximal processes of many AChE-positive neurons (Fig. 2D), thus resulting in an underestimation of the actual number of AchE-positive cells present in those sections. In order to achieve better visualization of AChE-positive cells, septal reaggregates were exposed to a 5-min pulse of 40 PM DFP 7 h before harvesting. Sections from septal reaggregates treated in this fashion contained well-defined multipolar

Effects of nerve growth factor on development of cholinergic neurons

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Table 1. Effects of nerve growth factor on choline a~tyitmnsferase activity in cultures of septal cell reaggregatesf NGF concentrations (ng/mU 0 (control) 10

Number of experiments

ChAT activity Cpmol ACh fo~~/min/mg protein)

6 6 3 3

310 f 1020 + 1213 f 1204 +

65 50* 48* 32*

tCuitures were grown in the presence of various con~ntmtions of NGF beginning on day 6 of cuiture. Reaggregates were harvested following 17 days in culture and were processed for the measurement of ChAT activity. Values represent the mean & S.E.M. *Differs from the control at P < 0.0005 (onetailed f test).

AChE-positive neurons with proximal dendrites (Fig. 3). when the reaggregates were grown in the presence of NGF (Fig. 3B), the resulting sections

contained more intense AChE staining than those grown without NGF (Fig. 3A). Despite the more intense staining, individual AChE-positive cells could still be identified. Treatment with DFP therefore allowed for a more accurate estimation of AChE-cell number. Initial studies using lOng/mf of NGF demjonstrat~ a significant increase in AChE-ceif number (Table 3). The specificity of the effect of NGF on increasing AChE-cell number was examined by adding antibodies to NGF (1: 200 dilution) to culture medium containing 10 ng/mf of NGF. After 17 days in culture, the septal reaggregates were treated with DFP before being processed for AChE histochemistry (Fig. 3). The results indicated that the effect of NGF on increasing AChE-cell number could be blocked by NGF antiserum but not by non-immune serum (Table 3). Similar results of increased AChEcell number were also obtained in a separate experiment when 40 ng/ml of NGF were employed (data not shown). Effects of nerve growth factor antiserum on septd-

hippocurnpal coaggregutes Recent studies in the CNS have suggested a possible role for NGF as a target-derived cholinergic trophic factor.23-25 However, most of these studies

utilized the administration of exogenous NGF. In order to evaluate the possible role of “endog~ous NGF” in mediating the observed target-cell effects on the development of septaf chofinergic neurons, antiNGF was added to the medium containing ~ptai-hip~campa1 (S-H) coaggregates after 24 h in culture. After growing in culture for 21 days, the S-H coaggregates were processed for measurement of ChAT activity and AChE histochemistry. Results of our study revealed that anti-NGF antibodies reduced the ChAT activity of the S-H coaggregates by 20% when compared to the values of the controls (P < 0.05) (Table 4). Cell counts of AChE-stained sections revealed that the number of AChE-positive cells in the anti-NGF-treated S-H coaggregates was lower than the media-only control value by 50% (P < 0.01) and 30% lower when compared to the non-immune serum controls (P < 0.05) (Table 5). Visual examination of the sections from the !!LH coaggregates treated with antibodies to NGF revealed the same pattern of cells and fibers as that of the controls, i.e. multipolar AChE-positive cells possessing fibers which were well-defined, of fine-caliber and with varicosities (Fig. 4). Examination of serial sections from multiple aggregates under the electron microscope revealed numerous AChE-labeled synapses in the hip~campaf target areas of the S-H coaggregates treated with anti-NGF (Fig. 4H). These qualitative observations suggest that NGF antibodies

Table 2. Effect of nerve growth factor and nerve growth factor antiserum on choline acetyltransferase activity in cultures of septal cell reaggregatest Culture conditions 0 ng/ml NGF (control) 10 &ml NGF 10 ng/ml NGF + anti-NGF antiserum 10 ng/ml NGF + non-immune serum

Number of experiments 3 3 3 2

ChAT activity (pmol ACh fo~ed/m~n/mg protein) --

240&42 868 & 52’ 172&34$ 192 5 46*

Effects of NGF and NGF antiserum on ChAT activity within septal reaggregates. jCuItures were grown for 17 days in the following conditions: culture medium only; 10 ng/ml NGF added to the medium (beginning at day 6 of culture); 10 ng/ml NGF plus NGF antiserum (1:200 dilution) added; or 10 ng/ml NGF plus non-immune serum (1:200 dilution) added. Values represent the mean I!IS.E.M. Assays were performed in triplicate from each flask. *Differs from the control at P < 0.001 (one-tailed t test). fNo significant difference from the control group (Student’s f test). NSC 29/l-I

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Fia. I. Photomicrograohs of sections of 17-day-old, AchEstained septal reaggregates with or without N&F treatment. (As Septal reaggregates without NGF treatment. Area marked by the asterisk is shown at hirrher ma~ifi~ation in B. (B) High-uower magnitication of AChE-positive f&em in A. These fibers are $orIy de&red and often d&mti&tdus. (C) Se$al reaggregates cultured in the presence of IO0ngjml NGF from day 6 in culture. Dense heavily-stained AChE fibers can be observed when compared to A. Staining is especially promjnent at the periphery of the reaggregate section. Area marked by the asterisk is shown at higher magnification in D. (D) High-power magnification of AChE-positive fibers in C. Aithough the density of fiber staining increases, these fibers do not exhibit the well-defined ‘*axon-like” ap~aran~ similar to those observed in the presence of target cells (Fig. 4A). This result suggests that NGF does not mimic all the effects of hippocampal target cells on septal cholinergic fiber proliferation. Scale bars: A, C = 100 pm; B, D = 50pm.

Effects of nerve growth factor on development of chohnergic neurons

Fig. 2. Specificity of NGF effects on AChEstaining in 17day-old septal reaggregate. NGF was added beginning at day 6 of culture. (A) Septal maggregate grown in a culture medium without NGF. (B) Septal reaggregate cultured in the presence of lOng/ml NGF. The AChE histochemistry reveals dense, heaviiy-stained AChE fibers which are most prominent at the periphery of the reaggregate section (similar to Fig. IC). (C) Septal reaggregate cultured in the presence of 10 ngjml NGF and NGF antisernm (1: 200 dilution). AChE histochemistry reveals a fiber density similar to A, indicating that the effect of NGF on fiber proliferation is antagonized. Arrow indicates a cell situated at the edge of the reaggregate section which has prominent proximal fibers. (D) Septal reaggregate cultured in the presence of IO t&ml NGF and non-immune serum (1: 200 dilution). AChE histochemistry demonstrates that the fiber density is the same as that of the NGF treated confirming the specificity of the NGF effect on fiber proliferation. A cell body (arrow) situated at the edge of the reaggregate section is obscured by the dense plexus of heavily-stained fibers. Scale bars = 100 pm.

215

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Fig. 3. Photomicrographs of sections of 17-day-old, DFP-treated and AChE-stained septal reaggregates. (A) Reaggregate grown in a culture medium without NGF. (B) Reaggregate cultured in the presence of NGF. (C) Reaggregate cultured in the presence of NGF and NGF antiserum. (D) Reaggregate cultured in the presence of NGF and normal rabbit serum. These reaggregates were cultured under the same conditions as those in Fig. 2A, B, C and D, respectively, except that they were exposed to DFP on the day of harvest and allowed to survive for 67 h before harvesting. When compared with their counterparts in Fig. 2, well-defined multipolar AChE-positive neurons with proximal dendrites can be identified (arrowheads in A and C; arrows in B and D). Note the AChE-positive cells (arrows) situated near the periphery of the reaggregate section in B and D. Without DFP treatment these cells would probably have been obscured by the dense, heavily-stained fibers, and therefore would be difficult to discern and count. Scale bars = 100 pm.

Effects of nerve growth factor on development of cholinergic neurons

217

Table 3. Effects of nerve growth factor and nerve growth factor antiserum on the number of choliner~c neurons in ~isopropyl~uorophosphate-tr~ted septal reaggregates Conditions in flasks No NGF 10 ng/ml 10 ng/ml IOn&ml

Number of AChE-positive neurons per flaskt

---.-

added (control) NGF added NGF + NGF antiserum added NGF + non-immune serum added

-

3904 13,181* 3392$ 12.082*

Septal reaggregates were cultured for 17 days in media containing no NGF (control), NGF (10 ng/ml). NGF plus anti-NGF (1: 200 dilution), or NGF plus non-immune serum (I:200 dilution). On the day of harvest, the reaggregates were treated with 1pmof DFP for Smin and subsequently stained for AChE. The numbers of AChE cells per flask were estimated as described in text. tl0 x IO6 septal cells were added to each flask. *Significantly differs from control at P < 0.001 (Poisson compa~son test). $Does not significantly differ from control (Poisson comparison test).

did not block synapse formation

between cholinergic axons and hippocampal target cells. The target cell areas in these sections were easily identified because the respective cell populations (septai versus hippo-

campal) sort out,20 and the hippocampal cells have a characteristic ultrastructural morphology.2’ In order to ensure full penetration of immunoglobulin molecules into the reaggregates, immunohistochemical staining using goat anti-rabbit IgG was performed on S-H coaggregates cultured in the presence of NGF antiserum which is derived from rabbit. The results revealed positive staining even at the center of the reaggzegate sections {data not shown), suggesting that NGF antibody molecules are able to penetrate into the reaggregates. DISCUSSION

Eflect of exogenous nerve growth factor on devetoping septal c~o~ineTgi~ neurons

Evidence that NGF is a neurotrophic factor for central cholinergic neurons has been provided by both in v&o 12X37+43 and in vitro’“~*s*‘9*33 studies. The results of the present study concerning the effects of NGF on developing septal cholinergic neurons in reaggregate cultures are consistent, in part, with this evidence. Our results demonstrate that NGF not only enhances septal cholinergic cells’ ChAT activity, but

also increases their fiber staining and the number of cells positive for AChE. The present findings are, however, in contrast to the earlier report by Hefti et aLts who showed that NGF had no effect on cholinergic fiber proliferation or cell number in monolayer cultures of septal cholinergic neruons from embryonic day 17 (E17) rat embryos. In a more recent study employing postnatal rat septal monolayer cultures,‘3 it was found that NGF treatment resulted in increased number of AChE-positive cells in postnatal day (P) 8-14 cells but not in culture from P1-4. In both instances NGF enhanced ChAT activity. The authors concluded that there was a developmental change in NGF actions and cell-saving effects were only evident at a later period. In the present experiments NGF treatment was performed on cells harvested at a much earlier developmental period but cultured for a time interval that includes the period of active synaptogenesis within the hippocampus (see below). It should be appreciated that the observation of increased number of AChE-positive cells foIIowing NGF treatment does not necessarily prove a “cell surviving” effect since NGF may merely induce the expression of AChE in developing cells. This question has been previously addressed by our laboratory with respect to the trophic actions of hippocampal target cells on septal cholinergic development. “J’ In this case the increased numbers

Table 4. Effects of nerve growth factor antiserum on choline acetyltransferase activity in cultures of 21-day-old septal-hippocampal coaggregates Cuiture conditions Control Anti-NGF serum (1:200 dilution) Non-immune serum (I : 200 dilution)

Number of ChAT activity experiments (% of control) 2 2 2

lOO;t2 80 If: lO* 110&3

Cultures were grown in media alone (control), media conta~njng NGF antiserum, or media containing non-immune serum from day 2 in culture. Values represent the percentage of control (lOO%, absolute value = 161 pmol ACh formed/min/mg protein) as mean fi S.E.M. Assays were performed in triplicate from each flask. *Differs from control at P < 0.05; differs from the non-immune serum treatment at P cO.02.

218

J. HSIANG et al.

of cholinergic cells observed under the light microscope” were shown to result from enhancement of cell survival as determined under the electron microscope.*’ An ultrastructural study of the NGF-treated septal reaggregates at different time points would provide useful information in regard to this issue. Such experiments are currently in progress and preliminary data from these ultrastructural studies suggests that NGF treatment increases the number of AChE-positive cells observed in septal aggregates by enhancing the expression of AChE activity.50

The role of hippocampus-derived in the development

of central

nerve growth factor cholinergic

system

Although numerous studies have described the effects of NGF on central cholinergic neurons, whether or not the responsiveness of these neurons to exogenous NGF is of physiological significance remains unclear. NGF has been recently reported to be a hippocampus-derived neurotrophic factor.24.25 However, attempts to demonstrate deleterious effects of NGF antibodies on developing cholinergic neu-

Fig. 4.

Fig. 4. Effect of anti-NGF on S-H coaggregates. Photomicrographs of sections of 21-day-old AChEstained S-H coaggregates cultured in media alone, media containing non-immune serum, or media containing NGF antiserum beginning at day 2 in culture. (A) S-H coaggregate grown in culture medium alone. AChE histochemistry reveals a typical target cell-induced pattern of fibers and cells similar to that described in previous studies.‘OThe boxed area is shown at higher magnification in C. (B) S-H coaggregate cultured in the presence of non-immune serum (1: 200 dilution). AChE histochemistry reveals a pattern of cells and fibers similar to those in A. The boxed area is shown at higher magnification in D. (C) Higher-power photomicrograph of boxed area of AChE-positive cells and fibers in A. These fine caliber fibers are well-defined with extensive arborizations and varicosities. (D) Higher-power photomicrograph of boxed area of AchE-positive cells and fibers in B. The appearance of these cells and fibers does not differ from those shown in C. (E) S-H coaggregate cultured in the presence of NGF antiserum (1: 200 dilution). AChE histochemistry reveals fibers and cells similar to those in A. The boxed areas are shown at higher magnification in F and G. (F) and (G) Higher-power photomicrographs of boxed areas of AChE-positive cells (G) and fibers (F) in E. The appearance of these cells and fibers are similar to those shown in A and B suggesting that NGF antibodies do not inhibit chohnergic fiber proliferation in S-H coaggregates. (H) High-power electron micrographs showing AChE-labeled synapse (arrowheads) and unlabeled synapse (arrows) in a 21-day-old S-H coaggregate which was cultured in the presence of NGF antiserum beginning on the second day of culture. The identification of labeled synapses in this case suggests that anti-NGF does not inhibit the process of synaptogenesis between septal cholinergic terminals and hippocampal target cells. Scale bars: A, B, E = 200 pm; C, D, F; G = 50 pm; H = 0.5 pm. 219

J. HSIANG ef al.

220

Table 5. Effects of nerve growth factor antiserum in seDtal-hiDDocamnai Conditions

in

flasks Culture media only (control) NGF antiserum added (1: 200 dilution) Non-immune serum added (1: 200 dilution)

on the number coaggregates

Number of experiments

of cholinergic

neurons

Number of AChE-positive neurons per flask?

I

7155

2 2

3907 + 715’ 5638 k 12

Twenty-one-day-old S-H coaggregates were cultured in media containing NGF antiserum or non-immune serum. For control cultures, neither NGF antiserum nor non-immune serum was added to the media. After the coaggregates were harvested, they were stained for AChE, and the number of AChE cells was then estimated as described in text. t5 x lo6 septal cells and 5 x lo6 hippocampal cells were added to each flask. *Differs from control at P < 0.01;differs from non-immune serum treatment at P i 0.05 (Poisson comparison test).

rons, which would support a role in development for endogenous NGF, have been inconclusive. Earlier experiments involving intraventricular and intracortical injections of NGF antibodies in neonatal rats did not result in a decrease in ChAT activity of the cortex, septum and hippocampus.’ Contrary to these studies, intrauterine injection of anti-NGF antibodies directly into the E15.5 rat embryos resulted in a significant decrease in ChAT levels of the nucleus basalis region, septum and hippocampus. The reduction of ChAT levels in the latter experiment was presumably due to better penetration of the injected antibodies at earlier developmental stages, however no quantitation of cholinergic cell survival was made. In a more recent study, a reduction of septal fibers projecting to the hippocampus was demonstrated when anti-NGF antiserum was applied to organotypic co-cultures from 7-day-old rat septum and hippocampus.” The results of the present study regarding effects of anti-NGF antibodies on S-H coaggregates argue in part for a physiological function of endogenous NGF in the development of central cholinergic neurons. We were able to detect effects of anti-NGF on ChAT levels (20% reduction) and cholinergic cell number (3&50% reductions). The magnitudes of these effects were considerably less than the greater than threefold increases in cholinergic markers observed following addition of exogenous NGF to septal cells grown alone in reaggregate culture. As discussed above, these changes may not reflect changes in neuronal degeneration or survival, but rather alterations in the levels of the neurochemical markers expressed by the cells. In contrast to the report of GIhwiler er al.,” we did not observe any obvious reduction in the density of cholinergic fibers, and synapse formation appeared to proceed normally in the S-H coaggregates treated with anti-BGF. This finding suggests that NGF is not required in the initial process of synapse formation. Since the number of cholinergic synapses in the coaggregates was not quantified, the possibility that there was a small decrease in the number of cholinergic synapses in S-H coaggregates treated with. antiNGF antibodies cannot be ruled out. There is a major difference between the present study and that

of GHhwiler et al. The tissues used in our experiments originated from El5 mouse embryos, a stage well before any septal innervation to hippocampus can be detected.35 The tissues used in Glhwiler’s experiments were obtained from 7-day-old rats, a stage at which septal cholinergic fibers have already invaded their hippocampal target. 35 Therefore, in order to obtain septal and hippocampal explants at this stage, one would anticipate injury occuring to the septohippocampal connections. As a consequence, the observed effect of anti-NGF in the latter study may have resulted from antagonism of an NGF effect elaborated in response to injury. One approach to examine the functions of NGF in developing cholinergic neurons is to compare the normal development of the septohippocampal pathprofile of NGF expresway,” and the developmental sion in the hippocampus. Previous studies have shown that neurons in rat basal forebrain are born between El3 and El7,* but their axons do not invade the dentate gyrus of the hippocampus until P3.35 Only by Pl I, do the axons become distributed in their mature laminar arrangement.34 During this period of septal innervation (P3-l l), synaptogenesis is most advanced in the dentate gyrus,‘j but the specific activity of ChAT is low and gradually increases to the adult level at around P17.38 Based on earlier studies which indicated that cholinergic synaptic development is the major contributor to the ontogenic increase in ChAT activity,4,32 the finding that ChAT levels are the highest at PI7 suggests develoment of functional cholinergic terminals at this age. This suggestion is further supported by the finding that high affinity choline uptake, a marker for the presence of cholinergic presynaptic membrane, develops most rapidly during the period around 16-l 7 days of age, similar to the ontogenesis of ChAT activity.42 The developmental profile of NGF in rat hippocampus has been investigated recently by Large et a1.28 who have demonstrated that the levels of both NGF mRNA and protein are barely detectable at birth, increase rapidly after PlO, and then reach peak levels by P21. After this age, the level of NGF mRNA, but not NGF protein, declines three-fold by P35.

Effects of nerve growth factor on development of cholinergic neurons

Considering the studies cited above, a possible role for NGF in the developing central cholinergic system can be suggested. Because the amount of NGF is relatively low and relatively uniform throughout the brain during the first 10 postnatal days, NGF seems unlikely to be involved in the initial guidance of septal cholinergic axons to their hippocampal target cells.**It is also not certain whether or not NGF plays an essential role in the initial process of synapse formation, which is most active between P3 and Pl 1. On the other hand, hippocampus-derived NGF is very likely involved in the differentiation of functional cholinergic synapses after the synaptic connections are made. This notion is supported by the fact that a rapid development of high-affinity choline uptake occurs during the period of peak NGF levels.42 In addition, there is a close correlation between changes in NGF levels over time and ChAT activity found in rat hippocampus.’ Finally, the accumulation of NGF protein precedes the rise in ChAT activity in the basal forebrain.28 Moreover, NGF may also participate in the stabilization and long-term maintenance of the innervating cholinergic neurons since a high level of NGF is maintained in the hippocampus throughout adulthood. This suggestion is further supported by two recent observations. First, following the transection of the septohippocampal pathway in adult rats, intraventricular injection of NGF has been reported to rescue septal cholinergic neurons which otherwise would degenerate.16 Second, a higher level of NGF in the hippocampal formation can be measured after the native septal innervation has been destroyed.*“” This increase in NGF level is probably due to the accumulation of NGF which would have been retrogradely transported by the presynaptic fibers. Enhanced NGF synthesis from the hippocampal cells in response to ~mbrial tran~~on is unlikely since there is no increase in NGF mRNA in the hippocampus after fimbrial lesions. 26These proposed functions of NGF in the development of the central cholinergic system are congruent with recent observations on its function in the periphery.* Using the El0 mouse trigeminal ganglion and part of its cutaneous targetfield, Davies et al. showed that NGF does not attract sensory nerve fibers from the trigeminal ganglion to their target-field by chemotaxis, but that it is involved in the target-controlled development of sensory neurons.’

221

In NGF-treated septal reaggregates, while there is clearly enhanced cholinergic fiber staining, these fibers do not exhibit the well-defined “axon-like” appearance that we have observed in the presence of target cells.2o This obse~ation strongly suggests that “target effects” are not totally mimicked by exposure to NGF. Furthermore, application of anti-NGF antiserum to S-H coaggregates does not produce a fiber pattern that resembles the one grown in the absence of their target cells.2o In fact, antibodies to NGF produce no grossly detectable alterations in the morphology of cholinergic cells and fibers as compared with untreated S-H coaggregates. Evidence for additional factors has recently been reported. First, it has been demonstrated that two distinct hippocampal growth factors can be identified in the rat hippocampal formation.’ One of these factors probably is NGF since it promotes neurite outgrowth from sympathetic neurons and is blocked by antisera to NGF. The other factor, however, accelerates neurite extension from parasympathetic neurons and is not affected by the NGF antisera. Second, it has been shown that rat hippocampus contains a macromolecular factor which promotes the survival of peripheral cholinergic neurons.i4 This factor is suggested to be of glial origin and its con~ntration appears to be regulated by non-cholinergic afferents to the hippocampus. Finally, Ojika and Appe13’ have demonstrated that soluble extracts from rat hippocampus enhance cholinergic activities (ChAT level, high affinity choline uptake, and ACh synthesis) of the rat medial septal nucleus, and the responsible factor in the extracts is not neutralized by anti-NGF antisera. CONCLUSION

Endogenous NGF may play a physiological role in central cho~iner~c development by promoting the neurochemical differentiation of cholinergic cells and processes. It appears less likely that NGF participates in the process of axonal guidance or initial synapse formation. In addition to NGF, there are probably other hippocampal-derived trophic factors involved in the development of cholinergic circuitry. Future studies should further clarify the exact mode of action of endogenous NGF in the regulation of cholinergic development. Equally important is the investigation of its interactions with other hippocampal-derived trophic factors in governing this process. ~ck~owledge~n~~-his research was supported by NS25787(BHW), ND-04583 (BHW), MC28942 (AH), the

The results of the present investigation suggest that hippocampal-derived trophic signals other than NGF may be essentia1 for septal choline&c development.

Brain Research Foundation (BHW, The University of Chicago), NS-24054 (WCM) and NS-01015 (WCM). The authors wish to express their appreciation to Eligia Buhay and Steve Price for their technical assistance.

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(Accepted 1 August 1988)