Cholinergic Differentiation in Neurogenic Basal Forebrain Cultures Marjana Martinic, Mary P. Lambert, Sherwin Hua, and William L. Klein*

Department of Neurobiology and Physiology, Institute for Neuroscience, Northwestern University, Evanston, Illinois 60208

SUMMARY To study early events in the central nervous system (CNS) cholinergicdevelopment, cells from rat basal forebrain tissue were placed in culture at an age when neurogenesis in vivo is still active [embryonic day (E) 151. The rapid mortality of these cells in defined medium, with 50% mortality after 5-10 h, was blocked completely by soluble proteins from the olfactory bulb ( a basal forebrain target), extending earlier observations (Lambert, Megerian, Garden, and Klein, 1988)- Treated cultures were capable of incorporating thymidine into DNA, and most cells incorporating 'H-thymidine (>90%) also stained positive for neurofilament, confirming neuronal proliferation in the supplemented cultures. A small percentage of 3H-thymidine labelled cells were glial fibrillary acidic protein (GFAP) positive, but growth factors that support astroglial proliferation [epidermal growth

factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1 )] were not sufficient for neuronal support. After 5 culture days with supplemented medium, almost 50% of the cells showed choline acetyltransferase ( ChAT) immunofluorescence.The cholinergic neurons typically formed clusters separate from noncholinergic cells. These mature cultures did not develop if young cultures were treated with aphidicolin to block DNA synthesis. The data show that cultures of very young rat basal forebrain cells can be neurogenic, giving rise to abundant cholinergic neurons, and that early cell proliferation is essential for long-term culture survival. Keywords: neurogenic basal forebrain, choline acetyltransferase, aphidicolin, olfactory bulb, trophic.

INTRODUCTION

mann, Schwab, and Thoenen, 1983). Much of the work with basal forebrain cholinergic circuits has focussed on later developmental stages. In the Ch3 region, for example, it is known that about 10%20% of the cells become cholinergic (Rye et al., 1984), and these provide most of the cholinergic input to the olfactory bulb (Mesulam et al., 1983a,b). Ch3 neurons reach their cholinoceptive targets about the time of birth (Schwob and Price, 1984), although, at this age, choline acetytransferase (ChAT) activity in the target area is low (Large, Lambert, Gremillion, and Klein, 1986). After a 1-week lag, the pre- and postsynaptic cholinergic markers within the olfactory bulb develop in synchrony, during the second and third postnatal weeks (Large et al., 1986). Studies of earlier development have shown that cells in the basal forebrain leave their mitotic cycle

Cholinergic neurons of the rat basal forebrain, which project to major targets in the olfactory bulb, hippocampus, and cerebral cortex (Meisami and Firoozi, 1985; Mesulam, Mufson, Wainer, and Levey, 1983a,b; Rye, Wainer, Mesulam, Mufson, and Saper, 1984) have provided a focus for studies of central nervous system (CNS) cholinergic differentiation (Williams, Jodelis, and Donald, 1989; Montero and Hefti, 1988; Hefti, 1986; Gage, Bjorklund, and Stenevi, 1984; Gnahn, Hefti, HeuReceived September 25, 1991; accepted January 13, 1992. Journal of Neurobiology, Vol. 23, No. 3, pp. 252-269 (1992) 0 1992 John Wiley & Sons, Inc. CCC 0022-3034/92/030252-18$04.00 * To whom correspondence should be addressed.

252

A Neurogenic Cholinergic CNS Syslem

and are identifiable as ChAT-positive between embryonic days (E) 12 and 17 (Bayer, 1985; Semba and Fibiger, 1988; Brady, Phelps, and Vaughn, 1989). Cultured cells from this stage can exhibit long-term survival and differentiation when grown in the presence of soluble proteins from the olfactory bulb (Lambert et al., 1988), suggesting either that differentiated neurons were maintained or that cells were neurogenic. T h e current work was designed to distinguish between these possibilities, as well as to further characterize the cholinergic differentiation shown by this culture system. The data show that ( 1 ) a large fraction of the cultured cells become cholinergic neurons, which have a marked tendency toward self-segregation; ( 2 ) DNA synthesis occurs in cells that become neurons as well as glia; and ( 3 ) when DNA synthesis is blocked, the cultures do not survive. This culture system should be of use for studies of cholinergic neurogenesis and the transition from neuroblast to cholinergic neuron.

METHODS

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combinant human insulin-like growth factor (IGF-I ) were obtained in sterile, lyophilized form from Collaborative Research, Inc.. 3H-Thymidine was obtained from New England Nuclear (specific activity = 77.9 Ci/ mmol). Aphidicolin, used at final concentrations of 5 and 10 pM, and 3-( 4,5-dimethythiazol-2-y1)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma. All cultures used for immunocytochemistry and incorporation were grown in 24-well culture plates obtained from Nunc. Nunc 96-well culture dishes coated with poly-L-lysine were used for MTT assays. NTB2 emulsion was obtained from Kodak. All other chemicals were obtained from Sigma.

Dissection Timed-pregnant Holtzman Sprague-Dawley rats were sacrificed by C 0 2asphyxiation. Embryos (E l 5-16) were removed under sterile conditions, decapitated, and the heads were placed into drops of cold HCMF on sterile Sylgard-coated plates. Embryonic basal forebrain tissue was dissected according to the method of Bjorklund, Stenevi, Schmidt, Cunnett, and Gage ( 1983) using Dumont no. 5 forceps and iridectomy scissors. Tissue pieces were pooled in 1 ml EBSS until dissociation.

Media

Dissociation and Plating

High-glucose Dulbecco's minimal Eagle medium (DMEM) supplemented with gentamicin (50 mg/l) and the N2 supplements of Bottenstein and Sat0 ( 1979)were used as control culture medium. The N2 supplements were used at the following concentrations: insulin ( 5 pg/ ml), transferrin ( 5 pg/ml), progesterone (20 n M ) , putrescene ( 100 p M ) , and selenium dioxide (30 n M ) . Earle's basic salt solution (EBSS), supplemented with 3.0 mg/ml bovine serum albumin (BSA) and 0.277 mg/ ml MgSO,, was used for tissue collection. Hanks' calcium- and magnesium-free salt solution (HCMF) was used for dissection and preparation of extracts. (DMEM, EBSS, and HCMF were obtained from Gibco/BRL Laboratories, Grand Island, NY.)

The pooled tissue was transferred into 1 ml0.1% trypsin (Worthington 3x) in HCMF, incubated for 8-10 min at 37"C, and briefly (1-2 min) spun down in a clinical centrifuge. The supernatant was carefully removed and replaced by 2 ml of a solution of DNase and soybean trypsin inhibitor in EBSS. After an 8- to 10-min incubation at 37"C, the cells were briefly centrifuged and resuspended in 1 ml DMEM. After careful trituration, the number of viable cells was counted on a hemacytometer in the presence of nigrosin (0.5% in HCMF). The viability was routinely >go%. The cells were plated at 250,000-270,000 viable cells per milliliter. Prior to plating, the culture dishes were coated with poly-L-lysine (200 pg/ml) and sterilized overnight under ultraviolet ( U V ) light.

Other Reagents Rabbit anti-rat GFAP was obtained from Accurate Chemical, and FITC-conjugated goat anti-rabbit IgG was purchased from ICN. Mouse anti-pig monoclonal antibody to ChAT was obtained from BoehringerMannheim. Antiserum to neuron-specific enolase was obtained from Polysciences. Fetal calf serum was purchased from Hyclone and rat serum was drawn in the laboratory by cardiac puncture. A mouse anti-pig monoclonal antibody to neurofilament 200 was obtained from Sigma Chemical Company. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and re-

Extract Preparation Olfactory bulbs and cerebella were dissected from adult female rat brains. The tissue was washed with cold HCMF and frozen at -20°C until extract preparation. Extract was prepared by homogenizing thawed tissue with 1 ml of HCMF per 70 mg wet weight of brain tissue. After centrifugation at 4000g for 20 min, the supernatant was filtered through a sterile 0.22-pm filter and frozen until use. Protein content was determined by Lowry protein assay (Lowry, Rosebrough, Farr, and Randall, 1951).

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MTT Survival Assay The assay was performed according to the method of Manthorpe, Fagnani, Skaper, and Varon (1986), with modifications described below. Cells were plated on poly-L-lysine at four densities (75,000, 105,000, 150,000, and 225,000 cells/cm2) in a volume of 110 pl. Different plating densities were used to compensate for any effects of plating density on survival. Only the same plating density was compared in each MTT assay. Media was Dulbecco's modified Eagle medium without phenol red (Gibco), with the N2 supplements of Bottenstein and Sat0 (1979). Incubation time with MTT was 6 h. The volume of isopropanol/HCI added was 200 111, and the mixing volume was 100 pl. Plates were read on a Dynatech MR600 microplate reader (graciously made available by S. Pierce). Wells with no cells were used as a control, and control values were automatically subtracted out by the plate reader. Data from quadruplicate wells of the same density were averaged and plotted using Microsoft Excel and CricketGraph software. In preliminary experiments, the intensity of the blue color (tonm - control o.d.5,0-630 ",,) of the formazan tal o.d.570-630 product was found to be proportional to the number of living cells, as determined by cell counting of fixed cultures at 1.5 and 17 h after plating (data not shown). Living cells have large, phase-dark, rounded cell bodies and are attached to the substrate.

3H-Thymidine Incorporation Incorporation was determined according to methods described by Han, Lauder, and D'Ercole (1987) with the modifications noted below. Cells were plated and grown as described. The time of exposure to 'H-thymidine (0.66 pCi/ml, specific activity = 77.9 Ci/mmol) was 23 h. Cultures were washed three times with room temperature DMEM before being fixed with cold 10%trichloroacetic acid for 20 min at 4°C. The solution was removed and fixation was repeated. After removal of the second solution, cells were allowed to air dry and then solubilized with 0.4 ml of 1 % sodium dodecyl sulfate (SDS) in 0.3 N NaOH. After 5 min, the solution was removed to an Eppendorf tube. The well was washed with 0.1 ml of the SDS solution, and the wash was added to the tube. A 50-pl aliquot ofsolution was added to the 10-ml scintillation cocktail (0.4% 2,5-diphenyloxazole (PPO) in toluene/triton/2:1), shaken, and counted after 24 h. Data from six wells were averaged for each point. Protein was determined according to the method of Lowry et al. ( 1951 ) on duplicate 150-111 samples of each solubilized solution.

0. I mg/ml olfactory bulb (OLB) extract. On culture day 3 (C3), the medium was exchanged for an identical medium containing 4 pCi/ml 3H-thymidine. After 10 min, the cultures were washed and fixed with ethanol/acetone (3: 1 ) (Stein and Yanishevski, 1979). Cultures were processed for GFAP staining as described; subsequently, coverslips, cell-side up, were mounted on slides and processed for autoradiography with NTB2 emulsion (Stein and Yanishevski, 1979). Exposure time was 3-5 weeks at 4°C. Slides were observed with a Nikon DiaphotTMD inverted microscope equipped for epifluorescence.

Acetylcholinesterase Staining Cultures were grown with various additions, then fixed on C5 with 1% glutaraldehyde. Staining for AChE was performed according to the method of Karnovsky and Roots ( 1964), with the following modification. Butyrylcholinesterase was inhibited by the presence of 0.1 m M tetraisopropyl pyrophosphoramide (iso-OMPA). Cultures were fixed with 1% glutaraldehyde in 0. l Msodium maleate buffer (pH 6.0) for 30 min on ice. Cells were washed three times with maleate buffer to remove any glutaraldehyde and incubated with the staining solution ( 5 mg acetylthiocholine chloride, 6.5 ml 0.1 M sodium maleate buffer, 0.5 mlO.1 Msodium citrate, 1 ml0.03 A4 CuSO,, 1 ml water, 1 ml 0.005 M K3Fe(CN),), and iso-OMPA for 3 h at 37°C. After washing five times with maleate buffer and twice with H,O, cells were dehydrated with 70% ethanol, 95% ethanol, and 100% ethanol, mounted with glycerol, and viewed.

Choline Acetyltransferase lmmunocytochemistry Cultures were grown with the appropriate additions for 5 days and subsequently labelled for ChAT according to the method of Hefti et al. (1985). After removal of the medium, the cells were washed three times with phosphate-buffered saline (PBS). Fixation was with 4% paraformaldehyde for 30 min at 20°C. After removal of the paraformaldehyde, the cultures were blocked for 48 h in 0.1 M Na,HPO, ( p H 7.4), containing 50 g/1 sucrose, and 50 g/1 BSA. After a 15-min permeabilization with 0.1% Triton X-100 in PBS at 20"C, the cultures were incubated with antibody to ChAT ( 1:500 dilution in PBS) for 12 h at 20°C. The cultures were washed with PBS and incubated with a 1:40 dilution of FITC-conjugated goat anti-mouse IgG for 3 h at 20°C. The cells were washed one final time in PBS, and the labelling was visualized by fluorescence microscopy.

Autoradiography

Glial Fibrillary Acidic Protein Labelling

Basal forebrain cultures were prepared as described and plated on polylysine-coated coverslips in the presence of

Cultures were grown as specified, and subsequently fixed and labelled with rabbit anti-rat antibody to GFAP, ac-

A Neurogenic Cholinergic CNS System

cording to the methods of Hefti et al. (1985) and Manthorpe et al. (1979). Cultures were washed twice with EBSS at room temperature. Cells were fixed with 3.7% formaldehyde for 30 min at room temperature, and subsequently washed once with EBSS, and once with PBS. Incubation with anti-GFAP primary antibody (150 dilution in PBS) was for 30 min, 37°C. After three washes ( 5 minutes each) with PBS at room temperature, the cultures were mounted with Citifluor to prevent bleaching, and viewed with fluorescencemicroscopy.

Neuron-Specific Enolase (NSE) Labelling Basal forebrain cells were grown under appropriate conditions for the desired number of days and labelled for NSE by the method of Morrison, Sharma, de Vellis, and Bradshaw ( 1986). The medium was removed and the cells were fixed by successive incubations in 3.7% formaldehyde and 0.2% Triton X-100 in 3.7% formaldehyde for 10 min at room temperature. The cultures were washed with Tris/KCl buffer and incubated for 3 h at 37°C with rabbit anti-rat NSE antiserum. After washing with Tris/KCI buffer, the cells were incubated with goat anti-rabbit FITC for 1 h, and washed exhaustively with Tris/ KC1 before viewing.

Neurofilament Labelling of Mitotic Cells Cells were grown with olfactory bulb extract, as described, for 24 h. At this time, the medium was replaced by an identical medium containing 0.2 pCifml 3H-thymidine. After 24 h, the thymidine was removed by changing the medium and washing it three times with warm (37°C) DMEM. The cells were allowed to grow with OLB protein for an additional 24 h before fixing with ethanol/acetone ( 3:2). Cultures were treated with antibody to neurofilament for 2 h at 37°C and visualized by IgG-rhodamine. Coverslips were then attached to slides with Permount and dipped into NTB2 emulsion in total darkness. Slides were developed after 3, 7, or 10 days. Pictures of phase, bright-field, and fluorescent light were made of each microscope field.

Statistics Data are presented as mean 2 standard deviation.

RESULTS Cell Death in Defined Medium is Rapid Basal forebrain cells, a region rich in potential cholinergic neurons, were cultured in defined medium to simplify the study of factors that influence early CNS cholinergic development. These cells (from

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rat embryonic basal forebrain, E 15- 16) deteriorated morphologically within 2 days when grown in defined medium. Quantitative assessment of this deterioration was made using a metabolic cell viability assay developed by Manthorpe et al. ( 1986). The basis of the assay is the appearance of a blue color in living cells from the reduction of the tetrazolium salt, MTT, to a formazan product. The amount of color product is measured as a difference in optical density between 570 and 630 nm, the absorbances of reaction product and substrate, respectively. Basal forebrain cells were plated on poly-L-lysine at several plating densities ranging from 75,000 to 225,000 cells/cm*. When grown in defined medium, cultures showed a drop in optical density within 2 h, the earliest time point tested (Fig. 1). Although the shape of the mortality curve varied among experiments, cultures showed early and extensive loss of viability. The average of three experiments indicated that 50% mortality occurred at approximately 5- 10 h after plating (data not shown). This result extends previous observations that basal forebrain cells plated with no addition at a density of 55,000 cells/cm2 or less deteriorated morphologically after 1-2 days in culture (Lambert et al., 1988).

Inhibition of Cell Death by Olfactory Bulb Proteins: Requirement for Protein Synthesis Trophic factors from target tissue have been shown to stimulate survival in CNS cultures. A previous observation indicates that basal forebrain cell survival and neurite outgrowth are supported for periods of at least 3 weeks by soluble proteins from a target, the olfactory bulb (Lambert et al., 1988). Therefore, the efficacy of olfactory proteins during early culture time was assessed in the MTT assay. Cultures grown with a 100 pg/ ml final concentration of olfactory bulb proteins showed little decrease in optical density during the first 48 h after plating (Fig. 1 ). This result is consistent with the previous observation. Stimulation of survival by trophic factors in some cell culture systems has been hypothesized to be due to suppression of an endogenous cell death program (Martin et al., 1988; Koike, Martin, and Johnson, I989 ) . T o determine if olfactory proteins were suppressing the induction of cytotoxic proteins in basal forebrain cultures, the effects of inhibiting protein or mRNA synthesis were studied for the first 24 h of culture. Cycloheximide ( 1 pg/

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Figure 1 Olfactory bulb proteins enhance cell survival. Basal forebrain cells were grown with and without 0.1 mg/ml olfactory bulb proteins at a density of 75,000 cells per well. At the indicated times, MTT was added to the culture medium. The difference in optical density at 570 and 630 nm was determined 6 h later. The filled squares represent cultures grown with olfactory proteins. The open squares are control cultures. Standard deviation among quadruplicates is shown by error bars.

ml) or 5,6-dichlorobenzimidazole (DRB) (50 or minus olfactory proteins, were introduced into the medium at plating and at 3, 6, and 9 h after growth with olfactory proteins. Cell viability was assessed with the MTT assay 22 h after plating. In all cases, the presence of inhibitor reduced the optical density to ~ 1 2 % of that seen without inhibitor. The optical density in cultures with inhibitor was similar to that in no-addition controls. Morphologically, cultures with inhibitor also resembled controls. although neurite outgrowth was not observed. When protein or mRNA synthesis was inhibited, cells died, even in the presence of olfactory proteins. These results imply that death in defined medium was not due exclusively to the induction of cytotoxic proteins; furthermore, cell survival in the presence of olfactory proteins required protein synthesis. p M ) , respectively, plus

3H-Thymidine Incorporation Maintained with Olfactory Bulb Extract

In situ, cells of the basal forebrain are still proliferating at El 5 (Brady et al., 1989; Semba and Fi-

biger, 1988). When cultures were tested for their ability to incorporate 3H-thymidine into DNA, rates of incorporation were found to depend both on the supplements in which they were growing and on their time in culture. Cells growing in defined medium (control) incorporated thymidine at a rate that decreased rapidly with time in culture (Table I ). After 72 h, essentially no 3H-thymidine was incorporated into DNA. At this time, the cells had shrunken somata and no neurites. Decreased DNA synthesis in control cultures is consistent with the results of the MTT assay. When cultures were grown with olfactory bulb proteins, initial incorporation of 3H-thymidine during the first 23 h was nearly double that of controls (Table 1 ). Dose-response analysis indicates that maximal stimulation of 3H-thymidine incorporation was achieved at 0.05 mg/ml. Cultures showed 3H-thymidine incorporation for at least 9 days (Table 1 ) and, although incorporation per microgram of protein decreased gradually to 25% ofthe original incorporation rate by C9, total incorporation did not decrease until after 7 days in culture. Supplementing basal forebrain cultures with

A Neurogenic Cholinergic CNS System

257

Table 1 DNA Synthesis Is Maintained in the Presence of Olfactory Bulb Proteins Time of 'HThymidine Addition ~-

Control

OLB Extract Total cpm (Average)

cPm/Pg

%

I146 ( 5 ) 988 ( 5 ) 863 (5) 667 (2) 274 (3)

100 86 75 58 24

Total cpm (Average)

cpm/pg

9 0

4185 720 65

624 (4) 128 (6) 1 1 (2)

54 I1 1

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9768 11,462 10,500 10,375 5395

4h 24 h 3 days 7 days 9 days

-

Note: cpm = counts per minute. Determination of incorporation was 23 h after 'H-thymidine addition. The numbers in parentheses represent the number of experiments used to determine the average counts per minute per microgram.

proteins from the olfactory bulb thus had a strong positive influence on the maintenance of DNA synthesis. Olfactory Bulb Proteins and Fetal Calf Serum (FCS) Stimulate Different Cell Populations

Since fetal calf serum is typically used to maintain nerve cell cultures, its effect on 3H-thymidine incorporation in basal forebrain cultures was assessed. Basal forebrain cells cultured in the presence of 10%FCS also maintained 3H-thymidineincorporation (Table 2). The amount of incorporation on culture day 2 was similar to that seen with olfactory bulb supplement. To determine if FCS and olfactory bulb proteins support DNA synthesis in the same population of cells, the additive properties of the two supplements were tested. 3H-Thymidine incorporation was measured in the presence of both olfactory bulb proteins (0.1 mg/ml, twice the dose producing maximum response) and fetal calf serum. The results show that the effects of the two supplements on total 3H-thymidine incorporation on culture day 2 were the sum of each added alone

(Table 2). Incorporation per microgram of protein was also additive at this time. After 9 days in culture with both additions, total incorporation was still greatly increased over either addition alone. The data indicate that different populations of cells responded to the addition of FCS and olfactory bulb proteins. Consistent with thymidine uptake data, cultures grown with FCS were also found to differ morphologically from cultures supplemented with olfactory bulb proteins. There was little neurite extension for the first 24 h, after which shorter processes (30%cell diameter, as opposed to 200% with olfactory bulb supplement) could be seen. By C2, proliferation of flat, fibronectinimmunoreactive cells was observed (data not shown). Further experiments identified a network of neuron-specific enolase-immunoreactive cells growing on the lawn of flat cells. Cultures grown in the presence of FCS and olfactory bulb proteins showed a morphology characteristic of both supplements. As previously seen in cultures grown with FCS alone, a lawn of flat cells developed. Topping the flat cells were NSE-positive aggregates interconnected by fascicles, characteristic of cultures grown with olfactory bulb supplements. The morphology of the cultures further indicated that FCS

Table 2 3H-ThymidineIncorporation Is Increased in Cultures Grown with FCS and Olfactory Bulb Proteins ~

Culture Day 24 h

Addition

Total cpm

Total pg

cpm/ccg

OLB FCS alone OLB FCS OLB FCS alone OLB FCS

6600 4880 1 1,435 4710 32,320 6 1,935

17.2 14.5 16.8 29.3 69.6 106.5

384 335 68 1 233 465 582

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Note: Abbreviations as per Table 1 and text. Culture day refers to the time at which 'H-thymidine was added. Determination of incorporation was 23 h later. The concentration of fetal calf serum was 10%.Olfactory bulb proteins were added at 0.1 mg/ml.

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and olfactory proteins affect different populations of cells. Astrocyte Proliferation Insufficient for Trophic Effect

Astrocytes are known to produce trophic activity that can support neurons in culture, raising the possibility that the neuronal effects of olfactory bulb proteins may be consequent to an effect on the astrocytes that may be present. Therefore, cells incorporating 3H-thymidine were characterized using autoradiography and immunochemical staining. The majority of cells incorporating thymidine and those expressing GFAP were found to be different [Fig. 2(A,C)] , although some cells that incorporated thymidine were also GFAP positive [Fig. 2( B,D)] . Trophic factors with known supportive and mitogenic effects on astrocytes were also tested and compared with olfactory proteins. Cells were grown in the presence of EGF, IGF-1, and bFGF. EGF is known to preferentially support astrocytes in CNS cultures ( Monnet-Tschudi and Honneger, 1989).IGF- 1 and bFGF also support astrocyte proliferation and have recently been shown to influence neuronal survival (Aizenman and de Vellis, 1987; DiCicco-Bloom and Black, 1988; Walicke, 1989a; Walicke, Cowan, Ueno, Baird, and Guillemin, 1986). Basal forebrain cultures were plated in the presence of EGF at a concentration of 50 ng/ ml. Only isolated patches of process-bearing cells survived for 5 days in culture, while most of the cells deteriorated [Fig. 3 (D)] . Characterization of these residual cells showed that 90% were GFAP immunoreactive ( not shown ) . IGF- 1 ( 20- 100 ng/ ml) and bFGF (1-5 ng/ml) were even less effective at maintaining survival. In each case, fewer than 7% of the cells survived the first 3 culture days [Fig. 3 (B,C)]. These results indicate that known astrocyte growth factors are not adequate for maintaining neurons either as a downstream or primary consequence. Most Proliferative Cells are Neuroblasts

Although proliferation of astrocytes was supported to some extent by olfactory proteins, the majority of cells incorporating 3H-thymidine were not astroglia. These cells were most likely neurons, since >90% became neuron-specific enolase positive (see below). To verify if mitosis of neuroblasts was supported by our culture conditions, a second dou-

ble-label experiment was run using 3H-thymidine and antibody to neurofilament (NF). This protein is produced by neurons soon after their final mitotic division as processes are being formed (Cochard and Paulin, 1984; Raju, Bignami, and Dahl, 1981). Cultures grown on coverslips were exposed to 'H-thymidine from 24 to 48 h after plating. The radioactivity was removed by careful washing, and cells were allowed to grow for an additional 24 h before being fixed. Cells were then treated with antibody to neurofilament visualized with rhodamine, affixed to slides, and processed for autoradiography with Kodak NTB2 emulsion. Cells were assessed for the presence of neurofilament and/or silver grains after exposure times of 7-10 days (Fig. 4). Of the 385 cells examined, 24% incorporated 3H-thymidine and 78% produced neurofilament. Of the cells positive for 3H-thymidine, 90% were also neurofilament positive. These data show that our culture system supports both neuroblast division and the terminal step of neurogenesis, in which neuroblasts exit the cell cycle to differentiate into neurons. Differentiation of Older Cultures: Cholinergic Aggregates

Neuronal differentiation in these cultures was studied further by examining morphology and neurochemical phenotype. Cultures grown with olfactory proteins adopted a characteristic morphology that developed in three phases: ( 1 ) single cell adhesion to the substrate within 4 h after plating and initial neurite extension up to 200% of the cell diameter by 24 h; ( 2 ) migration of cell bodies into aggregates within 48 h of plating; ( 3 ) fasciculation of neurites interconnecting aggregates by C5 [Fig. 5 (A)]. Immunocytochemical labelling to determine the cellular composition of the cultures showed that over 90% (93% -+ 4%; n = 2406 cells) of the total cells in C5 cultures were positive for the neuronal marker NSE, while fewer than 10% (7% t 2%; n = 196 cells) produced GFAP, a marker for astrocytes, extending a previous study (Lambert et al., 1988 ). The aggregates were predominantly NSE positive, while isolated GFAP-immunoreactive cells were found randomly dispersed throughout the cultures. In vivo, between 10%and 75% of basal forebrain neurons have been reported as cholinergic, depending on the region studied (Mesulam et al., 1983a; Rye et al., 1984). Basal forebrain cells in culture with olfactory bulb proteins were, there-

Figure 2 Two cell types incorporate 3H-thymidine. A, C and B, D are pairs of photographs showing identical microscopic fields. ( A , B) Bright field. highlighting emulsion grains over cells that incorporated 3H-thymidine. (C, D) Fluorescent light, identifying labelled cells that express GFAP. The arrowheads in B and D indicate cells with 3H-thymidine incorporation that are also GFAP positive.

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Figure 3 Growth factors bFGF, IGF-I, and EGF do not mimic the effect of olfactory bulb proteins. ( A ) Olfactory bulb proteins, 0.1 mg/ml. (€3) bFGF, 5 ng/ml. ( C ) IGF-I. 20 ng/ml. ( D ) EGF, 50 ng/ml. (A-C) were photographed on C2. ( D ) was photographed on C5.

A Nmrogenic Cholinrrgic CNS System

Figure 4 Cells that incorporate 'H-thymidine become neurofilament positive. Basal forebrain cells were grown with olfactory proteins and 'H-thymidine as described in Methods. Cultures were fixed on C3 and processed for autoradiography and immunocytochemistry for neurofilament visualized with rhodamine. (A-C) and (D-F) Micrographs of identical cells, respectively: ( A , D) phase illumination, (B, E ) fluorescence, (C, F) bright field. In ( A-C) five cells are shown. Three of the cells (arrows) have incorporated 'H-thymidine and are also neurofilament positive. One cell is neurofilament positive only (open arrowhead), and one cell is 'H-thymidine positive alone (filled arrowhead). Nuclei of these cells appear to be pushed to one side of the cell body. In (D-F), four cells are present, two of which have incorporated 'H-thymidine and are neurofilament positive (arrows). Note the partitioned arrangement of the cytoplasm and nucleus, seen by neurofilament staining (small arrowheads) and silver grains (large arrowheads), respectively. Two cells are 'H-thymidine positive only. More mature cells in (C) show no 3H-thymidine incorporation and typical neurofilament staining in processes.

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comprised entire aggregates [Fig. 6 ( A)]. Conversely, other aggregates were completely ChAT negative. Neurite fascicles connected ChAT-positive aggregates to each other or to aggregates consisting exclusively of ChAT-negative cells. In some instances, ChAT-positive cells were not segregated into discrete aggregates but formed immunoreactive clusters within larger aggregates that also contained unlabelled areas [Fig. 6(B)]. In all cases, self-segregation of ChAT-immunoreactive cells in the presence of olfactory bulb supplement was prominent.

Lack of Long-Term Support by Cerebellar Proteins

Figure 5 Survival. aggregation. and AChE expression are enhanced by the addition of olfactory proteins. E 15C5 basal forebrain cells plated with 0.1 mg/ rnl final concentrations of olfactory bulb extract ( A ) and cerebellar extract ( B ) were fixed and stained for the presence of AChE by the method of Karnovsky and Roots ( 1964). Individual cells as well as large clusters show positive reaction product.

fore, characterized with respect to the extent of cholinergic differentiation. Staining for acetylcholinesterase by the method of Karnovsky and Roots ( 1964)in CS cultures supplemented with olfactory bulb proteins showed the presence of predominantly AChE-positive cells [Fig. 5 ( A)]. Staining was found in 93% 2% ( n = 1706) of the total cell population. Cultures were analyzed further using a monoclonal antibody to ChAT. In CS cultures grown with olfactory bulb proteins, there were abundant ChAT-positive cells (43% t- 5%; IZ = 348). The distribution of Ch AT-positive cells showed a characteristic organization that was nonrandom ( Fig. 6 ) . ChAT-immunoreactive cells were segregated into discrete clusters in which positive cells were in contact with each other rather than being interspersed with unlabelled cells. The clusters often

*

The effects of olfactory bulb supplements were not mimicked by the addition of soluble supplements from the cerebellum, which receives no cholinergic input from the basal forebrain (Rye et a]., 1984). In the presence of cerebellar proteins at a final concentration of 100 pg/ml, cell survival could be maintained no longer than S days. and the cultures showed progressively increasing deterioration [Fig. S ( B)] . Dose-response analysis indicated that increasing the concentration of cerebellar proteins to as high as 400 pg/ml did not improve survival. Adhesion to the substrate occurred within 4 h, as in olfactory bulb-supplemented cultures, and neurite extension could be seen within the first 24 h after plating. Later phases of development, however, were not observed. While small clusters of cells were formed, there was no large-scale aggregation and no fasciculation of neurites. Examination of the cell population showed the predominance of NSE-positive cells. although GFAP immunoreactivity was found in 7% ? 0.4% of the cells ( n = 176). When CS cultures were analyzed for cholinergic markers, staining for AChE showed the enzyme to be present in 80% 8% ( M = 542) of individual cells (Fig. S ). Self-segregation of ChATpositive cells was not observed.

*

Long-Term Survival Requires DNA Synthesis in Young Cultures Because the cultures that showed long-term survival initially showed prominent DNA synthesis, an experiment was designed to test whether DNA synthesis was a requirement for long-term survival. Aphidicolin ( Spadaii, Sala, and Pedrali-Noy, 1982), an inhibitor of DNA polymerase alpha, was

Figure 6 Basal forebrain cultures grown with olfactory proteins show segregation of cells containing ChAT. Cultures grown in olfactory bulb extract for 5 days were labelled with mouse anti-pig monoclonal antibody to ChAT. Visualization of positive cells was achieved by means of FITC-conjugated secondary IgC. ChAT-positive cells may comprise entire aggregates ( A ) , or may be confined to discrete regions within larger aggregates ( B ) . Arrows mark segregated ChAT-positive clusters. ( C ) and ( D )are phase micrographs ofthe fields shown in ( A ) and ( B ) . respectively.

used to prevent DNA synthesis. As expected, the incorporation of 3H-thymidine was reduced in the presence of aphidicolin (Table 3). Approximately 90% of the 3H-thymidine incorporation in olfactory bulb protein-supplemented cultures was also eliminated within 24 h after the addition of inhibitor. Cultures with 10 pA4 aphidicolin showed a loss of processes and shrinkage of cell bodies by C2, and complete degeneration by C3. Decreasing the concentration of aphidicolin to I pM postponed the deterioration of cultures by 1-2 days (data not shown); concentrations of 0.1 p M or less appeared similar to cultures with no inhibitor. Olfactory bulb proteins, as well as FCS, were unable to block culture deterioration when the inhibitor was added at plating (Fig. 7). These results indicate that the initial survival of basal forebrain cultures mediated

by olfactory bulb proteins requires some DNA synthesis. When established cultures ( 7 days) were exposed to aphidicolin for 2 days, their morphology was similar to that of cultures not exposed to aphidicolin (Fig. 7 ) . Although 3H-thymidineincorporation was halted (Table 3), aggregates were formed and processes were maintained for at least the 2 days studied. Longer periods were not followed. DISCUSSION

Embryonic basal forebrain cells placed in culture were found to undergo very rapid death, with a half-life of 5-10 h. This rapid cell death was blocked by protein supplements from olfactory

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Table 3 3H-Thymidine Incorporation in the Presence of Olfactory Bulb Proteins Is Halted by Aphidicolin ~~~

Time of 3H-Thymidine Addition 4h

24 h 3 days 5 days 7 days

OLB Extract

-

~~

OLB Extract and AFD

Total cpm

CPmlPg

Total cpm

cpm/a

10,463 7180 741 5 10,358 5760

1113 780 506 666 388

942 1000 305 2035 1042

106 66

31 138 60

Note: AFD = aphidicolin; other abbreviations as per Table 1, Aphidicolin ( 5 p k f ) was added 24 h before the addition of 'H-thymidine, except for the 4-h point where aphidicolin was added at plating. Incorporation was determined 23 h after 'H-thymidine addition. These results represent the average of three experiments.

bulb, previously shown to promote long-term survival in culture (Lambert et al., 1988). The supplemented cultures, which were capable of DNA synthesis, gave rise to cell populations that were about 90% neuronal and 10%glial. Half the mature cells were ChAT positive. Factors known to promote glial proliferation (EGF, FGF, IGF- 1 ) provided little support for neurons. A key finding was that supplemented cultures did not survive when DNA synthesis was inhibited. The culture system should have potential for analyzing factors that influence proliferation as well as differentiation of basal forebrain neuroblasts, particularly with respect to the cholinergic phenotype. The transition from neuroblast to neuron in the basal forebrain culture system reflects a transition that also is occurring at the same time in v i v a Cholinergic differentiation in the basal forebrain proceeds on a caudal to rostra1 axis, beginning on E 1 1 and ending approximately on El7 (Brady et al., 1989; Semba and Fibiger, 1988). This differentiation essentially resembles overall neurogenesis for this area (Bayer, 1985), although other data have indicated that some neurogenesis continues postnatally (Creps, 1974). The cells used in these cultures are isolated in the middle of this wave of neurogenesis, which appears to continue in vitro under the conditions established here. A key finding of this work is that cells in these cultures that respond to olfactory proteins with DNA synthesis are primarily neuronal precursors. Factors that stimulate DNA synthesis in neuronal and glial precursors have been investigated previously by several groups. Early studies ( Asou, Iwasaki, Hirano, and Dahl, 1985; Kriegstein and Dichter, 1984) used serum to sustain embryonic rat brain cultures and reported that 6%- 1 3% of the NF-positive cells had undergone division in vitro. Other work has focused on testing various addi-

tions for their influence on 3H-thymidine incorporation. In cultured sympathetic neurons, IGF- 1 and insulin double the percentage of tyrosine hydroxylase-positive cells that incorporate 3H-thymidine ( DiCicco-Bloom and Black, 1988). IGF- 1 also stimulates DNA synthesis in aggregating brain cell cultures (Lenoir and Honegger, 1983). Meningeal cells and bFGF were found to double or quadruple, respectively, the percentage of neurofilament-positive CNS cells that are labelled by 'Hthymidine over a 24-h period (Gensburger, Labourdette, and Sensenbrenner, 1986, 1987). Basic FGF also is mitogenic for a number of cell types (Walicke, 1989b) and recently has been shown to increase neuronal survival in culture ( Walicke, 1988, I989b; Walicke et al., 1986; Morrison et at., 1986) as well as cholinergic survival in vivo (Anderson, Dam, Lee, and Cotman, 1988). The percentage of cells incorporating 3H-thymidine in the above experiments ranged from 24% to 30%,similar to levels seen here in the presence of olfactory proteins. In our culture system. however, only 7% of the plated cells survived in the presence of bFGF and ICF-I, and this survival was for