Nerve-growth-factor-dependent and cell-density-independent survival of septal cholinergic neurons in culture from postnatal rats

Neuroscience Research, 8 (1990) 69-82

69

Elsevier Scientific Publishers Ireland Ltd.

N E U R E S 00338

Research Articles

Nerve-growth-factor-dependent and cell-density-independent survival of septal cholinergic neurons in culture from postnatal rats H i r o s h i H a t a n a k a , C h i k a Nishio, Y o i c h i K u s h i m a a n d H i r o k o T s u k u i Division of Protein Biosynthesis, Institute for Protein Research, Osaka University, Osaka (Japan) (Received 22 December 1989; Accepted 25 January 1990)

Key words." Choline acetyltransferase; Neuronal survival; Acetylcholinesterase; Postnatal CNS neuron

SUMMARY We have established a primary neuronal cell culture technique from the postnatal ( P l l to P15) rat CNS to study the nerve growth factor ( N G F ) response to basal forebrain cholinergic neurons. The survival of septal cholinergic neurons in culture was monitored both by the determination of choline acetyltransferase activity and by counting acetylcholinesterase-positive cells. Cells obtained from postnatal septal regions were found to reqt,ire a plentiful oxygen supply during the dissociation of the cells. N G F - m e d i a t e d survival of the septal cholinergic neurons was similarly observed in the cultures by using different plating cell densities up to 12.5 × l0 s c e l l s / c m 2. These results suggest that the promotion by N G F of cell survival in culture is independent of plating cell density.

INTRODUCTION

Nerve growth factor ( N G F ) has been extensively investigated for its role in the regulation of neuronal development both in PNS and CNS 1~.35. N G F regulates the differentiation and survival of PNS sympathetic and sensory neurons, and also of CNS basal forebrain cholinergic neurons. In particular, the survival effect of N G F on CNS neurons is thought to be important in neuronal cell loss during senile dementia in the aged brain 2,2~. In fact, N G F appears to support cell survival after retrograde degeneration of cholinergic neurons in vivo 22,23,37. AS in these in vivo experiments, mature basal forebrain cholinergic neurons in culture can respond to N G F for the promotion of cell survival t8- 20 N G F and N G F receptors were reported to be expressed continually in the adult rat brain. N G F has been detected at high levels in the specific brain regions innervated by basal forebrain cholinergic n e u r o n s 2432. Furthermore, Auburger et al. 4 found a very sharp increase in the amount of N G F in the hippocampus and septum in the 12-14-dayold period of rat brain development. The continuous expression of a significant amount

Correspondence: Dr. H. Hatanaka, Division of Protein Biosynthesis, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan. Tel. 06-877-5111 (ext. 3871). Fax 06-876-2533.

Abbreviations: N G F = nerve growth factor; C h A T = c h o l i n e acetyltransferase; A C h E = acetylcholin'esterase; b F G F = basic fibroblast growth factor; IL-6 = interleukin 6; PNCS = precolostrum newborn calf serum; MAP2 = microtubule-associated protein 2. 0168-0102/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

70 of N G F m R N A in the hippocampus has been reported 5. The expression of N G F receptor in adult rat basal forebrain cholinergic neurons has been detected 29,30,k~3 N G F could thus work as an endogenous neurotrophic factor for basal forebrain cholinergic neurons in the rat brain even after neuronal development. We have developed primary cell cultures from the postnatal rat brain to study the cellular function of basal forebrain neurons and their response to N G F . Recently, Hartikka and Hefti 13 reported that plating cell density and glial cells modulated the effects of N G F on the survival of septal cholinergic neurons in culture from fetal (El7) and newborn (to P2) rat brain. They claimed that the cell survival effect of N G F occurred in low-density culture, but not in high-density culture. We now report the results of studies on basal forebrain cholinergic neurons, from postnatal ( P l l to P15) rat brains, grown under various culture conditions. N G F is found to promote cell survival in culture, independent of plating cell density. MATERIALS A N D M E T H O D S

Chemicals and determination of enzyme activity N G F was prepared as 2.5S-form from male mouse submandibular glands according to the methods of Bocchini and Angeletti 7 with some modifications by Suda et al. 34 Monoclonal mouse antibody MC192 8 against rat N G F receptor was a gift from Prof. E. Shooter (Stanford University, California, U.S.A.). Polyclonal rabbit antibodies 28 against mouse microtubule-associated protein-2 (MAP2) were a gift from Prof. K. Mikoshiba (Osaka University, Japan). A mouse monoclonal antibody P C T H 7 against rat tyrosine hydroxylase was prepared as described previously 16 Choline acetyltransferase (CHAT) activity was determined by the method of F o n n u m 9 with the modifications described previously iv

Preparation of cultured cells Figure 1 is an illustrated diagram of the preparation of cultured neurons from postnatal rat CNS. The septal regions of postnatal rat (Wistar ST, both sexes, Shizuoka) brains were dissected on ice under a dissecting Nikon stereomicroscope with a fiber optic light source. The location of the area was determined according to a developmental rat

ether - onest her ized PIO- P15 r o t s

cold L15

medium

Ipapain DNase I culture medium~"-~ ~.__~ plating 37 °C, 15 min 200 rpm

Fig. 1. Cell culture method for postnatal rat CNS neurons.

71

e-

papain (units~ml } Fig. 2. P12 septal culture: effect of papain concentration for digestion of septal tissue fragments on resulting ChAT activity in culture. Septal neurons were prepared from 12-day-old rats using the indicated various concentrations of papain, as described in the text. Cell yield was 7.2, 6.2, 6.4 and 4.7 x 105 cells in the preparation by using 6, 9, 12 and 15 units/ml papain, respectively, per 1 rat septum. Cells were plated at a density of 0.5 rat septal cells. Culture was carried out for 6 days without (open circles) and with (closed circles) NGF at a concentration of 100 ng/ml. ChAT activity was determined as described in the text. Values are means _+SD (n = 4). atlas 31. Postnatal ages of rats were designated with the birth day set as postnatal day 1 (PI). The preparation methods for cultured cells have been described previously ~s.~9. The dissected tissue fragments (small pieces, < 1 m m thick) were quickly transferred within 2 min into cold oxygen-bubbled L-15 m e d i u m (without serum). Cell culture Tissue fragments were served to 10 ml of a freshly prepared Ca 2+, Mg2+-free phosphate-buffered saline (PBS) containing papain (Cooper, 90 units except for Fig, 2), D N a s e I (Sigma, 2000 units), D,L-cysteine-HCl (Sigma, 2 mg), bovine serum albumin (recrystallized, Armour, 2 mg) and glucose (50 mg), and then incubated twice for 15 min at constant 200 r p m rotation in a 3 7 ° C incubator (New Brunswick Sci., G24 I n c u b a t o r Shaker). Figure 2 shows the effects of different a m o u n t s of papain on subsequent cultures of dissociated septal cholinergic neurons. The optimal concentration of papain was found to be 9 u n i t s / m l . Different lots of papain resulted in small changes in the culture. Papains obtained from C o o p e r and Pharmacia gave almost the same results, but the others were not suitable for our culture. Trypsin (Difco, 1 : 250) was usable for the culture of fetal rat C N S neurons, as shown previously 17 but was not suitable for the culture of postnatal C N S cholinergic neurons. After papain digestion, the tissue fragments were resuspended in a culture m e d i u m consisting of 5% ( v / v ) precolostrum n e w b o r n calf serum (PNCS, lot PF04, Mitsubishi Kasei), 5% ( v / v ) heat-inactivated horse serum (lot 32N3176, Gibco), 1% ( v / v ) heat-inactivated rat serum (prepared by ourselves) and 89% 1 : 1 mixture of Dulbecco's modified Eagle's and H a m ' s F12 m e d i u m (Gibco) containing 15 m M H E P E S buffer ( p H 7.2), 30 nM selenium, 1.9 m g / m l sodium bicarbonate, 50 u n i t s / m l penicillin G and 0.1 m g / m l streptomycin sulfate. Cells were then dissociated by gentle drawing through two different sizes of plastic tips (1.2 and 0.8 m m diameter). Cells were then c o u n t e d in a h e m o c y t o m e -

72 ter for viable cells which exclude nigrosin dye. The viability of the resulting dissociated cells was about 95%. Cell yield from one rat septum area was 6.5 + 1.7 × 105 cells ( J~ = 7) for P l l - P 1 5 . Cells were usually plated at a density of 0.5 rat septal cells (about 3.3 x lt) ~ cells)/cm 2 on a feeder layer of astroglial cells in 48-well plates (l cm 2 of culture surface area, Costar), noted otherwise. In the case of the experiment using the different cell density, cells were plated either on the feeder cells or directly on polyethyleneimine-coated surfaces in 48-well plates. In the high-density culture experiments, the dissociated cells were initially applied onto the PNCS serum lower layer, and centrifuged at 900 rpm for 5 min. In the case of fetal rat septal cholinergic neurons 17, brains were removed from fetal El7 rats, which were purchased as precisely timed pregnant Wistar ST rats. Cells were also digested by papain, which gave similar results in culture to those obtained by the trypsin digestion method. The dissociated cells were seeded onto a polyethyleneiminecoated surface in 48-well plates (Coatar). After they were cultivated for 1 day, the medium was substituted by a serum-free medium, namely T I P / D F medium i4 All cell cultures were carried out at 3 7 ° C in 95% ( v / v ) air and 5% ( v / v ) CO 2 humidified atmosphere in a CO 2 incubator (Tabai, BNA-110).

Cytochemical determination Immunohistochemistries for N G F receptor, MAP2 or tyrosine hydroxylase were performed as follows. Cells were washed by PBS fixed in 4% paraformaldehyde at room temperature. Immunochemical staining for the antigens using the corresponding antibodies was carried out by using avidin-biotin-peroxidase complex (Vector). Acetylcholinesterase (ACHE) cytochemistry was performed by the modified methods of Hefti et al. 23 as described previously 19 RESULTS

Cell culture from postnatal rats After papain digestion, the dissociated septal cells from P3-P25 postnatal rats served for the determination of specific ChAT activities. These values were compared with those in the rat septal area in vivo as shown in Figure 3. The specific C h A T activities before and after papain digestion showed almost the same pattern at various ages. These C h A T

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Fig. 3. In vivo C h A T activities of rat s e p t u m area at different ages (A) a n d the activities of p r e p l a t i n g septal cells after p a p a i n d i g e s t i o n (B). C h A T activities were d e t e r m i n e d as d e s c r i b e d in the text. Values are m e a n s 5: S D (n = 4).

73 TABLE I PII SEPTAL C U L T U R E FOR D I F F E R E N T C U L T I V A T I O N DAYS: EFFECTS OF N G F ON ChAT ACTIVITIES A N D N U M B E R OF AChE-POSITIVE N E U R O N S IN C U L T U R E Days in culture

Added NGF

Total ChAT activities ( p m o l / min/well)

AChE-positive neurons (cells/ well)

Cellular ChAT activities ( p m o l / min/well)

5 days

none NGF none NGF

2.19 ± 0.26 9.14±1.36 1.65 + 0.04 6.60±1.89

54.5 ± 8.7 167 ±13 33.2 ± 3.1 114 ±13

40 55 50 58

9 days

Septal neurons from ll-day-old rats were cultured with or without N G F at concentration of 100 n g / m l as described in the text. Cell yield was 5.2 X 10 s cells per 1 rat septum. Cells were plated at a density of 0.5 rat septal cells on an astroglial feeder layer in 48-well plates (culture surface was 1 cm2). Cultures were carried out for 5 and 9 days. Determination of ChAT activity and AChE staining was conducted as described in the text. Values are m e a n s ± SD (n = 4).

activities in the dissociated septal cells have already been shown to be sustained during the 1 week culture period in the presence of 100 n g / m l N G F , as described previously ts.t9. These results suggest that the C h A T activities of the cultured septal cells in the presence of N G F reflect in vivo ChAT activities. The viable AChE-positive cholinergic neurons from P14 rat septum were more numerous in the presence of N G F than in the absence of N G F during the different culture times, as shown in Table I. Table II shows the effects of the cell preparation on subsequent cultures with and without N G F . It was necessary to supply sufficient oxygen to the tissue fragments for reproducible cultures of postnatal rat septal cholinergic neurons. In the preparation step

TABLE II P14 A N D Pll SEPTAL CULTURES: EFFECT OF N G F ON ChAT ACTIVITY IN C U L T U R E U N D E R D I F F E R E N T C O N D I T I O N S IN THE CELL PREPARATIONS

Exp. I Without O2-bubbling With O2-bubbling Exp. II Using 27 rats Using 3 rats

Added NGF

Total ChAT activities (pmol/min/well)

none NGF none NGF

1.71 ± 0.15 3.39 ± 0.44 3.18 ± 0.60 8.44±2.21

none NGF none NGF

1.51±0.16 6.21±0.57 1.33±0.32 6.02±0.73

In Exp. I, septal neurons from 14-day-old rats were prepared as described in the text, except for correcting the tissue fragments in the oxygen-bubbling or non-oxygen bubbling L-15 medium. Cell yield was 9.2 × 105 cells (in the case of the experiment without O2-bubbling) and 5.8 x 105 cells (in the case with O2-bubbling ) per one rat septum. Cells were plated at a density of 0.5 rat septal cells on an astroglial feeder layer in 48-well plates (culture surface was 1 cm2). Culture was carried out for 6 days in the same manner as in legend of Table I. In Exp. II, septal neurons were prepared from ll-day-old rats (3 rats and 27 rats at the same time), as described in the text. Cell yield was 8.2 × l0 s cells (in the case using 27 rats) and 7.7 × 105 cells (in the case using 3 rats) per 1 rat septum. Cells were plated at a density of 0.4 rat septal cells. Culture was carried out for 6 days. ChAT activity was determined as described in the text. Values are means +_SD (n = 4).

74

Fig. 4. lmmunohistochemical staining of MAP2 (B), of N G F receptor (D) in P15 septal neurons for 6 days, and of tyrosine hydroxylase (F) in PI5 midbrain catecholamine neurons for 6 days in culture. Cells from postnatal rat septum and midbrain were cultured as described in the text. Cultures were carried out on an astroglial feeder layer in 48-well plates. In D, N G F added at concentration of 100 n g / m l in culture. Immunostaining was carried out as described in the text. A, C and E were phase contrast photomicrographs of the same fields as B. D and F, respectively. Scale bar: 50/Lm.

75 b e f o r e p l a c i n g the f r a g m e n t s into o x y g e n - b u b b l e d L15 m e d i u m , p o s t n a t a l rat b r a i n s n o r m a l l y r e m a i n in the ischemic state for nearly 2 rain after d e c a p i t a t i o n of the e t h e r - a n e s t h e t i z e d rats. W e f o u n d that the results of the culture f r o m the rats in the p r o l o n g e d ischemic state up to 15 min were a l m o s t the same (K. H i s a n a g a a n d H. H a t a n a k a , u n p u b l i s h e d ) . However, P14 septal n e u r o n s d i s s o c i a t e d from the b r a i n after s t a n d i n g for 30 min on ice s h o w e d only 60% of C h A T activity in the n o r m a l l y p r e p a r e d c u l t u r e ( < 2 min p r e p a r a t i o n , d a t a not shown). T h e s e results suggest that the brief e x p o s u r e of p o s t n a t a l rat b r a i n to a n o x i a ( < 15 rain) d a m a g e d the n e u r o n s slightly; nonetheless, these n e u r o n s c o u l d be c u l t u r e d a n d they showed n o r m a l responses to N G F in culture. As shown in T a b l e II, a lack of o x y g e n s u p p l y to the c o r r e c t i n g L-15 m e d i u m for tissue f r a g m e n t s severely d a m a g e d the resulting c u l t u r e d neurons. T a b l e II also shows the effect of the n u m b e r of rat b r a i n s c u l t u r e d at the s a m e time. In the case of the e x p e r i m e n t using 27 rats, the time for p l a c i n g the f r a g m e n t s in o x y g e n b u b b l e d L-15 m e d i u m was p r o l o n g e d , so the m e t a b o l i c energy c o n s u m p t i o n from the L-15 m e d i u m might be high. However, the results of cell cultures o b t a i n e d b y using 27 rats versus o n l y 3 rats were similar with respect to C h A T activities after 6 d a y s in culture, a n d their N G F responses were also the same. F i g u r e 4 shows c u l t u r e d cells from two different areas ( s e p t u m a n d m i d b r a i n ) of the p o s t n a t a l rat C N S . In a d d i t i o n , we were able to cultivate n e u r o n s from the h i p p o c a m p u s , a m y g d a l a , striatum, a n d spinal c o r d (H. H a t a n a k a et al., u n p u b l i s h e d ) .

'NGF-dependent survi~,al of cholinergic neurons even in high-density culture of postnatal rat septal neurons H a r t i k k a a n d Hefti ~3 recently r e p o r t e d that N G F was not necessary for the survival of fetal a n d n e w b o r n rat septal cholinergic n e u r o n s in h i g h - d e n s i t y cultures. W e t h e r e f o r e e x a m i n e d the p o s s i b l e N G F - d e p e n d e n t effect in different p l a t i n g densities of septal cells. F i g u r e 5 shows the c e l l - d e n s i t y - d e p e n d e n t changes in C h A T activities with a n d w i t h o u t

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Fig. 5. Pll septal cultures at various cell densities: effect of NGF on ChAT activities in culture. Cell yield of septal cells from ll-day-old rats was 8.2 x 105 cells per 1 rat septum. Cells were plated at densities of 8.2, 4.1, 2.1 and 1.0 x 105 cells, corresponding to 1.0, 0.5, 0.25 and 0.125 rat septal cells, respectively, on an astroglial feeder layer in 48-well plates (culture surface was 1 cm2). Culture was carried out for 6 days without (open circles) and with (closed circles) NGF at a concentration of 100 ng/ml. ChAT activity was determined as described in the text. Values are means+_SD (n = 4).

76 TABLE I!I P15 SEPTAL CULTURES AT VARIOUS CELL DENSITIES: EFFECTS OF NGF ON ('hAT ACI'IVITIfLS AND NUMBER OF AChE-POSITIVE NEURONS IN CULTURE Cell density

Added NGF

Total ChAT activities (pmol/ rain/well)

AChE-positive neurons (cells/ well)

Cellular ChAT activities (pmol/ rain/cell)

1.0 rat/well

none NGF none NGF none NGF

4.38 ± 0.24 12.2 + 1.5 2.42 _+0.37 6.17+_0.88 1.53 _+0.39 2.13_+0.59

44.7 ± 1.2 160 ± 19 48.0 _+ 7.6 143 +_ 8 16.7 ± 3.21 53.3_+ 0.58

98 76 50 43 92 40

0.5 rat/well 0.25 rat/well

Cell yield of septal cells from 15-day-old rats was 3.9x 105 cells per 1 rat septum. Cells were plated at a density of 3.9, 2.0 and 1.0x 105 cells, corresponding to 1.0, 0.5 and 0.25 rat septal cells, respectively, on an astroglial feeder layer in 48-well plates (culture surface was 1 cm2). Culture was carried out for 6 days. Determination of ChAT activity and AChE staining was conducted as described in the text. Values are means+SD (n=4). Cellular ChAT activity was obtained by dividing total activity by the number of AChE-positive neurons, as shown in Fig. 6.

N G F after 6 d a y s in culture o f P14 septal cholinergic n e u r o n s on an astroglial feeder layer. A high level of C h A T activity was o b s e r v e d in the presence of N G F . A t the m a x i m a l h i g h - d e n s i t y culture using 1.0 rat septal n e u r o n s (the d i s s o c i a t e d viable n u m b e r of cells was 8.2 x 10 5) p e r cm 2 culture surface, which was c o a t e d b y an astroglial feeder layer, C h A T activity in culture w i t h o u t N G F was o n l y 17% of that with N G F . T h e i n c r e a s e d level of C h A T activity in the culture with N G F was c o n f i r m e d to be due to the increased n u m b e r of viable A C h E - p o s i t i v e , i.e. cholinergic, n e u r o n s in the culture with N G F ( T a b l e III). In T a b l e III, cellular C h A T activity has been c a l c u l a t e d by d i v i d i n g each value of total C h A T activity by the n u m b e r of viable A C h E - p o s i t i v e neurons. These values (CHAT activity p e r cell) with a n d without N G F were m o s t l y the s a m e in the various cell d e n s i t y cultures. These d a t a clearly suggest that the N G F - p r o m o ted cell survival effect on c u l t u r e d p o s t n a t a l rat septal cholinergic n e u r o n s was not d e p e n d e n t on p l a t i n g cell density. F i g u r e 6 shows A C h E staining of cholinergic n e u r o n s in cultures with a n d without N G F at different p l a t i n g cell densities. In F i g u r e 7, an e x p e r i m e n t was c a r r i e d out like that in F i g u r e 5 except for direct p l a t i n g of a different a m o u n t of the cells o n t o a p o l y e t h y l e n e ± m i n e - c o a t e d s u b s t r a t u m with no astroglial feeder layer. A l t h o u g h the n o n - n e u r o n a l cells, which c o n s i s t e d m a i n l y of astroglial cells a n d o l i g o d e n d r o g l i a l cells, were c o - c u l t u r e d with n e u r o n s in these p o s t n a t a l rat C N S cultures, c y t o s i n e a r a b i n o s i d e a d d e d e x o g e n o u s l y to the m e d i u m was a b l e to s u p p r e s s these m i t o t i c cells in culture. T h e p o p u l a t i o n of glial cells was therefore e x p e c t e d to be r e d u c e d in the culture. T h e cell d e n s i t y d e p e n d e n c y on C h A T activities with a n d without N G F in culture was a l m o s t the s a m e as that in the e x p e r i m e n t using an astroglial feeder layer. T h e h i g h - d e n s i t y culture (12.5 × 10 5 c e l l s / c m 2) without an astroglial feeder layer was still N G F - r e s p o n s i v e with respect to C h A T activities in p o s t n a t a l septal culture. T h e results also suggest that N G F is a b l e to p r o m o t e cell survival i n d e p e n d e n t o f p l a t i n g cell density. However, c o u n t i n g of viable A C h E - p o s i t i v e n e u r o n s in the culture was very difficult, b e c a u s e c o n s i d e r a b l e n o n - s p e c i f i c staining o c c u r r e d in the a b s e n c e of an astroglial feeder layer.

77

Fig. 6. AChE staining of P15 septal cultures at various cell densities. Cell culture was the same as that in Table III. Cells were plated at densities of 1.0 (A, B), 0.5 (C, D) and 0.25 (E, F) rat septal cells on an astroglial feeder layer in 48-well plates (culture surface was 1 cruZ). Culture was carried out for 6 days without (A, C, E) and with N G F at a concentration of 100 n g / m l (B, D, F). AChE staining was performed as described in the text. Scale bar: 100 ~m.

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cell density ( r a t / w e l l ) Fig. 7. P13 septal cultures at various cell densities grown on culture surface without an astroglial feeder layer: effect of N G F on C h A T activities in culture. Cell yield of septal cells from 13-day-old ras was 8.3 x 105 cells per 1 rat septum. Cells were plated at densities of 12, 8.3, 4.2, 2.0 and 1.0 x 105 cells, corresponding to 1.5, 1:0, 0.5, 0.25 and 0.125 rat septal cells, respectively, on a polyethyleneimine-coated surface in 48-well plates (culture surface was 1 cm2). Culture was carried out for 6 days without (open circles) and with (closed circles) N G F at a concentration of 100 n g / m l . C h A T activity was determined as described in the text. Values are m e a n s + SD (n = 4).

NGF-mediated ChA T induction in fetal rat septal culture." plating cell density dependency For comparison on

NGF-mediated

with results obtained ChAT

induction

was

by using postnatal examined

=

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dependency

of fetal rat (El7)

septal

!

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rats, cell density

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1

6 0

0.25

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cell density ( ratwell ) Fig. 8. El7 septal cultures at various cell densities: effect of N G F on C h A T activities in culture. The preparation of fetal rat septal neurons was carried out as described in the text. Cell yield was 13.4x 105 cells per 1 rat septum. Cells were plated at the indicated density on a polyethyleneimine-coated surface in 48-well plates (culture surface was 1 cm2). Culture was carried out in a serum-free medium for 6 days without (open circles) and with (closed circles) N G F at a concentration of 100 n g / m l . C h A T activity was determined as described in the text. Values are means + SD (n = 4).

79 cholinergic neurons (Fig. 8). Cells were plated on a polyethyleneimine-coated surface and cultured in a serum-free, T I P / D F TM medium. N G F - m e d i a t e d induction of ChAT activity was observed throughout the different plating cell densities in the culture. DISCUSSION Basal forebrain magnocellular cholinergic neurons are thought to play an indispensable role in the functional neuronal pathways of learning and memory 6. Some correlations between death a n d / o r shrinking of these neurons and loss of the cholinergic neurons and senile dementia of the Alzheimer type have been pointed out. N G F , one of the well-characterized neurotrophic factors, has been known to play an important role in neuronal differentiation and cell survival of basal forebrain magnocellular cholinergic neurons m,23 A hypothesis that the projections of forebrain cholinergic neurons to the hippocampus and cortex are supported by N G F acting as a target-derived neurotrophic factor has been proposed.

Survival effect of NGF on cultured postnatal rat septal cholinergic neurons In the present paper, we were able to demonstrate that the survival effect by N G F on cultured rat septal neurons was not dependent on plating cell density, contrary to the recent report by Hartikka and Hefti 13. We have previously reported that, during the early postnatal day, the action of N G F changes from the induction of ChAT activity to the promotion of cholinergic neuronal cell survival in septal cholinergic neurons in culture 19 N G F appeared to support cell survival of basal forebrain cholinergic neurons obtained from P l l - P 1 4 rat septum 18 the vertical limb of the diagonal band of Broca 19 and the nucleus basalis of Meynert 2o. The role of N G F appeared to change from a differentiation factor to a survival-promoting factor during the early postnatal period, which is just coincident with the period of completion of the septal projection to the hippocampal formation 27 In the present experiments, we cannot exclude the possibility of a modulatory effect of astroglial cells which were preplated as a feeder layer and were also co-cultured with cells obtained from the postnatal rat septal area. The preliminary seeded astroglial cells as a feeder layer are not damaged so much in culture by 1 /~M cytosine arabinoside, because the feeder cells have already grown to confluence and have stopped their cell division. The large portion of astroglial cells which existed in the postnatal septal area were unable to survive in the presence of cytosine arabinoside. The effects of N G F on survival of postnatal rat septal cholinergic neurons in culture without an astroglial feeder layer were similar to those using these astroglial cells as a feeder layer. These results suggest that N G F could operate as an independent survival factor for astroglial cells. On the other hand, N G F elicits neural differentiation, i.e. an induction of C h A T activity, in fetal rat septal cholinergic neurons in culture. N G F - m e d i a t e d induction of C h A T activity was also observed even under high-density culture conditions. In agreement with the results of our previous report 17 and also with those of Hartikka and Hefti 13. The latter report showed that N G F - d e p e n d e n t cell survival occurred only under low-density culture conditions, but not under high-density conditions. As they mentioned, the physiological significance of N G F in promoting cell survival only in low-density culture is doubtful. It seems to be difficult to correlate in vitro, low-density cultures to the normal neuronal development of fetal CNS neurons in vivo. Regarding neuronal cell survival in vivo, the period of development may not be the only important factor for cell survival, since the postmitotic, mature cells continue to live for long intervals. Thus, some

80 survival conditions and factors could be expected to operate for the support of Iong-tcrm neuronal cell survival. In the present paper, we were able to demonstrate the effects of N G F on survival of cultured P l l - P 1 5 septal cholinergic neurons obtained from the rat CNS even after the developmental stage. Hefti 2: initially reported the N G F - m e d i a t e d promotion of in vivo survival of adult rat septal cholinergic neurons after fimbrial transsection. Also, Williams et al. ~7 and K r o m e r 25 independently found results similar to those of Hefti 22. They concluded that basal forebrain cholinergic neurons destined to die after axotomy can be rescued by N G F administration. Cells obtained by papain digestion of postnatal rat septal tissues seem to be similar in cultures to those of in vivo cholinergic neurons after axotomy. They must regenerate their neurites and recover from cellular damage under culture conditions. N G F might potentiate some regenerative cellular steps, and then result in rescue from cell death.

Various factors affected by the survival of cultured neurons Factors other than N G F are known to promote the survival of basal forebrain cholinergic neurons. The basic fibroblast growth factor ( b F G F ) promoted the survival of septal cholinergic neurons in vivo t. Interleukin-6 (IL-6) promoted the survival of septal cholinergic neurons in vitro11 The survival of tyrosine-hydroxylase-positive neurons obtained from the P15 rat midbrain area, including mainly substantia nigra, was supported by the addition of IL-6 in culture 12 (y. Kushima et al., unpublished). The cholinergic interneurons obtained from P10 rat striatum were supported in their survival in culture by N G F , but P7 rat spinal cholinergic motor neurons were not affected by the addition of N G F in culture (H. Hatanaka et al., unpublished). Neither b F G F nor IL-6 belongs to the type of target-derived factors, while N G F is thought to be a typical target-derived factor. These findings suggest that the survival of basal forebrain chotinergic neurons is influenced by various trophic factors. Many survival factor candidates have been reported to affect various kinds of CNS neurons in culture 36. In many cases, the in vitro assay system for cell survival was used by preparing CNS neurons from fetal rats. The low-density cultures of the dissociated CNS neurons are known to have difficulty in maintaining their cell viability even for several days in culture. Candidate factors could promote their cell survival in low-density CNS neuronal cell cultures 3s. Hartikka and Hefti 13 also reported the effects of N G F on cell survival in low-density septal cholinergic neuronal culture. In the present work, we suggest that the effects of trophic factors on cell survival in CNS culture are observed in tissues from postnatal rats. In PNS neuronal culture, several trophic factors including N G F are known to affect cell survival. Their effects seem to be different from those in CNS neuronal culture 26 Characteristics of cultured postnatal septal neurons In the present paper, we have established the neuronal culture method from postnatal ( P l l - P 1 5 ) rat CNS. As can be seen in Figure 1, this culture system has several unique characteristics: First, a sufficient supply of oxygen during the preparation of dissociated cells from postnatal rat brain tissue is necessary for their viability in culture. On the other hand, an oxygen supply for culture of fetal rat septal neurons is not necessary. Furthermore, we found better cell survival of postnatal rat forebrain cholinergic neurons in culture in a 50% oxygen atmosphere during cultivation in a CO z incubator than with normal air (20% oxygen atmosphere) (H. H a t a n a k a et al., manuscript in preparation). Also, low cholinergic cell survival was observed in a 10% oxygen atmosphere culture. These results strongly

81 s u g g e s t t h a t c u l t u r e d p o s t n a t a l rat septal c h o l i n e r g i c n e u r o n s r e q u i r e h i g h e r o x y g e n levels for s u r v i v a l d u r i n g d i s s o c i a t i o n a n d c u l t i v a t i o n p e r i o d s in a C O 2 i n c u b a t o r . Second, postnatal neurons, which have already been connected by synapse formation, c o u l d be l a b e l e d r e t r o g r a d e l y in v i v o w i t h f l u o r e s c e n t l a t e x m i c r o s p h e r e s b e f o r e c u l t u r e . W e f o u n d t h a t the s u r v i v a l of r e t r o g r a d e l y l a b e l e d n e u r o n s p r o j e c t i n g to the h i p p o c a m p u s f r o m b a s a l f o r e b r a i n n e u r o n s w a s p r o m o t e d b y the a d d i t i o n of N G F in c u l t u r e 3 T h i r d , p o s t n a t a l C N S n e u r o n s c o n s i s t o f v a r i o u s t y p e s of cells as well as d i f f e r e n t cell sizes. T h e e x t e n t of t h e d i f f e r e n c e in the cell size o f p o s t n a t a l m a t u r e d C N S n e u r o n s w a s e x p e c t e d to b e l a r g e r t h a n that o f fetal C N S n e u r o n s . W e p u r i f i e d the m a g n o c e l l u l a r b a s a l f o r e b r a i n c h o l i n e r g i c n e u r o n s b y the m e t h o d o f e l u t r i a t i o n c e n t r i f u g a t i o n ( H . H a t a n a k a et al,, m a n u s c r i p t in p r e p a r a t i o n ) . AC KNOWLEDGEM ENTS W e t h a n k Prof. E. S h o o t e r o f S t a n f o r d U n i v e r s i t y a n d Prof. K. M i k o s h i b a of O s a k a U n i v e r s i t y for k i n d l y s u p p l y i n g the a n t i b o d i e s . W e are i n d e b t e d to D r . E. M e y e r of U n i v e r s i t y o f F l o r i d a for his critical r e a d i n g of the m a n u s c r i p t . T h i s w o r k was s u p p o r t e d in p a r t b y a G r a n t - i n - A i d for S c i e n t i f i c R e s e a r c h o n P r i o r i t y A r e a s ( M o l e c u l a r Basis of N e u r a l C o n n e c t i o n ) o f the J a p a n e s e M i n i s t r y o f E d u c a t i o n , S c i e n c e a n d C u l t u r e ( N o . 038). REFERENCES 1 Anderson, K.J., Dam, D., Lee, S. and Cotman, C.W., Basic fibroblast growth factor prevents death of lesioned cholinergic neurons in vivo, Nature (Lond.) 332 (1988) 360-361. 2 Appel, S.H., A unifying hypothesis for the cause of amytrophic lateral sclerosis, Parkinsonism, and Alzheimer disease. Ann. Neurol., 10 (1981) 499-505. 3 Arimatsu, Y., Miyamoto, M., Tsukui, H. and Hatanaka, H., Nerve growth factor promotes survival of retrogradely labeled hippocampus-projecting neurons in the rat basal forebrain in vitro. Det~,. Brain Res., 45 (1989) 297-301. 4 Auburger, G., Heumann, R., Hellweg, R., Korsching, S. and Thoenen, H., Developmental changes of nerve growth factor and its mRNA in the rat hippocampus: comparison with choline acetyltransferase. Det,. BioZ, 120 (1987) 322 328. 5 Ayer-LeLievre, C., Olson, L., Ebendal, T., Seiger, A. and Persson, H., Expression of the /3-nerve growth factor gene in hippocampal neurons, Science, 240 (1988) 1339-1341. 6 Bartus, R.T., Dean III, R.L, Beer, B. and Lippa, A.S., The cholinergic hypothesis of geriatric memory dysfunction, Science, 217 (1982) 408-417. 7 Bocchini, V. and Angeletti, P.U., The nerve growth factor: purification as a 30,000-molecular weight protein, Proc. Natl. Acad. Sci. USA, 64 (1969) 787-794. 8 Chandler, C.E., Parsons, L.M., Hosang, M. and Shooter, E., A monoclonal antibody modulates the interaction of nerve growth factor with PC12 cells, J. BioL Chem., 259 (1984) 6882-6889. 9 Fonnum, F., A rapid radiochemical method for the determination of choline acetyhransferase, J. Neurochem., 24 (1975) 407-409. 10 Gnahn, H., Hefti, F., Heumann, R., Schwab, M.E. and Thoenen, H., NGF-mediated increase of choline acetyltransferase (CHAT) in the neonatal rat forebrain: evidence for a physiological role of NGF in the brain. Det~. Brain Res., 9 (1983) 45-52. 11 Hama, T., Miyamoto, M., Tsukui, H., Nishio, C. and Hatanaka, H., Interleukin-6 as a neurotrophic factor for promoting the survival of cultured basal forebrain cholinergic neurons from postnatal rats, Neurosci. Lett., 104 (1989) 340 344. 12 Hama, T., Miyamoto, M., Takei, N., Noguchi, K., Kushima, Y. and Hatanaka, H., Interleukin-6 promotes the survival of cultured brainstem catecholamine neurons from postnatal rats, Bull. Jpn. Neurochem. Soc., 28 (1989) 92 93. 13 Hartikka, J. and Hefti, F., Development of septal cbolinergic neurons in culture: plating density and glial cells modulate effects of NGF on survival, fiber growth and expression of transmitter-specific enzymes, J. Neurosci., 8 (1988) 2967-2985.

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