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Effects of nerve growth factor and basic fibroblast growth factor on survival of cultured septal cholinergic neurons from adult rats Yoichi Kushima, Chika Nishio, Takeshi Nonomura and Hiroshi Hatanaka Dicision o f Protein Biosynthesis, Institute for Protein Research, Osaka University, Osaka (Japan)

(Accepted 14 July 1992)

Key words: Neurotrophic factor; Septum; Acetylcholinesterase; Neuronal cell survival; Neuronal cell culture; Papain digestion

We have established a primary culture technique for neuronal cells from rat basal forebrain from postnatal day 58 (P58) to study the effects of neurotrophic factors on the neurons. The survival of acetylcholinesterase (AChE)-positive neurons of 2-week-old rat septum has already been reported to be strongly supported by nerve growth factor (NGF) in culture. In this culture study of neurons from adult rat brains, the survival of AChE-positive neurons from P58 rat septum was slightly improved by NGF, although low affinity NGF receptor expression was also observed on cultured P58 rat septum neurons as well as on those from 2-week-old rats. The addition of basic fibroblast growth factor (bFGF) improved the survival of AChE-positive neurons cultured from P58 rat septum, but did not promote the survivalof neurons from P12 rat septum. These results suggest that NGF changes to a maintenance factor in adult rat brain from a survival factor in postnatal 2-week-old rats. The survival of cholinergic neurons in culture of adult rat septum might be supported by factor(s) other than NGF, such as bFGF.

INTRODUCTION N e u r o n a l d i f f e r e n t i a t i o n a n d survival are s u p p o r t e d

cholinergic n e u r o n s in the adult rat after axotomy of s e p t o - h i p p o c a m p a l projection can be p r e v e n t e d by the a d m i n i s t r a t i o n of exogenous N G F 13'15"16'19 However,

by n e u r o t r o p h i c factors. Nerve growth factor ( N G F ) is

the removal of the entire p o p u l a t i o n of h i p p o c a m p a l

well characterized a n d has b e e n k n o w n to act o n cholinergic n e u r o n s in the basal forebrain. N G F is known to be c o n t i n u o u s l y p r o d u c e d a n d secreted, a n d

cells p r o d u c i n g N G F as the target area of septal cholinergic n e u r o n s did not cause the loss of septal cholinergic n e u r o n s 24.

is retrogradely t r a n s p o r t e d even in the adult rat C N S 2-4'22'29. N G F is c o n s i d e r e d to be related with

In addition, basic fibroblast growth factor ( b F G F ) is k n o w n to aid the survival of e m b r y o n i c rat n e u r o n s 4'8'28.

n e u r o n a l cell death, especially in senile animals, but the correlation has not b e e n clarified. W e have already d e m o n s t r a t e d changes in the neu-

However, the n e u r o t r o p h i c roles of N G F a n d b F G F in m a t u r e n e u r o n s have not b e e n clarified. W e have established a n e u r o n a l cell c u l t u r e t e c h n i q u e for adult (up to P58) rat brain, a n d studied the effects of N G F a n d b F G F o n cholinergic n e u r o n s c u l t u r e d from adult rat septum.

rotrophic role of N G F d u r i n g the early postnatal life of the rat: N G F acts as a d i f f e r e n t i a t i o n factor in the early stages a n d as a survival factor in the later ones 'j'"~'12. This indicated that N G F may rescue the n e u r o n s from cell d e a t h at the e n d stage of the formation of the septal cholinergic synapse into hippocampus. T h e n e u r o t r o p h i c effect of N G F on the f u r t h e r d e v e l o p m e n t of septal cholinergic n e u r o n s has not b e e n d e f i n e d in detail, b e c a u s e n e u r o n a l primary culture from adult rat CNS had not b e e n established. As d e m o n s t r a t e d in in vivo studies, the loss of septal

MATERIALS AND METHODS Chemicals

NGF was prepared as a 2.5S form from male mouse submandibular glands according to the methods of Suda et al. 25. Human recombinant bFGF 21 was the kind gift of Takeda Pharmaceutical Co. Polyclonal rabbit antibody against mouse microtubule-associated protein 2 (MAP2)z° was kindly given by Drs. M. Niinobe and K.

Correspondence: H. Hatanaka Division of Protein Biosynthesis, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan. Fax: (81) (6) 876-2533.

265 Mikoshiba (Institute for Protein Research, Osaka University). Monoclonal antibody MC192 against low affinity NGF receptor 6 was kindly provided by Dr. E. Shooter (Stanford University Medical School). Media for cell culture were obtained from Gibco. Other chemicals of analytical grade were purchased from Sigma, unless otherwise stated.

Preparation of dissociated cells front P58 rats The technique for culturing adult rat CNS neurons was modified from the previously published method for the culture of postnatal

2-week-old rat CNS neurons 8-I°. Briefly, tissue fragments of the septal area were dissected out from postnatal P58 rats (Wistar ST, both sexes, Shizuoka). The postnatal age of rats was designated with the birth date set as postnatal day 1. Dissection was carried out on ice under a Nikon stereomicroscope with a fiber optic light source to avoid warming the brain. The location of the area was determined by using a developmental rat atlas 23. The dissected tissue fragments were quickly transferred into ice cold oxygen-bubbled L-15 medium. The time from removal of the brain to placing the fragments into oxygen-bubbled L-15 medium did not exceed 2 min. Then, the tissue

Fig. 1. Immunohistochemical staining of MAP2 in P58 rat septum neurons. Cells from P58 rat septum were cultured as described in the text. Cultures were performed on astroglial feeder layers for 5 days. Cell density was 0.5 rat septum per cm 2, Immunostaining for MAP2 was carried out as described in the text, and visualized with an FITC-labelled anti-rabbit antibody. Bar = 100/xm.

266 fragments were added to 10 ml of freshly prepared Ca2+,Mg ~ +-free phosphate-buffered saline (PBS) containing papain (Worthington, 90 units), DNase I (2,000 units), D,L-cysteine-HC1 (2 mg), bovine serum albumin (recrystallized, Armour, 2 mg) and glucose (50 mg), and then incubated twice for 15 min at a constant rotation of 250 rpm in a 37°C incubator (New Brunswick Sci., G24 Incubator Shaker). After papain digestion, the tissue fragments were resuspended in a warmed culture medium consisting of 5% (v/v) precolostrum newborn calf serum (PNCS, Mitsubishi Kasei), 5%, (v/v) heat-inactivated horse serum (Gibco), 1% (v/v) heat-inactivated rat serum (prepared by ourselves) and a 89% 1 : 1 mixture of Dulbecco's modified Eagle's and Ham's F12 medium containing 15 mM HEPES buffer, pH 7.2, 30 nM selenium, 1.9 m g / m l sodium bicarbonate, 50 units/ml penicillin G and 0.1 m g / m l streptomycin sulfate (Meiji Seika). Cells were then dissociated by gently drawing the fragments through three sizes of plastic tips, 1.6 mm in diameter, 1.2 mm and 0.8 mm, in that order. Dissociated cells were applied to a PNCS layer and centrifuged, then the cells were resuspended in the serum-containing medium and plated on a feeder layer of astroglial cells in 48-well plates (Sumitomo Bakelite, cultured surface is 0.66 cm2). Cells were plated at a density of 0.5 rat septum per cm 2. Astroglial cells were prepared from 19- or 20-day fetal rat cortex as described previously x2. After the cells were cultured for 3-6 h in humidified 5% CO2-95% air at 37°C in the serum-containing medium, half of the medium was changed. The serum-free culture was obtained by changing the medium to a chemically defined medium, namely T I P / D F medium, which consists of the same constituents as the serum-containing medium except for the addition of 5 / z g / m l human transferrin, 5 p~g/ml bovine insulin (Colab. Res.) and 20 nM progesterone. The culture medium was changed every 2 or 3 days. Cytosine

arabinoside was added to the medium at a concentration of 1 # M every other time the medium was changed.

Histochemistry Acetylcholinesterase (ACHE) staining was performed on cultured septal cholinergic neurons according to the method of Tago et al. ~" Briefly, cells were fixed with 4% paraformaldehyde in sodium phosphate buffer (pH 7.2) for 30 min at room temperature. Then, cells were incubated in a reaction mixture containing acetylcholine iodide as substrate and tetraisopropyl pyrophosphoramide as the pseudocholinesterase inhibitor. AChE-positive neurons were visualized with diaminobenzidine and hydrogen peroxide. Neuronal cell staining was done by using a polyclonal anti-MAP2 antibody with fluorescein-conjugated anti-rabbit IgG (Capped as a second antibody.

RESULTS

Neuronal cell culture from adult rats We have previously reported that the culture of neurons from postnatal 2-week-old rat brains is possible without the separation of living cells after papain digestion. However, when dissociated adult rat septal neurons were plated directly on the culture plates without the purification step, few neurons survived. There was much debris in the culture of P58 rat

Fig. 2. MAP2 and AChE double staining of P58 rat septal cultures. Cell culture of P58 rat septum was conducted as described in the text. Immunohistochemistry of MAP2 (A,C) and AChE staining (B,D) were carried out as described in the text. Cells were cultured without NGF (A,B) or with NGF (100 ng/ml) (C,D) for 5 days in T I P / D F medium on an astroglial feeder layer. A and C are photomicrographs of the same field as B,D. Bar = 50/zm.

267 septum prepared without the purification step, and it seemed that the debris inhibited the attachment of living cells to astroglial feeder layers. After centrifugalion of the cell suspension onto a PNCS layer, viable ceils without broken pieces of tissue were obtained and they grew well. The viable number of the MAP2-posilive neurons in culture from P58 rats was almost half of that of neurons from P14, as shown in Table I. Fig. 1 shows the immunohistochemical staining of MAP2positive neurons cultured from P58 rat septum for 5 days in T I P / D F medium. These MAP2-positive neurons had some neurites, and fusiform and multipolar cells were observed.

Effects of NGF and bFGF on survival of cultured adult neurons Cultured septal cells of P58 rats in T I P / D F medium contained AChE-positive neurons, as shown in Fig. 2. The AChE-positive neurons grew well, and they were more well-developed in culture with NGF. The numbers of viable AChE-positive and MAP2-positive neurons in the presence and absence of NGF are presented in Table I. NGF did not affect the number of viable MAP2-positive neurons in culture from adult and P14 rats. The survival of cultured AChE-positive cells from adult rats was slightly promoted by NGF at a concentration of 100 ng/ml, as shown in Table I. On the other hand, NGF strongly promoted the survival of cultured AChE-positive neurons from P12 and P14 rats. Fig. 3 shows the response of cultured septal cholinergic neurons from P12, P38 and P58 rats to NGF a n d / o r bFGF by depicting the number of viable AChE-positive neurons. In the culture of P12 rat septum cells with NGF, AChE-positive neurons were sigTABLE I

Effect o f N G F on surcical o f MAP2- and AChE-positit'e neurons from PI2, PI4 and P58 rat septum Cell cultures of PI2, PI4 and P58 rat septum were performed as described in the text. lmmunohistochemistry of MAP2 and AChE staining were carried out as described in the text.. Cells were grown with or without N G F (100 n g / m l ) for 5 days and stained. N,D., not determined. Values are means 5: S.D., n = 4.

MAP2-positice cells / cm 2 P12 in serum-free medium control N.D. +NGF N.D. P14 in serum-containing medium control 5,8135:1,010 +NGF 5,818± 172 P58 in serum-free medium control 2,690:t: 301 + NGF 2,505 ___ 455

A ChE-positice cells / cm 2 67.3 5:10.6 221.95:36.7 ** 85.85:3.0 210.0± 17.0 ** 20.05:4.6 29.0 ± 11.5

** P < 0.01 versus the control culture (ANOVA).

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AChE-positive neurons (cells/crn=) Fig. 3. Effects of N G F and b F G F on survival of AChE-positive neurons from P12, P38 and P58 rat septum. Cultures were grown on astroglial feeder layers, as described in the text. After the cells were grown for 5 days, AChE-positive cells were visualized as described in the text and counted under microscopic observation. N G F and b F G F were added at concentrations of 100 n g / m i . As a control, cells were cultured without the growth factors. Values are means 5: S.D., n = 4; • P < 0.05 and * * P < 0.01 versus the culture without growth factors (ANOVA).

nificantly more numerous than in the control, but the number was not changed by the addition of bFGF. The effect of NGF on survival, however, was less in P38 and P58. The survival effect of NGF was not evident even if the concentration of 1-500 ng/mi were tested on P35 rat septal neurons in culture (data not shown). Unlike NGF, the effect of bFGF on survival became evident in cultures of P38 and P58 rat septum. The maximal dose of bFGF on postnatal 8-week-old rat septum was 100 ng/ m l (data not shown). As shown in Fig. 3, additional NGF and bFGF reduced the effect of NGF on survival in cultures of P12 rat septum. In cultures of P38, the effect of bFGF on the survival of AChE-positive neurons was slightly decreased by NGF. However, the improvement of the survival of AChE-positive neurons with bFGF was not suppressed by NGF in culture of P58 rat septum. These effects of NGF and bFGF on survival did not change even if lower doses were supplied (data not shown).

268 The reason for the reduction in the effect of NGF on survival in AChE-positive neurons cultured from P38 and P58 rat septum has not been clarified. As a possible explanation, N G F receptor expression may have disappeared in P38 and P58 rat septal cultures. As shown in Fig. 4, we tested the presence of low affinity NGF receptor on cultured septal cells from P58 rat. Immunohistochemical staining by monoclonal antibody MC192 showed expression of the receptor in cultures from P58 (Fig. 4) and from P12 rats (data not shown). Thus, the decreased effect of NGF on survival in P58 cultures was not due to the absence of the receptor.

but does not induce this activity in cultures from PI4 septum. In this study, we measured the effect of NGF on survival in older rats. Although the effect of NGF

DISCUSSION We have established a primary culture technique for neuronal cells from adult rat brains in order to study the effects of neurotrophic factors on mature neurons. We first investigated the effect of NGF and b F G F on the survival of adult basal forebrain cholinergic neurons in culture. The physiological role of N G F in adult rat CNS has been examined following exogenous administration in vivo. However, there have been no in vitro studies on the effect of NGF on the survival of mature septal cholinergic neurons. The method of preparation resulted in good cell viability. The purification of living cells was done by centrifuging the cell suspension on the PNCS lower layer. Without the purification step, neuronal cell culture was successful for 2-week-old rat septum, but not for P38 septum. Thus, the elimination of debris from the cell suspension may help to allow the dissociated neurons to attach to the astroglial feeder layers and grow in vitro. The immunohistochemical staining of MAP2 indicated that the neuronal cells prepared from P58 rat septum developed well in serum-free culture medium for one week (Fig. 1). The number of viable MAP2-positive neurons from P58 rat septum was half that from P14 septum (Table I). The number of AChE-positive neurons from P58 rat septum was a quarter that from P12 septum (Table I). The different degrees of survival of these cells mean that projecting massive neurons such as cholinergic neurons are vulnerable to the steps of preparation. However, the morphologies of the cultured MAP2- and AChE-positive neurons demonstrated that the living neurons were vital (Figs. 1 and 2). Thus, living neurons from P58 rat septum were not seriously damaged in culture. We have already reported that NGF supports the survival of basal forebrain cholinergic neurons obtained from P l l - P 1 4 rats T M . NGF induces cellular ChAT activity in culture from P1-P4 rat septal regions,

Fig. 4. Immunohistochemical staining of low affinity N G F receptor in cultured P58 rat septum. P58 rat septum cells were cultured for 7 days without factors (A), with 100 n g / m l of N G F (B), and with I(X] n g / m l bFGF (C). At day 7, cells were fixed and stained by monoclonal antibody MC192 against low affinity N G F receptor with a rhodamine-labelled second antibody. Bar = 50 p~m.

269 on the survival of cultured P38 and P58 rat septum cholinergic neurons was less than that on P12 and P14 neurons (Fig. 3 and Table I), NGF improved the growth of P58 rat septal cholinergic neurons (Fig. 2). An effect of NGF on the maintenance of adult rat septum cholinergic neurons was also suggested from studies on theadministration of NGF 14 and anti-NGF antibody 27 in vivo. These data suggest changes in the neurotrophic role of NGF: it acts as a differentiation and growth factor in the embryonic stage, as a survival factor in 2 weeks postnatal, and as a maintenance factor in adult rat brain. It has been reported that chronic intraventricular injections of NGF into adult rats with septo-hippocampal transection prevents the disappearance of cholinergic neurons in basal forebrain 13'15'16'19. These data demonstrate that administration of exogenous NGF after CNS lesions can inhibit retrograde degeneration of axotomized septai cholinergic neurons in vivo. In contrast, Sofroniew et al. showed that uninjured basal forebrain cholinergic neurons of young adult rats did not die after excitotoxic ablation of their target hippocampal neurons 24. They reported that after Nmethyl-o-aspartic acid (NMDA) lesioning of hippocampus, septum cholinergic neurons were shrunken and showed reduced staining of ChAT rather than cell loss. In the present study, we have shown that the effect of NGF on survival was less on mature septum cholinergic neurons in culture (Fig. 3 and Table I). Immunohistochemical staining by monoclonal antibody MC192, which is specific for the rat low affinity NGF receptor 6, demonstrated that P58 rat septum cholinergic neurons had NGF receptor molecules, like P12 neurons (Fig. 4). This observation showed that receptor expression was not suppressed in cultures from P58 rat septum. Thus, uninjured or restored septal cholinergic neurons of adult rats do not require NGF for survival. Pharmacological administration of NGF, however, may prevent degenerative changes and improve development. It has been reported that bFGF can promote the in vitro survival of embryonic neurons, including those from the hippocampus, cerebral cortex, striatum, septum, and thalamus of El8 rats 4's'28, and from mesencephalon 7. In our cultures from P12 rat septum, bFGF did not support the survival of AChE-positive neurons (Fig, 3). bFGF, however, strongly supported the survival of P38 and P58 rat septal cholinergic neurons in vitro. The possibility that bFGF acted via other types of cells than neurons cannot be ruled out. Although many non-neuronal cells contaminated the cultures from P12 rat septum, bFGF did not improve the survival of cholinergic neurons. Thus, bFGF must directly

support the survival of cholinergic neurons from adult rat septum, bFGF also rescues septal cholinergic neurons from degeneration by axotomy 1'5. These results suggest that bFGF physiologically supports the survival of septal cholinergic neurons in adult rat brain. In contrast with our results, Matsuda et al. reported that bFGF increased the survival of cultured neurons from P15 rat septum TM,but their immunohistochemical staining with anti-neurofilament antibody showed the positive neurons only on astroglial cells and the distinction of viable neurons seemed to be impossible for the specificity. Although bFGF alone did not affect the survival of P12 cholinergic neurons, the effect of bFGF plus NGF on survival was less than the effect of NGF alone (Fig. 3). In cultures of P38 rat septum, the survival-promoting effect of bFGF plus NGF was somewhat reduced in comparison with that of bFGF alone. The reasons are unclear why NGF and bFGF together suppressed the enhancement of survival of each alone. We observed that the intensity of immunohistochemical staining of NGF receptors by MC192 antibody in cultures from P15 rat septum was decreased by the addition of bFGF during cultivation (data not shown). It has also been reported that the enhancement of ChAT molecule expression by NGF is suppressed by bFGF 17. Thus, it is possible that these factors down-regulate the expression of each other's receptors, or suppress the induction of intracellular signals. Further studies are required to clarify the physiological role of bFGF in the CNS.

Acknowledgements.

We thank Prof. E. Shooter of Stanford University and Dr. M. Niinobe and Prof. K. Mikoshiba both of Osaka University for kindly supplying the antibodies. We also thank Takeda Pharmaceutical Co. for their kind gift of human recombinant bFGF. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas of the Japanese Ministry of Education, Science and Culture.

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