Brain Research 868 (2000) 113–118www.elsevier.com/ locate /bres

Short communication

Trophic interactions between brain-derived neurotrophic factor andS100b on cultured serotonergic neurons

a,b , a b*Mayumi Nishi , Mitsuhiro Kawata , Efrain C. AzmitiaaDepartment of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan

bDepartment of Biology, New York University, 1009 Main Building, 100 Washington Square East, New York, NY 10003, USA

Accepted 23 February 2000

Abstract

Brain-derived neurotrophic factor (BDNF) and S100b stimulate serotonergic neurons in fetal rat raphe primary cultures grown underserum-free conditions. BDNF (50 ng/ml) treatment for 3 h enhanced S100b immunoreactivity in both raphe and hippocampal glial cells.Combined treatment with BDNF and S100b for 3 days increased the soma area of 5-HT neurons, but not the neurite length. Our resultssuggest that BDNF and S100b, which regulate different signal transduction cascades, interact to exert complimentary effects on neuronalmaturation by acting sequentially, not concurrently. 2000 Elsevier Science B.V. All rights reserved.

Theme: Development and regeneration

Topic: Neurotrophic factors: biological effects

Keywords: Tissue culture; Raphe; Neuronal–glial interaction; MAP kinase; BDNF

Few protein growth factors have trophic activity on BDNF to p-chloroamphetamine (PCA)-treated rats pro-3serotonergic (5-HT) neurons. The most active protein duced an increase in sprouting, 5-HT synthesis and [ H]5-

trophic factors on 5-HT neurons identified to date are HT uptake [12]. Surprisingly, the increased innervationS100b [2,11] and BDNF [6,12,16]. S100b, a calcium- persisted for months after BDNF infusion was terminated.binding protein synthesized and stored in CNS glial cells, This long-term action may involve activation of the trophicacts mainly on neurite extension [2,10]. S100b stabilizes activity of glial cells. We investigated the interactionsmicrotubules by inhibiting phosphorylation of brain cyto- between BDNF and S100b in primary raphe cultures andskeletal proteins such as MAP2, tau and GAP-43 [4,8,18], hippocampal glial cultures to elucidate the hypothesis thatalthough direct action on tubulin polymerization is possible the trophic effects exerted by BDNF on 5-HT neurons[5]. The release of S100b from glial cells is induced by involve the glial derived protein S100b. Initially, we5-HT agonists [19]. examined the direct actions of BDNF on S100b immuno-1A

BDNF effects on 5-HT neurons may result from the cytochemistry in cultured glial cells. In addition, wedirect action of BDNF on 5-HT neurons [16]. There is a measured the trophic interactions between BDNF and

3trkB receptor with a cytoplasmic tyrosine kinase domain S100b on [ H]5-HT uptake, soma area and neurite lengthtrkB(gp145 ) [9] expressed on raphe neurons [14]. In of 5-HT-immunoreactivity (IR) positive neurons in pri-

addition, cultured cortical glial cells, which predominantly mary raphe cultures. We used antibodies against S100b totrkBexpress gp95 lacking the tyrosine kinase domain [7], block the actions of the glial factor and the tyrosine kinase

also respond to BDNF [17]. Direct cortical infusion of inhibitor, K252a, to block the actions of BDNF. Ourresults show that BDNF has a direct action on the somaarea and metabolism of neurons, but an indirect action on

*Corresponding author. Department of Anatomy and Neurobiology, neurite length which requires S100b released from glialKyoto Prefectural University of Medicine, Kawaramachi Hirokoji,

cells.Kamigyo-ku, Kyoto 602-8566, Japan. Tel.: 181-75-251-5301; fax: 181-Dissociated raphe primary cultures and hippocampal75-251-5306.

E-mail address: nmayuni@koto.kpu-m.ac.jp (M. Nishi) mixed glial cultures were prepared from 14-day-old [3]

0006-8993/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 00 )02201-0

114 M. Nishi et al. / Brain Research 868 (2000) 113 –118

and 18-day-old Sprague-Dawley rat fetuses [13], respec- before drug treatment. More than 95% of the cells in thetively. Briefly, brain tissue was mechanically dissociated secondary hippocampal glial cultures were GFAP immuno-by triturating through a fire-polished glass pipette and cytochemically positive. In all experiments, BDNF (Prom-

5 2plated at a cell density of 5310 /cm . The cultures were ega, WI, USA) was used at a concentration of 50 ng/mlmaintained in complete neuronal medium (CNM), consist- and S100b (East Acres Biologicals, MA, USA) at aing of 92.5% (v/v) Eagle’s minimum essential medium concentration of 10 ng/ml. A specific anti-S100b antibody(MEM, Sigma, St. Louis, MO, USA), 1% (w/v) non- (purified IgG fraction; a gift from Dr. L. VanEldik) wasessential amino acids (Gibco, USA), 0.16% (w/v) glucose employed at a dilution of 1 /10,000. As a control antibody,and 5% (v/v) fetal bovine serum (Sigma) for 24 h. After normal rabbit IgG (1/10,000 dilution) was used. Some24 h, the CNM was removed and the culture medium was cultures were preincubated with fungus-derived kinasechanged to serum-free media (MEM with 0.16% glucose, inhibitor, K252a (100 nM; Sigma).

31% non-essential amino acids, 20 mM putrescine, 15 nM [ H]5-HT uptake was performed as described previouslysodium selenite, 5 mg/ml insulin, and 100 mg/ml trans- [3]. Briefly, the medium was removed from the culturesferrin). Hippocampal glial cells were cultured in CNM for and the cells washed twice in fresh MEM. MEM (378C)the first 7 days and then sub-cultured with trypsin-EDTA. was added to each well and the reaction was allowed to

3The obtained secondary glial cultures were maintained in proceed in a solution containing 50 nM [ H]5-HT (19–27CNM for another 7 days and in serum-free media for 1 day Ci /mmol; DuPont-New England Nuclear, MA, USA) and

Fig. 1. Effects of BDNF on S100b immunoreactivity in raphe culture and hippocampal glial culture. (A) Control culture for raphe was grown in CNM forthe first 24 h, then in the absence of serotonin nor steroids for the last 3 days before fixation. (B) Raphe culture was treated with BDNF (50 ng/ml) for thelast 3 h before fixation. (C) Control culture of hippocampal glial cells was grown in CNM for the first 7 days and then sub-cultured. The obtainedsecondary glial cultures were maintained in CNM for another 7 days, then in the absence of serotonin nor steroids for the last 3 days before fixation. (D)Hippocampal glial culture was treated with BDNF (50 ng/ml) for the last 3 h before fixation. After fixation, the cells were subjected to S100b

immunocytochemistry. In the culture treated with BDNF for 3 h, S100b immunoreactivity increased within and around the glial cells (arrow). Bar: 50 mm.

M. Nishi et al. / Brain Research 868 (2000) 113 –118 115

2510 M pargyline for 30 min at 378C. Nonspecific For immunocytochemistry, cultured cells were fixed for3accumulation and retention of [ H]5-HT was examined by 20 min at 378C in 4% paraformaldehyde in phosphate

incubating the cultures as described, but in the presence of buffer (PB, pH 7.4). After blocking with 2% bovine serum2510 M fluoxetine, which is a recognized specific uptake in PB for 1 h at RT, fixed cells were incubated with rabbit

blocker for 5-HT neurons. The radioactivity of each well antisera directed against 5-HT (1/4000 dilution; Incstar,was measured in a Beckman liquid scintillation counter. MI, USA) and S100b (1 /10,000 dilution) for 48 h at 48C.

Fig. 2. Effects of various treatments on the soma area and the process length of 5-HT-IR neurons. Each photomicrograph shows a typical 5-HT-IR neuronin each treatment. (A) Control. (B) S100b (10 ng/ml). (C) BDNF (50 ng/ml). (D) BDNF (50 ng/ml)1S100b (10 ng/ml). (E) BDNF (50ng/ml)1anti-S100b antibody (1 /10,000 dilution). (F) BDNF (50 ng/ml)1K252a (100 nM) were added for the last 3 days before fixation. The treatmentwith S100b promoted the extension of 5-HT-IR processes (arrow), but not soma area (B). In the culture treated with BDNF (C) and BDNF1S100b (D),5-HT-IR neurons with a larger soma area (asterisk) were observed. An antibody to S100b blocked the increase in the soma area induced by BDNF(arrowhead). Bar: 50 mm.

116 M. Nishi et al. / Brain Research 868 (2000) 113 –118

The cultures were reacted with biotinylated goat anti-rabbitantibody (1 /250 dilution; Boehringer Mannheim, IN,USA) for 1 h at RT, followed by the avidin–biotin–peroxidase complex (Vector, CA, USA). The cells werevisualized with 0.02% 3,39-diaminobenzidine (Sigma) and0.006% H O in 0.1 M Tris–HCl buffered (pH 7.6) saline.2 2

Slides were subjected to morphometric analyses using theOptimas (ver.4.0) image acquiring software program(Bioscan, Inc., WA, USA) according to the publishedprotocol [15]. The mean values were determined for theaverages of three wells for each experiment from threeindependent experiments performed with different animalson separate days. Statistical significance was determinedusing ANOVA and the post-hoc Tukey test.

S100b-IR was observed in both midbrain and hippocam-pal glial cells under our culture conditions. In BDNF-treated raphe cultures and hippocampal glial cultures, thestaining intensity of S100b-IR cells increased after 3 h(Fig. 1B and D) and 2 days (data not shown) of treatment.In the cultures treated with BDNF for 3 h, the intensity ofS100b-IR increased not only within the glial cells but alsoaround the glial cells (Fig. 1B and D). Interestingly, in theBDNF-treated glial cells, we also observed an apparent Fig. 3. Effects of BDNF, S100b, BDNF1S100b, BDNF1anti-S100b,increase in membrane blebbing, which showed intense and BDNF1K252a on the soma area of 5-HT-IR cells. The cells were

treated for 3 days, at which time they were fixed and subjected to 5-HTS100b-IR staining.immunocytochemistry. The mean values obtained from three independentThe effects of interactions of BDNF and S100b on theexperiments were calculated. The results were analyzed using ANOVAsoma area and the process length of 5-HT-IR neurons werefollowed by the Tukey honest test. Data are presented as the mean1

examined in 4-day-old raphe cultures. In cultures treated S.E.M. *P , 0.05, statistically significant compared with the controlwith BDNF for 3 days, the soma area of 5-HT-IR cells was value; **P , 0.05, statistically significant compared with the BDNF-

treated group.significantly increased (135%, P , 0.05), while S100b

did not show a significant increase in the soma area (Figs.2B, 2C and 3). For analyses of the length of 5-HT-IRprocesses, all the processes branching directly from the cellbody (primary process) were measured. BDNF showed no control rabbit IgG did not block the effect of BDNF on the

3significant change in the length of processes for individual increase in the soma area of 5-HT-IR neurons or [ H]5-HT5-HT-IR neurons (Figs. 2C and 4). In contrast, S100b uptake.significantly promoted the total process length (1110%, The last treatment we explored was to inhibit the actionsP , 0.01), especially the length of the longest process of BDNF on its downstream target, tyrosine kinase.(Figs. 2B and 4). Combined treatment of BDNF and Addition of K252a (100 nM) to normal raphe cultures did

3S100b induced an increase in the soma area (134%, not induce significant changes in soma size or [ H]5-HTP , 0.05), but not in the total process length (120%, not uptake. Pretreatment with K252a (100 nM) for 30 minsignificant) (Figs. 2D, 3 and 4). inhibited the increase in soma area described above (221%

3The measure of the high-affinity uptake of [ H]5-HT compared with BDNF treatment, P , 0.05) (Figs. 2F andprovides one of the most reliable biochemical estimates of 3). This blockade of BDNF stimulation by K252a was also

3the surface area of a specific neurotransmitter cell [1]. A observed with [ H]5-HT uptake (226% compared withprevious study indicated that the uptake is a more reliable BDNF treatment, P , 0.05) (Fig. 5).indicator of cell number and size than measures of Immunocytochemical studies showed that BDNF treat-transmitter or enzyme levels [3]. Both BDNF and S100b ment for 3 h and 2 days increased the S100b level both

3increased the uptake of [ H]5-HT in raphe cultures by 48 inside and around the glial cells. This pattern has beenand 33%, respectively, compared with the control value observed in vivo and may indicate released S100b [21].(Fig. 5). The glial cells after BDNF treatment show S100b in blebs

The interactions of S100b and BDNF were studied by evaginating from the plasma membrane. These resultsbinding the circulating S100b in the cultures. An antibody suggest that BDNF promotes the process releasing S100b

against S100b blocked the effects of BDNF on the soma by membrane evagination, a mechanism not previouslyarea (224% compared with BDNF treatment, P , 0.05) suggested. In fact, the release of S100b, which has been

3(Figs. 2E and 3) and [ H]5-HT uptake (248% compared measured, is somewhat of a puzzle since the gene for thiswith BDNF treatment, P , 0.05) (Fig. 5). The addition of molecule lacks a typical release signaling domain.

M. Nishi et al. / Brain Research 868 (2000) 113 –118 117

The direct action of BDNF effects on glial cells iscontroversial. Cultured cortical glial cells respond toBDNF [17]. BDNF produced a marked increase in MAPkinase activity, intracellular calcium concentration and c-fos expression in glial cells. The mechanism of BDNF-induced signal transduction in glial cells may involve a

trkBrelatively small amount of gp145 , but involvement oftrkBthe more abundant gp95 cannot be excluded. A previ-

ous study showed that the release of S100b from as-trocytes was induced by 5-HT agonists [19]. The trophic1A

actions of S100b do not require neuronal support [21]. Inour studies, the direct action of BDNF on glial cells wastested. Hippocampal glial cultures are established in theabsence of steroids and serotonin as described previously.Addition of BDNF increased S100b staining in the glialcells in these cultures. This is strong evidence for a directglial response to BDNF without the intervention of neu-rons or of serotonin.

We addressed the question of possible trophic interac-tions between BDNF and S100b by using an antibodyblocking approach. An antibody to S100b, not normal

Fig. 4. Effects of BDNF, S100b, BDNF1S100b, and BDNF1anti- rabbit IgG, blocked the BDNF-induced increase in theS100b on the total length of 5-HT-IR cells. The cells were treated for 3 3soma area of 5-HT-IR and [ H]5-HT uptake. These resultsdays, at which time they were fixed and subjected to 5-HT immuno-

support the speculation. BDNF acts on 5-HT-IR neuronscytochemistry. The mean values obtained from three independent experi-ments were calculated. The results were analyzed using ANOVA followed by two mechanisms: (1) increases glial S100b, a proteinby the Tukey honest test. Data are presented as the mean1S.E.M. which stabilizes cytoskeletal proteins; (2) promotes kinase*P , 0.05, statistically significant compared with the control value. activities, a process which initiates the arborization and/or

sprouting of neurites. One possibility suggested by ourexperiments is that BDNF, synthesized by neurons, firststimulates the initialization of sprouting of 5-HT-IR neu-rons by enhancing the kinase activities. Then S100b,synthesized in glial cells, promotes neurite extension byinhibiting kinase activities. There is the question of whyBDNF applied in cultures can stimulate S100b, but notresult in neurite elongation, while S100b applied alonedoes produce neurite elongation. In fact, when we appliedboth BDNF and S100b in raphe cultures, there was noincrease in neurite extension. We propose that concurrentapplication is less effective since BDNF actions on kinaseenzymes oppose the S100b-induced neurite elongation,because this step depends on reduced kinase activity [20].

In conclusion, the results of the present study indicatethat complete and long-lasting trophic activity of BDNFmay require sequential, not concurrent, action with S100b.Further studies are needed to explore the details of thistemporal interaction.

Acknowledgements

We are grateful to Dr. L. Mamounas for helpful discus-Fig. 5. Effects of BDNF, S100b, BDNF1anti-S100b, BDNF1K252a sions during the course of this work. We would like to

3and BDNF1IgG on [ H]5-HT uptake by primary cultured serotonergic thank Xia Ping Hou for excellent technical assistance. Thiscells. The mean values obtained from three independent experiments were work was supported by funds from MH55250 to ECA andcalculated. The results were analyzed using ANOVA followed by the

Grants-in-Aid for Scientific Research from the Ministry ofTukey honest test. Data are presented as the mean1S.E.M. *P , 0.05,Education, Science, Sports and Culture, Japan, to MN andstatistically significant compared with the control value; **P , 0.05,

statistically significant compared with the BDNF-treated group. MK.

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