Introduction enhancement of neurite extension [39] and acceleration of

Brain Research 885 2000 240–250 www.elsevier.com locate bres Research report Distinct signaling pathways involved in multiple effects of basic fibroblast growth factor on cultured rat hippocampal neurons a , b b Hiroshi Katsuki , Yuko Itsukaichi , Norio Matsuki a Department of Pharmacology , Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan b Laboratory of Chemical Pharmacology , Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan Accepted 12 September 2000 Abstract 21 We investigated possible involvement of voltage-dependent Ca channels VDCCs and several intracellular signaling mechanisms in multiple actions of basic fibroblast growth factor bFGF, such as survival promotion, induction of calbindin D expression as well as 28k acceleration of neuritic branch formation of cultured rat hippocampal neurons. Immunocytochemical staining with anti-g-aminobutyric acid GABA antibody showed that the promotion of neuron survival by bFGF in high cell-density cultures were exerted exclusively on GABA-negative neurons. Nicardipine 5 mM attenuated the effect of bFGF on neuronal survival and formation of neurite branches, suggesting that the activity of L-type VDCCs is required for these effects. In contrast, stimulation of calbindin expression by bFGF was not attenuated by nicardipine. A phospholipase C inhibitor U73122 1 mM prevented the effect of bFGF on neurite branch formation, but not on neuronal survival or calbindin expression. On the other hand, chronic application of phorbol-12-myristate-13-acetate 1 mM inhibited the effect of bFGF on neuronal survival, without inhibiting the other bFGF actions. Forskolin 100 mM attenuated the effect of bFGF on neuronal survival and neurite branch formation, indicating that cyclic AMP plays negative regulatory roles in these actions of bFGF. Taken together, these results suggest that multiple biological actions of bFGF on hippocampal neurons are exerted through, and modulated by, distinct signaling pathways.  2000 Elsevier Science B.V. All rights reserved. Theme : Development and regeneration Topic : Neurotrophic factors: biological effects Keywords : FGF-2; Hippocampus; Neuronal differentiation; Development; Calcium channel; Intracellular signaling 1. Introduction enhancement of neurite extension [39] and acceleration of neuritic branching [2]. bFGF also regulates the expression Basic fibroblast growth factor bFGF, originally iso- of enzymes and proteins with specific functions, such as lated and identified from bovine brain and pituitary based calbindin [33]. Little information is available, however, on the stimulatory activity on fibroblast proliferation, is concerning the cellular mechanisms leading to various now considered as a potent neurotrophic factor in the consequences of biological actions of bFGF. brain. bFGF and its high-affinity receptors are abundantly Manifestation of diverse biological actions of bFGF may present in the embryonic as well as the adult central be attributable to the mobilization of distinct intracellular nervous system [10,19,44], suggesting that they play a signaling pathways. Activation of membrane-associated pivotal role in neuronal development and functions. In- high-affinity FGF receptors, possessing tyrosine kinase deed, bFGF has been shown to exert various biological activity, is the initial trigger to exert the effects of bFGF effects on neuronal cells, such as promotion of progenitor [11,13]. Activated receptor tyrosine kinases are known to cell proliferation [25], support for cell survival [36], mobilize several downstream effectors. A small GTP- binding protein Ras is converted to its active form in response to growth factor receptor stimulation [22,32]. Corresponding author. Tel.: 181-75-753-4536; fax: 181-75-753- Activation of FGF receptors also leads to tyrosine phos- 4579. E-mail address : hkatsukipharm.kyoto-u.ac.jp H. Katsuki. phorylation of phospholipase C PLC-g to enhance its 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 5 3 - X H . Katsuki et al. Brain Research 885 2000 240 –250 241 enzymatic activity [3,23]. Moreover, phosphatidylinositol in the case of calbindin immunocytochemistry was added PI 3-kinase, which catalyzes phosphoinositide phos- for 1 h, then avidin–biotin peroxidase complex method phorylation at the D-3 position of the myo-inositol ring, is was performed with Vectastain Elite ABC kit Vector activated by growth factor receptor stimulation via interac- according to the manufacturer’s instructions. tions of SH2 domains of the kinase regulatory subunit with Cell counting was performed as described previously 2 the receptor [24]. These signal transduction mechanisms [1]. Four areas of 1 mm , the total of which corresponds to 2 may be differentially involved in diverse biological conse- 4 of the whole area 1 cm , were chosen at random quences of receptor tyrosine kinase activation in various from each well and the number of neurons in the areas cell types [12,18,43]. 400–1300 cells in each well was counted under a In the present study we set out for the experiments to microscope. Number of surviving neurons in each well 2 discriminate which signaling pathways play an important was expressed as cells cm and data from four culture role in the bFGF-induced biological responses in fetal rat wells from the same sister culture were presented in figures hippocampal neurons. We have previously demonstrated as mean6S.E.M. Reproducibility of results was confirmed that increase in the amount of L-type voltage-dependent by using different sister cultures. In the present cultures, 21 21 Ca channels VDCCs and consequent increases in Ca more than 90 of the cells were labeled by antibodies to influx may play a critical role in the acceleration of neurofilament and microtubule-associated protein-2 and neuritic branch formation by bFGF [27]. Accordingly, the identified as neurons by their morphology. Therefore, potential involvement of VDCCs in promotion of neuronal possible contaminating non-neuronal cells including as- survival and enhancement of calbindin expression by troglia and fibroblast cells were estimated to be less than bFGF was also investigated. 10. Cultures on 35-mm dishes were used for the evaluation of neurite branching, the methods essentially according to 2. Materials and methods those described previously [2,27]. Twenty-four hours after