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. 2020 Jun 4;14:521. doi: 10.3389/fnins.2020.00521

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Copyright © 2020 Takeuchi, Kawanai, Yamauchi, Chen, Miyaoka, Yamada, Asano, Hayata-Takano, Nakazawa, Yano, Horiguchi, Nakagawa, Takuma, Waschek, Hashimoto and Ago.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Effects of Ro25-1553, VIP, and PACAP on axon outgrowth in cultured cortical neurons. (A) RT-PCR analysis showed that all VIP/PACAP receptors PAC1, VPAC1, and VPAC2 were expressed in primary cultured cortical neurons. (B) Representative pNF- and MAP2-immunostained images of cultured cortical neurons are shown. Cells were cultured with Ro25-1553 (10 nM), VIP (10 nM), or PACAP38 (10 nM) for 3 days in vitro and double-immunostained for pNF (red) and MAP2 (green). Scale bar, 50 μm. (C) Quantitative analysis of changes in axon length was shown. VIP (F₄,₂₁₅ = 16.542, P < 0.0001) and Ro25-1553 (F_4,215 = 25.257, P < 0.0001), but not PACAP (F_4,215 = 2.383, P > 0.05), reduced the axon length. Values represent mean ± SD of 40–60 neurons from three independent experiments. **P < 0.01 vs. control.