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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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VPAC2 receptor-mediated reductions in axon and dendritic outgrowth in cultured cortical neurons. Primary cortical neurons were cultured with Ro25-1553 (10 nM) or VIP (10 nM) for 3 days in vitro and double-immunostained for pNF and MAP2. (A) PG99-465 (100 nM) was treated 30 min before the treatment with Ro25-1553 or VIP. PG99-465 blocked Ro25-1553- and VIP-induced reductions in axon length (main effects: F_2,215 = 8.338, P < 0.001 for Ro25-1553/VIP, F_1,215 = 129.957, P < 0.0001 for PG99-465; interaction: F_2,215 = 24.711, P < 0.0001), total numbers (main effects: F_2,215 = 8.231, P < 0.001 for Ro25-1553/VIP, F_1,215 = 127.826, P < 0.0001 for PG99-465; interaction: F_2,215 = 10.525, P < 0.0001) and length (main effects: F_2,215 = 3.701, P < 0.05 for Ro25-1553/VIP, F_1,215 = 160.683, P < 0.0001 for PG99-465; interaction: F_2,215 = 11.789, P < 0.0001) of dendrites, and dendritic complexity (main effects: F_2,215 = 3.744, P < 0.05 for Ro25-1553/VIP, F_1,215 = 61.073, P < 0.0001 for PG99-465; interaction: F_2,215 = 3.003, P < 0.05). (B) Primary cortical neurons were prepared from VPAC2 receptor knockout (VPAC2-KO) mice and littermate wild-type mice. Ro25-1553-induced reductions in axon length (main effects: F_1,156 = 12.655 P < 0.001 for treatment, F_1,156 = 17.717, P < 0.0001 for genotype; interaction: F_1,156 = 4.182, P < 0.05), total numbers (main effects: F_1,156 = 7.274, P < 0.01 for treatment, F_1,156 = 15.414, P < 0.001 for genotype; interaction: F_1,156 = 15.414, P < 0.001) and length (main effects: F_1,156 = 15.814, P < 0.001 for treatment, F_1,156 = 14.262, P < 0.001 for genotype; interaction: F_1,156 = 20.342, P < 0.0001) of dendrites, and dendritic complexity (main effects: F_1,156 = 2.175, P > 0.05 for treatment, F_1,156 = 19.468, P < 0.0001 for genotype; interaction: F_1,156 = 5.576, P < 0.05) were abolished in cortical neurons derived from VPAC2-KO mice. Values represent mean ± SD of 40 neurons from three independent experiments. **P < 0.01 vs. control, n.s.; not significant.