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. 2020 Jan 7;11:72. doi: 10.1038/s41467-019-13809-8

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Schematic of experimental setup and imaging location. Right, an in vivo two-photon image of GCaMP6f-expressing apical dendritic spines in the superficial layer of Cg1/M2. b Protocol for the RSC stimulation. c Fluorescence transients in dendritic spines in Cg1/M2 in response to electrical stimulation of the retrosplenial cortex (RSC). Each trace shows a single 32-pulse-stimulation trial during pre-injection (left) or post-injection (right) period. One example spine was plotted for saline injection (black), and another example spine was plotted for ketamine injection (red). Dashed line, time of stimulation onset. d Trial-averaged spine calcium responses as a function of the number of stimulation pulses applied in a trial, for pre-injection (dashed line) vs. post-injection period (solid line) and for saline (black line) vs. ketamine (10 mg.kg) injection (red line). A three-way mixed ANOVA was performed with drug (saline, ketamine) as a between-subjects factor, and stimulation levels (1, 2, 4, 8, 16, 32, 64) and epoch (pre-injection, post-injection) as within-subjects factors. A three-way interaction was significant (F(6,2364) = 5.6, P = 9 × 10⁻⁶). A subsequent two-way mixed ANOVA was performed separately on the saline and ketamine datasets, which showed a significant two-way interaction between epoch (pre vs. post) and stimulation levels for ketamine (F(6,1146) = 6.6, P = 7 × 10⁻⁷), but not for saline (F(6,1218) = 20.0, P = 0.06). Post-hoc Tukey-Kramer test on the ketamine dataset indicated significantly elevated spine responses for post-injection vs. pre-injection for stimulation pulse numbers of 4 (P = 0.01), 8 (P = 0.007), 16 (P = 0.008), 32 (P = 0.02), and 64 (P = 9 × 10⁻⁵). Line, mean ± s.e.m. For saline, n = 204 dendritic spines from 4 animals. For ketamine, n = 192 dendritic spines from 4 animals. Refer to Supplementary Fig. 5f for full distribution. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant. Error bars, ±s.e.m.