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. 2020 Jan 31;23(2):100874. doi: 10.1016/j.isci.2020.100874

Figure 4.

Figure 4

PKC Is Involved in Both FSS-Induced 5-HT2A Receptor Internalization In Vitro and PHM-Attenuated HTR In Vivo

(A and B) MARCKS was phosphorylated in neuronal cells after FSS, depending on PKC activity. Neuro2A cells, either left unexposed or exposed to FSS (average 0.91 Pa, 0.5 Hz, 30 min) with and without PKC inhibitor pretreatment (Ro 31-8220; 4 μM, 1 h), were subjected to anti-TUJ-1 (green) and anti-phospho-MARCKS (red in A) or anti-PKCγ (red in B) immunostaining. Nuclei were stained with DAPI. Higher-magnification images of anti-PKCγ immunostaining of arrow-pointed cells are presented with cell margins (white dashed lines) as insets (B). Scale bars, 20 μm. Images are representative of three independent experiments with similar results.

(C) PKC inhibition hampered both 5-HT- and FSS-induced 5-HT2A receptor internalization. Neuro2A cells with combinations of Ro 31-8220 pretreatment and 5-HT administration (10 μM, 15 min) or FSS application (average 0.91 Pa, 0.5 Hz, 30 min) were fixed and stained for 5-HT2A receptor (5-HT2AR; red) and TUJ-1 (green). Scale bars, 20 μm. Images are representative of three independent experiments with similar results.

(D) Quantification of 5-HT2A receptor internalization (top left chart: p < 0.001, top right chart: p = 0.001, bottom left chart: p = 0.013, bottom right chart: p = 0.041, unpaired t test; 20 cells analyzed in each sample, n = 3 for each group).

(E) Schematic representation of the experimental protocol for PHM with PKC inhibition. Ro 31-8220 or vehicle was injected just before each bout of PHM.

(F and G) PKC inhibition nullified the effect of PHM on 5-HTP-induced HTR. Head twitching was counted as in Figures 1B and 1C. (F) Count of head twitching in 5-min blocks. Asterisks (*) indicate statistical significance between PHM with (green line) and without (red line) Ro 31-8220 (10–15 min: p = 0.019, 15–20 min: p = 0.012, 20–25 min: p = 0.049, 25–30 min: p = 0.024, unpaired t test; n = 8 mice for each group), whereas there were no significant differences at any time point between Ro 31-8220 with (green line) and without (black line) PHM (p > 0.05, unpaired t test; n = 5 mice for group of Ro 31-8220 without PHM). (G) Total count of head twitching for 30 min after 5-HTP administration (column 1 versus 2: p = 0.016, column 1 versus 3: p = 0.041, column 2 versus 3: p = 0.98, unpaired t test).

Data are represented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. See also Figures S2 and S7.