Figure 1.

Treadmill Running and Passive Head Motion (PHM) Similarly Modulate Behavioral and Neuronal Response to 5-HT

(A) Schematic representation of experimental protocol for analysis of the effects of treadmill running on head-twitch response (HTR).

(B and C) Treadmill running alleviated 5-HTP-induced HTR. Count of head twitching in 5-min blocks (B) and for 30 min (C) post-5-HTP administration (p = 0.027, unpaired t test; n = 10 mice for each group). Control 1 in (B, C, G, H, I, and J) represents mice that were placed in the treadmill machine without turning it on (30 min per day, 7 days).

(D) Schematic representation of experimental protocol for analysis of the effects of PHM on HTR. PHM (cyclical 5-mm head drop) was applied to generate vertical accelerations of 1.0 × g peaks at the heads of mice (2 Hz, 30 min per day, 7 days).

(E and F) PHM alleviated 5-HTP-induced HTR. Head twitching was counted as in (B) and (C), respectively (p = 0.035, unpaired t test; n = 10 mice for each group). Control 2 in (E–J) represents mice that were anesthetized and placed in a prone position with their heads on the platform that was left unoscillated (30 min/day, 7 days).

(G) Micrographic images of anti-c-Fos (green), anti-5-HT_2A receptor (red), and anti-NeuN (blue) immunostaining of the PFC of mice administered 5-HTP (or vehicle) after a week of daily treadmill running or PHM. Scale bar, 100 μm. Images are representative of four to five mice.

(H) Both treadmill running and PHM decreased c-Fos expression in 5-HT_2A receptor-positive neurons of mouse PFC. Relative population (%) of c-Fos-positive cells of 300 NeuN- and 5-HT_2A receptor-positive cells is shown (left chart: p < 0.001, one-way ANOVA with post hoc Bonferroni test; right chart: p < 0.001, unpaired t test; n = 4 mice for columns 1, n = 5 mice for columns 2 to 5).

(I) Micrographic images of anti-5-HT_2A receptor (5-HT_2AR; red) and anti-NeuN (green) immunostaining of the PFC of mice injected with 5-HTP (or vehicle) after a week of daily treadmill running or PHM. Higher-magnification images of anti-5-HT_2A receptor immunostaining of arrow-pointed cells are presented with a gray scale. Yellow lines indicate the margins of somas outlined by NeuN-positive signals, and cyan arrowheads point to internalized anti-5-HT_2A receptor immunosignals. Scale bars, 20 μm. Images are representative of five mice.

(J) Quantification of internalized and membrane-associated 5-HT_2A receptor-positive area relative to NeuN-positive area in mouse PFC. Thirty-five to forty NeuN-positive neuronal somas were analyzed for each mouse (Internalized: left chart, p < 0.001, one-way ANOVA with post hoc Bonferroni test; right chart, p = 0.0027, unpaired t test; Membrane-associated: left chart, p < 0.001, one-way ANOVA with post hoc Bonferroni test; right chart, p = 0.0025, unpaired t test; n = 5 mice for each group). Data for 5-HTP(+) samples in (G–J) were obtained from mice infused with 4% paraformaldehyde/PBS immediately after HTR test shown in (B, C, E, and F).

Data are represented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. See also Figures S1, S2, S3, and S4.