Figure 3. Effects of short-term administration of FN1040 on AR2 mice.

(A) Behavioral changes. FN1040, FN58, or vehicle was intracerebroventricularly infused through surgically implanted cannula under isoflurane anesthesia and Food and Drug Administration-approved α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid antagonist perampanel was administered orally. AR2 mice infused with FN1040 at a concentration of 30 μM (n = 5; red) exhibited active locomotion (moving and walking) in the same level as vehicle-treated AR2 mice (n = 5; green) during 24 h after surgery. In contrast, mice receiving perampanel at an effective dose (20 mg/kg, n = 5; purple) exhibited marked inactivity. AR2 mice infused with FN58 at a concentration of 20 μM (n = 5, blue) exhibited the delay in recovery compared with FN1040, and the sedation prolonged to the following day. Left: total time mice moved and walked during 180 s of observation. Right: the number of rearing (standing on hindlimbs) during 180 s of observation. Mean ± SEM. Two-way ANOVA followed by Tukey’s multiple-comparisons test. **P < 0.01, ****P < 0.0001 (Tables S2 and S3). (B) Representative image of TDP-43 immunohistochemistry (green: anti-TDP-43 antibody positive area). TDP-43 localized within the nucleus of motor neurons (Nuc) of normal mice, whereas it localized in the cytoplasm (Nuc/Cyto and Cyto) or disappeared (Absent) in AR2 mice. Scale bar = 20 μm. (C) Anterior horns of the cervical spinal cord double stained with anti-ChAT (red) and anti-TDP-43 (green). Scale bar = 100 μm. (D) The number of ChAT-positive cells in the anterior horn of the cervical spinal cord was significantly larger in the 20 μM of FN1040 group compared to that in the control (artificial CSF) group and lower FN1040 concentration groups. Six anterior horn cells per mouse, three mice per group. (E) Mice treated with FN1040 showed a dose-dependent increase in the diameter of ChAT-positive cells. (F) The proportion of ChAT-positive cells with nuclear localization (Nuc/Cyto + Nuc) was higher in the FN1040-treated mice compared to that in vehicle-treated mice (n = 3 in each group). Nuc, nucleo; Nuc/Cyto, nucleo-cytoplasmic; Cyto, cytoplasmic; Absent, lack of TDP-43 immunoreactivity. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Mean ± SEM, two-way ANOVA followed by Tukey’s multiple-comparisons test (Table S4).

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