Deletion of Aqp9 protects against MPP⁺ toxicity in vitro.

A) Photomicrograph (left) and schematic representation (right) of a representative midbrain slice. Site of MPP⁺ application is indicated in red. B-E) Immunofluorescence staining of representative midbrain slices from WT (B, C) and Aqp9^-/- mice (D, E) showing TH-positive neurons in the MPP⁺ treated side (ipsilateral; B, D), and in the control side (contralateral, C, E). Note the extensive loss of ipsilateral TH-positive neurons in the WT slice (B). F-H) Quantitative analyses of the TH-positive cell count in slices treated with 30 μM MPP⁺ (F, G) show significant loss of TH-positive neurons in the ipsilateral hemisphere of the WT slice (n = 8) (F). No significant difference was observed between the ipsi- and contralateral hemisphere of Aqp9^-/- slices treated with 30 μM MPP⁺ (n = 7) (G), WT slices treated with a combination of 60 μM MPP⁺ and 100 μM phloretine (n = 11) (H), or WT slices treated with saline (n = 8) (G). In WT mice, the TH-positive cell count contralateral to MPP⁺ application (F) was lower than the TH-positive cell count in saline treated slices (H), suggesting spillover of MPP⁺ from the ipsilateral side. RRF: retrorubral field; Aq: aqueduct; V: ventricle; A9: population of dopaminergic neurons. Bars are mean ± SEM; ***p<0.001. Scale bar: 100 μm; scale bar inset: 50 μm.