Anoctamin 2-chloride channels reduce simple spike activity and mediate inhibition at elevated calcium concentration in cerebellar Purkinje cells
Fig 2
Neuroanatomy of the cerebellar cortex is comparable between wildtype and Ano2-/- mice.
(A) Representative immunohistochemical stainings for calbindin (green) and parvalbumin (red) on sagittal cerebellar slices of wildtype (left) and Ano2-/- mice (right). Stainings were used to compare Purkinje cell density (B) and size (C) as well as the density of molecular layer interneurons (D) between wildtype and Ano2-/- mice. DAPI (blue) was used to visualize cell nuclei. (B)–(D) No significant differences were detected in Purkinje cell density (B; wt 41.1 ± 6.4 per mm PCL, n = 61 images from N = 6 animals, Ano2-/- 38.5 ± 7.5 per mm PCL, n = 42 N = 6; Student’s t-test p = 0.0811) and diameter (C; wt 20.7 ± 3.4 μm, n = 342 cells in 61 images from N = 6 animals, Ano2-/- 21.1 ± 3.7 μm, n = 246 in 42 images N = 6; Student’s t-test p = 0.2091), as well as in the density of molecular layer interneurons (D; wt 15.9 per 105 μm3 ML IQR 13.3–20.7, n = 62 images from N = 6 animals; Ano2-/- 17.1 per 105 μm3 ML IQR 13.3–23.6, n = 45 N = 6; Wilcoxon-Mann-Whitney test p = 0.501) between wildtype and Ano2-/- mice. (E) Immunohistochemical staining for calbindin (blue), VGluT2 (green) and VGAT (red) on sagittal cerebellar slices. As expected, GABAergic synapses (red) cover the entire molecular layer, whereas climbing fiber synapses (green) only span the proximal two thirds of the molecular layer. (F) Detail of a Purkinje cell with primary and secondary dendrites (calbindin; blue) with climbing fiber synapses (VGluT2; green) and GABAergic synapses (VGAT; red). (G), (H) No significant differences were detected in density of GABAergic (G; wt 22.0 ± 6.8 per 102 μm2 ML, n = 42 images from N = 5 animals, Ano2-/- 21.8 ± 5.5 per 102 μm2 ML, n = 47 N = 5; Student’s t-test p = 0.8788) and climbing fiber synapses (H; wt 3.5 ± 1.0 per 102 μm2 ML, n = 42 images from N = 5 animals; Ano2-/- 3.2 ± 1.0 per 102 μm2 ML, n = 48 N = 5; Student’s t-test p = 0.1989) in the proximal half of the molecular layer between wildtype and Ano2-/- mice. (I) Histograms of the distances measured between adjacent climbing fiber synapses on a Purkinje cell dendrite reveal a comparable distribution of climbing fiber synapses between wildtype and Ano2-/- mice (bin size = 0.5 μm; wt n = 1698 distances in 35 images from N = 9 animals, Ano2-/- n = 1762 in 30 images N = 6). ML = molecular layer; PCL = Purkinje cell layer; GL = granule layer. Scale bars = 20 μm.