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. 2019 Sep 5;8:e45590. doi: 10.7554/eLife.45590

Figure 1. TRPC3 is predominantly expressed in the cerebellum in a zebrin-related pattern.

(A) Representative image and magnification (right) of sagittal cryosection of an adult mouse brain stained with anti-TRPC3. Inset, plane of section. (B) Coronal immunofluorescence images with anti-TRPC3 (red), anti-Zebrin II/Aldolase C (green) and anti-calbindin (blue) staining of the cerebellar cortex (left), with magnifications (right). TRPC3 is expressed in the cerebellar PCs and UBCs (triangles), in a pattern that in the vermis complements that of zebrin and appears more uniform in the hemispheres. Inset, plane of section. (C) Posterior coronal section of the cerebellar cortex (top) used to performed a quantification of the relative intensity of immunofluorescence staining of TRPC3, Zebrin II/Aldolase C and calbindin for PCs in the vermis (ventral lob. VIII, middle) and the hemisphere (ventral PM, bottom) (values normalized to the respective means). (D) Similar analysis of dorsal lob. III (middle) and sulcus of Sim to Crus I (bottom) in anterior section (top). TRPC3 expression is largely complementary to Zebrin II in the vermis and parts of the hemispheres (black arrow heads), but more uniform in other hemispheric areas. In general, TRPC3 expression demonstrates a weaker differentiation between low and high levels than Zebrin II. I-X, cerebellar lobules I-X; Sim, Simplex lobule; Cr II, Crus II; PM, paramedian lobule; Cop, Copula Pyramidis; gcl, granule cell layer; pcl, Purkinje cell layer; ml, molecular layer; D, dorsal; V, ventral; M, medial; L, lateral.

Figure 1.

Figure 1—figure supplement 1. Overview and local patterns of TRPC3 expression.

Figure 1—figure supplement 1.

(A) Confocal immunofluorescent images of coronal sections of wild-type mouse cerebellar cortex, from rostral to caudal, stained with anti-TRPC3 (red) and anti-Zebrin II/Aldolase C (green). TRPC3 is expressed in evident parasagittal bands, and is complementary to the expression of Zebrin II/Aldolase C (left) in the vermis, but more uniform in the hemispheres. (B–D) Immunofluorescent images of coronal sections of wild-type cerebellum, illustrating the distribution of TRPC3 in the anterior vermis (B), posterior vermis (C) and hemispheres (D–E). Demarcated areas in top rows are magnified underneath. Note that TRPC3 immunoreactivity is moderately higher in the Z– PCs than that in the Z+ PCs in the vermis. In the hemispheres the TRPC3-labeled bands are less well defined and can either be complementary to (D) or virtually indistinguishable from (E) Zebrin II bands. Cr II, Crus II; PM, paramedian lobule; Cop, copula of the pyramis; Sim, simple lobule; PFL, paraflocculus; FL, flocculus; ml, molecular layer; gcl, granule cell layer; pcl, Purkinje cell layer.
Figure 1—figure supplement 2. Quantification of TRPC3 expression compared to Zebrin II and calbindin.

Figure 1—figure supplement 2.

(A–D) Left, individual confocal immunofluorescent images of anti-TRPC3, anti-Zebrin II/Aldolase C and anti-calbindin staining, with the quantified section demarcated in the merged image. Examples (A) and (C) are similar to Figure 1C and D, respectively, but here presented with all individual images. Right, analysis of the relative intensity of the fluorescent image in the indicated section for TRPC3 (red), Zebrin II/Aldolase C (green) and calbindin (blue). Intensity values were normalized to their respective means and filtered by a moving average filter over 15 µm to reduce noise. Note that 1) TRPC3 expression appears complementary to Zebrin II in most of the vermis (see arrowheads) and hemispheric parts, while it appears to be expressed more uniformly in other areas of the hemispheres and 2) that Zebrin II expression differentiates the subpopulations more clearly than TRPC3, under these conditions. (E–F) Cross-correlation of staining intensities for ZebrinII/Aldolase C vs. TRPC3 (top) and ZebrinII/Aldolase C vs. Calbindin (bottom) in the vermis (left) and hemispheres (right) for examples in A (E) and in C (F). Whereas ZebrinII/Aldolase C positively correlates to Calbindin intensity (suggesting general fluctuations in staining intensity), the correlation with TRPC3 is consistently negative, that is when ZebrinII/Aldolase C intensity is high, TRPC3 is low and vice versa.
Figure 1—figure supplement 3. Western blot and immunostaining of pcp2Cre;TRPC3fl/fl mice.

Figure 1—figure supplement 3.

(A–B) Quantification (bottom) of TRPC3 levels in the anterior (left) and posterior (right) cerebellum in pcp2Cre;TRPC3fl/fl mice. The level of TRPC3 was lower in tissue from the anterior part (lobules I-III) (t7 = 2.63, p=0.034) and posterior part (lobules X) (t8 = 2.67, p=0.028) of the cerebellum of pcp2Cre;TRPC3fl/fl mice compared to controls. Note the residual TRPC3 present in the posterior cerebellum, presumably due to the presence of unaffected TRPC3-expressing UBCs, which are virtually absent in the anterior cerebellum. (B) Example images of full-length western blots. (C) Schematic for synaptic protein extraction protocol. (D) Subcellular localizations by western blots in the anterior (top) and posterior (bottom) cerebellum. TRPC3 is abundantly present in the membrane (P1) and synaptosomes (P2), but less so in the cytosol (S2). pcp2Cre;TRPC3fl/fl mice were devoid of TRPC3 completely in both anterior and posterior cerebellar fractions. (E–F) Coronal immunofluorescence images of immunohistochemical analysis of TRPC3 (red) and Zebrin II/Aldolase C (green) expression in the posterior cerebellar cortex of pcp2Cre;TRPC3fl/fl mutants (F) and normal mice (E) with a higher magnifications of the squared areas (right). In contrast to the TPRC3 staining in control mice there is no longer a banding pattern visible for TRPC3 in mutant mice, while aldolase C is still clearly present in bands. The presence of TRPC3 staining in the UBCs, see for example the example indicated by the arrowhead, confirms that the antibody worked and that the loss of TRPC3 is specific for PCs (marked by asterisks).
Figure 1—video 1. Light sheet imaging reconstruction of whole-mount immunolabeling for TRPC3 (white signal), cleared with iDISCO protocol and scanned in the horizontal plane of an adult mouse brain from dorsal to ventral (see Materials and methods).
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DOI: 10.7554/eLife.45590.006
For comparison, a subregion in the anterior vermis is compared side-by-side to the same region in a brain immunolabeled for EAAT4, which has an expression pattern similar to Zebrin II/Aldolase C.