Characterization of multiple bistratified retinal ganglion cells in a purkinje cell protein 2-Cre transgenic mouse line

Abstract

Retinal ganglion cells are categorized into multiple classes, including multiple types of bistratified ganglion cells (BGCs). The recent use of transgenic mouse lines with specific type(s) of ganglion cells that are labeled by fluorescent markers has facilitated the morphological and physiological studies of BGCs, particularly the directional-selective BGCs. The most important benefit from using transgenic animals is the capability to perform in vivo gene manipulation. In particular, the Cre/LoxP recombination system has become a powerful tool, allowing gene deletion, overexpression, and ectopic expression in a cell type-specific and temporally controlled fashion. The key to this tool is the availability of Cre mouse lines with cell or tissue type-specific expression of Cre recombinase. In this study we characterized the Cre-positive retinal ganglion cells in a PCP2 (Purkinje cell protein 2)-cre mouse line. We found that all of the Cre-positive retinal ganglion cells were BGCs. Based on morphological criteria, we determined that they can be grouped into five types. The On- and Off-dendrites of three of these types stratified outside of the cholinergic bands and differed from directional selective ganglion cells (DSGCs) morphologically. These cells were negative for Brn-3b and positive for both calretinin and CART retina markers. The remaining two types were identified as putative On-Off and On-DSGCs. This Cre mouse line could be useful for further studies of the molecular and functional properties of BGCs in mice.

Fig. 1

Distribution of PCP2-positive GCs in a retinal whole mount and a vertical section. A: Retinal whole-mount with tdTomato labeled GCs photographed at the GCL. White box shows region enlarged in B. B: High magnification of a retinal whole-mount viewed at the GCL. Most the PCP2-positive GC somas were evenly distributed but occasionally two or three somas were found in close proximity (shown by arrows). C: In a density recovery profile, a horizontal bar marks an average cell density (~185 cells / mm²), vertical bar indicates effective radius (~24 μm). D: Nearest neighbor analysis shows the most frequent distances between the neighboring cells (~40 μm). E: In a retinal vertical section, tdTomato fluorescence was detected in photoreceptors, bipolar cells, and a PCP2-positive GC (arrow). F: In a retinal whole mount, the virus-mediated expression of mCherry can be found in Cre-positive GCs. G: In the enlarged area from F, the somas, dendrites, and axons of the mCherry-expressing individual GCs can be clearly seen. ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bar = 1000 μm in A and F; 100 μm in B; 25 μm in E; 200 μm in G.

Fig. 2

Morphology and CART immunostaining of PCP2-GCs. A: Projection of the optical sections was taken from GCL through On-sublamina of the IPL. B: Projection of the optical sections in the same region as in A was taken through Off-sublamina. C-E: PCP2-tdTomato positive GCs (C) were stained for CART (D). All tdTomato positive GCs were found to express CART (E). Scale bar = 100 μm in A (applies to B); 50 μm in E (applies to C and D).

Fig. 3

Cluster dendrogram. The linkage distance shows the relative similarities of the cells. The insert shows the linkage distance of each clustering step. The dashed line on the main image and the insert marks abrupt increases in dissimilarity. Cells linked together below the dashed line are defined as cell types: On-Off DSGCs, On-DSGCs, Big-, Small-, and Middle-BGCs.

Fig. 4

Dendritic morphology and stratification of five types of PCP2-GCs. A, G, M, S, and Y: Projection of the optical sections were taken from GCL through On-sublamina of the IPL. The axons are indicated by arrows. The origins of the dendrites which ascend to the Off-sublamina are labeled by circles; the dendrites which dive back from the Off-sublamina into the On-sublamina are marked by triangles. B, H, N, T, and Z: Dendritic trees of the same cells shown in A, G, M, S, and Y, respectively, also stratified in the outer half of the IPL. The positions of the somas are indicated by asterisks. C, I, O, U, and AA: Normalized fluorescence intensity plotted against the levels of the IPL (green line for ChAT dendrites, magenta line for the PCP2-GC dendrites). The On- and Off-ChAT bands were assigned to 40% and 70% of the IPL, respectively. D, J, P, V, and BB: Z-stack projections taken from GCL through Off-sublamina for the areas marked by dashed areas on both dendritic tiers. The number of optical sections for each projection was 33, 31, 20, 20, and 20, respectively. E, K, Q, W, and CC: Rotated 90 degree along x-axis projections shown on D, J, P, V, and BB respectively. The same projections (magenta) superimposed with the corresponded images of the ChaT bands (green) are shown in F, L, R, X, and DD. Scale bar: 100 μm. Scale bar = 100 μm in Z (applies to A, B, G, H, M, N, S, T, and Y); 100 μm in BB (applies to D, J, P, and V); 10 μm in F, L, R, X, and DD.

Fig. 5

Comparison of dendritic stratification of two types of the putative DSGCs with the On-cholinergic plexus. A, F: On-dendritic tier of On-Off DSGC (A) and On-DSGC (F). B, G: Both cells shown in A and F (magenta, in focus) stratified at the On-ChAT band and followed the cholinergic plexus (green, in focus). C-E, H-J: Z-stack projection rotated 90 degree along the x-axis from the boxed area shown in B and G respectively. The number of optical sections for each projection was 20. Scale bar = 10 μm in G (applies to A, B, and F); 10 μm in E and J.

Fig. 6

Stratification of the dendrites of m-, s-, and b-BGCs in the On-sublamina. A, F, and K: On-dendritic tier of m-BGC (A), s-BGC (F), and b-BGC (K). B, G, and L: Cells shown in A, F, and K (magenta, in focus) stratified below the On-ChAT (green, out of focus). C-E, H-J, and M-O: Z-stack projection rotated 90 degree along the x-axis from the boxed area shown in B, G, and L respectively. The number of optical sections for each projection was 33, 31, and 17, respectively. Scale bar = 10 μm in L (applies to A, B, F, G, and K); 10 μm in E, J, and O.

Fig. 7

Immunostaining of PCP2-BGCs for Brn-3b and calretinin. A-B: The nucleus of m-BGC (A, arrow) was negative for Brn-3b (B, arrow). C-D: The nucleus of the s-BGC (C, arrow) was sometimes weakly stained with the antibody against Brn-3b (D, arrow). E-F: b-BGCs were negative for Brn-3b (arrow). G-H: On-DSGCs were positive for Brn-3b (arrows). I-O: The somas of m-BGC (I-J, arrow), s-BGC (K-L, arrow), and b-BGC (M-O, arrow) were positive for calretinin. P-Q: The somas of the two putative On-DSGCs (arrows) were negative for calretinin. bv - blood vessel. Scale bar = 50 μm in Q (applies to all panels).

Fig. 8

Central projection patterns of PCP2-GCs. All sections were photographed in the brain of the same mouse. The axon terminals of all GCs in both eyes were labeled by cholera toxin b subunit coupled to Alexa 488 (CTB, A, D, G, and J). Four different cross-section levels of the brain, LGN, SC, MTN, and SCN, contralateral to the eye injected with the viral vectors carrying a cre-excisable reporter gene (mCherry, B, E, H, and K) are shown. A-C: In the coronal section through the LGN, the projections of the PCP2-GCs were observed in the lateral part of the vLGN and ventro-lateral part of the dLGN. They were absent from the IGL and the OPN (C). D-F: The projections of the PCP2-GCs were located predominantly within a narrow superficial sublamina of the SC. G-I: Very weak mCherry-fluorescence was detected in the MTN. J-L: No mCherry-positive fibers were detected in the SCN region but were observed in the nearby OT. vLGN – ventral lateral geniculate nucleus; dLGN - dorsal lateral geniculate nucleus; SC – superior colliculus; IGL – intergeniculate leaflet of the lateral geniculate complex; OPN – olivary pretectal nucleus; MTN – medial terminal nucleus; SCN – suprachiasmatic nucleus; OT – optic tract. Scale bar = 450 μm in C and F; 200 μm in I and L.