Plasticity of TRPM1 expression and localization in the wild type and degenerating mouse retina

Abstract

The light response in retinal ON bipolar cells is associated with disinhibition of current flow through cation channels recently identified as type 1 members of the melastatin transient receptor potential (TRPM) family. We determined the developmental expression of Trpm1 in the wild type C57BL/6, DBA/2J, DBA2J-Gpnmb mouse retinas and in Pde6brd1 retinas characterized by degeneration of rod photoreceptors. Trpm1 mRNA in wild type retinas was low at birth but exhibited progressive increases in abundance up to early adulthood at postnatal day 21 (P21). Retinal Trpm1 mRNA content did not decrease following loss of photoreceptors. At P21, TRPM1-immunopositive perikarya migrated into the outer nuclear layer. The TRPM1 protein was trafficked to discrete postsynaptic puncta in wild type retinas whereas in adult Pde6brd1 mouse retinas, TRPM1 translocated to bipolar perikarya and bar-like structures in the distal inner nuclear layer. These findings show that expression and localization of the TRPM1 in the mouse retina is plastic, modulated by use-dependence and availability of sustained excitatory input.

Figure 1

Trpm1 gene expression in C57BL/6 wild type and Pde6b^rd1 retina. (A) Real Time PCR (RT-PCR) analysis for Trpm1 in wild type and rd1 retinas at different postnatal ages. RT-PCR signals are plotted relative to the value in a single WT animal at P2 (assigned the value of 1). ISH for Trpm1 in wild type (B) and Pde6br^d1 (C) retinas. The reaction product is confined to bipolar perikarya. Scale bar =50 μm. (D – F) RT-PCR analysis for Cav1.4 (CACNA1F), Rho and Grm6 genes in wild type and Pde6br^d1 retinas. *P<0.05; **P<0.01 for WT compared to rd1 at each age. Abbreviations: OS, outer segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; RGCL, retinal ganglion cell layer.

Figure 2

TRPM1 localization in C57BL/6 wild type and Pde6b^rd1 retina. (A–D) P21 WT retinas. (A) Expression is WT retinas is confined to cells with bipolar morphology. Scale bar = 20 μm. (B) Double labeling with SV2. TRPM1-ir puncta (arrowheads) are proximal to SV2-ir photoreceptor terminals. Scale bar = 20 μm. (C) TRPM1-ir puncta colocalized with PNA; PNA-negative puncta (arrowheads in Ciii) are presumably localized to rod bipolar cells. Scale bar = 5 μm. (D) Double labeling with SV2; scale bar = 2 μm. (E–H) P21 Pde6b^rd1 retinas. (E–F) Double labeling for TRPM1 and the perikarya marker propidium iodide (PI). The ONL and OPL are shrunk, reflecting loss of ONL perikarya. TRPM1-ir perikarya have migrated (arrowheads) or appear in the process of migration (arrows) into the remaining ONL. Scale bar = 20 μm in E, 10 μm in F. (G) Double labeling with SV2. TRPM1-ir puncta are still observed in P21 rd1 OPL. Scale bar = 2 μm. (H) Double labeling with rhodopsin (Rho). Displaced TRPM1-ir cells (arrows) are not rods. Scale bar = 10 μm. (I–K) P90 Pde6b^rd1 retinas. (I) ONL and OPL have disappeared. The TRPM1 antibody strongly labels the cell bodies of remaining bipolar neurons. Scale bar = 20 μm. (J & K) Some of the few perikarya displaced into space previously occupied by the ONL are TRPM1-ir (arrowhead in J). TRPM1 signals label cell bodies and horizontal bar-like processes in the distal INL (arrows). Scale bar = 5 μm.

Figure 3

(A) Vglut2 gene expression in C57BL/6 and DBA/2J retinas shows gradual loss of the RGC marker in the DBA/2J strain; *P<0.05; ** P<0.01. (B & C) Trpm1 and Grm6 mRNA levels in DBA/2J are not statistically different compared to Gpnmb-DBA/2J control retinas.