TRPM1 is required for the depolarizing light response in retinal ON-bipolar cells

Abstract

The ON pathway of the visual system, which detects increases in light intensity, is established at the first retinal synapse between photoreceptors and ON-bipolar cells. Photoreceptors hyperpolarize in response to light and reduce the rate of glutamate release, which in turn causes the depolarization of ON-bipolar cells. This ON-bipolar cell response is mediated by the metabotropic glutamate receptor, mGluR6, which controls the activity of a depolarizing current. Despite intensive research over the past two decades, the molecular identity of the channel that generates this depolarizing current has remained elusive. Here, we present evidence indicating that TRPM1 is necessary for the depolarizing light response of ON-bipolar cells, and further that TRPM1 is a component of the channel that generates this light response. Gene expression profiling revealed that TRPM1 is highly enriched in ON-bipolar cells. In situ hybridization experiments confirmed that TRPM1 mRNA is found in cells of the retinal inner nuclear layer, and immunofluorescent confocal microscopy showed that TRPM1 is localized in the dendrites of ON-bipolar cells in both mouse and macaque retina. The electroretinogram (ERG) of TRPM1-deficient (TRPM1(-/-)) mice had a normal a-wave, but no b-wave, indicating a loss of bipolar cell response. Finally, whole-cell patch-clamp recording from ON-bipolar cells in mouse retinal slices demonstrated that genetic deletion of TRPM1 abolished chemically simulated light responses from rod bipolar cells and dramatically altered the responses of cone ON-bipolar cells. Identification of TRPM1 as a mGluR6-coupled cation channel reveals a key step in vision, expands the role of the TRP channel family in sensory perception, and presents insights into the evolution of vertebrate vision.

Fig. 1.

TRPM1 is expressed by ON-bipolar cells in the mouse retina. (A) In situ hybridization of vertical sections of mouse retina with antisense (Left) and sense control (Right) TRPM1 probes. A hybridization signal is detected in many cell somata in the INL, where bipolar cell nuclei and somata are located (black arrows). Occasional unlabeled cell somata are likely horizontal cells (white arrowhead). (B) Vertical sections from a wild-type (Top) and TRPM1^−/− (Bottom) retina were immunofluorescently labeled by antibodies directed against TRPM1 (green) and PKCα (red). Areas of colocalization appear yellow in the merged images (Right). onl, Outer nuclear layer; opl, outer plexiform layer; inl, inner nuclear layer; ipl, inner plexiform layer.

Fig. 2.

TRPM1 is associated with both rod and cone ON-bipolar cells in the primate retina. (A and B) Vertical section of macaque retina double labeled for TRPM1 (green, A and B) and PKCα (red, B). Arrowheads indicate putative cone ON-bipolar cells (TRPM1 positive, PKCα negative cells). (C and D) Horizontal optical section in the plane of the inner nuclear layer from a whole mount macaque retina double labeled for TRPM1 (green, C and D) and PKCα (red, D). Asterisks indicate putative cone ON-bipolar cells. (E–G) Vertical section through the outer plexiform layer of the macaque retina double labeled with an antibody against TRPM1 (red, E and G) and the cone marker, peanut agglutinin-Alexafluor (green, F and G).

Fig. 3.

ERG b-waves and oscillatory potentials are absent in TRPM1-deficient mice. Rod isolated ERGs (A and C) and photopic cone-mediated ERGs (B and D) from combined wild-type and TRPM1^+/− mice (n = 4; gray lines) compared with TRPM1^−/− mice (n = 5; solid black lines). Numbers indicate scotopic (A and C) and photopic (B and D) flash intensities (log candela-seconds/meter²). Note that scales vary between scotopic and photopic graphs.

Fig. 4.

Chemically simulated ON-bipolar cell light responses from wild-type and TRPM1^−/− retina. Patch clamp recording of a rod (A) and a cone (C) ON-bipolar cell in a mouse retinal slice. The patch electrode was filled with internal solution containing Alexa488 hydrazide, and the puffer pipette (visible in A) was filled with Ames media containing 600 μM CPPG and Alexa-594. Rod bipolar cell synaptic terminal is indicated by an arrow in A. Current traces at −60 mV from rod (B) and cone (D) ON-bipolar cells from wild-type and TRPM1^−/− retinas; CPPG was applied to the outer plexiform layer as indicated.

Fig. 5.

Comparison of capsaicin-activated currents in ON-bipolar cells of wild-type and TRPM1^−/− mice. Patch-clamp recordings were made in the whole-cell mode from ON-bipolar cells in the retinal slice preparation at a holding potential of −60 mV. Capsaicin (100 μM) was applied to the OPL at the time indicated via a “puffer” pipet. These traces are representative of >10 recordings from each genetic background.