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TRPM3 expression in mouse retina.

Brown RL, Xiong WH, Peters JH, Tekmen-Clark M, Strycharska-Orczyk I, Reed BT, Morgans CW, Duvoisin RM - PLoS ONE (2015)

Bottom Line: Within the retina, two closely related members of the melastatin TRP family, TRPM1 and TRPM3, are highly expressed.TRPM1 has been shown to be required for the depolarizing response to light of ON-bipolar cells, but the role of TRPM3 in the retina is unknown.Within the IPL, TRPM3 immunofluorescence was markedly stronger in the OFF sublamina than in the ON sublamina.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Physiology and Neuroscience, Washington State University, Pullman, Washington, United States of America; WWAMI Medical Education Program, Washington State University, Pullman, Washington, United States of America.

ABSTRACT
Transient receptor potential (TRP) channels constitute a large family of cation permeable ion channels that serve crucial functions in sensory systems by transducing environmental changes into cellular voltage and calcium signals. Within the retina, two closely related members of the melastatin TRP family, TRPM1 and TRPM3, are highly expressed. TRPM1 has been shown to be required for the depolarizing response to light of ON-bipolar cells, but the role of TRPM3 in the retina is unknown. Immunohistochemical staining of mouse retina with an antibody directed against the C-terminus of TRPM3 labeled the inner plexiform layer (IPL) and a subset of cells in the ganglion cell layer. Within the IPL, TRPM3 immunofluorescence was markedly stronger in the OFF sublamina than in the ON sublamina. Electroretinogram recordings showed that the scotopic and photopic a- and b-waves of TRPM3(-/-) mice are normal indicating that TRPM3 does not play a major role in visual processing in the outer retina. TRPM3 activity was measured by calcium imaging and patch-clamp recording of immunopurified retinal ganglion cells. Application of the TRPM3 agonist, pregnenolone sulfate (PS), stimulated increases in intracellular calcium in ~40% of cells from wild type and TRPM1(‑/‑) mice, and the PS-stimulated increases in calcium were blocked by co-application of mefenamic acid, a TRPM3 antagonist. No PS-stimulated changes in fluorescence were observed in ganglion cells from TRPM3(-/-) mice. Similarly, PS-stimulated currents that could be blocked by mefenamic acid were recorded from wild type retinal ganglion cells but were absent in ganglion cells from TRPM3-/- mice.

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TRPM3 is expressed in the IPL and GCL of the mouse retina.Mouse retina sections were labeled by immunofluorescence for TRPM3. A) Transmitted light DIC image showing the retinal cell layers. B) TRPM3 immunoreactivity is observed in the IPL and GCL of a WT mouse retina. C) No immunoreactivity is detected in a retinal section from a TRPM3-/- mouse. D) Transverse retinal sections of the inner retina double-labeled for TRPM3 (top, red) and calretinin (middle, green). Visible in the merged image (bottom), the outer, OFF half of the IPL (sublamina a) is more strongly labeled than the inner, ON, sublamina b. E) Imaging of the ganglion cell layer, double-labeled for TRPM3 (top, red) and Brn3a (middle, green). The merged image (bottom) shows that Brn3a-positive ganglion cells express TRPM3. Abbreviations are as follows: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars = 20 μm.
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pone.0117615.g003: TRPM3 is expressed in the IPL and GCL of the mouse retina.Mouse retina sections were labeled by immunofluorescence for TRPM3. A) Transmitted light DIC image showing the retinal cell layers. B) TRPM3 immunoreactivity is observed in the IPL and GCL of a WT mouse retina. C) No immunoreactivity is detected in a retinal section from a TRPM3-/- mouse. D) Transverse retinal sections of the inner retina double-labeled for TRPM3 (top, red) and calretinin (middle, green). Visible in the merged image (bottom), the outer, OFF half of the IPL (sublamina a) is more strongly labeled than the inner, ON, sublamina b. E) Imaging of the ganglion cell layer, double-labeled for TRPM3 (top, red) and Brn3a (middle, green). The merged image (bottom) shows that Brn3a-positive ganglion cells express TRPM3. Abbreviations are as follows: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars = 20 μm.

Mentions: For immunofluorescent localization of the TRPM3 channel in the retina, antiserum was generated against a peptide corresponding to C-terminus of mouse TRPM3. Reactivity of the antiserum with TRPM3 was confirmed by western blotting and immunofluorescent labeling of HEK293 cells transfected with a plasmid encoding a GFP-TRPM3 fusion protein (not shown). TRPM3 antiserum was applied to retina sections from WT and TRPM3-/- mice. In the WT retina, immunofluorescence was strong over the inner plexiform and ganglion cell layers (Fig. 3B). Weak immunofluorescence was observed in the inner nuclear layer and outer plexiform layer. No immunoreactivity was observed in the TRPM3-/- retina (Fig. 3C). Within the inner plexiform layer (IPL), TRPM3 immunoreactivity was stronger in the OFF sublamina compared to the ON sublamina. This is apparent in double labeling for TRPM3 and calretinin, which labels three bands in the IPL (Fig. 3D) [30]. Within the ganglion cell layer, a subset of cells (~40%) were intensely labeled. From the large size of the cell bodies and their double labeling with Brn3a these are most likely ganglion cells (Fig. 3E). There are however a few TRPM3-positive cells that are not labeled by Brn3a. These could be displaced amacrine cells, or Brn3a-negative ganglion cells.


TRPM3 expression in mouse retina.

Brown RL, Xiong WH, Peters JH, Tekmen-Clark M, Strycharska-Orczyk I, Reed BT, Morgans CW, Duvoisin RM - PLoS ONE (2015)

TRPM3 is expressed in the IPL and GCL of the mouse retina.Mouse retina sections were labeled by immunofluorescence for TRPM3. A) Transmitted light DIC image showing the retinal cell layers. B) TRPM3 immunoreactivity is observed in the IPL and GCL of a WT mouse retina. C) No immunoreactivity is detected in a retinal section from a TRPM3-/- mouse. D) Transverse retinal sections of the inner retina double-labeled for TRPM3 (top, red) and calretinin (middle, green). Visible in the merged image (bottom), the outer, OFF half of the IPL (sublamina a) is more strongly labeled than the inner, ON, sublamina b. E) Imaging of the ganglion cell layer, double-labeled for TRPM3 (top, red) and Brn3a (middle, green). The merged image (bottom) shows that Brn3a-positive ganglion cells express TRPM3. Abbreviations are as follows: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars = 20 μm.
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pone.0117615.g003: TRPM3 is expressed in the IPL and GCL of the mouse retina.Mouse retina sections were labeled by immunofluorescence for TRPM3. A) Transmitted light DIC image showing the retinal cell layers. B) TRPM3 immunoreactivity is observed in the IPL and GCL of a WT mouse retina. C) No immunoreactivity is detected in a retinal section from a TRPM3-/- mouse. D) Transverse retinal sections of the inner retina double-labeled for TRPM3 (top, red) and calretinin (middle, green). Visible in the merged image (bottom), the outer, OFF half of the IPL (sublamina a) is more strongly labeled than the inner, ON, sublamina b. E) Imaging of the ganglion cell layer, double-labeled for TRPM3 (top, red) and Brn3a (middle, green). The merged image (bottom) shows that Brn3a-positive ganglion cells express TRPM3. Abbreviations are as follows: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars = 20 μm.
Mentions: For immunofluorescent localization of the TRPM3 channel in the retina, antiserum was generated against a peptide corresponding to C-terminus of mouse TRPM3. Reactivity of the antiserum with TRPM3 was confirmed by western blotting and immunofluorescent labeling of HEK293 cells transfected with a plasmid encoding a GFP-TRPM3 fusion protein (not shown). TRPM3 antiserum was applied to retina sections from WT and TRPM3-/- mice. In the WT retina, immunofluorescence was strong over the inner plexiform and ganglion cell layers (Fig. 3B). Weak immunofluorescence was observed in the inner nuclear layer and outer plexiform layer. No immunoreactivity was observed in the TRPM3-/- retina (Fig. 3C). Within the inner plexiform layer (IPL), TRPM3 immunoreactivity was stronger in the OFF sublamina compared to the ON sublamina. This is apparent in double labeling for TRPM3 and calretinin, which labels three bands in the IPL (Fig. 3D) [30]. Within the ganglion cell layer, a subset of cells (~40%) were intensely labeled. From the large size of the cell bodies and their double labeling with Brn3a these are most likely ganglion cells (Fig. 3E). There are however a few TRPM3-positive cells that are not labeled by Brn3a. These could be displaced amacrine cells, or Brn3a-negative ganglion cells.

Bottom Line: Within the retina, two closely related members of the melastatin TRP family, TRPM1 and TRPM3, are highly expressed.TRPM1 has been shown to be required for the depolarizing response to light of ON-bipolar cells, but the role of TRPM3 in the retina is unknown.Within the IPL, TRPM3 immunofluorescence was markedly stronger in the OFF sublamina than in the ON sublamina.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Physiology and Neuroscience, Washington State University, Pullman, Washington, United States of America; WWAMI Medical Education Program, Washington State University, Pullman, Washington, United States of America.

ABSTRACT
Transient receptor potential (TRP) channels constitute a large family of cation permeable ion channels that serve crucial functions in sensory systems by transducing environmental changes into cellular voltage and calcium signals. Within the retina, two closely related members of the melastatin TRP family, TRPM1 and TRPM3, are highly expressed. TRPM1 has been shown to be required for the depolarizing response to light of ON-bipolar cells, but the role of TRPM3 in the retina is unknown. Immunohistochemical staining of mouse retina with an antibody directed against the C-terminus of TRPM3 labeled the inner plexiform layer (IPL) and a subset of cells in the ganglion cell layer. Within the IPL, TRPM3 immunofluorescence was markedly stronger in the OFF sublamina than in the ON sublamina. Electroretinogram recordings showed that the scotopic and photopic a- and b-waves of TRPM3(-/-) mice are normal indicating that TRPM3 does not play a major role in visual processing in the outer retina. TRPM3 activity was measured by calcium imaging and patch-clamp recording of immunopurified retinal ganglion cells. Application of the TRPM3 agonist, pregnenolone sulfate (PS), stimulated increases in intracellular calcium in ~40% of cells from wild type and TRPM1(‑/‑) mice, and the PS-stimulated increases in calcium were blocked by co-application of mefenamic acid, a TRPM3 antagonist. No PS-stimulated changes in fluorescence were observed in ganglion cells from TRPM3(-/-) mice. Similarly, PS-stimulated currents that could be blocked by mefenamic acid were recorded from wild type retinal ganglion cells but were absent in ganglion cells from TRPM3-/- mice.

Show MeSH
Related in: MedlinePlus