Abstract
Purpose: To identify the cellular source of connexin36 (Cx36) expression in the outer nuclear (ONL) and outer plexiform layer (OPL) of the mouse retina.
Methods: Transgenic mice were generated that express (i) a fusion protein with EGFP targeted to the C-terminal domain of Cx36, and (ii) lacZ under the control of the Cx36 promoter. To determine the cellular location of Cx36, we identified retinal neurons immunocytochemically with established retinal markers and by intracellular injection of Alexa594. Cx36 could then be localized by targeting expression of EGFP and lacZ to the appropriate cell types in vertical sections and dissociated cells. In addition, we performed single cell RT-PCR with cytoplasm harvested from rod photoreceptors, horizontal cells, Müller cells, and pigment epithelial cells.
Results: EGFP labeling in the OPL appears in discrete clusters, which are aligned but not coincident with cone pedicles. Cx36 is expressed by a population of OFF bipolar cells that are immunoreactive for neurokinin3 (NK3), and by a population of ON cone bipolar cells identified by the presence of the -subunit of the G-protein Go. We found a close spatial relationship between Cx36 and the ionotropic glutamate receptor subunit GluR1, but not with GluR2/3, GluR4 and the kainate receptor subunits GluR5/6/7. Cx36 was never co-localized with CaBP 28K and protein kinase C, excluding expression of Cx36 in horizontal cells and rod bipolar cells, respectively. EGFP labeling in the ONL is restricted to cones, whereas rods, Müller cells and pigment epithelial cells do not express Cx36. The finding that only cones and cone bipolar cells express Cx36 was supported by the lacZ staining of isolated neurons and subsequently confirmed by the PCR data of identified cells.
Conclusions: We have shown that Cx36 is expressed in dendrites of OFF bipolar cells which are aligned with cone pedicles. Interestingly, Cx36 and GluR1 appear to be closely associated in flat contacts made by OFF cone bipolar cells. Cx36 is also expressed in a population of ON cone bipolar cells. In the ONL, Cx36 is exclusively expressed by cones.
Methods: Transgenic mice were generated that express (i) a fusion protein with EGFP targeted to the C-terminal domain of Cx36, and (ii) lacZ under the control of the Cx36 promoter. To determine the cellular location of Cx36, we identified retinal neurons immunocytochemically with established retinal markers and by intracellular injection of Alexa594. Cx36 could then be localized by targeting expression of EGFP and lacZ to the appropriate cell types in vertical sections and dissociated cells. In addition, we performed single cell RT-PCR with cytoplasm harvested from rod photoreceptors, horizontal cells, Müller cells, and pigment epithelial cells.
Results: EGFP labeling in the OPL appears in discrete clusters, which are aligned but not coincident with cone pedicles. Cx36 is expressed by a population of OFF bipolar cells that are immunoreactive for neurokinin3 (NK3), and by a population of ON cone bipolar cells identified by the presence of the -subunit of the G-protein Go. We found a close spatial relationship between Cx36 and the ionotropic glutamate receptor subunit GluR1, but not with GluR2/3, GluR4 and the kainate receptor subunits GluR5/6/7. Cx36 was never co-localized with CaBP 28K and protein kinase C, excluding expression of Cx36 in horizontal cells and rod bipolar cells, respectively. EGFP labeling in the ONL is restricted to cones, whereas rods, Müller cells and pigment epithelial cells do not express Cx36. The finding that only cones and cone bipolar cells express Cx36 was supported by the lacZ staining of isolated neurons and subsequently confirmed by the PCR data of identified cells.
Conclusions: We have shown that Cx36 is expressed in dendrites of OFF bipolar cells which are aligned with cone pedicles. Interestingly, Cx36 and GluR1 appear to be closely associated in flat contacts made by OFF cone bipolar cells. Cx36 is also expressed in a population of ON cone bipolar cells. In the ONL, Cx36 is exclusively expressed by cones.
| Original language | English |
|---|---|
| Article number | 1069 |
| Journal | Investigative Ophthalmology & Visual Science |
| Volume | 44 |
| Publication status | Published - May 2003 |
| Event | The Association for Research in Vision and Ophthalmology (ARVO) Annual Meeting - Fort Lauderdale, United States Duration: 4 May 2003 → 9 May 2003 |