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Cone and rod cells have different target preferences in vitro as revealed by optical tweezers.

Clarke RJ, Högnason K, Brimacombe M, Townes-Anderson E - Mol. Vis. (2008)

Bottom Line: Cell orientation of the photoreceptor also did not affect preferences: Cells oriented away from dendritic processes could reorient their axonal pole toward the target cell.Cone cells preferred normal partners, and rod cells preferred novel partners.Further,these differences may help explain the patterns of photoreceptor sprouting seen in retinal degeneration in which rod, but not cone, cells invade the inner retinal layers where third-order neurons are located.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Neuroscience, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103, USA.

ABSTRACT

Purpose: When neural circuits are damaged in adulthood, regenerating and sprouting processes must distinguish appropriate targets to recreate the normal circuitry. We tested the ability of adult nerve cells to target specific cells in culture using the retina as a model system.

Methods: Under sterile culture conditions, retinal cells, isolated from tiger salamander retina, were micromanipulated with optical tweezers to create pairs of first-order photoreceptor cells with second- or third-order retinal neurons. The development of cell contact and presynaptic varicosities, the direction and amount of neuritic growth, and nerve cell polarity were assessed after seven days in vitro. Cultures were labeled for rod opsin to distinguish rod from cone cells and for the alpha subunit of the trimeric G protein Go (Go alpha) to identify cone-dominated and mixed rod-cone ON bipolar cells.

Results: Quantitative analysis of growth demonstrated that target preferences were cell-specific: Cone cells preferred second-order bipolar cells, whereas rod cells grew toward third-order neurons, which include amacrine and ganglion cells. In addition, when rod cells grew toward bipolar cells, they chose an abnormally high number of Go alpha-positive bipolar cells. These growth patterns were not affected by tweezers manipulation or the amount of growth. Cell orientation of the photoreceptor also did not affect preferences: Cells oriented away from dendritic processes could reorient their axonal pole toward the target cell.

Conclusions: Cone cells preferred normal partners, and rod cells preferred novel partners. These intrinsic preferences indicate that adult nerve cells can have differing capacities for targeting even if they come from the same cell class. Further,these differences may help explain the patterns of photoreceptor sprouting seen in retinal degeneration in which rod, but not cone, cells invade the inner retinal layers where third-order neurons are located.

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Photoreceptors paired with bipolar cells, analyzed for ON and OFF subtype. A: Schematic diagram of the types of bipolar cells and their inputs in salamander retina (based on [26,30,31]). ON bipolar cells with cone input stain positive for Goα (red). All cone cells contact Goα-positive bipolar cells. In contrast, for rod cells, only 30% contact Goα-positive cells. Thus, some of the ON bipolars contacted by rod cells are not Goα-positive. Both cone and rod cells also contact OFF bipolar cells. B-G: Detection of Goα-positive bipolar cells: B: A pair consisting of a rod and a bipolar cell was classified as undetermined after 7 days in culture, i.e., the photoreceptor did not show more growth either toward or away from the target bipolar cell. C: The rod cell stained immunopositive for rod opsin confirming its identification as a rod cell. D: The same pair, after staining for Goα, showed that the bipolar cell stained immunopositive for Goα. Goα immunolabel is present along the cytoplasmic surface of the plasma membrane in all parts of the cell as has been described for salamander bipolar cells [26]. E: A pair consisting of a cone cell and a bipolar cell was classified as attractive after 7 days in culture. The attraction was evident due to more neuritic growth toward than away from the target. F: After staining for rod opsin, the cone was immunonegative confirming its identification. G: After Goα staining, the bipolar cell was immunopositive whereas the cone cell retained only background staining. Scale bar equals 20 µm. H: Goα-positive and -negative bipolar cells were distinguished in pairs made with cones (n=49) and rods (n=64) for the attracted, repulsed and undetermined categories (see I for the actual numbers in each group). Cone and rod cells were both attracted to and repulsed by Goα-positive bipolar cells. However, for both cone and rod cells, Goα-positive bipolar cells were significantly more attractive than repulsive (p<0.03 and p<0.02, respectively). I: In vivo about 30% of rod cells contact Goα-positive bipolar cells [26]. In cultured pairs, the number of rod cells that were attracted to Goα-positive bipolar cells was significantly higher than expected: about 82% (14 of 17 cells) of Goα-positive cells were attractive to rod cells (p=0.001, tested with the exact binomial test). Pairs classified as undetermined were not included in the analysis. The proportion of Goα-positive bipolar cells of the total bipolar cell population is 41% in vivo. The proportion of Goα-positive to Goα-negative cells presented to rod cells was similar, 38% (24/64) but for cone cells it was 63% (31/49). See text for discussion.
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f4: Photoreceptors paired with bipolar cells, analyzed for ON and OFF subtype. A: Schematic diagram of the types of bipolar cells and their inputs in salamander retina (based on [26,30,31]). ON bipolar cells with cone input stain positive for Goα (red). All cone cells contact Goα-positive bipolar cells. In contrast, for rod cells, only 30% contact Goα-positive cells. Thus, some of the ON bipolars contacted by rod cells are not Goα-positive. Both cone and rod cells also contact OFF bipolar cells. B-G: Detection of Goα-positive bipolar cells: B: A pair consisting of a rod and a bipolar cell was classified as undetermined after 7 days in culture, i.e., the photoreceptor did not show more growth either toward or away from the target bipolar cell. C: The rod cell stained immunopositive for rod opsin confirming its identification as a rod cell. D: The same pair, after staining for Goα, showed that the bipolar cell stained immunopositive for Goα. Goα immunolabel is present along the cytoplasmic surface of the plasma membrane in all parts of the cell as has been described for salamander bipolar cells [26]. E: A pair consisting of a cone cell and a bipolar cell was classified as attractive after 7 days in culture. The attraction was evident due to more neuritic growth toward than away from the target. F: After staining for rod opsin, the cone was immunonegative confirming its identification. G: After Goα staining, the bipolar cell was immunopositive whereas the cone cell retained only background staining. Scale bar equals 20 µm. H: Goα-positive and -negative bipolar cells were distinguished in pairs made with cones (n=49) and rods (n=64) for the attracted, repulsed and undetermined categories (see I for the actual numbers in each group). Cone and rod cells were both attracted to and repulsed by Goα-positive bipolar cells. However, for both cone and rod cells, Goα-positive bipolar cells were significantly more attractive than repulsive (p<0.03 and p<0.02, respectively). I: In vivo about 30% of rod cells contact Goα-positive bipolar cells [26]. In cultured pairs, the number of rod cells that were attracted to Goα-positive bipolar cells was significantly higher than expected: about 82% (14 of 17 cells) of Goα-positive cells were attractive to rod cells (p=0.001, tested with the exact binomial test). Pairs classified as undetermined were not included in the analysis. The proportion of Goα-positive bipolar cells of the total bipolar cell population is 41% in vivo. The proportion of Goα-positive to Goα-negative cells presented to rod cells was similar, 38% (24/64) but for cone cells it was 63% (31/49). See text for discussion.

Mentions: The same cultures as described were reexamined for bipolar subtype interaction by immunolabeling. In salamander retina, ON bipolar cells have either predominantly cone input, more equally mixed rod and cone input, or predominantly rod input [31] (Figure 4A). The presence of a unique receptor-associated G protein, Go, allowed us to positively distinguish most ON subtype cells. Staining for the alpha subunit of Go protein (Goα) is present in cone-dominated bipolars and mixed rod-cone bipolars, together comprising about 41% of all bipolar cells [26] (Figure 4A). The other 59% of bipolar cells, which are not immunoreactive for Goα, consists of the rod-dominated ON bipolars and all OFF cells. After fixation, cultures, which had been immunostained for rod opsin to distinguish rod from cone cells, were restained for Goα to distinguish ON and OFF bipolar cells (Figure 4B-G). There were 113 photoreceptor-bipolar pairs. For each category, attraction, repulsion, and undetermined, the number of Goα-positive and -negative cells were counted. Both Goα-positive and -negative bipolar cells were present in each category; however, Goα-positive cells were more attractive than repulsive (Figure 4H). For cones, 58% of Goα-positive cells were attractive, 29% were undetermined, and 13% were repulsive. For rod cells, 58% of Goα-positive cells were attractive, 23% were undetermined, and 19% were repulsive. χ2 analysis confirmed that cone and rod cells were more attracted than repulsed by Goα-positive cells (p<0.03 and p<0.02 respectively). In contrast, Goα-negative cells were approximately equally attractive and repulsive for both cone and rod cells (for cone cells, 44% versus 39%; for rod cells, 45% versus 35%). The data suggest a dependence upon bipolar cell subtype in neurite targeting, with the ON bipolar subtype providing a significantly attractive target for growth arising from both cone and rod cells. Thus, cell subtypes were not equally involved in targeting.


Cone and rod cells have different target preferences in vitro as revealed by optical tweezers.

Clarke RJ, Högnason K, Brimacombe M, Townes-Anderson E - Mol. Vis. (2008)

Photoreceptors paired with bipolar cells, analyzed for ON and OFF subtype. A: Schematic diagram of the types of bipolar cells and their inputs in salamander retina (based on [26,30,31]). ON bipolar cells with cone input stain positive for Goα (red). All cone cells contact Goα-positive bipolar cells. In contrast, for rod cells, only 30% contact Goα-positive cells. Thus, some of the ON bipolars contacted by rod cells are not Goα-positive. Both cone and rod cells also contact OFF bipolar cells. B-G: Detection of Goα-positive bipolar cells: B: A pair consisting of a rod and a bipolar cell was classified as undetermined after 7 days in culture, i.e., the photoreceptor did not show more growth either toward or away from the target bipolar cell. C: The rod cell stained immunopositive for rod opsin confirming its identification as a rod cell. D: The same pair, after staining for Goα, showed that the bipolar cell stained immunopositive for Goα. Goα immunolabel is present along the cytoplasmic surface of the plasma membrane in all parts of the cell as has been described for salamander bipolar cells [26]. E: A pair consisting of a cone cell and a bipolar cell was classified as attractive after 7 days in culture. The attraction was evident due to more neuritic growth toward than away from the target. F: After staining for rod opsin, the cone was immunonegative confirming its identification. G: After Goα staining, the bipolar cell was immunopositive whereas the cone cell retained only background staining. Scale bar equals 20 µm. H: Goα-positive and -negative bipolar cells were distinguished in pairs made with cones (n=49) and rods (n=64) for the attracted, repulsed and undetermined categories (see I for the actual numbers in each group). Cone and rod cells were both attracted to and repulsed by Goα-positive bipolar cells. However, for both cone and rod cells, Goα-positive bipolar cells were significantly more attractive than repulsive (p<0.03 and p<0.02, respectively). I: In vivo about 30% of rod cells contact Goα-positive bipolar cells [26]. In cultured pairs, the number of rod cells that were attracted to Goα-positive bipolar cells was significantly higher than expected: about 82% (14 of 17 cells) of Goα-positive cells were attractive to rod cells (p=0.001, tested with the exact binomial test). Pairs classified as undetermined were not included in the analysis. The proportion of Goα-positive bipolar cells of the total bipolar cell population is 41% in vivo. The proportion of Goα-positive to Goα-negative cells presented to rod cells was similar, 38% (24/64) but for cone cells it was 63% (31/49). See text for discussion.
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Related In: Results  -  Collection

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Show All Figures
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f4: Photoreceptors paired with bipolar cells, analyzed for ON and OFF subtype. A: Schematic diagram of the types of bipolar cells and their inputs in salamander retina (based on [26,30,31]). ON bipolar cells with cone input stain positive for Goα (red). All cone cells contact Goα-positive bipolar cells. In contrast, for rod cells, only 30% contact Goα-positive cells. Thus, some of the ON bipolars contacted by rod cells are not Goα-positive. Both cone and rod cells also contact OFF bipolar cells. B-G: Detection of Goα-positive bipolar cells: B: A pair consisting of a rod and a bipolar cell was classified as undetermined after 7 days in culture, i.e., the photoreceptor did not show more growth either toward or away from the target bipolar cell. C: The rod cell stained immunopositive for rod opsin confirming its identification as a rod cell. D: The same pair, after staining for Goα, showed that the bipolar cell stained immunopositive for Goα. Goα immunolabel is present along the cytoplasmic surface of the plasma membrane in all parts of the cell as has been described for salamander bipolar cells [26]. E: A pair consisting of a cone cell and a bipolar cell was classified as attractive after 7 days in culture. The attraction was evident due to more neuritic growth toward than away from the target. F: After staining for rod opsin, the cone was immunonegative confirming its identification. G: After Goα staining, the bipolar cell was immunopositive whereas the cone cell retained only background staining. Scale bar equals 20 µm. H: Goα-positive and -negative bipolar cells were distinguished in pairs made with cones (n=49) and rods (n=64) for the attracted, repulsed and undetermined categories (see I for the actual numbers in each group). Cone and rod cells were both attracted to and repulsed by Goα-positive bipolar cells. However, for both cone and rod cells, Goα-positive bipolar cells were significantly more attractive than repulsive (p<0.03 and p<0.02, respectively). I: In vivo about 30% of rod cells contact Goα-positive bipolar cells [26]. In cultured pairs, the number of rod cells that were attracted to Goα-positive bipolar cells was significantly higher than expected: about 82% (14 of 17 cells) of Goα-positive cells were attractive to rod cells (p=0.001, tested with the exact binomial test). Pairs classified as undetermined were not included in the analysis. The proportion of Goα-positive bipolar cells of the total bipolar cell population is 41% in vivo. The proportion of Goα-positive to Goα-negative cells presented to rod cells was similar, 38% (24/64) but for cone cells it was 63% (31/49). See text for discussion.
Mentions: The same cultures as described were reexamined for bipolar subtype interaction by immunolabeling. In salamander retina, ON bipolar cells have either predominantly cone input, more equally mixed rod and cone input, or predominantly rod input [31] (Figure 4A). The presence of a unique receptor-associated G protein, Go, allowed us to positively distinguish most ON subtype cells. Staining for the alpha subunit of Go protein (Goα) is present in cone-dominated bipolars and mixed rod-cone bipolars, together comprising about 41% of all bipolar cells [26] (Figure 4A). The other 59% of bipolar cells, which are not immunoreactive for Goα, consists of the rod-dominated ON bipolars and all OFF cells. After fixation, cultures, which had been immunostained for rod opsin to distinguish rod from cone cells, were restained for Goα to distinguish ON and OFF bipolar cells (Figure 4B-G). There were 113 photoreceptor-bipolar pairs. For each category, attraction, repulsion, and undetermined, the number of Goα-positive and -negative cells were counted. Both Goα-positive and -negative bipolar cells were present in each category; however, Goα-positive cells were more attractive than repulsive (Figure 4H). For cones, 58% of Goα-positive cells were attractive, 29% were undetermined, and 13% were repulsive. For rod cells, 58% of Goα-positive cells were attractive, 23% were undetermined, and 19% were repulsive. χ2 analysis confirmed that cone and rod cells were more attracted than repulsed by Goα-positive cells (p<0.03 and p<0.02 respectively). In contrast, Goα-negative cells were approximately equally attractive and repulsive for both cone and rod cells (for cone cells, 44% versus 39%; for rod cells, 45% versus 35%). The data suggest a dependence upon bipolar cell subtype in neurite targeting, with the ON bipolar subtype providing a significantly attractive target for growth arising from both cone and rod cells. Thus, cell subtypes were not equally involved in targeting.

Bottom Line: Cell orientation of the photoreceptor also did not affect preferences: Cells oriented away from dendritic processes could reorient their axonal pole toward the target cell.Cone cells preferred normal partners, and rod cells preferred novel partners.Further,these differences may help explain the patterns of photoreceptor sprouting seen in retinal degeneration in which rod, but not cone, cells invade the inner retinal layers where third-order neurons are located.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Neuroscience, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103, USA.

ABSTRACT

Purpose: When neural circuits are damaged in adulthood, regenerating and sprouting processes must distinguish appropriate targets to recreate the normal circuitry. We tested the ability of adult nerve cells to target specific cells in culture using the retina as a model system.

Methods: Under sterile culture conditions, retinal cells, isolated from tiger salamander retina, were micromanipulated with optical tweezers to create pairs of first-order photoreceptor cells with second- or third-order retinal neurons. The development of cell contact and presynaptic varicosities, the direction and amount of neuritic growth, and nerve cell polarity were assessed after seven days in vitro. Cultures were labeled for rod opsin to distinguish rod from cone cells and for the alpha subunit of the trimeric G protein Go (Go alpha) to identify cone-dominated and mixed rod-cone ON bipolar cells.

Results: Quantitative analysis of growth demonstrated that target preferences were cell-specific: Cone cells preferred second-order bipolar cells, whereas rod cells grew toward third-order neurons, which include amacrine and ganglion cells. In addition, when rod cells grew toward bipolar cells, they chose an abnormally high number of Go alpha-positive bipolar cells. These growth patterns were not affected by tweezers manipulation or the amount of growth. Cell orientation of the photoreceptor also did not affect preferences: Cells oriented away from dendritic processes could reorient their axonal pole toward the target cell.

Conclusions: Cone cells preferred normal partners, and rod cells preferred novel partners. These intrinsic preferences indicate that adult nerve cells can have differing capacities for targeting even if they come from the same cell class. Further,these differences may help explain the patterns of photoreceptor sprouting seen in retinal degeneration in which rod, but not cone, cells invade the inner retinal layers where third-order neurons are located.

Show MeSH
Related in: MedlinePlus