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Targeted destruction of photosensitive retinal ganglion cells with a saporin conjugate alters the effects of light on mouse circadian rhythms.

Göz D, Studholme K, Lappi DA, Rollag MD, Provencio I, Morin LP - PLoS ONE (2008)

Bottom Line: Intravitreal injection of this immunotoxin results in targeted destruction of melanopsin cells.In particular, the photosensitivity of the circadian system is significantly attenuated.This approach can be applied to any species subject to the existence of appropriate anti-melanopsin antibodies.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Virginia, Charlottesville, Virginia, United States of America.

ABSTRACT
Non-image related responses to light, such as the synchronization of circadian rhythms to the day/night cycle, are mediated by classical rod/cone photoreceptors and by a small subset of retinal ganglion cells that are intrinsically photosensitive, expressing the photopigment, melanopsin. This raises the possibility that the melanopsin cells may be serving as a conduit for photic information detected by the rods and/or cones. To test this idea, we developed a specific immunotoxin consisting of an anti-melanopsin antibody conjugated to the ribosome-inactivating protein, saporin. Intravitreal injection of this immunotoxin results in targeted destruction of melanopsin cells. We find that the specific loss of these cells in the adult mouse retina alters the effects of light on circadian rhythms. In particular, the photosensitivity of the circadian system is significantly attenuated. A subset of animals becomes non-responsive to the light/dark cycle, a characteristic previously observed in mice lacking rods, cones, and functional melanopsin cells. Mice lacking melanopsin cells are also unable to show light induced negative masking, a phenomenon known to be mediated by such cells, but both visual cliff and light/dark preference responses are normal. These data suggest that cells containing melanopsin do indeed function as a conduit for rod and/or cone information for certain non-image forming visual responses. Furthermore, we have developed a technique to specifically ablate melanopsin cells in the fully developed adult retina. This approach can be applied to any species subject to the existence of appropriate anti-melanopsin antibodies.

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Depletion of ipRGCs greatly alters entrainment, period lengthening in LL and masking by LL.Running records of a control and 3 UF008/SAP-treated mice showing phase angle of entrainment adopted in response to the gradual offset photoperiod, period during DD, during LL and re-entrainment (animals #139, 135) or failure to re-entrain (animals #122, 133) to LD12∶12 (grayed area indicates darkness). The phase angle of entrainment (Φ) during the gradual offset photoperiod is indicated for each individual (ZT12 = light completely off). B) Daily irradiance pattern recorded with a Gigahertz-Optik P-9710-2 universal optometer measured at cage level during the gradual offset light-dark paradigm. C) Relationship between remaining ipRGC density and the stable phase angle of entrainment of adopted by UF008/SAP injected mice and controls during the gradual offset photoperiod. Note the cluster of controls and the outlier animal (green-filled circle), which was injected with the saporin conjugate, but has not lost its ipRGCs. A second outlier animal (red-filled circle) had a normal phase angle of entrainment despite greatly reduced ipRGC density. D) There was no difference in period in circadian period during DD between UF008/SAP and IgG/SAP injected mice, but in LL, IgG/SAP injected mice significantly lengthened their periods (p<0.001; paired t test), becoming significantly different from UF008/SAP injected animals (p<.001, unpaired t test) which did not show any period lengthening in response to LL. E) LL induced masking was absent in UF008/SAP injected mice. Revolutions per day for the last 5 days in DD were compared to those during the initial 5 days of LL. For UF008/SAP mice, revolutions during DD and LL did not differ; for controls, revolutions/24 hr dropped by about 40% (p<.001; paired t tests).
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pone-0003153-g007: Depletion of ipRGCs greatly alters entrainment, period lengthening in LL and masking by LL.Running records of a control and 3 UF008/SAP-treated mice showing phase angle of entrainment adopted in response to the gradual offset photoperiod, period during DD, during LL and re-entrainment (animals #139, 135) or failure to re-entrain (animals #122, 133) to LD12∶12 (grayed area indicates darkness). The phase angle of entrainment (Φ) during the gradual offset photoperiod is indicated for each individual (ZT12 = light completely off). B) Daily irradiance pattern recorded with a Gigahertz-Optik P-9710-2 universal optometer measured at cage level during the gradual offset light-dark paradigm. C) Relationship between remaining ipRGC density and the stable phase angle of entrainment of adopted by UF008/SAP injected mice and controls during the gradual offset photoperiod. Note the cluster of controls and the outlier animal (green-filled circle), which was injected with the saporin conjugate, but has not lost its ipRGCs. A second outlier animal (red-filled circle) had a normal phase angle of entrainment despite greatly reduced ipRGC density. D) There was no difference in period in circadian period during DD between UF008/SAP and IgG/SAP injected mice, but in LL, IgG/SAP injected mice significantly lengthened their periods (p<0.001; paired t test), becoming significantly different from UF008/SAP injected animals (p<.001, unpaired t test) which did not show any period lengthening in response to LL. E) LL induced masking was absent in UF008/SAP injected mice. Revolutions per day for the last 5 days in DD were compared to those during the initial 5 days of LL. For UF008/SAP mice, revolutions during DD and LL did not differ; for controls, revolutions/24 hr dropped by about 40% (p<.001; paired t tests).

Mentions: Mice in the rhythm regulation studies were unilaterally injected with 400 ng UF008/SAP or IgG/SAP and contralaterally enucleated (to reduce overall variability in ipRGC ablation) under LD12∶12 conditions before being subjected to various lighting regimes. Figure 7A shows actograms representative of behavior across four different lighting conditions. None of the mice lost entrainment in LD12∶12 as an immediate consequence of UF008/SAP treatment. When switched to a graded photoperiod in which the irradiance gradually declined to zero (LD15G∶9; Figure 7B), all animals entrained, but with markedly different phase angles between groups (Figure 7C). Large differences in phase angle of entrainment were related to the correspondingly large differences in irradiance at the time of activity onset (Figure 7B). In constant darkness (DD), the circadian periods of the two groups did not differ. In constant light (LL), the circadian period of UF008/SAP mice did not change, whereas for control mice the period lengthened, as expected (Figure 7D). When the mice were returned from LL to the original LD12∶12 (without regard to the phase of the individual mice), 9 of 10 UF008/SAP mice required 16 or more days to re-entrain (median = 24 days), whereas every control animal required 14 or fewer days (median = 3 days). Subsequent histology showed that, on the average, UF008/SAP treatment yielded approximately 80% loss of melanopsin cells. Among the UF008/SAP-treated mice, the 3 individuals with the highest densities of remaining ipRGCs required 0, 16 and 16 days to re-entrain (corresponding to 81, 27 and 21 cells/mm2, respectively). Four of the UF008/SAP-injected mice never re-entrained. The density of melanopsin expressing RGCs averaged 15.2 cells/mm2 in these four animals.


Targeted destruction of photosensitive retinal ganglion cells with a saporin conjugate alters the effects of light on mouse circadian rhythms.

Göz D, Studholme K, Lappi DA, Rollag MD, Provencio I, Morin LP - PLoS ONE (2008)

Depletion of ipRGCs greatly alters entrainment, period lengthening in LL and masking by LL.Running records of a control and 3 UF008/SAP-treated mice showing phase angle of entrainment adopted in response to the gradual offset photoperiod, period during DD, during LL and re-entrainment (animals #139, 135) or failure to re-entrain (animals #122, 133) to LD12∶12 (grayed area indicates darkness). The phase angle of entrainment (Φ) during the gradual offset photoperiod is indicated for each individual (ZT12 = light completely off). B) Daily irradiance pattern recorded with a Gigahertz-Optik P-9710-2 universal optometer measured at cage level during the gradual offset light-dark paradigm. C) Relationship between remaining ipRGC density and the stable phase angle of entrainment of adopted by UF008/SAP injected mice and controls during the gradual offset photoperiod. Note the cluster of controls and the outlier animal (green-filled circle), which was injected with the saporin conjugate, but has not lost its ipRGCs. A second outlier animal (red-filled circle) had a normal phase angle of entrainment despite greatly reduced ipRGC density. D) There was no difference in period in circadian period during DD between UF008/SAP and IgG/SAP injected mice, but in LL, IgG/SAP injected mice significantly lengthened their periods (p<0.001; paired t test), becoming significantly different from UF008/SAP injected animals (p<.001, unpaired t test) which did not show any period lengthening in response to LL. E) LL induced masking was absent in UF008/SAP injected mice. Revolutions per day for the last 5 days in DD were compared to those during the initial 5 days of LL. For UF008/SAP mice, revolutions during DD and LL did not differ; for controls, revolutions/24 hr dropped by about 40% (p<.001; paired t tests).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2519834&req=5

pone-0003153-g007: Depletion of ipRGCs greatly alters entrainment, period lengthening in LL and masking by LL.Running records of a control and 3 UF008/SAP-treated mice showing phase angle of entrainment adopted in response to the gradual offset photoperiod, period during DD, during LL and re-entrainment (animals #139, 135) or failure to re-entrain (animals #122, 133) to LD12∶12 (grayed area indicates darkness). The phase angle of entrainment (Φ) during the gradual offset photoperiod is indicated for each individual (ZT12 = light completely off). B) Daily irradiance pattern recorded with a Gigahertz-Optik P-9710-2 universal optometer measured at cage level during the gradual offset light-dark paradigm. C) Relationship between remaining ipRGC density and the stable phase angle of entrainment of adopted by UF008/SAP injected mice and controls during the gradual offset photoperiod. Note the cluster of controls and the outlier animal (green-filled circle), which was injected with the saporin conjugate, but has not lost its ipRGCs. A second outlier animal (red-filled circle) had a normal phase angle of entrainment despite greatly reduced ipRGC density. D) There was no difference in period in circadian period during DD between UF008/SAP and IgG/SAP injected mice, but in LL, IgG/SAP injected mice significantly lengthened their periods (p<0.001; paired t test), becoming significantly different from UF008/SAP injected animals (p<.001, unpaired t test) which did not show any period lengthening in response to LL. E) LL induced masking was absent in UF008/SAP injected mice. Revolutions per day for the last 5 days in DD were compared to those during the initial 5 days of LL. For UF008/SAP mice, revolutions during DD and LL did not differ; for controls, revolutions/24 hr dropped by about 40% (p<.001; paired t tests).
Mentions: Mice in the rhythm regulation studies were unilaterally injected with 400 ng UF008/SAP or IgG/SAP and contralaterally enucleated (to reduce overall variability in ipRGC ablation) under LD12∶12 conditions before being subjected to various lighting regimes. Figure 7A shows actograms representative of behavior across four different lighting conditions. None of the mice lost entrainment in LD12∶12 as an immediate consequence of UF008/SAP treatment. When switched to a graded photoperiod in which the irradiance gradually declined to zero (LD15G∶9; Figure 7B), all animals entrained, but with markedly different phase angles between groups (Figure 7C). Large differences in phase angle of entrainment were related to the correspondingly large differences in irradiance at the time of activity onset (Figure 7B). In constant darkness (DD), the circadian periods of the two groups did not differ. In constant light (LL), the circadian period of UF008/SAP mice did not change, whereas for control mice the period lengthened, as expected (Figure 7D). When the mice were returned from LL to the original LD12∶12 (without regard to the phase of the individual mice), 9 of 10 UF008/SAP mice required 16 or more days to re-entrain (median = 24 days), whereas every control animal required 14 or fewer days (median = 3 days). Subsequent histology showed that, on the average, UF008/SAP treatment yielded approximately 80% loss of melanopsin cells. Among the UF008/SAP-treated mice, the 3 individuals with the highest densities of remaining ipRGCs required 0, 16 and 16 days to re-entrain (corresponding to 81, 27 and 21 cells/mm2, respectively). Four of the UF008/SAP-injected mice never re-entrained. The density of melanopsin expressing RGCs averaged 15.2 cells/mm2 in these four animals.

Bottom Line: Intravitreal injection of this immunotoxin results in targeted destruction of melanopsin cells.In particular, the photosensitivity of the circadian system is significantly attenuated.This approach can be applied to any species subject to the existence of appropriate anti-melanopsin antibodies.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Virginia, Charlottesville, Virginia, United States of America.

ABSTRACT
Non-image related responses to light, such as the synchronization of circadian rhythms to the day/night cycle, are mediated by classical rod/cone photoreceptors and by a small subset of retinal ganglion cells that are intrinsically photosensitive, expressing the photopigment, melanopsin. This raises the possibility that the melanopsin cells may be serving as a conduit for photic information detected by the rods and/or cones. To test this idea, we developed a specific immunotoxin consisting of an anti-melanopsin antibody conjugated to the ribosome-inactivating protein, saporin. Intravitreal injection of this immunotoxin results in targeted destruction of melanopsin cells. We find that the specific loss of these cells in the adult mouse retina alters the effects of light on circadian rhythms. In particular, the photosensitivity of the circadian system is significantly attenuated. A subset of animals becomes non-responsive to the light/dark cycle, a characteristic previously observed in mice lacking rods, cones, and functional melanopsin cells. Mice lacking melanopsin cells are also unable to show light induced negative masking, a phenomenon known to be mediated by such cells, but both visual cliff and light/dark preference responses are normal. These data suggest that cells containing melanopsin do indeed function as a conduit for rod and/or cone information for certain non-image forming visual responses. Furthermore, we have developed a technique to specifically ablate melanopsin cells in the fully developed adult retina. This approach can be applied to any species subject to the existence of appropriate anti-melanopsin antibodies.

Show MeSH
Related in: MedlinePlus