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Melanopsin-dependent light avoidance in neonatal mice - PubMed

  • ️Fri Jan 01 2010

Melanopsin-dependent light avoidance in neonatal mice

Juliette Johnson et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) form a light-sensitive system separate from rods and cones. Direct light stimulation of ipRGCs can regulate many nonimage-forming visual functions such as photoentrainment of circadian rhythms and pupil responses, and can intensify migraine headache in adults. In mice, ipRGCs are light responsive as early as the day of birth. In contrast, their eyelids do not open until 12-13 d after birth (P12-13), and light signaling from rods and cones does not begin until approximately P10. No physiological or behavioral function is established for ipRGCs in neonates before the onset of rod and cone signaling. Here we report that mouse pups as young as P6 will completely turn away from a light. Light-induced responses of ipRGCs could be readily recorded in retinas of pups younger than P9, and we found no evidence for rod- and cone-mediated visual signaling to the RGCs of these younger mice. These results confirm that negative phototaxis is evident before the onset of rod- and cone-mediated visual signaling, and well before the onset of image-forming vision. Negative phototaxis was absent in mice lacking melanopsin. We conclude that light activation of melanopsin ipRGCs is necessary and sufficient for negative phototaxis. These results strongly suggest that light activation of ipRGCs may regulate physiological functions such as sleep/wake cycles in preterm and neonatal infants.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

Phototaxis assay for neonatal mouse pups. Schematic diagram of test chamber, recording instrument and light stimulator (described in detail in

SI Materials and Methods

).

Fig. 2.
Fig. 2.

Negative phototaxis is evident as early as postnatal day 6 (P6) in WT mice. (A) Average latencies to first complete turn for P6-, P7-, P8-, and P9-aged pups. (B) Average time that pups faced in their original orientation (detailed descriptions of protocols, measurements, and statistical analyses in

SI Materials and Methods

) Dark and light bars show data from dark and light-stimulation, respectively. Statistical differences (t test) between light–dark latencies were significant: P < 0.0001 at P6; P < 0.003 at P7; P < 0.0001 at P8 and P < 0.005 at P9. The statistical differences in original position duration in the same animals were also significant at all ages: P < 0.0002 (P6; n = 6), P < 0.001 (P7; n = 9), P < 0.0001 (P8; n = 15), and P < 0.0001 (P9; n = 5). ***P < 0.001; **P < 0.01. Error bars represent SEM.

Fig. 3.
Fig. 3.

Multielectrode array (MEA) recordings of retinal ganglion cell spiking demonstrate that rod and cone-mediated light responses do not drive ipRGCs or nonipRGCs in mice younger than P10. (A) Histograms of light-evoked spikes from RGCs in response to a 40 s step of light. Top trace: P16 WT mouse; middle trace: P8 WT mouse, and bottom trace: P8 Opn4−/− mouse. (Note: although spontaneous waves of spiking were observed in P8 retinas, none of the spike activity shown here was induced by light.) Only the P16 recording (top record) shows short latency responses attributable to rod and cone signals. Top, middle, and bottom traces are averages of 16, 26, and 5 RGCs that responded to the onset of light, respectively. (B) Histograms of light-evoked spikes recorded from P16 WT mouse. Light stimuli were 6 s in duration, and the timescale is expanded compared with A. Top trace: Responses in control saline. Bottom trace: Recordings from same retina in synaptic blocker mixture of NBQX (20 μM, AMPA/KA glutamate receptor antagonist), DL-AP5 (100 μM, NMDA receptor antagonist), DL-AP4 (20 μM, mGluR6 agonist that blocks light responses in ON bipolar cells) and DHβE (2 μM, an agonist for nicotinic ACh receptors). All light-evoked spiking was eliminated in this mixture. Top and bottom traces are averages of 102 and 85 RGCs, respectively, recorded in the same retina. (C) Histograms of longer latency light-evoked spike responses from ipRGCs in P8 mouse. Top trace: control saline. Bottom trace: Synaptic blocker mixture. Same synaptic blockers used in B did not eliminate the light-evoked ipRGC responses. Top and bottom traces are averages of 66 and 62 RGCs, respectively, recorded in the same retina. Preservation of the long latency sustained light responses attributable to ipRGCs was observed in four of four retinas treated with the drug mixture (P8 and P9). (D) Histograms of light-evoked spikes in P16 Opn4−/− retina. Top trace: Short latency rod- and cone-mediated ON and OFF responses recorded in control saline. Middle trace: recordings in synaptic blocker mixture used in B and C. Bottom trace: spiking activity recorded in response to 20 mM K+ in the presence of the synaptic blocker mixture. Top, middle and bottom traces are averages of 56, 12, and 45 RGCs, respectively, recorded in the same retina. These records demonstrate that rod- and cone-mediated light responses are present in older Opn4−/− mice, and that all this activity is blocked with synaptic blockers, yet the RGCs themselves are still capable of spiking when depolarized with potassium.

Fig. 4.
Fig. 4.

Negative phototaxis is absent in neonatal melanopsin null (Opn4−/−) mice. Experiments are identical to those described for Fig. 2. All times are reported in minutes ± SEM. Latencies to first turn in dark averaged 3.56 ± 0.85, 4.83 ± 0.16, and 3.66 ± 0.49, at P7, P8, and P9, respectively. In light, latencies were 2.62 ± 0.58, 3.26 ± 0.77, and 4.15 ± 0.48 at the same ages. Durations in original orientations in the dark averaged 4.82 ± 0.16 min, 3.56 ± 0.85, and 3.97 ± 1.56, respectively at P7, P8, and P9, respectively. In light, average durations were 3.88 ± 0.58, 2.62 ± 0.16, and 4.13 ± 0.48 at the same ages. No light–dark time differences of significance were detected in the latencies to the first complete body turn or for the duration of times spent in the original orientation. P7, n = 7; P8, n = 7; P9, n = 13. Error bars represent SEM.

Fig. 5.
Fig. 5.

Light stimulates movement in Opn4+/+ and Opn4+/− pups, but not in Opn4−/− littermates. Movement was quantified as the distance the centroid of the pup's image traveled during the 60 s test periods (Materials and Methods). Mice were placed in the chamber for periods of 2–5 min in darkness. Distances during the 60-s period before (dark bars) and after light onset (light bars) are plotted for Opn4+/+ (n = 11), Opn4+/− (n = 20), and Opn4−/− (n = 9) mice. Results from P7–P9 aged animals are grouped together. Statistically significant differences between light and dark were observed in the Opn4+/+ and Opn4+/− mice (P < 0.001). No statistically significant light–dark differences were observed in the Opn4−/− mice (P > 0.05). Posttest analysis (Bonferroni) revealed no statistical differences between distances traversed in darkness of +/+, +/−, or −/− littermates. Error bars represent SEM.

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References

    1. Berson DM, Dunn FA, Takao M. Phototransduction by retinal ganglion cells that set the circadian clock. Science. 2002;295:1070–1073. - PubMed
    1. Van Gelder RN. Non-visual photoreception: sensing light without sight. Curr Biol. 2008;18:R38–R39. - PubMed
    1. Hattar S, et al. Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice. Nature. 2003;424:76–81. - PMC - PubMed
    1. Mrosovsky N, Hattar S. Impaired masking responses to light in melanopsin-knockout mice. Chronobiol Int. 2003;20:989–999. - PubMed
    1. Lucas RJ, Freedman MS, Muñoz M, Garcia-Fernández JM, Foster RG. Regulation of the mammalian pineal by non-rod, non-cone, ocular photoreceptors. Science. 1999;284:505–507. - PubMed

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