Figure 1.
Role of melanopsin in the behavioural aversion to light in mice.
(A) Open field apparatus: animals were placed into the front-half (FH) of the arena and remained there for 30 minutes. Time spent in the back-half (BH) of the arena was recorded. (B) and (C) Average (±SEM) percentage of time spent in the dark BH of the arena during the 30-minute trial. The FH is either illuminated, white bars (light FH), or in darkness, black bars (dark FH). (B) In untreated animals photophobic behaviour is evident in wildtype (WT), melanopsin only (MO) rd/rd cl mice, and melanopsin knockout (MKO) mice. Triple knockout (TKO) mice, lacking melanopsin and functional rods and cones show no aversion to light. (C) Atropine significantly increases aversive behaviour in WT, MO, and TKO mice. In MKO mice, atropine increases the average aversive behaviour but this does not reach significance. Atropine does not significantly affect behaviour when the FH is in darkness in any of the genotypes. Stars (*) indicate significance levels (Student's t-test): * p<0.05; ** p<0.01; *** p<0.001.
Figure 2.
Temporal kinetics of the behavioural aversion to light in mice.
Graphs showing time spent in the dark back-half (BH) of the arena over the course of the 30-minute trial. Data are binned into 6, 5-minute bins throughout the trial, with y-axis showing average (±SEM) percentage time spent in the dark BH. (A–D) shows data from untreated animals, and (E–H) after bilateral application of atropine drops. (A) and (E) WT, (B) and (F) MO (rd/rd cl), (C) and (G) MKO (Opn4−/−) and (D) and (H) TKO (Opn4−/− Gnat1−/− Cnga3−/−). White triangles, trials when the front-half (FH) is in light, black triangles, trials when the FH is in darkness. Results of the regression analyses are shown in table 1. Stars (*) indicate significance levels (Bonferroni post tests, light FH v dark FH at each time point): * p<0.05; ** p<0.01; *** p<0.001.
Table 1.
Regression analysis of temporal kinetics of light aversion in mice.
Figure 3.
Effect of atropine on pupil size.
In (A) MO (rd/rd cl), and (B) TKO (Opn4−/− Gnat1−/− Cnga3−/−) mice. Images below each graph illustrate pupil size pre- and post- light stimulation with atropine (Atr.) or without atropine (No Atr.) application in the two genotypes.
Figure 4.
Atropine augments an ERG b-wave preserved in TKO mice.
(A) b-wave amplitude of flash ERG responses in the presence and absence of atropine drops. A small but significant increase in the ERG b-wave amplitude was apparent following application of atropine drops (data presented as mean±SEM; n = 5 for each group). This is demonstrated in the average of all ERG responses in each group, shown in (B), atropine treated shown in grey, and untreated in black (scale bars: y-axis = 25 µV, x-axis = 50 ms; n = 5 for each group).
Figure 5.
Axotomy abolishes the atropine-induced light aversion response in TKO mice.
(A) Diagramatic illustration of the axotomy technique (image modified from [75] ), a swift back and forth movement of the needle severs both the optic nerve and central retinal artery. (B) Immunoreactivity for calretinin positive retinal afferents (red) is abolished in the superficial gray (SuG) and the optic nerve (Op) layers of the superior colliculus of a bilaterally axotomised TKO (bottom) compared to an unoperated contol (top). Collicular sections are −3.88 mm from bregma [76], scale bar is 200 µm. (C) Behavioural aversion to light in atropine-treated TKO mice is abolished in bilaterally axotomised animals. (D) Time spent in the dark back half (BH) of the arena over the course of the 30-minute trial. White triangles, from trials when the front-half (FH) is in light, black triangles when the FH is in darkness. Abbreviations: bsc, brachium of the superior colliculus; Ch-RPE, choroid retinal pigment epithelium; CRA, central retinal artery; ns, not significant; OA, ophthalmic artery, ON, optic nerve; Sc, sclera.
Figure 6.
Channelrhodopsin-2 expression in the inner retina of TKO mice causes the induction of behavioural light aversion.
(A) and (B) AAV2 transduced expression of Channelrhodopsin-2/Venus fusion (ChR2V) protein (green) in the ganglion cell layer of a TKO retina (AAV2-ChR2V TKO). (A) Transverse retinal section, ChR2V is visualised in many cells of the ganglion cell layer. Scale bar 20 µm (B) Immunohistochemistry on flat mount retina (focussing on the ganglion cell layer) for β-galactosidase (red) with ChR2V in green. Scale bar 100 µm. (C–D) Light aversion behaviour in the AAV2-ChR2V TKO. In these two graphs the comparison is between transduced and untreated animals when there is illumination in the front-half (FH). (C) Time spent in the dark back-half (BH) of the arena during the total 30 minutes of the trial. (D) Time spent in the dark (BH) of the arena over the course of the 30-minute trial. Stars (*) indicate significances (** p<0.01; *** p<0.001).