The extreme weight loss profile in <i>Opn4</i>^<i>-/-</i> mice could not be attributed to any gross-metabolic changes that occur early during the ketogenic diet—blue or red LD challenge.

<p>Solid bars stand for WT animals, striped bars stand for <i>Opn4</i>^{<b><i>-/-</i></b>} mice. Bars are also color-coded: blue stands for blue LD, red stands for red LD. All mice were fed <i>ad libitum</i> ketogenic diet during the course of the experiment (n = 4 mice / group). All groups, regardless of their genotype and the light conditions they were in, consumed similar amounts of food (A) and water (B) (t-tests: p>0.05). Activity was measured as infrared beam crossings and expressed as number of counts per 15 minute intervals. C) Activity levels were similar in all groups (t-test: p>0.05). There were no significant differences between groups in metabolic parameters such as oxygen consumption (D) and respiratory exchange rate (RER) (E). Respiratory exchange rate was calculated for each animal by taking the carbon dioxide production / oxygen consumption ratio. F) Heat production also did not differ significantly between groups. All values represent group means with ±SEM. (Under red and blue LD cycle: <i>Opn4</i>^{<b><i>-/-</i></b>} n = 4, WT n = 4)</p

Analysis of all genotypes and light groups reveals significant effects of light regime (p<0.01) and genotype (p<0.0001) on weight change.

<p>Several genotypes and light groups demonstrate pronounced differences in the second half of the experiment. Overall analysis indicates <i>Opn4</i>^{<b><i>-/-</i></b>} mice are significantly different in their weight change pattern than WTs (p<0.0001), but they are also different from <i>Opn4</i>^{<b><i>-/-</i></b>}<i>; Pde6</i>^{<b><i>rd1/rd1</i></b>} animals (p<0.05). Red LD condition causes more weight change than constant darkness over 5 weeks on the ketogenic diet challenge (p<0.01). A week by week analysis reveals the following differences in addition to the ones already mentioned in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127031#pone.0127031.g001" target="_blank">Fig 1</a>: A) All genotypes under 12h:12h blue LD condition (end of week 2: WT vs <i>Opn4</i>^{<b><i>-/-</i></b>}, p = 0.0002; end of week 3: WT vs <i>Opn4</i>^{<b><i>-/-</i></b>}, p = 0.0001; end of week 4 and 5: WT vs <i>Opn4</i>^{<b><i>-/-</i></b>}, p<0.0001). B) All genotypes under 12h:12h red LD condition (end of week 4: <i>Opn4</i>^{<b><i>-/-</i></b>} vs. <i>Opn4</i>^{<b><i>-/-</i></b>}<i>; Pde6</i>^{<b><i>rd1/rd1</i></b>}, p = 0.001; end of week 5: <i>Opn4</i>^{<b><i>-/-</i></b>} vs. <i>Opn4</i>^{<b><i>-/-</i></b>}<i>; Pde6</i>^{<b><i>rd1/rd1</i></b>}, p<0.0001; <i>Opn4</i>^{<b><i>-/-</i></b>} vs. <i>Pde6b rd1/rd1</i>, p = 0.0002). C) All genotypes in constant darkness (end of week 4: <i>Opn4</i>^{<b><i>-/-</i></b>} vs. WT, p = 0.0016; end of week 5: <i>Opn4</i>^{<b><i>-/-</i></b>} vs. WT, p = 0.0002). <i>Opn4</i>^{<b><i>-/-</i></b>} mice in constant darkness lose significantly less weight than the ones in red LD by the end of week 5 (p = 0.0004). RM-ANOVA, followed by Tukey-Kramer <i>post hoc</i> analysis. (For the weekly contrasts, alpha value was adjusted to 0.0017—Dunn-Sidak correction for contrasts. Fig 2 includes data replotted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127031#pone.0127031.g001" target="_blank">Fig 1</a> to demonstrate the full effects of all genotypes and light conditions. Under both blue and red LD cycles: <i>Opn4</i>^{<b><i>-/-</i></b>} n = 9, WT n = 12, <i>Pde6</i>^{<b><i>rd1/rd1</i></b>} n = 7, <i>Opn4</i>^{<b><i>-/-</i></b>}<i>; Pde6</i>^{<b><i>rd1/rd1</i></b>} n = 5. Under constant darkness: <i>Opn4</i>^{<b><i>-/-</i></b>} n = 8, WT n = 8, <i>Pde6</i>^{<b><i>rd1/rd1</i></b>} n = 8, <i>Opn4</i>^{<b><i>-/-</i></b>}<i>; Pde6</i>^{<b><i>rd1/rd1</i></b>} n = 6.)</p

<i>Opn4</i>^<i>-/-</i> mice lose significantly more weight as compared to the WT controls over the course of 5 weeks of ketogenic diet exposure A) under a 12h:12h blue LD and B) under a 12h:12h red LD cycle.

<p>RM-ANOVA followed by Tukey-Kramer post hoc analysis: <i>Opn4</i>^{<b><i>-/-</i></b>} vs. WT in blue LD p = 0.0009; in red LD p = 0.0005. (<i>Opn4</i>^{<b><i>-/-</i></b>} vs. WT at the end of week 2: in blue LD p = 0.0002, in red LD p = 0.0009; end of week 3: in blue LD p = 0.0001; in red LD p = 0.0003; end of week 4: in blue LD p<0.0001, in red LD p<0.0001; end of week 5: in blue LD p<0.0001, in red LD p<0.0001. For the weekly contrasts, alpha value was adjusted to 0.0017—Dunn-Sidak correction for contrasts. Under both blue and red LD cycles: <i>Opn4</i>^{<b><i>-/-</i></b>} n = 9, WT n = 12)</p

Both <i>Opn4</i>^<i>-/-</i> mice and the wild type controls entrain to a 12h:12h fluorescent LD cycle (yellow block corresponds to fluorescent lights on) as measured by radio-telemetry.

<p>After two weeks, animals are transferred to a 12h:12h blue or red LD cycle (red and blue blocks corresponds to lights on) and concurrently placed on a KD. A and B) Representative actograms of WT animals. C and D) Representative actograms of <i>Opn4</i>^{<b><i>-/-</i></b>} animals. Arrows indicate the day animals are switched to the KD. All actograms have been double-plotted with a 10-minute bin size. (Under red LD cycle: <i>Opn4</i>^{<b><i>-/-</i></b>} n = 4, WT n = 5. Under blue LD cycle: <i>Opn4</i>^{<b><i>-/-</i></b>} n = 4, WT n = 4.)</p

Destruction of ipRGCs by UF008/SAP depends on dose, but not on location within the retina.

<p>Ablation of ipRGCs by UF008/SAP is dose-dependent, but not related to retinal eccentricity. Each triplet of bars represents data from “peripheral” (P), ”middle” (M), and “central” (C) fields, respectively. (2-way ANOVA with Bonferroni post-tests).</p

Depletion of ipRGCs greatly alters entrainment, period lengthening in LL and masking by LL.

<p>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).</p

UF008/SAP kills ipRGCs without apparent damage to general retinal morphology.

<p>UF008/SAP does not induce changes in gross retinal morphology. Eyes injected with 800 ng/eye of UF008/SAP conjugate did not differ ( 2-way ANOVA with Bonferroni post-test) from the PBS-injected control eyes in their relative (A) ONL or (B) INL thickness. (open bars, control eyes; black bars, UF008/SAP-injected). C) Cryostat sections through control retina (left column) and through UF008/SAP treated retina (right column). Red label (all images) - Melanopsin immunoreactive (IR) cells (*) and processes (arrowheads); Green label (Upper Row) - GFAP-IR glia at the base of the ganglion cell layer; Green label (Middle Row) - CALB-IR cells (arrows) in the outer and inner nuclear layers; Green label (Bottom row) - ChAT-IR amacrine cells (arrows) in the inner nuclear and ganglion cell layers between which are the two ChAT-IR terminal zones of the inner plexiform layer. Blue label - The nuclear stain, DAPI, most clearly reveals cells in the outer nuclear layer of the upper and lower images. Note the absence of melanopsin-IR staining in treated retinas (right column). Abbreviations of retinal layers: g – ganglion cell; inl – inner nuclear; ipl – inner plexiform (on and off sublayers); onl – outer nuclear; opl – outer plexiform; p – photoreceptor. Bar = 20 µm.</p

UF008/SAP greatly reduces retinal projections to the suprachiasmatic nucleus and intergeniculate leaflet.

<p>CT-B tracing of RHT projections remaining after intravitreal UF008/SAP injection shows a terminal field densest along the lateral, ventrolateral and ventral SCN border, but greatly reduced in, or absent from, the dorso-central SCN. B) In the IGL, the contralateral projection from the UF008/SAP-injected retina is largely absent. C) There are no readily apparent differences with respect to the remaining retinal innervation of the OPT. The red label in all parts of the figure identifies terminals from the contralateral, undamaged retina. Abbreviations: APT – anterior pretectal n.; CPT – commissural pretectal n.; DLGc – dorsal lateral geniculate n;, contralateral; IGLc – intergeniculate leaflet, contralateral; IGLi – intergeniculate leaflet, ipsilateral; MPT – medial pretectal n.; NOT – nucleus of the optic tract; PPT – posterior pretectal n.; RHT – retinohypothalamic tract; VLGc – ventral lateral geniculate n., contralateral. (Ipsi- and contra- are referenced with respect to the injected with UF008/SAP).</p

Masking response to a light pulse is absent in mice that lose entrainment to LD12∶12.

<p>Masking is nearly normal by UF008/SAP injected mice in response to a 1 hr light pulse. However, the animals recovered more slowly than controls. There is no effect of treatment or of time, but there is a significant interaction (1 way ANOVA with repeated measures, p<.004). In mice that lost entrainment subsequent to UF008/SAP treatment, a 1 hr light pulse failed to induce masking.</p

UF008/SAP treatment has little effect on visual cliff performance.

<p>Both UF008/SAP and IgG/SAP injected groups perform quite accurately in the visual cliff test, but there is a small deficit in the UF008/SAP treated group (p<0.05, Mann-Whitney test).</p