Attenuated PLRs in Vglut2-cKO mice.

<p>(A) Images of control and Vglut2-CKO mice pupils before and during exposure to high intensity (3.8 mW/cm²⁾ white light stimuli. (B) Summary of PLRs measured in control mice. Consensual PLRs were measured in control (n = 8) and Vglut2-cKO (n = 10) mice. Vglut2-cKO mice show severe deficits in PLRs under low (4 µW/cm²⁾ and high (3.8 mW/cm²⁾ intensity white light stimuli (** p<0.05). Light stimuli were delivered for 20 s and maximum pupil area was measured before and during the light stimulus. Percent of pupil area following the light stimulus is shown normalized to the pupil area during dark conditions.</p

Ablation of VGLUT2 expression in Vglut2-cKO mouse retinas.

<p>(A) Diagram of gene targeting strategy. Mouse with exon 2 of <i>Vglut2</i> gene flanked by LoxP sites was bred with the Opn4-Cre mouse line in which Cre was inserted in the Opn4 gene locus. Progeny contained mice with deletion of Vglut2 exon 2 in ipRGCs (Vglut2-cKO mice). (B) Retinal sections of Vglut2-cKO and control littermate stained for Opn4, VGLUT2. Notice the co-localization of VGLUT2 with Opn 4 (arrows) in the control but not in the Vglut2-cKO mice retinas. ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar  = 50 µm.</p

Mice Deficient of Glutamatergic Signaling from Intrinsically Photosensitive Retinal Ganglion Cells Exhibit Abnormal Circadian Photoentrainment

<div><p>Several aspects of behavior and physiology, such as sleep and wakefulness, blood pressure, body temperature, and hormone secretion exhibit daily oscillations known as circadian rhythms. These circadian rhythms are orchestrated by an intrinsic biological clock in the suprachiasmatic nuclei (SCN) of the hypothalamus which is adjusted to the daily environmental cycles of day and night by the process of photoentrainment. In mammals, the neuronal signal for photoentrainment arises from a small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) that send a direct projection to the SCN. ipRGCs also mediate other non-image-forming (NIF) visual responses such as negative masking of locomotor activity by light, and the pupillary light reflex (PLR) via co-release of neurotransmitters glutamate and pituitary adenylate cyclase-activating peptide (PACAP) from their synaptic terminals. The relative contribution of each neurotransmitter system for the circadian photoentrainment and other NIF visual responses is still unresolved. We investigated the role of glutamatergic neurotransmission for circadian photoentrainment and NIF behaviors by selective ablation of ipRGC glutamatergic synaptic transmission in mice. Mutant mice displayed delayed re-entrainment to a 6 h phase shift (advance or delay) in the light cycle and incomplete photoentrainment in a symmetrical skeleton photoperiod regimen (1 h light pulses between 11 h dark periods). Circadian rhythmicity in constant darkness also was reduced in some mutant mice. Other NIF responses such as the PLR and negative masking responses to light were also partially attenuated. Overall, these results suggest that glutamate from ipRGCs drives circadian photoentrainment and negative masking responses to light.</p></div

Circadian locomotor activity for the Vglut2-cKO and control mice under LD and DD conditions.

<p>(A–C) Representative activity records from animals initially held in a 12:12 LD cycle then transferred to DD. Control (Ctrl)(A) and mutant (Vglut2-cKO)(B–C) animals demonstrate entrainment in LD as indicated by enhanced activity in the dark period. In DD the animals show a free running activity rhythm with some Vglut2-cKO mice exhibiting low-amplitude rhythms (B). In these animals, although the majority of activity was confined to the dark phase under LD, activity onset in DD was variable. Shaded regions indicate periods of darkness mirrored in LD bars above the actograms. (D–E) Amplitude of locomotor activity in DD was more variable among Vglut2-cKO (n = 17) than control (n = 9) mice. (F) Period of locomotor activity was comparable between control (n = 9) and Vglut2-cKO (n = 17) mice.</p

Negative masking responses to light are impaired in the Vglut2-cKO mice.

<p>Control and Vglut2-cKO mice were subjected to 3.5 h light: 3.5 h dark cycles for 14 days. (A) Control mice show robust activity during dark period reflecting strong aversion to locomotor activity under bright light conditions. The Vglut2-cKO mice, on the other hand, exhibited activity in both dark and light periods indicative of less pronounced negative masking responses to light. Shaded regions indicate periods of darkness. (B) Activity in the dark normalized to the total activity is higher in the control (n = 7) than the Vglut2-cKO (n = 8) mice (** p<0.05). (C) Analysis of the individual mice shows more variable masking responses among the Vglut2-cKO mice (n = 9) than control littermates (n = 7).</p

Vglut2-cKO mice show impaired re-entrainment to phase shifts in the LD cycle.

<p>(A) Representative double-plotted actograms of mice subjected to 6 hr phase advance on days marked. Top bars indicate initial LD cycle; bottom bars below indicate shifted cycle. (B) Representative double-plotted actograms of mice subjected to 6 hr phase delay on days marked. Top bars indicate initial LD cycle; bottom bars below indicate shifted cycle. (C) Number of days required for reentrainment after the 6 hr phase advance or phase delay in control (n = 7) and Vglut2-cKO (n = 4) mice. Vglut2 mice showed delayed re-entrainment to either phase advances or phase delays in the LD cycles (** p<0.05)).</p

Intrinsically Photosensitive Retinal Ganglion Cells (ipRGCs) Are Necessary for Light Entrainment of Peripheral Clocks

<div><p>Light is a powerful entrainer of circadian clocks in almost all eukaryotic organisms promoting synchronization of internal circadian rhythms with external environmental light-dark (LD) cycles. In mammals, the circadian system is organized in a hierarchical manner, in which a central pacemaker in the suprachiasmatic nucleus (SCN) synchronizes oscillators in peripheral tissues. Recent evidence demonstrates that photoentrainment of the SCN proceeds via signaling from a subpopulation of retinal ganglion cells (RGCs) which are melanopsin-expressing and intrinsically photosensitive (ipRGCs). However, it is still unclear whether photoentrainment of peripheral clocks is mediated exclusively by the ipRGC system or if signaling from RGCs that do not express melanopsin also plays a role. Here we have used genetic “silencing” of ipRGC neurotransmission in mice to investigate whether this photoreceptive system is obligatory for the photoentrainment of peripheral circadian clocks. Genetic silencing of ipRGC neurotransmission in mice was achieved by expression of tetanus toxin light chain in melanopsin-expressing cells (<i>Opn4</i>::<i>TeNT</i> mouse line). Rhythms of the clock gene <i>Period 2</i> in various peripheral tissues were measured by crossbreeding <i>Opn4</i>::<i>TeNT</i> mice with <i>PER2</i> luciferase knock-in mice (<i>mPER2</i>^<i>Luc</i>). We found that in <i>Opn4</i>::<i>TeNT</i> mice the pupillary light reflex, light modulation of activity, and circadian photoentrainment of locomotor activity were severely impaired. Furthermore, <i>ex vivo</i> cultures from <i>Opn4</i>::<i>TeNT</i>, <i>mPER2</i>^<i>Luc</i> mice of the adrenal gland, cornea, lung, liver, pituitary and spleen exhibited robust circadian rhythms of PER2::LUC bioluminescence. However, their peak bioluminescence rhythms were not aligned to the projected LD cycles indicating their lack of photic entrainment <i>in vivo</i>. Finally, we found that the circadian rhythm in adrenal corticosterone in <i>Opn4</i>::<i>TeNT</i> mice, as monitored by <i>in vivo</i> subcutaneous microdialysis, was desynchronized from environmental LD cycles. Our findings reveal a non-redundant role of ipRGCs for photic entrainment of peripheral tissues, highlighting the importance of this photoreceptive system for the organismal adaptation to daily environmental LD cycles.</p></div

PER2::LUC rhythms of peripheral tissues in <i>Opn4</i>::<i>TeNT</i> mice are not entrained by light.

<p>Circular plots of peak bioluminescence rhythms in peripheral tissues presented in control (A) and mutant (B) mouse tissues. Mice were housed in the absence of running wheels. Notice the phases of peak bioluminescence rhythms are significantly dispersed among tissues in the <i>Opn4</i>::<i>TeNT</i> mice indicating their lack of entrainment to environmental light-dark cycles. ADR, adrenal (n = 12–17); COR, cornea (n = 15–20); LIV, liver (n = 12–14); LUN, lung (n = 14–16); PIT, anterior pituitary (n = 14–19; SPL, spleen (n = 7–11).</p

TeNT expression doesn’t alter ipRGCs responses to light.

<p>(A-C) Average ipRGCs responses to 1min light stimulations (480nm) of increasing irradiance (5.10¹⁰ photons/cm²/s to 5.10¹³ photons/cm²/s) recorded from retinas of <i>Opn4</i>^<i>cre/+</i>, <i>R26</i>^<i>+/+</i> (A, n = 123), <i>Opn4</i>^<i>cre/+</i>, <i>R26</i>^{<i>+/TeNT</i>} (B, n = 87), <i>Opn4</i>^<i>cre/+</i>, <i>R26</i>^{<i>TeNT/TeNT</i>} (C, n = 158) mice and the corresponding dose response curves (D).</p

Lack of c-FOS expression in the <i>Opn4</i>::<i>TeNT</i> suprachiasmatic nucleus following light pulse at CT 16.

<p>(A) Immunocytochemical staining for c-FOS in the SCN of control and <i>Opn4</i>::<i>TeNT</i> mice exposed to a light pulse. Notice the diminished number of c-FOS positive cells in the mutant mouse. Scale bar = 100 um. (B) Quantification of c-FOS expression in the suprachiasmatic nucleus in control mice exposed to a light pulse (+) or no light pulse (-) and in mutant mice exposed to a light pulse. Expression is reflected by the number of c-FOS positive cells per suprachiasmatic nucleus per slice. n = 3–8. *P<0.004 by Kruskal-Wallis non-parametric ANOVA, followed by Dunn’s multiple comparison test.</p