Figure 1.
Ablation of VGLUT2 expression in Vglut2-cKO mouse retinas.
(A) Diagram of gene targeting strategy. Mouse with exon 2 of Vglut2 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.
Figure 2.
Circadian locomotor activity for the Vglut2-cKO and control mice under LD and DD conditions.
(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.
Figure 3.
Vglut2-cKO mice show impaired re-entrainment to phase shifts in the LD cycle.
(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)).
Figure 4.
Vglut2-cKO mice exhibit abnormal entrainment to a skeleton photoperiod.
Representative actograms from control (A) and Vglut2-cKO animals (B) under skeleton photoperiod light cycles (1 hour light pulses, 12 h apart). Control mice photoentrain to the 1-h light pulses and restrict their activity to one of the dark periods. Vglut2-cKO mice showed a variable degree of photoentrainment with some activity in both dark periods. Shaded regions of the activity records indicate periods of darkness.
Figure 5.
Attenuated PLRs in Vglut2-cKO mice.
(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.
Figure 6.
Negative masking responses to light are impaired in the Vglut2-cKO mice.
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).