The amplitude of the a wave (A–C) and b wave (D–F) of the scotopic ERG steadily decreased during aging, and the daily rhythm in the a and b wave amplitude was only present in mice at 3 and 6 months (Two-way ANOVA, P<0.001, followed by Tukey tests). No significant difference in the amplitude of the a and b waves was detected in 12-month-old mice (Two-way ANOVA, P>0.1) White circles indicate the average amplitude at ZT6, and black circles indicate the average values at ZT18. Each point represents the mean ± SEM, where N = 4–14 for each point. Figures G through I, show representative traces of the dark-adapted ERGs at ZT18 (black lines) and at ZT6 (grey lines) for the different ages (G = 3 months; H = 6 months; I = 12 months).

(A) No differences were observed at ZT6 in the STR among young and old mice (t-tests, P>0.5 in all cases). Black circles, white circles, and black triangles indicate 3-, 6-, and 12-month-old mice, respectively. Each point represents the mean ± SEM, N = 6–8 for each point. (B) At ZT18 the STR significantly increased (−4.60 log log cd*s/m² vs −3.60 log cd*s/m²n) in 12-month-old mice (t-tests, P<0.05). Figures C through E show representative traces of the ERG at the different ages (C = 3 months; D = 6 months; E = 12 months).

(A) The amplitude of b wave of the photopic ERG at ZT6 was not affected by age at ZT6 (Two-way ANOVA, P>0.1). (B) It showed a significant decrease at ZT18 (Two-way ANOVA, P, 0.01, followed by Tukey tests, P<0.05) A significant difference in the amplitude of the b wave between ZT6 and ZT18 was only detected in 3-month-old mice (Two-way ANOVA, P>0.1 followed by Tukey tests, P<0.05). Closed circles, open circles, and closed triangle indicate 3-, 6- and 12-month-old mice, respectively. Each data point represents 4 to 8 animals, and the mean ± SEM, where N = 6–8 for each point. Figures C through E show representative wave forms of each age group at ZT18 (C = 3 months; D = 6 months and E = 12 months).

TOTO-3 staining of nuclear layers (red in A and blue in B) reveals an intact retinal layering in C3H-f^+/+ control 12-month-old mice. (A) Rod bipolar cells labelled by protein kinase c (PKCα) antibodies (green) have normal morphology and layering. (B) PKCα antibodies show again RB (red) with normally distributed bassoon puncta (green) decorating their dendritic tips in the opl. (C) Rod bipolar cells (RB) (red) have dendritic tips associated to green puncta, representing kinesins II positive synaptic ribbons. Most puncta are appropriately confined in the opl, but some of them are displaced in the onl (arrows). (D) PKCα labelling of Rod bipolar cells (red) and PSD-95 (green). Most presynaptic endings of photoreceptors (labelled by PSD-95) are confined to the OPL, but a few are clearly seen in the onl (arrow). (E) Antibodies against Goα (green), specific for depolarizing bipolar cells, in combination with PKCα (red) allow visualization of rod bipolar cells (yellow-orange) and depolarizing cone bipolars (green, CB). Dendrites of both categories of cells are clearly visible (arrows). (F) Calbindin (red) in combination with neurofilament (green) antibodies show a normal pattern of staining of horizontal cell bodies (HC), their axonal endings (yellow profiles in the opl) and calbindin positive amacrine cells (AC). Neurofilament antibodies also stains ganglion cells (GC, arrow). (G) One tyrosine hydroxylase (TH) positive amacrine cell with the typical large size body (arrow) and main dendritic plexus in the outermost part of the ipl. (H) Antibodies against the enzyme glutamine synthase show the fine morphology of Müller glial cells (green). Anti-glial fibrillary acidic protein (GFAP) antibodies show astrocytes regularly placed at the innermost retinal margin (red, arrow). Scale bars are 20 μm. Ph: Photoreceptors; onl: outer nuclear; opl: outer plexiform; inl: inner nuclear; ipl: inner plexiform; gcl: ganglion cell layer.

Melatonin injection (1 mg/Kg) increased the amplitude of the a (A, B) and b wave (D, E) of the scotopic ERG in 3- and 6-month-old mice (Two-way ANOVA P<0.01, followed by Tukey tests, P<0.05) with respect to the values obtained in control mice. The same dose did not increase the amplitude of the a and b wave in 12 months old mice (Two-way ANOVA, P>0.01, C, F). Melatonin injection (0.1 mg/Kg) increased the amplitude of the a and b waves of the scotopic ERG in 3-month-old mice (Two-way ANOVA, P<0.01, followed by Tukey tests, P<0.05 A, D), but not in 6 month-old mice (Two-way ANOVA, P>0.05; B, E, C, F). White circles indicate control groups; black triangles indicate melatonin 0.01 mg/kg; white squares indicate melatonin 0.1 mg/kg; and black circles indicate melatonin 1 mg/kg. Each value represents the mean ± SEM, N = 5–6 for each point.