Loss of circadian photoentrainment and abnormal retinal electrophysiology in Math5 mutant mice

Abstract

Purpose: To determine how the absence of retinal ganglion cells (RGCs) in Math5 (Atoh7) mutant mice affects circadian behavior and retinal function.

Methods: The wheel-running behavior of wild-type and Math5 mutant mice was measured under various light-dark cycle conditions. To evaluate retinal input to the suprachiasmatic nuclei (SCN) anatomically, the retinohypothalamic tracts were labeled in vivo. To assess changes in retinal function, corneal flash electroretinograms (ERGs) from mutant and wild-type mice were compared under dark- and light-adapted conditions. Alterations in retinal neuron populations were evaluated quantitatively and with cell-type-specific markers.

Results: The Math5-null mice did not entrain to light and exhibited free-running circadian behavior with a mean period (23.6 +/- 0.15 hours) that was indistinguishable from that of wild-type mice (23.4 +/- 0.19 hours). The SCN showed no anterograde labeling with a horseradish peroxidase-conjugated cholera toxin B (CT-HRP) tracer. ERGs recorded from mutant mice had diminished scotopic a- and b-wave and photopic b-wave amplitudes. The scotopic b-wave was more severely affected than the a-wave. The oscillatory potentials (OPs) and scotopic threshold response (STR) were also reduced. Consistent with these ERG findings, a pan-specific reduction in the number of bipolar cells and a smaller relative decrease in the number of rods in mutant mice were observed.

Conclusions: Math5-null mice are clock-blind and have no RGC projections to the SCN. RGCs are thus essential for photoentrainment in mice, but are not necessary for the development or intrinsic function of the SCN clock. RGCs are not required to generate any of the major ERG waveforms in mice, including the STR, which is produced by ganglion cells in some other species. The diminished amplitude of b-wave, OPs, and STR components in Math5 mutants is most likely caused by the decreased abundance of retinal interneurons.

Figure 1

Math5-null mice exhibit free-running circadian behavior. Actograms show wheel-running behavior for representative Math5^+/+ (A) and Math5^−/− (B) mice. Local clock time is double-plotted across the horizontal axis with the light stimulus indicated. The amplitude of the tick marks shows the intensity of wheel-running activity. The data were recorded during a single 100-day experiment, as indicated on the vertical axis. (A) Wild-type mice had wheel-running patterns that started and finished at the same clock time each day under LD conditions and were therefore photoentrained. When the LD cycle was advanced 6 hours, wild-type mice entrained to the new cycle within a few days, advancing their behavior by 6 hours. In constant darkness (DD), the wild-type mice were free-running with a 23.4-hour period (n = 17). On returning to LD, the mice were re-entrained, with a 24-hour period. In LL, the mice were free-running with a 24.7-hour period (n = 17). (B) Math5^−/− mice were free-running under all light conditions, with a 23.6-hour period (n = 7). They resembled wild-type mice in DD. Their SCN clocks were intrinsically normal but did not photoentrain. (C) Free-running periods for each animal under DD and LL conditions, with the group averages ± SD (red). LD, 12 hours light-dark.

Figure 2

Math5-null mice lack retinohypothalamic tracts. (A) CT-HRP was injected into one eye, labeling its optic nerve and projections in the brain, including the optic chiasm (OC) and suprachiasmatic nuclei (SCN). Coronal brain sections were stained with TMB to visualize RGC projections. (B) In wild-type mice, the ipsilateral RHT (arrowhead) and both SCN (arrow) were labeled, whereas the contralateral RHT was unstained. (C) In Math5^−/− mice, the SCN (arrow) and the RHT were unstained. The ventral surface is at the bottom of each panel. 3V, third ventricle. Scale bar, 100 μm.

Figure 3

Math5-null mice have diminished flash ERGs. (A) Representative scotopic ERG recordings. Math5^−/− mice have decreased a- and b-wave amplitudes (note the difference in scale bars). The STR (arrows) and OPs (arrowheads) are indicated. (B) Photopic ERG recordings. Math5^−/− mice have decreased b-wave amplitudes (note the difference in scale bars). (C) STRs recorded from 10 wild-type (top) and 10 Math5^−/− (bottom) eyes. Waveforms were elicited by a −5.9 and −4.4 log cd-s/m² light stimulus. Averaged responses for wild-type (blue) and Math5^−/− (red) mice are superimposed in the center. Math5 mutants required a 30-fold greater stimulus intensity to generate an STR comparable to wild-type mice. (D) Scotopic ERGs averaged from 10 wild-type and 10 Math5^−/− eyes at the maximum stimulus (0.6 log cd-s/m²) are normalized and superimposed to compare the timing and relative amplitude of a- and b-waves. The b-wave amplitude is decreased more than the a-wave.

Figure 4

Quantitative ERG analysis of wild-type and Math5-null mice. (A) Intensity vs. response (V − log I) plots for the scotopic b- and a-wave. (B) Intensity vs. response plots for the photopic b-wave. The intensity necessary to produce a 30-μV b-wave response is indicated in each panel by the gray line crossing the horizontal axis. The amplitudes are significantly reduced in Math5^−/− retinas at all light intensities (t-test, *P < 0.05), except for scotopic a-waves recorded at −2.4 and −2.9 log cd-s/m². Each point shows the average data recorded from 10 eyes (5 mice) ± SEM. (C) The implicit times for the scotopic a-, b- and OP waves recorded from wild-type (▪) and Math5^−/− (□) mice at maximum stimulus intensity. (D) Comparison of a-wave, b-wave, and oscillatory potential amplitudes in wild-type (▪) and Math5^−/− (□) mice. ∑OP is the sum of the four largest oscillatory waves. The bottom right diagram shows how amplitudes and implicit times were measured. Error bars: SD. (E) Plot showing the altered relationship between scotopic maximum a-wave (Va_max) and b-wave (Vb_max) amplitudes in Math5^−/− mice. Each symbol represents a different eye. The least-squares regression lines and equations are indicated. The b-wave was disproportionately reduced in the mutants. (○) Math5^−/− (•) wild-type mice.

Figure 5

Math5 mutant retinas have thinner nuclear and synaptic layers. Sections from wild-type (A) and Math5^−/− (B) eyes were stained with H&E. The GCL is hypocellular due to the absence of RGCs. (C) Diagram showing 250-μm fields (rectangles) used for quantitative analysis of the retina. (D) Histogram comparing the thickness of wild-type (▪) and Math5^−/− (□) retinal laminae. (E) Histogram comparing normalized planimetric cell density of wild-type (▪) and Math5^−/− (□) ONL and INL. There were significantly fewer cells in the Math5^−/− retinas, particularly within the INL. Scale bar, 50 μm; *P < 0.05; **P < 0.001. Error bars, ±SEM.

Figure 6

Math5-null mice have fewer rods. Rod photoreceptors in wild-type (A) and Math5^−/− (B) retinas were immunostained with RetP1, which labels rod outer (OS) and inner (IS) segments, and perikarya in the ONL. Math5^−/− have persistent hyaloid vasculature (arrow) in the vitreous. (C) Rod perikarya counts per field, normalized to wild-type. Scale bar, 50 μm; **P < 0.001. Error bars, ±SEM.

Figure 7

Confocal immunofluorescence micrographs showing that all bipolar subtypes are decreased in Math5 mutants. (A, B) PKCα staining of rod bipolar cells. Somata (arrows) and axon termini (arrowheads) are indicated. (C, D) G_oα staining of cone ON and rod bipolar cells. Arrows: G_oα-positive somata. (E, F) Recoverin staining of cone OFF bipolar cells (arrows). Their axon termini are located in the OFF sublamina of the IPL (arrowheads). (G, H) Neurofilament 160-kDa staining of RGCs (arrowhead) and horizontal cells (arrows). The labeling of horizontal cells is equivalent in both retinas, but RGCs are absent in the Math5^−/− animals. Scale bar, 50 μm.