Loss of photic entrainment and altered free-running circadian rhythms in math5-/- mice

Abstract

Mammalian free-running circadian rhythms are entrained to the external light/dark cycle by photic signaling to the suprachiasmatic nuclei via the retinohypothalamic tract (RHT). We investigated the circadian entrainment and clock properties of math5-/- mutant mice. math5 is a critical regulator of retinal ganglion cell development; math5-/- mice show severe optic nerve hypoplasia. By anterograde cholera toxin B tracing, we find that math5-/- mice do not develop an identifiable RHT pathway. This appears to be attributable to agenesis or dysgenesis of the majority of RHT-projecting retinal ganglion cells. math5-/- mice display free-running circadian rhythms with a period approximately 1 hr longer than B6/129 controls (24.43 +/- 0.10 vs 23.62 +/- 0.19 hr; p < 0.00001). The free-running period of heterozygote mice is indistinguishable from that of controls. math5-/- mice show no entrainment to light/dark cycles, whereas heterozygote mice show normal entrainment to both 12 hr light/dark cycles and to a 1 hr skeletal photoperiod. math5-/- mice show reduced ability to entrain their rhythms to the nonphotic time cue of restricted running wheel access but demonstrate both free-running behavior and entrained anticipation of wheel unlocking in these conditions, suggesting the presence of a second diurnal oscillatory system in math5-/- animals. These results demonstrate that retinal ganglion cell input is not necessary for the development of a free-running circadian timekeeping system in the suprachiasmatic nucleus but is important for both photic entrainment and determination of the free-running period.

Fig. 1.

Anterograde cholera toxin B staining for the retinohypothalamic tract in control (B6/129; left) andmath5^−/− (right) mice. Note the minimal optic chiasm inmath5^−/−mice. No staining was seen in any math5^−/− SCN.

Fig. 2.

Representative whole-mount retinal immunohistochemical specimens for melanopsin in control B6/129 andmath5^−/− retinas. One retinal quadrant is shown in each image.

Fig. 3.

math5^−/− mice do not entrain to a light/dark cycle. Top panels, Mice were allowed to run freely for 1 week and were then subjected to LD 12:12 (lights on represented by gray bars) for 14 d, followed by 1 week of DD conditions. All data are shown as double-plotted wheel-running raster plots. Bottom panel, Comparison of math5^−/− andmath5^+/− mice for entrainment during 50 d of LD 12:12. No entrainment was seen in any of sevenmath5^−/− mice tested.

Fig. 4.

math5^+/− mice entrain to a skeletal photoperiod and show normal photic phase shifting responses. Top, Representative double-plotted wheel running actograms for math5^+/−(left) and control (right) mice kept in LD 1:23 (lights on represented by gray bars).Bottom, Phase responses for photic phase shifting inmath5^+/− and control B6/129 mice. Phase responses were calculated on the basis of the change in the free-running wheel activity phase after a 1 hr 100 lux light stimulus given at the indicated phase. n = 7 formath5^+/− mice; n= 4 for control mice. Differences between genotypes were not statistically significant (p > 0.4 for each).

Fig. 5.

math5^−/− mice fail to synchronize activity to limited wheel availability. Double-plotted drinking activity actograms are shown for threemath5^+/− (left) and three control (right) mice. Mice were housed in DD conditions. Wheel access was limited to 2 hr/d (gray bars) for 46 d.

Fig. 6.

math5^−/− mice show 24 hr anticipatory behavior during limited wheel availability. Mean drinking monitor activity per hour was summated over the six nonentraining math5^−/− mice during 46 d of limited wheel availability (n = 276 d for each point). Data are shown as mean ± SE. Thegray bar represents the period of wheel availability.Points represent succeeding hours (i.e., thepoint at 0 hr represents time 0–1).