Developmental Loss of ipRGCs Causes a Lengthened Free-running Circadian Period

Abstract

Purpose: : It is well established that retinal input during development is required for establishing proper circuitry and function of some retinal targets in the brain. We hypothesized that ipRGC innervation of the master clock in the suprachiasmatic nucleus (SCN) could influence the basic properties of circadian rhythms.

Methods: : We created two knock-in alleles that express either diphtheria toxin (DTA) or Cre, both under the control of the melanopsin promoter (Opn4^DTA and Opn4^Cre). We utilized a Cre-dependent alkaline phosphatase (AP) reporter (Z/AP) mouse line in combination with our Opn4^Cre and Opn4^DTA alleles to determine the target innervation of ipRGCs in the brain. We harvested retinas and brains from Opn4^Cre/DTA; Z/AP mice and their control counterparts (Opn4^Cre/+; Z/AP) at various stages of development and stained for AP. We compared ipRGC density (cells/mm²) in Opn4^Cre/DTA; Z/AP mice to Opn4^Cre/+; Z/AP (control) mice at postnatal days 0, 3, 5, 9, and 14. We recorded wheel-running behavior under the following light paradigms: 12/12 light/dark, constant dark, constant light, and ultradian (3.5 hours of dark followed by 3.5 hours of light).

Results: : We found that a significant number of ipRGCs are ablated before P0, due the presence of DTA, verifying that DTA expression is sufficient to kill ipRGCs during development. By P14, the number of remaining ipRGCs is similar to adult numbers. Consistent with the killing of ipRGCs, we show that innervation of the SCN by ipRGCs is prevented and this lack of innervation persists in adults. Using wheel-running activity, we show that Opn4^DTA/DTA mice cannot photoentrain and have severely impaired phase shifting and PLR, similar to our published report on an attenuated version of DTA (aDTA) driven by the melanopsin promoter (Opn4^aDTA/aDTA mice). In contrast to our published Opn4^aDTA/aDTA mice, Opn4^DTA/DTA mice free-run with a significantly longer period (about 24.25 hours) than wild type mice, which normally free run with a period of 23.5 hours.

Conclusions: : The data indicates that input from ipRGCs to the SCN at a critical developmental stage is required for setting the intrinsic period length of the circadian clock. A possible reason for the difference between the two diphtheria toxin mice is that in Opn4^aDTA/aDTA mice, the majority of ipRGCs are retained during development and are only progressively lost throughout adulthood. The lengthened free running period seen in Opn4^DTA/DTA mice is also seen in mice whose eyes were enucleated at birth and homozygous Math5 knockout mice, which lack the majority of RGCs.

Keywords: circadian rhythms • photoreceptors • retinal development