CBA/J mice have attenuated phase-shifting responses to light compared to CBA/N controls [6, 7]. These results are surprising because CBA/J mice have greater numbers of ipRGCs . A reasonable hypothesis is that the increased number of ipRGCs would result in enhanced behavioral responses to light. The work described here attempts to reconcile these findings by examining changes in central processing that could explain the differences in behavior.
To identify the relationship between the increased numbers of ipRGCs and the attenuated behaviors in CBA/J mice, we examined potential changes in neuropeptide expression in the SCN. VIP is expressed by many SCN neurons and is often used as a marker for the ventral SCN. Mice lacking VIP or its receptor, VPAC2, have weak circadian rhythms in DD, while over-expressing the VPAC2 receptors shortens the free-running period in DD [21, 22]. Genetic deletion of VIP or the VPAC2 receptor disrupts rhythmic clock gene expression, the rhythmic action potential firing of SCN neurons, and behavioral circadian rhythms . Synchrony of individual oscillators and behavioral rhythmicity can be rescued by application of VIP or the VPAC2 receptor agonist RO 25-1553 [24, 25]. In addition, VIP is known to play a role in phase-shifting because injection of VIP into hamster SCN causes phase-shifts in locomotor activity that are similar to those produced by light [26, 27]. Together these data suggest an important role for VIP in light transmission and coordination in the SCN and in circadian behaviors.
In these experiments, we sought to determine whether there are differences in VIP expression in SCN neurons that might account for the changes of photic entrainment. We found a greater number of VIP-positive cells in the SCN of CBA/J mice. This finding was unexpected because it suggests that greater numbers of light responsive cells are associated with attenuated circadian behaviors. This finding mirrors that of our previous work, which showed that CBA/J mice have greater numbers of light responsive cells in the retina compared to controls .
To further explore changes in the SCN, we looked at neurons that function to output circadian timing signals, which might be influenced by VIP. VP is expressed in neurons of the dorsal-lateral SCN and is released in a circadian manner and relies on input from VIP-expressing cells for synchronization, implicating its role in circadian output. VIP also induces phase shifts in VP expression, and mice with a null mutation in the gene for the VIP receptor, VPAC2, do not express rhythms in VP expression [22, 27]. We now report that CBA/J mice have greater numbers of detectable cells expressing VP compared to controls. This finding shows that in addition to greater numbers of input cells, CBA/J mice also have greater numbers of neurons involved in output pathways.
Retinal input to the SCN is necessary for light entrainment; therefore changes in retinal innervation could explain differences in circadian behavior. We measured light-induced FOS expression in the SCN of the CBA/J mice at a time when a light pulse was shown to affect an attenuated phase-shift in circadian behavior. Given that our previous work demonstrates that outer retinal degeneration during development results in greater numbers of ipRGCs, and that there are greater numbers of VIP-positive cells in the SCN, we hypothesized that there would be a greater degree of retinal innervation in CBA/J mice compared to controls. We found that there were greater numbers of FOS positive cells in CBA/J mice compared to controls. In both strains, the FOS positive cells were located throughout the SCN, which is in agreement with previous studies [18–20]. These data suggest that there are greater numbers of light sensitive cells in the SCN of CBA/J mice. Whether FOS is involved in the entrainment pathway or if it is simply a marker for an early response to light is unclear. However, a change in the number of cells expressing FOS in response to light would suggest differences in functional innervation of the SCN, which could underlie changes in central processing. These data support the conclusion that the alterations in retinal and SCN anatomy observed in CBA/J mice also reflect a functional change.
In attempting to reconcile the observations that CBA/J mice have greater numbers of cells involved in circadian entrainment, but have attenuated phase changes, it is important to note that the SCN is a heterogeneous structure made up of many neurons expressing different peptides. One molecule of great importance is γ-aminobutyric acid (GABA), which is expressed in most SCN neurons and plays a major role in neurotransmission. The exact mechanism by which GABAergic synaptic transmission translates into a behavior is unclear, however, GABA can synchronize and phase-shift clock cells . In hamsters, injection of the GABA receptor agonist, baclofen, into the SCN decreases the animals' phase-shifting responses to light in wheel running paradigms . This suggests that activation of GABA receptors plays a role in attenuating behavioral responses to light. In mouse SCN, GABA is expressed within approximately 70% of VIP-expressing cells . VIP enhances inhibitory transmission by increasing the frequency of IPSCs mediated by GABA in the SCN . As a result, the increase in VIP-positive cell numbers in CBA/J mice might lead to an increased modulation of GABAergic activity.
VP is also colocalized with GABA . It is possible that the increase in VP cells could play a role in the behaviors of CBA/J mice if the effects are mediated by an increase in GABA levels. Further work is needed to better understand how communication among VIP, VP, GABA and other neuropeptides within the SCN influences circadian function.
Retinal innervation is an important part of establishing proper pathways during development. The number of cells and connections they make are dependent on intrinsic properties of the cells and the target being innervated. The loss of target tissues can lead to an increase in cell death of the innervating cells, suggesting that the cell depends on the target for survival cues. Experimentally increasing the number of targets decreases the degree of developmental cell death [33, 34]. In addition, the reduction of afferent input to the target results in a significant increase in neuronal death. This has been shown in the retina and superior cervical ganglion of the rat [34–36]. Together these data suggest that retinal innervation plays a role in determining cell number of the target cell as well as the neuron reaching it.
If developmental changes in retinal innervation affect the development of the SCN, this could explain why CBA/J mice have attenuated phase-shifting responses, while other animal models of retinal degenration, such as the rd/rd mouse and RCS rat do not [37, 38]. These animals lose photoreceptors later in development, after the retina has matured, unlike the CBA/J mice, which lose outer retinal layers during postnatal development. Changes in the degree of innervation during development could explain the differences in ipRGC number as well as differences in the SCN.