The marmosets were submitted to two behavioral tests. In the first one, a non-smelling object (such as a pen or a key) was placed two centimeters away from each animal's face. The normal marmosets directed their sight to the object and tried to grab and bite it; the blind animals did not react to the objects at all. The same test was repeated with the objects in movement, and again only the normal marmosets reacted by directing their sight to the moving object. The second test took place in a room with dim light (10 lux). A spotlight was placed on one side of the animals and directed to their faces. The normal marmosets turned their faces towards the light, while the blind ones did not.
As shown in Figure 1, blind marmosets were clearly synchronized to the external cycles. Like normal animals, blind animals showed a normal biphasic activity circadian rhythm, with a more intense bout of activity at the beginning of the light phase and a second bout near the end. However, this bimodal pattern was less prominent in blind animals (Figure 2). Additionally, blind marmosets showed a shorter active phase compared to normal animals. After we shifted the light phase by 6 hours (first a delay and then an advance), blind marmosets were entrained to the new light-dark cycle, but their entrainment was much slower compared to the normal marmosets (Figures 1 and 2). The blind animals synchronized only after 12–14 days, while the normal animals did so after 3–4 days. During entrainment, the phase angle of activity onset in relation to the LD cycle was different in blind and normal marmosets (see Figure 2).
The animals were then placed in constant light conditions in order to determine if they had functional circadian clocks. As show in Figure 1, the marmosets showed free-running circadian rhythms. Free-running periods were significantly different between the two groups: blind marmosets showed a 23.2-hour period while normal marmosets free-ran showed a period of 23.6 hours. This shorter period in blind marmosets could be explained by their lower activity and, consequently, decreased motor activity feedback to the circadian system, or by the participation of classical photoreceptors (rods and cones) in the generation of free-running circadian rhythms. However, it could also be explained by the suggestion that the loss of rods and cones has an impact on the nature of light information reaching the SCN [8].
For photic entrainment to occur, the circadian oscillator must respond differently to light at different phases of its cycle. Phase response curves (PRC) are useful descriptions of these phase-dependent responses. A number of non-photic stimuli, both pharmacological and non-pharmacological, have been identified as able to induce phase shifts in mammalian circadian clocks as a function of the circadian phase that the stimulus is presented [14]. The PRC of non-photic stimuli (including dark pulses presented to animals kept in constant light) is 180° out of phase with photic stimuli. We tested the phase-shift response of blind marmosets using dark pulses of 2-hour duration. When the dark pulse was given in the early subjective day, it caused a phase delay; when given in the late subjective day, it caused a phase advance. This result is an important contribution to the discussion about the non-photic phase shift in this species. Our results agree with Glass et al [14], in whose studies the qualitative similarities between the phase responses to entraining photic and non-photic stimuli in marmosets and nocturnal mammals were demonstrated.
Many of the non-photic stimuli that induce phase shifts in the circadian clocks also induce an acute increase in locomotor activity in nocturnal mammals, and it appears that at least some of the phase shifting effects of these agents is due to the induction of activity and/or arousal [15]. In the present study in marmosets, the phase shifts produced by dark pulses were not due to the inhibition of activity. The dark pulses produced an inhibition of activity (negative masking) in the sighted marmosets but not in the blind ones, despite the fact that both groups showed phase shifts with dark pulses.
The response of the circadian system to different stimuli, photic and non-photic, is of great importance because implies that circadian systems are in fact able to use many sources of information. As the marmoset is a social animal, we also investigated social synchronization between these animals. Blind marmosets showed different activity onset during the free-running phase, but they showed a stable phase angle. The two normal marmosets showed the same behavior but with different free-running periods from the blind marmosets. Therefore, despite the fact that the four animals were in the same room, the blind marmosets were not synchronized with the normal ones.
One limitation of this study is the small number of animals, but the results of the two normal marmosets are similar to other studies that were conducted in our laboratory [16]. Considering studies previously carried out in rodents along with our present results, it is possible to infer that the blind marmosets had normal retinal ganglion cells, which are required to synchronize their circadian clocks to the LD cycle. In the absence of classical photoreceptors, photosensitive ganglion cells are sufficient for photic entrainment [17].
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