Entrainment to the light-dark cycle

As described above (see 1.2) photic information is perceived by visual and nonvisual photoreceptors of the retina and passed on to the SCN via the retinohypothalamic tract (RHT). However, since the circadian system responds to photic signals of much higher intensities and durations than the visual system [158], [159], as well as despite visual blindness (loss of rod and cone photoreceptors) [160], [161], entrainment stimuli seem to differ from photic information conveying visual light perception. Melanopsin expressing intrinsically photosensitive retinal ganglion cells (ipRGC) have shown to project to the SCN, the intergeniculate leaflet (IGL), and the olivary pretectal nucleus (OPN) [162]. However, neither loss of rods and cones, nor of ipRGCs photoreceptors alone [163] abolishes circadian entrainment to light, indicating that all of these cell

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types are involved in passing on photic information to the SCN. Nevertheless, the importance of the RHT for circadian entrainment has been demonstrated by lesion and electrical stimulation experiments [164], [165]. Innervation of SCN core neurons by efferent RHT projections has been shown to result in the induction of the core clock genes Per1/2, as well as phase resetting of the SCN clock (for details see 1.2). Daily resetting of the SCN is transmitted to peripheral circadian clocks in order to entrain the entire organism to the environmental light-dark cycle. Interestingly, light induced phase resetting by transcriptional activation is almost immediate in SCN core neurons, while changes in shell neurons follow gradually, generating “phase waves” during SCN entrainment [166].

Historically, two concepts of (photic) entrainment have been developed: (i) non-parametric entrainment due to daily phase shifts induced by light-dark transitions, as well as (ii) parametric entrainment due to de- or acceleration of the circadian clock period induced by sustained light exposure [167]. Nevertheless, ultimately changes of the circadian period will result in phase changes of the circadian cycle. Therefore, both, parametric and non-parametric entrainment describe how phase changes serve to adapt the free-running circadian period to the Zeitgeber period and establish phase coherence between internal and external cycles. Non-parametric entrainment can be described by so-called phase response curves (PRC), defined by times at which single Zeitgeber pulses induce phase delays, phase advances, or no phase change (also referred to as “dead-zone”). Parametric entrainment on the other hand is described by velocity response curves (VRC), which can be estimated from the PRC [22]. Today, phase response curves exist for a multitude of Zeitgebers in various species, model organisms, tissues or even cell lines (PRC Atlas:

https://as.vanderbilt.edu/johnsonlab/prcatlas/). PRCs are graphical representations of phase shifts in response to Zeitgeber stimuli as a function of when the stimulus was given (can be circadian time, Zeitgeber time or similar) (Figure 1-6). Thus, they are defined by unique shapes and amplitudes that help to deduce information about temporal gating of the Zeitgeber responses, i.e. how oscillators respond to the same signal at different times of the day, as well as about underlying mechanisms of phase adjustments. Photic PRCs are commonly characterized by phase shifts during the subjective night (CT12-24), i.e. the part of the circadian cycle under constant conditions, which corresponds to night in the light-dark cycle [168]. Oppositely,

non-photic PRCs are often characterized by phase shifts during the subjective day (CT0-12), i.e. the part of the circadian cycle under constant conditions, which corresponds to day in the light-dark cycle. For example, responses to forced activity cycles or social interaction elicit such non-photic PRC profiles [169]. In addition to the kind of Zeitgeber, PRCs can be distinguished by the magnitude of phase shifts induced by a Zeitgeber stimulus. While type-0 PRCs are characterized by large phase responses (≥ 12 hours) resulting in abrupt switches between delaying and advancing shifts (the “break point”), type-1 PRCs are characterized by smaller phase shifts (< 6 hours) and gradual transitions between delays and advances (Figure 1-6) [170]. An alternative way of plotting phase responses to Zeitgeber stimuli are so-called phase transition curves (PTC), a plot of circadian phase prior to a stimulus versus circadian phase following a stimulus. Thus, type-0 and type-1 PRCs are derived from the slopes of such PTCs: if a Zeitgeber always resets the oscillator to the same phase, the slope of the PTC will be zero; it will be around 1 if the stimulus shifts the oscillator by a certain amount and in a time-dependent manner [171]. Whether type-0 or type-1 phase responses are induced usually depends on the strength of the Zeitgeber stimulus but can also be influenced by intercellular coupling among single cell oscillators within a network.

Consistent with theoretical concepts of entrainment, intercellular coupling has been shown to regulate Zeitgeber responses by altering oscillator amplitudes, as well as amplitude relaxation rates [57], [172].

Figure 1-6: Types of phase response curves (PRCs)

In type-1 PRCs (solid line) small phase responses are occurring during the subjective night: phase delays at the early and phase advances at the late subjective night gradually transition from one to

another. Type-0 PRCs (dashed line) are characterized by large phase responses leading to a point of break point between phase delaying and phase advancing portion. Times at which no phase changes in response to a stimulus are occurring are referred to as dead zone and occur during the subjective day for photic PRCs. (adapted from [96])

Photic phase responses in mammals

For the mammalian circadian system light is the most dominant external Zeitgeber.

Phase responses to light stimuli are characterized by type-1 photic PRCs. As described above (see 1.2), Per1 and Per2 induction appear to be the underlying mechanism of photic phase adjustment. Therefore, Per1/2 phase of expression has been related to the magnitude of light induced phase changes. Indeed, this is true for nocturnal rodents [96]. During the subjective day Per1/2 expression is high, resulting in a dead-zone of light induced phase shifts of locomotor activity rhythms. During the early subjective night Per1/2 expression is declining, resulting in phase delays. During the late subjective night Per1/2 expression is inclining again, resulting in phase advances (Figure 1-5). Interestingly, even though diurnal mammals display a reversal in their activity pattern, phase responses to light stimuli resembles that of nocturnal mammals with regard to clock gene expression and locomotor activity [173]–[175].

Even today it is not clear which mechanisms, downstream of photic resetting, regulate the switch from nocturnality to diurnality but differences at the level of SCN output pathways have been suggested [176]–[178].

In mammals, immediate early induction of Per1 expression by light stimulation and phase shifts of locomotor activity have been shown to correlate, with rapid (~0.5-1 hour) and strong responses at CT12-CT20 (subjective night) [179]. In the case of Per2, response to photic stimuli is more variable, with slower increases (~1.5-3 hours) in gene expression after light pulses given between CT12-CT16 (early subjective night) [94], [180]. Expression of Per3, a third Period homologue, has been shown to remain unaltered in response to light pulses during the subjective night [181]. Due to the distinct transcriptional responses of Per1 and Per2 to light stimuli, it has been proposed that Per1 functions as primary target of photic entrainment in the SCN, while Per2 induction may be mediated by secondarily effects, e.g. transcriptional activity of the immediate early expressed genes c-Fos and c-Jun [94], [180]. Additionally, lack of Per3 induction by photic stimuli has strengthened the idea that the three Period homologues fulfill tissue-specific functions in Zeitgeber induced phase resetting and circadian time keeping [181], [182]. Moreover, even though time-of-day differences in

Per1 and Per2 expression have been associated with dualistic responses (advance versus delay) of the SCN to light pulses during the subjective day and the subjective night, precise mechanisms remain unclear. Induction of other clock components, e.g.

Clock at CT10 [183], spatiotemporal patterns of synchronization between light-sensing SCN core SCN and shell neurons [166], as well as “gating” of light responsiveness in the SCN [184] have been proposed as alternative mechanism.