We first asked whether Rai1 plays a role in circadian clock function and entrainment by assessing patterns of spontaneous locomotor activity both under standard light-dark conditions (LD12:12) and constant dark (DD) conditions. Under LD12:12 conditions, mice of both genotypes were more active during the dark compared to the light period, reaching highest activity levels in the first 6 hr after dark onset (ZT12-18; Figure 1A,B), consistent with the mouse being a nocturnal species. Despite this overall similarity of the distribution of locomotor activity over the 24 day, we observed a number of striking differences between the two genotypes. Whereas during the light period Rai1^+/+ mice displayed occasional activity bouts, especially immediately preceding dark onset and following light onset, activity in Rai1^+/- mice was restricted to the dark with an abrupt increase in activity immediately following dark onset and an equally abrupt suppression of activity after light onset, leaving the light period almost devoid of any activity (Figure 1A,C). The number of movements during the light phase was reduced by 67% in Rai1^+/- mice, which is substantial considering that in the wild-type littermate controls locomotor activity reaches its lowest levels during this period (Figure 1B).

Figure 1 with 1 supplement see all

Download asset Open asset

Figure 1—source data 1

Excel file with data sheets corresponding to each figure panel containing the numerical values on which Figure 1 and Figure 1—figure supplement 1 are based.

For Figure 1 panels D-F and Figure 1—figure supplement 1 individual data are provided as well.

https://doi.org/10.7554/eLife.23292.003

Download elife-23292-fig1-data1-v3.xlsx

The overall 24 hr activity levels (both under LD and DD) were lower in Rai1^+/- mice compared to wild-type (Rai1^+/+ vs. Rai1^+/-: LD12:12: 545 ± 79 vs. 317 ± 36; p=0.019; DD: 473 ± 77 vs. 268 ± 35 movements/24 hr; p=0.006, t-tests). These lower overall activity levels could not, however, explain the extremely low activity levels observed during the light period in Rai1^+/- mice. Even after taking these overall genotype differences into account by expressing activity during the light as a percentage of each animal’s individual average 24 hr activity level, activity in Rai1^+/- mice remained greatly reduced compared to wild-type littermates (p=0.022, t-test; Figure 1D, see also Figure 5—figure supplement 2). Activity during the light phase was also lower when compared to the average activity levels reached in the rest phase (rho) under DD conditions in Rai1^+/- mice (p=0.007, paired t-test; Figure 1D) but not in Rai1^+/+ mice. Moreover, relative activity levels (again expressed as % of each animal’s 24 hr activity under LD12:12) during rho under DD, did no longer differ between genotypes (Figure 1B,D; p=0.3, t-test), constituting further evidence that the loss of one Rai1 allele leads to a marked suppression of activity during light exposure.

The suppression of activity by light depends on its intensity (Aschoff, 1960) and the extreme suppression by light observed in Rai1^+/- mice might occur at higher light intensities only. We therefore exposed a subset of mice to a low light intensity (i.e., 0.6 cds/m²) compared to our habitual 6.6 cds/m². Although at these lower light levels Rai1^+/- mice were more active compared to the higher light level condition, relative locomotor activity during the 12 hr light phase (as % of each animal’s 24 hr activity under LD12:12 at 6.6 cdm/m²) was still greatly suppressed in Rai1^+/- mice compared to their wild-type littermates (p<0.05; t-test; Figure 1—figure supplement 1A). Interestingly, activity levels of wild-type mice during the 12 hr light period did not differ between the two light intensities.

After release into DD conditions, Rai1^+/- mice maintained a circadian organization of activity and rhythms free-ran with a period length slightly shorter than 24 hr as in wild-type mice (23.8 ± 0.03 hr for both genotypes; p=0.82; t-test). In addition, no significant difference in the robustness of the circadian distribution of locomotor activity was observed during free-run (maximum values of the X²-statistic; Rai1^+/+ vs. Rai1^+/-: 1458 ± 242 vs. 976 ± 84; p=0.086; t-test), while analyses of the day-to-day variability of activity onset under these conditions (i.e., individual r-values for the linear regression of activity onset under DD) suggest a more precise circadian timing of overt behaviors in Rai1^+/- compared to wild-type littermates (r = 0.96 ± 0.01 and 0.88 ± 0.03, respectively; p<0.05; t-test; see Materials and methods for details). Also, the phase angle of entrainment, which quantifies the relationship between the endogenously generated circadian timing of activity and the timing of light exposure (see Materials and methods for details), differed between the two genotypes. While activity onset in wild-type mice under DD conditions occurred later than predicted by the mean activity onset under the preceding LD12:12 (−0.49 ± 0.17 hr; p<0.005; paired t-test), Rai1^+/- mice exhibited a positive phase angle (+0.36 ± 0.14 hr; p<0.05; paired t-test), which significantly differed from wild-type (p<0.005; t-test). This relative advancement of activity onset in Rai1^+/- mice was accompanied by an abrupt, 2.5 hr ‘expansion’ of the active phase (alpha), while in Rai1^+/+ mice alpha did not significantly change from that observed under LD12:12 (+18 min; Figure 1A,E). The alpha expansion was already present on the first day under DD and was due to both an advance of activity onset (consistent with the positive phase angle of entrainment) and a delay of activity offset compared to the timing of these two events on the preceding day under LD12:12 (Figure 1A). These observations indicate that besides the altered light-suppression of activity (or ‘masking’ as it is referred to in the circadian literature), circadian entrainment by light is also affected in Rai1^+/- mice. Even at low intensity (i.e., 0.6 cds/m² vs. the habitual 6.6 cds/m²), light was still able to confine activity to the dark period of an LD12:12 light-dark cycle and alpha did not increase in Rai1^+/- mice (Figure 1—figure supplement 1B).

To further investigate photic entrainment, we assessed the capacity of single, 1 hr light pulses in shifting the phase of activity onset under DD conditions. Light pulses were given at either CT16 or −22, when C57BL/6 mice show maximum phase delays and advances, respectively (Schwartz and Zimmerman, 1990; Daan and Pittendrigh, 1976). Although both light pulses resulted in the expected change in activity onset of the free-running activity rhythm, the magnitude of the phase advance after light pulses administrated at CT22, was more than two times larger in Rai1^+/- mice than in wild-type (Figure 1F).

Because one of the hallmarks of SMS is disturbed sleep, we next investigated the role of Rai1 in sleep-wake regulation by recording EEG and EMG signals, concomitant with locomotor activity under various lighting conditions.

Time course analyses under LD12:12 revealed that Rai1^+/- and Rai1^+/+ mice showed very similar distributions of sleep and wakefulness over the 24 h day (Figure 2A; see also Figure 6A) and for none of the three sleep-wake states (i.e., waking, NREM sleep, and REM sleep) did the 24 hr values significantly differ (Figure 2—source data 1). Nonetheless, significant differences were observed between genotypes in the sleep-wake distribution. Rai1^+/- mice displayed more wakefulness (and less NREM sleep) immediately after dark onset and before light onset. Moreover, during the light period, time-spent-awake was slightly but significantly reduced compared to wild-type littermates (Figure 2B). Even so, the lower overall activity levels as well as the near complete suppression of locomotor activity by light observed in Rai1^+/- mice, were not accompanied by similar changes in time-spent-awake. This dissociation resulted in a striking 15-fold reduction in locomotor activity when expressed per unit of time-spent-awake during the light period (Figure 2C). Behavioral observation using video recordings confirmed that when Rai1^+/- mice were behaviorally awake during the light period they did not leave the nest or only very briefly to drink. Because overall activity levels, but not waking (Figure 2B), were reduced also in the dark period, activity expressed relative to time-spent-awake was also reduced during this period albeit to a much smaller degree (twofold; Figure 2C). Thus, Rai1-haploinsufficiency, while suppressing gross body movements during light exposure, leaves general wakefulness largely unaffected.

Figure 2

Download asset Open asset

Figure 2—source data 1

Twenty-four hour values for total wakefulness, theta-dominated wakefulness (TDW), NREM sleep, and REM sleep under LD12:12 conditions.

n = 4/genotype.

https://doi.org/10.7554/eLife.23292.006

Download elife-23292-fig2-data1-v3.docx

Figure 2—source data 2

Excel file with data sheets corresponding to each figure panel containing the numerical values on which the Figure 2 graphs are based.

For all panels individual data are provided as well.

https://doi.org/10.7554/eLife.23292.007

Download elife-23292-fig2-data2-v3.xlsx

We then verified whether the immobility in Rai1^+/- mice was accompanied by altered brain activity during wakefulness. Quantitative analyses of the waking EEG revealed increased activity within the delta band (2–3 Hz) in Rai1^+/- mice compared to Rai1^+/+ mice during the light period but not during the dark period (Figure 3A). This contrast between light and dark could result from differences in the relative contribution of distinct waking behaviors to the overall waking spectra, specifically theta-dominated wakefulness (TDW; [Welsh et al., 1985; Vassalli and Franken, 2017]) which is considered an active state of wakefulness associated with exploratory behavior and locomotor activity (Buzsáki, 2002) (see Materials and methods for the quantification of TDW). Accordingly, time spent in TDW in Rai1^+/- mice was reduced 2.3-fold in the light period compared to that in wild-type mice (Figure 2B), also relative to time-spent-awake (1.9-fold; Figure 2C). Thus, in the presence of light, the lack of one Rai1 allele specifically suppresses active wake behaviors, while leaving total time-spent-awake unaffected.

Figure 3

Download asset Open asset

The EEG spectra during TDW showed the expected theta peak and no differences between genotypes, while for the EEG spectra in the remainder of wakefulness ('quiet' wakefulness or ‘non-TDW’), we again observed an increase in EEG power density in the 2–3 Hz range (Figure 3A). This indicates that the reduced contribution of TDW to total wakefulness in Rai1^+/- mice did not underlie this EEG difference. Moreover, the observed increase in the 2–3 Hz range of the EEG power density appeared strictly light driven and not a consequence of for example circadian time, because it was equally observed under LD1:1 conditions when light is presented at all circadian phases (see below and Figure 3C). The increase in delta activity in the waking EEG might suggest that when in non-TDW and exposed to light, Rai1^+/- mice are drowsier. Delta activity in the waking EEG did not only differ between genotypes but was generally higher in the 12 hr light period than in the dark period (2–5 Hz), irrespective of waking state (wakefulness, TDW, and non-TDW) and genotype (Figure 3B). However, only in non-TDW was this light-dark delta power difference larger in Rai1^+/- mice than in Rai1^+/+ mice (Figure 3B), suggesting that Rai1^+/- mice are also more sensitive to light at the level of the EEG. This analysis also revealed that EEG activity in the high-beta / low-gamma activity range (25–40 Hz) was increased in the light versus the dark period and that this increase was again larger in Rai1^+/- mice (Figure 3B).

We next assessed whether the suppressing effect of light on locomotor activity and TDW could be sustained beyond 12 hr. When Rai1^+/- mice were submitted to 60 hr of constant light (LL for 48 hr starting at the end of a ‘habitual’ 12 hr light period), mean activity levels remained continuously suppressed without marked circadian modulation (Figure 4A). Overall activity was reduced to one-fourth of the 24 hr levels under LD12:12 (Figure 4B). This is in stark contrast to wild-type mice in which the circadian modulation of locomotor activity was preserved under LL and overall levels did not significantly differ from LD12:12 (Figure 4A,B). Interestingly, in neither genotype was the overall time-spent-awake changed under LL (Figure 4B). The sustained and important reduction in locomotor activity while leaving wakefulness largely unaffected again attests to the clear dissociation between these behaviors when one Rai1 allele is missing. Although total wakefulness was not affected by LL, TDW was reduced by one third in Rai1 heterozygous mice compared to LD12:12, and unchanged in their wild-type littermates (Figure 4A,B). Although under LL a clear circadian modulation in wakefulness, TDW, and (in wild-type mice only) locomotor activity could still be discerned, the onset of the first wake/active phase was delayed more relative to that under LD12:12 in Rai1^+/- mice compared to wild-type (6.9 ± 0.7 vs. 2.2 ± 0.5 hr, respectively; p<0.0001, t-test).

Figure 4

Download asset Open asset

Figure 4—source data 1

Excel file with data sheets corresponding to figure panels A and B containing the numerical values on which the Figure 4 graphs are based.

For both panels individual data are provided as well.

https://doi.org/10.7554/eLife.23292.010

Download elife-23292-fig4-data1-v3.xlsx

After having established that light suppression of active wake behaviors can be sustained for at least 60 hr in Rai1^+/- mice, we assessed the acute effects of light. To further investigate the acute effects of light identified under LD12:12 (Figure 1C), we implemented a 48 hr LD1:1 light-dark protocol which, in addition, allowed us to determine whether light suppression of activity was modulated by circadian time. In Rai1^+/- mice, activity levels were rapidly suppressed and reached near zero levels during each of the repeated 1-hr-light pulses, irrespective of time-of-day (Figure 5A,B), giving the changes in activity a distinct ‘on-off’ or ‘switch-like’ character (Figure 5A, Figure 5—figure supplement 1). In contrast, while wild-type mice were also less active during the light pulses, their activity levels remained well above zero and still varied according to circadian time with highest values reached in the subjective dark phase (Figure 5A,B). Indeed, in contrast to wild-type mice, activity levels reached during the light pulses in Rai1^+/- mice were, on average, similar to the activity (or lack thereof) they exhibited during the light phase under LD12:12 (Figure 5—figure supplement 2).

Figure 5 with 2 supplements see all

Download asset Open asset

Figure 5—source data 1

Excel file with data sheets corresponding to figure panels A and B containing the numerical values on which the Figure 4 graphs are based.

For both panels individual data are provided as well.

https://doi.org/10.7554/eLife.23292.012

Download elife-23292-fig5-data1-v3.xlsx

During 1-hr-dark pulses, activity levels varied according to circadian time in both genotypes, but were generally lower in Rai1^+/- mice than in Rai1^+/+ mice (Figure 5A), consistent with the overall lower activity levels observed during LD12:12. When expressed relative to each animal’s mean activity level observed under LD12:12 conditions, Rai1^+/- mice reached higher values during the 1-hr-dark pulses compared to wild-type (Figure 5B), suggesting that the acute activating effect of a dark pulse was also amplified in Rai1^+/- mice, which is consistent with the pronounced increase in activity at the light-to-dark transitions under LD12:12 (Figure 1C).

Effects of light, time-of-day, and genotype on TDW largely paralleled the effects on activity, although values did not differ between genotypes during the dark pulses (Figure 5A,B). Also, wakefulness was suppressed by the light pulses and this suppression was stronger in Rai1^+/- mice compared to wild-type (Figure 5B). This markedly differs from the lack of suppression of wakefulness by light under LD12:12 and LL, demonstrating that the direct effects of light differ between acute and sustained light administration. Evidence of a stronger, acute activation by the dark pulses was also observed for wakefulness (Figure 5B), which in combination with the stronger acute suppression by light resulted in a larger amplitude of change in Rai^+/- mice between light and dark pulses, resulting in a distinct saw-tooth pattern (Figure 5A).

Next, we verified whether genotype-driven differences in the homeostatic aspect of sleep regulation could have contributed to the differences in wake behavior. We therefore challenged mice of both genotypes with a 6-hr sleep deprivation during the first half of the light period (ZT0-6) and compared their compensatory sleep responses. Compared to baseline levels, mice of both genotypes spent more time asleep during the 18 hr for which recovery sleep was monitored and this extra time asleep did not differ between genotypes (Figure 6A,B). Similarly, the dynamics of EEG delta power over the 72 hr recording period, including its levels immediately after the sleep deprivation, did not differ between genotypes (Figure 6A). EEG delta power during NREM sleep quantifies the amplitude and prevalence of delta oscillations (1–4 Hz) and is thought to reflect a sleep homeostatic process in both humans and mice (Daan et al., 1984; Franken et al., 2001). In sum, at least in the mouse, altered sleep homeostasis does not seem to contribute to the sleep-wake phenotypes associated with Rai1 haplo-insufficiency.

Figure 6

Download asset Open asset

Rai1 is widely expressed throughout the brain with high levels of expression observed in the olfactory bulb, cerebral cortex, hippocampus, striatum, thalamus, hypothalamus, cerebellum, and brainstem (Toulouse et al., 2003; Bi et al., 2007; Fragoso et al., 2015; Huang et al., 2016). Because circadian and/or light-dependent changes in Rai1 expression in the brain may contribute to the phenotypes we observed in Rai1-haploinsufficient mice, we sampled brain tissue under LD12:12 conditions at 3-hr intervals in wild-type mice. Rai1 expression levels in the forebrain were remarkably stable over the day, in contrast to the well-known nychthemeral changes in steady state mRNA levels of the established core circadian clock genes Bmal1 and Per2, which were quantified in the same samples (Figure 7). A database search confirmed that Rai1 is present also in the SCN and that its expression does not change with time-of-day (CircaDB; http://circadb.hogeneschlab.org/).

Figure 7

Download asset Open asset

In a further step to dissect the pathways by which locomotor activity was so strongly suppressed in Rai1^+/- mice, we quantified the retinal response to light pulses in dark-adapted mice. Scotopic ERG recordings revealed that retinal activation was importantly reduced, as evidenced by a decreased amplitude of the a-wave, reflecting the membrane hyperpolarization of the photoreceptors (Penn and Hagins, 1969), and the b-wave , reflecting the light response of retinal cells post-synaptic to photoreceptors (Stockton and Slaughter, 1989; Miller and Dowling, 1970) (Figure 8A,B). Together, these two effects led to a marked flattening of the ERG response in Rai1^+/- mice. Since the b-wave is a consequence of the activity of the components measured by the a-wave (photoreceptors), we cannot conclude whether the decreased b-wave is exclusively due to altered photoreceptor function or also to intrinsic interneuron defects.

Figure 8

Download asset Open asset

Figure 8—source data 1

Excel file with two data sheets corresponding to Figure 8 panel B a-wave and b-wave values, respectively.

For both panels individual data are provided.

https://doi.org/10.7554/eLife.23292.018

Download elife-23292-fig8-data1-v3.xlsx

The general retinal morphology and histology analysis did not show any salient differences between Rai1^+/+ and Rai1^+/- mice (Figure 9). No decrease in layer thickness of photoreceptors or interneurons was noticed that might have been indicative of retinal degeneration explaining the reduced ERG response to light. The lack of a difference in the rod marker rhodopsin, the cone marker GNAT2, the bipolar ON marker PKCalpha, and the active Müller glial marker GFAP, further supported that retinal composition was not noticeably impacted (Figure 9A–H). Moreover, the normal dendritic ends of Rai1^+/- bipolar cells, labeled by PKCalpha, suggest that the connections between photoreceptors and bipolar cells were not affected, as well as the general circuitry as shown by the calbindin, ChAT, and GABA labeling of the horizontal and/or amacrine cells and the different strata of connections in the inner plexiform layer (Figure 9I–N). Consistent with the histological results, qPCR analyses of retinal extracts did not reveal any difference in rhodopsin, melanopsin, and cone opsin mRNA levels between Rai^+/- and Rai^+/+ mice (Figure 9O). We did, however, observe a twofold decrease in Rai1 expression in Rai1^+/- mice, as could be expected from the haplo-insufficiency (Figure 9O). Taken together, these experiments did not reveal any anatomical or molecular factors contributing to the reduced ERG response.

Figure 9

Download asset Open asset

Next, we verified whether a light pulse in Rai1^+/- mice is also less efficient in activating the SCN by quantifying c-FOS immunoreactivity. In line with previous work (e.g. [Ruby et al., 2002]), a 1-hr-light pulse administered at ZT15-induced c-FOS immunoreactivity in the SCN of both genotypes (Figure 10B,C; p<0.0001, t-tests). The induction was, however, significantly reduced by ~40% in Rai1^+/- mice (Figure 10B,C; p<0.005, t-test). Although the decreased retinal activation upon a light pulse is consistent with the reduced downstream response in the SCN, both observations seem to contradict the acute and sustained behavioral hypersensitivity to light we observed in Rai1^+/- mice. This profound reduction in the activation of the SCN seems also at odds with the lack of a difference between the two genotypes in the ensuing phase delay of overt behavior after a light pulse (Figure 1F). However, at the level of the ventral subparaventricular zone (vSPVZ), a region immediately dorsal to the SCN, the opposite result was observed. The SPVZ extends dorsally of the SCN, from bregma −0.71 mm to bregma −0.83 mm (Paxinos and Franklin, 2013) and we focused on its ventral part (Figure 10A) because neuronal activity in this area was found to be in phase with locomotor activity rhythm (Todd et al., 2012) and because stimulation of this area inhibits locomotor activity specifically (Kramer et al., 2001). We found that compared to mice that did not receive a light pulse, light induced a significant increase in c-FOS immunoreactivity only in Rai1^+/- mice (p<0.05; t-test) and that the number of c-FOS-positive cells after the light pulse was higher in Rai1^+/- mice compared to their wild-type littermates (p<0.05, t-test; Figure 10C). The differential activation by light of the frontal vSPVZ in Rai1^+/- mice could thus represent the neuronal substrate underlying light’s marked suppression of active waking behaviors (Figure 11).

Figure 10

Download asset Open asset

Figure 10—source data 1

Excel file with two data sheets corresponding to Figure 10 panel C SCN and vSPVZ number of cFOS-positive cells, respectively.

For both structures individual data are provided.

https://doi.org/10.7554/eLife.23292.021

Download elife-23292-fig10-data1-v3.xlsx

Figure 11

Download asset Open asset

Our data revealed that Rai1 haplo-insufficiency in the mouse resulted in both circadian and light-induced phenotypes while leaving the homeostatic regulation of sleep unaffected. The most striking effect of the lack of one Rai1 allele was the all but complete abolishment of locomotor activity by light. The overall time-spent-awake and its diurnal distribution under standard LD12:12 conditions did not greatly differ between the two genotypes, underscoring that locomotor activity in the mouse can be a poor indicator of sleep-wake state.

Although overall wakefulness was little affected, the profound suppression by light of locomotor activity was accompanied by a number of changes in wake-related phenotypes indicating reduced alertness and attention while awake during the light in Rai1^+/- mice compared to their wild-type littermates. These include a reduction in time spent in TDW, a sub-state of wakefulness rich in EEG theta activity and associated with active, exploratory, and goal-oriented behavioral repertoires (Buzsáki, 2002), and an increase in delta activity in the waking EEG during the light. The latter observation suggests that Rai1^+/- mice are less alert and more drowsy when awake in the light, and is consistent with reports of delta activity increasing progressively during prolonged waking periods in human and rat, which in human is paralleled by decrements in cognitive performance (Cajochen et al., 2002; Franken et al., 1991; Dijk et al., 1992). Also, the high beta/low gamma frequency band was more strongly activated by light in Rai1^+/- mice than in wild-type. This frequency range also increases progressively during prolonged waking in both human and mouse (Cajochen et al., 2002; Grønli et al., 2016) and has been associated with behavioral arrest and slower movements (Zheng et al., 2015), as well as with the motor deficits accompanying Parkinson disease (Weinberger et al., 2009; Mallet et al., 2008), suggesting that processes related to motor control are reflected in the waking EEG and might be relevant also for the light-induced suppression of locomotor activity in the SMS model.

Results from the LD12:12, LD1:1, and LL experiments also allowed us to differentiate between sustained and acute direct effects of light on sleep-wake state. In the context of the current set of experiments, we refer to acute effects as those light effects that can be observed immediately after light onset; that is, within the first hour, while sustained refers to effects of longer light exposures; that is, 12 hr and longer. Results from the LD12:12 and LL experiments revealed that genotype did not alter the effects of sustained light exposure on total time-spent-awake (and -asleep), whereas the LD1:1 experiment provided evidence that the acute suppressing effects of light on time-spent-wake time were stronger in Rai1^+/- mice, as was the case for locomotor activity and TDW. In contrast to the almost complete suppression of locomotor activity and TDW during LD1:1, the effect on total time-spent-awake was weaker and depended on circadian time, with highest levels of wakefulness attained (or lowest suppression by light) when light pulses were given during the subjective dark phase. This suggests that the sleep-wake switching by light competes with the alerting signal provided by the circadian system, as it is more difficult for light to acutely switch sleep-wake state than to rapidly alter the quality of one state (e.g. active vs. quiet wakefulness).

In addition, the LD1:1 protocol revealed a stronger induction of wakefulness during the 1-hr-dark pulses in Rai1^+/- mice compared to wild-type. Also this acute, wake-promoting effect of the absence of light varied with circadian time and was largest at the start of the subjective dark phase. In comparison with the temporary overshoot in wakefulness and activity at the same time under LD12:12, which could be interpreted as ‘rebound’ from the suppression in the preceding 12 hr of light, the LD1:1 results instead suggests a circadian mechanism that could relate to the longer circadian active period observed in Rai1^+/- mice under DD conditions.

In the present study, circadian organization of locomotor activity under DD was not largely impacted by Rai1 haplo-insufficiency, and no differences in period length were observed. Our results contrast with those from one earlier study in which a 13 min shortening of the free-running period was reported (Lacaria et al., 2013). Despite this difference, both studies clearly demonstrate that circadian rhythm generation under DD is not compromised in Rai1^+/- mice. This is surprising given that Rai1 haplo-insufficiency results in the transcriptional dysregulation of core clock genes such as Clock, Per2, Per3, Cry1, and Bmal1 in SMS fibroblasts and in mouse hypothalamic tissue (Williams et al., 2012). Such extensive disruption of the core clock machinery could have been expected to impact the circadian organization of overt behaviors in vivo. If anything, our results show that circadian timing under constant dark conditions was more accurate in Rai1^+/- mice than in wild-type mice.

We did, however, identify two circadian phenotypes related to entrainment. Rai1^+/- mice showed a pronounced lengthening of the active phase (alpha) when released into DD. This expansion of alpha was due to both an earlier onset and a later cessation of activity compared to the timing of these two events under the preceding LD12:12 conditions. Given their hypersensitivity to light, the preferred (i.e. endogenously determined by the circadian system) earlier activity onset in Rai1^+/- mice seems thus ‘masked’ by light even of low intensity. Evidence of masking of activity onset in Rai1^+/- mice can also be deduced from the lack of transients during re-entrainment to LD12:12 after a period of DD (Figure 1A). The second and related circadian phenotype observed in Rai1^+/- mice is the twofold larger phase advance. We did not construct a full ‘phase response curve’ (PRC) but assuming a still larger phase advance 2 hr later (i.e. at light onset under entrained conditions) might have contributed to the earlier preferred activity onset in Rai1^+/- mice and also to the compression of alpha under entrained, LD12:12 conditions (compared to DD). Alpha compression during re-entrainment is accompanied by memory impairments (Ruby et al., 2015) and, assuming that in Rai1^+/- mice alpha is being compressed by 2.5 hr on a daily basis under LD12:12 conditions, might also have contributed to cognitive deficits reported for Rai1-compromised mice kept under these same lighting conditions (Bi et al., 2007; Huang et al., 2016). It is therefore of interest to verify whether memory deficits persist under LD regimens with shorter than 12 hr photoperiods better matching the longer endogenous active period observed in Rai1^+/- mice.

The sleep-wake disturbances, including excessive daytime sleepiness and attention deficits (Smith et al., 1986), together with the inverted melatonin rhythm observed in SMS patients (De Leersnyder, 2006; Potocki et al., 2000), strongly suggest impaired non-visual light perception. The reduced response to light at the level of the retina and the SCN we report here for Rai1^+/- mice, supports this idea and indicates that already at the input level, processing of light information is altered by the loss of one Rai1 allele. However, at the behavioral output level, the murine SMS model clearly differs from SMS patients because mice display a striking hypersensitivity to light with respect to active waking behaviors. These findings seem contradictory not only with regard to the human-mouse species difference, but also concerning the differences in the effects of light at the input level and its downstream behavioral consequences in our SMS mouse model. This contradiction might relate to the mechanisms that determine nocturnality versus diurnality which are thought to act downstream from the SCN.

Based on the neuroanatomical differences in the innervation of the vSPVZ by the SCN and the retina between nocturnal and diurnal rodents, Todd and colleagues Todd et al., 2012 proposed that in diurnal mammals light information reaches the vSPVZ predominantly through the SCN relay, which inhibits the vSPVZ and is thus alerting. If also true in humans, a weaker inhibition of the vSPVZ by the SCN might contribute to the lower levels of alertness and attention, as well as increased day-time sleepiness observed in SMS during the day (Figure 11C,D) (Elsea and Girirajan, 2008). In nocturnal species, light information encoded in the retina directly stimulates the vSPVZ via the retinohypothalamic tract (RHT) and, overcoming the simultaneous inhibition provided by the SCN, causes inactivity and sleep (Todd et al., 2012; Schwartz et al., 2004; Watts et al., 1987; Colwell, 2011; Hermes et al., 2009) (summarized in the Figure 11 schematic). According to this scenario, the reduced activation of the SCN observed in our SMS mouse model might have led to a reduced inhibition of vSPVZ neurons, which, in turn, explains both the stronger induction of vSPVZ c-FOS immunoreactivity we observed, as well as the exacerbated suppression of locomotor activity by light (Figure 11A,B). In the intact mouse, RAI1-dependend signaling thus seems to limit the light-induced activation of the vSPVZ thereby allowing animals to display activity during the light phase.

Much in the above scenario remains, however, to be demonstrated and species differences rather than the nocturnal versus diurnal niche differentiation might underlie the discrepancy in light-sensitivity between humans and mice lacking one Rai1 allele. Moreover, a number of other signaling pathways are likely to be involved. Specifically, pathways regulating active waking behaviors might be good alternate candidates. TGFα (transforming growth factor α) activates vSPVZ neurons thereby suppressing locomotor activity without affecting overall time-spent-awake (Kramer et al., 2001; Tournier et al., 2007), reminiscent of the profound and sustained effects of light in Rai1^+/- mice. Likewise, CLC (cardiotrophin-like cytokine) suppresses locomotor activity via CLC receptors on neurons localized in the periventricular region of the hypothalamus (Kraves and Weitz, 2006). Both TGF-α and CLC are produced in the SCN in antiphase with the daily (circadian) changes in locomotor activity. Moreover, CLC production is stimulated by light. As was the case for Rai1^+/- mice, suppression of locomotor activity by CLC is associated with normal waking behaviors although time-spent-awake and -asleep were not quantified (Kraves and Weitz, 2006). Finally, other structures than the vSPVZ have been shown to relay the direct effects of light on behavior such as the intergeniculate leaflet and olivary pretectal nuclei (Gall et al., 2016).

RAI1 is thought to be key to normal brain development and function due to its association with a number of neurobehavioral disorders which, besides SMS, include Potocki–Lupski syndrome (Potocki et al., 2007), schizophrenia (Joober et al., 1999), autism (van der Zwaag et al., 2009), Parkinson disease (Do et al., 2011), and cerebellar ataxia (Hayes et al., 2000). RAI1 is a highly conserved transcriptional regulator (Carmona-Mora et al., 2010) with high neuronal expression (Toulouse et al., 2003; Fragoso et al., 2015), and recent evidence shows that it acts as a positive transcriptional regulator by binding to active promotor and enhancer regions (Huang et al., 2016). Among RAI1 target genes are Bdnf (brain-derived neurotrophic factor) and Htr2c (serotonin receptor 2c) (Huang et al., 2016; Burns et al., 2010), both of which are implicated in hyperphagia leading to obesity, a phenotype accompanying SMS, concomitant with altered locomotor activity (Huang et al., 2016; Burns et al., 2010; Kernie et al., 2000; Nonogaki et al., 2003). Moreover, as its name indicates, Rai1 expression is induced by retinoic acid (Imai et al., 1995). Because retinoic acid signaling is a major pathway involved in eye formation (Cvekl and Wang, 2009) and other developmental processes, and because RAI1 appears to help assemble and maintain neuronal circuitry (Huang et al., 2016), the lack of one Rai1 allele is likely to impact eye development and light signaling pathways, maybe through its target gene Bdnf, which is known to be important for retinal development and synaptic connectivity (Grishanin et al., 2008). While we found no apparent structural differences in the retina, the ERG results did reveal a profound attenuation of the response to light pointing to altered retinal function. A more detailed examination of, for example, the morphology of synaptic terminals and expression levels of other photo-transduction proteins could therefore still yield new insights.

The SMS mouse model revealed that both circadian and direct light reception are affected. Our study thus brought new insights into the light-signaling pathways regulating behavior and suggests that Rai1 is a key regulator in non-visual light perception. Whether these effects are developmental, acute or both, remains to be determined. Given our results, investigating the functional integrity of cells and pathways related to photoreception and light signaling in SMS patients is an obvious next step.

C57BL/6Tyrc−Brd mice heterozygous for the Rai1 null mutation (Rai1^+/-) were generously provided by James R. Lupski (Baylor College of Medicine, Houston TX, USA). The mutation concerns a 3910 bp deletion due to the insertion of a neoR-expression cassette and an Escherichia coli lacZ coding sequence into Rai1 exon 2 (Bi et al., 2005). This mutation prevents the encoded protein from entering the nucleus thus resulting in a functionally inactive allele (Bi et al., 2005). In the vast majority of cases, homozygosity for this mutation results in embryonic lethality. In all experiments, 10- to 12-week-old male Rai1^+/- mice along with their male wild-type littermate controls (Rai1^+/+) were used. Rai1^+/- mice develop obesity by week 20 (Burns et al., 2010), but in the current experiments, they were not heavier than their wild-type littermates (Rai1^+/-: 29 ± 0.7; Rai1^+/+: 31 ± 0.3 g). All animals were kept under a 12 hr light/12 hr dark cycle (LD12:12, fluorescent lights, intensity 6.6 cds/m² or 0.97 µW/cm²) and were singly housed for EEG/EMG and locomotor activity recordings with food and water available ad libitum. Under LD12:12 we use Zeitgeber time (ZT) with ZT0 and ZT12 marking light and dark onset, respectively, while under constant dark (DD) conditions circadian time 12 (CT12) was defined as activity onset. Room temperature was kept at 25°C and humidity between 50% and 60%. All experiments were approved by the Ethical Committee of the State of Vaud Veterinary Office, Switzerland.

Animals were housed individually and placed in recording cabinets equipped with passive infra-red (PIR) sensors (Visonic Ltd, Tel Aviv, Israel). ClockLab (ActiMetrics, IL, USA) was used to record and analyze locomotor activity patterns and to schedule the light-dark regimens. Data were sampled at 5-min resolution, and period length (X2-periodogram) and onset of the main daily activity phase were extracted to calculate the duration of the active (alpha or α) and rest (rho or ρ) phase under DD conditions. Rai1^+/+ (n = 11) and Rai1^+/- mice (n = 12; recorded in five separate experimental cohorts from a total of eight different litters) were studied for >10 days under entrained conditions (LD12:12), followed by >20 days of DD ‘free-running’ conditions, followed again by LD12:12 (>10 days). The expected activity onset of the last day under LD was extrapolated back by linear regression of the DD activity onset times. The difference between the average activity onset under the preceding LD12:12 and this expected activity onset determined the phase angle of entrainment. As a measure of precision of activity onset under DD conditions, we compared the individual correlation coefficients (r) of the linear regression of activity onsets explained above to estimate the phase angle of entrainment between genotypes. The same number of observations contributed to each r-value, that is 16 circadian days. Correlation coefficients were Fisher's Z-transformed prior to statistical analysis. To estimate robustness of the circadian rhythm under DD, the individually obtained maximal amplitude of the X²-statistic was compared between genotypes as this statistic indexes how much of the total variance in the locomotor activity signal can be attributed to the circadian rhythm. For this analysis, the number of cycles contributing to the analysis was identical for each individual (n = 10/genotype), that is 18.

Evaluation of the phase-shifting capacity of light was performed in a separate group of mice (Rai1^+/+: n = 5; Rai1^+/-: n = 4; one experimental cohort from a total of two litters). For this experiment, mice were exposed to a 1-hr-light pulse (6.6 cds/m²) at circadian times (CT)16 and −22 (with CT12 designating activity onset). Both light pulses occurred 4 days after the mice were released in DD. The phase shifts were calculated as the difference, in circadian hours, between the average activity onset over the 3 days prior to the light pulse and the average activity onset over the 10 days following the light pulse. Activity onset on the first day after the light pulse was not taken into account.

In a third group of mice (Rai1^+/+: n = 5; Rai1+/-: n = 3; two experimental cohorts from a total of 3 litters), we tested whether mice are equally sensitive to light of low illumination, that is, 0.6 cds/m² versus the habitual 6.6 cds/m², both under a LD12:12 protocol. Mice were exposed to at least 5 days under 6.6 cds/m² followed by at least 6 days under 0.6 cds/m². The first day under the new light environment was discarded from the analysis.

EEG/EMG surgeries were performed under deep anesthesia as previously described (Mang and Franken, 2012). Rai1^+/+ (n = 8) and Rai1^+/- mice (n = 7; six experimental cohorts from a total of eight litters) were allowed to recover for >10 days before experiments started. EEG and EMG signals were amplified, filtered, analog-to-digital converted, and stored using EMBLA (A10 recorder) and Somnologica software (Medcare Flaga, Thornton, CO). The sleep-wake states wakefulness (W), rapid-eye-movement (REM) sleep, and non-REM (NREM) sleep were visually assigned to consecutive 4 s epochs as previously described (Mang and Franken, 2012). Four-second epochs scored as wakefulness in which the EEG was dominated by theta activity (EEG power in the 6.5–9.5 Hz range) were labeled as theta dominated wakefulness (TDW) using an algorithm with the following criteria: if the frequency bin showing highest power density (theta peak frequency or TPF) across the 3.5–15.0 Hz range was within the 6.5–12.0 Hz range, and the ratio of theta power in the ±1 Hz range surrounding the TPF over the total power across 3.5–45.0 Hz was above 0.228, then that epoch would score as TDW, if an additional set of three contextual criteria for that epoch were met: (1) the epoch immediately preceding a TDW epoch was scored as 'W' or 'TDW'; (2) the epoch immediately following a TDW epoch was not scored as NREM sleep; (3) isolated TDW epochs preceded and followed by 3 'W' epochs were excluded. Recordings of three C57BL/6J male mice were assessed visually for TDW state scoring and the 0.228 theta/total power ratio threshold was chosen because it yielded the best match with visually identified TDW. Please see (Vassalli and Franken, 2017) for details and validation. The term TDW was previously coined to describe a theta-rich waking sub-state associated with locomotor activity in the mouse (Welsh et al., 1985).

EEG/EMG signals were recorded continuously for a 3-day period, the first two of which served as baseline (48 hr) under LD12:12. During the first 6 hr of day 3 (ZT0-6), animals were sleep deprived by ‘gentle handling’ (Mang and Franken, 2012). Locomotor activity was recorded in parallel using the same PIR sensors. In a separate cohort (n = 4/genotype from a total of 2 litters), activity and sleep-wake patterns were investigated under short light-dark cycles (LD1:1; 1 day under LD12:12 followed by 2 days of LD1:1) and constant light (LL; 1 day under LD12:12 followed by 2 days under LL) conditions. The two experiments were separated by 3 days under LD12:12. In two individuals per genotype, behavior was further verified using infra-red video recordings.

For scotopic conditions, mice (Rai1^+/+: n = 5; Rai1^+/-: n = 6; 15 weeks old; two experimental cohorts from a total of four litters) were dark-adapted overnight and experiments were done under dim-red light. Corneal ERG was recorded with an active ring electrode and responses to single flashes of green light (520 nm; half-bandwidth 35 nm; at 10–4, 10–3, 10–2, 3 × 10–2, 10–1, 3 × 10–1, 1, 3, 10, and 30 cds/m²), generated by a stroboscope in the upper part of a Ganzfeld stimulator (Espion E3 apparatus; Diagnosys LLC, Lowell, MA), were quantified. For each stimulus intensity, 10 to 15 traces were averaged for each eye separately. The a-wave amplitude (photoreceptor response) was defined as the difference between the peak of the a-wave and baseline at the time of stimulation. The b-wave amplitude (second-order neurons) was defined as the difference between the peak of the b-wave and the peak of the a-wave.

Eye collection, retina processing, and immunolabeling were performed as previously described (Bemelmans et al., 2006). Specifically, antibodies directed against Rhodopsin (1:500, #MA5-11741, Pierce), G-Protein Subunit Alpha Transducin 2 (GNAT2, 1:200, RRID:AB_2279097, #Sc-390, Santa Cruz, Santa Cruz, CA), Protein kinase C alpha (PKCα, 1:200, RRID:AB_2168560, #sc-10800, Santa Cruz), Glial fibrillary acidic protein (GFAP, 1:500, RRID:AB_10013382, #Z0334, Dako, Zug, Switzerland), melanopsin (1:500, RRID:AB_444884, #Ab19383, Abcam, Cambridge, UK), calbindin (1:5000, RRID:AB_10000340, CB-38a, Swant, Marly, Switzerland), ChAT (1:2500, polyclonal goat, gift from Jean-Pierre Hornung, University of Lausanne), and GABA (1:500, RRID:AB_477652, #2052, Sigma, Buchs, Switzerland) were used and visualized with the appropriate secondary antibodies coupled to Alexa Fluor488 or Alexa633 (1:2000, Molecular Probes, Eugene, OR).

Mice were either exposed to a 1-hr-light pulse from ZT14.5 to ZT15.5 (6.6 cds/m²; n = 6 and 7 for wild-type and heterozygous mice, respectively) or left undisturbed (controls; n = 3/genotype) and sacrificed immediately at ZT15.5. Four experimental cohorts were used comprising nine litters. Mice were deeply anesthetized with pentobarbital sodium (300 mg/ml diluted at 1:15) administered intraperitoneally in a volume of 10 ml/kg body weight. Animals were then perfused transcardially with heparin/PBS followed by a 15 min fixation with 4% paraformaldehyde in PBS. In control mice, this was performed under dim-red light. Brains were carefully removed and post-fixed overnight in the fixative at 4°C. Cryoprotection of brains was achieved by using graded concentrations of sucrose (10-, 20-, and 30%). After 2 days of dehydration at 4°C in sucrose, brains were rapidly frozen in isopentane (Sigma-Aldrich Co, MO) vials placed on dry ice and stored at −80°C. Coronal sections (25 µm) were cut at −20°C using a cryostat. SCN sections were collected (0.22–0.83 mm posterior to bregma). Cryostat sections were mounted and left to air dry for at least 1 hr before being washed in PBS/0.25% Triton and incubated overnight with an anti-c-FOS antiserum (1:250; rabbit polyclonal, Santa Cruz Biotechnology Inc, CA). An Alexa568-conjugated goat anti-rabbit antibody was used as secondary antibody (1:1000, Molecular Probes, Thermo Fisher Scientific Inc, MA) to visualize c-FOS staining. DAPI staining (1:10000, Sigma-Aldrich Co, MO) was used to visualize cell nuclei. Sections were examined and photographed with a Zeiss Axio imager M1 microscope. The number of c-FOS-positive neurons as well as the intensity of the immunoreactivity was quantified using the ImageJ/FIJI plugin (National Institute of Health, MD) for the left and right hemispheres separately.

Pooled mRNA from left and right retina was isolated (n = 5/genotype; one experimental cohort, a total of 3 litters) and purified using the TRIzol RNA Isolation Reagents protocol (Invitrogen, Carlsbad, CA). RNA quantity was assessed with a NanoDrop ND-1000 spectrophotometer (ThermoScientific, Wilmington, DE). For cDNA synthesis, 0.5 μg of total RNA was treated with DNaseI (Amplification Grade, Invitrogen, Carlsbad, CA), reverse-transcribed using random hexamers and Superscript II reverse transcriptase (Invitrogen, Basel, Switzerland) according to standard procedures. The cDNA was diluted 10 times and 1 μl was used as template in a 10 μl TaqMan reaction on an ABI PRISM HT 7900 detection system in technical triplicates. Forward and reverse primers, as well as probes sequences were chosen from the coding regions of genes in Invitrogen website (Rai1, Opn1mw, Opn4, Rho, and Thy1). The expression of each gene was normalized against four control reference transcripts (ActinB, Eef1a1, Rps9, and TubA) using Qbase (Hellemans et al., 2007).

To assess the 24 hr time course of Rai1 brain expression under LD12:12 conditions, samples were collected at 3 hr intervals. At each interval, three male C57BL/6J mice were sacrificed by decapitation after being anesthetized with isoflurane, and the forebrain (whole brain minus brainstem and cerebellum) was dissected. Sampling was performed under normal light conditions during the light phase (ZT0, −6, and −9), and under dim red light during the dark (ZT12, −15, and −18). For each time point, a separate experimental cohort was used originating from four different litters. The tissue was placed in an Eppendorf tube, immediately flash frozen in liquid nitrogen and kept at −80°C until RNA extraction. RNA was isolated and purified with the RNeasy Lipid Tissue Midi kit (Qiagen, Hombrechtikon, Switzerland) and treated with DNaseI. Assessment of RNA quantity, cDNA synthesis, and amplification as described above. As reference transcripts Eef1a1, Gapdh, Gusb, and Rps9 were used. Time courses of Arntl (Bmal1) and Per2 expression were used as positive control.

Statistical analyses were performed using R Statistical Software (Foundation for Statistical Computing, Vienna, Austria) and SAS (SAS Institute Inc, Cary, NC). Significant effects of genotype and time-of-day and their interactions were assessed using analysis-of-variance (ANOVA) and decomposed using post-hoc t-tests. Simple two-group genotype contrasts were assessed using t-tests. Circadian rhythmicity in locomotor activity was evaluated using chi-square periodogram analysis (ClockLab, Actimetrics). Statistical significance was set to p<0.05 and results are reported as mean ± standard error of the mean (SEM). SigmaPlot 11 (Systat Software Inc., Chicago, IL) was used to generate graphs.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We thank Mirjam Münch for her constructive and important input on the project, Alexandre Reymond for housing the mice and sharing his expertise on genomic structural changes and their resulting pathologies, James R Lupski for generously providing the Rai1^+/- mice, Yann Emmenegger, Sylvain Crippa, and Catherine Moret for excellent technical support, Jean-Pierre Hornung for providing the ChaT antibodies, H Craig Heller for helpful discussions, and Charlotte Hor for editing the manuscript.

Animal experimentation: All experiments were approved by the Ethical Committee of the State of Vaud Veterinary Office, Switzerland (# VD2545).

© 2017, Diessler et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.