The input pathways to the circadian clock: from nocturnality to diurnality

Most organisms on Earth anticipate changes between day and night using an internal biological clock. This clock is localized in a small brain area: the suprachiasmatic nucleus (SCN). The SCN generates rhythms of approximately 24 hours (circadian) which are synchronized to light. We investigated how light signals reach the SCN and how the effects of light and behavioural activity on the SCN differ between diurnal and nocturnal species. In this thesis, we show that cone photoreceptors, involved in colour vision, contribute to the light responses of the circadian clock. As observed in fMRI experiments, the human SCN was also sensitive to different colours of light. Furthermore, the SCN of diurnal species was partly inhibited by light, whereas the SCN of nocturnal species was mostly excited. Finally, we observed that behavioural activity activates the SCN of diurnal species, whereas in nocturnal species the SCN is inhibited. We concluded that the input pathways to the circadian clock differ between diurnal and nocturnal species and hypothesize that these differences could contribute to the temporal activity profiles. Together, our findings facilitate the translation of circadian research from nocturnal laboratory animals to diurnal humans, for the benefit of biological rhythms and health in general.Velux StiftungLUMC / Geneeskund

Distinct contribution of cone photoreceptor subtypes to the mammalian biological clock

Ambient light detection is important for the synchronization of the circadian clock to the external solar cycle. Light signals are sent to the suprachiasmatic nuclei (SCN), the site of the major circadian pacemaker. It has been assumed that cone photoreceptors con-tribute minimally to synchronization. Here, however, we find that cone photoreceptors are sufficient for mediating entrainment and transmitting photic information to the SCN, as evaluated in mice that have only cones as functional photoreceptors. Using in vivo electrophysiological recordings in the SCN of freely moving cone-only mice, we observed light responses in SCN neuronal activity in response to 60-s pulses of both ultraviolet (UV) (lambda(max) 365 nm) and green (lambda(max) 505 nm) light. Higher irradiances of UV light led to irradiance-dependent enhancements in SCN neuronal activity, whereas higher irradiances of green light led to a reduction in the sustained response with only the transient response remain-ing. Responses in SCN neuronal activity decayed with a half-max time of similar to 9 min for UV light and less than a minute for green light, indicating differential input between short-wavelength-sensitive and mid-wavelength-sensitive cones for the SCN responsiveness. Furthermore, we show that UV light is more effective for photo-entrainment than green light. Based on the lack of a full sustained response in cone-only mice, we confirmed that rapidly alternating light levels, rather than slowly alternating light, caused substantial phase shifts. Together, our data provide strong evidence that cone types contribute to photoentrainment and differentially affect the electrical activity levels of the SCN.Circadian clocks in health and diseas

Reply to Sharifpour et al.: light response measurement of the human SCN by 7T fMRI

Stress and Psychopatholog

Hippocampal glucocorticoid target genes associated with enhancement of memory consolidation

Glucocorticoids enhance memory consolidation of emotionally arousing events via largely unknown molecular mechanisms. This glucocorticoid effect on the consolidation process also requires central noradrenergic neurotransmission. The intracellular pathways of these two stress mediators converge on two transcription factors: the glucocorticoid receptor (GR) and phosphorylated cAMP response element-binding protein (pCREB). We therefore investigated, in male rats, whether glucocorticoid effects on memory are associated with genomic interactions between the GR and pCREB in the hippocampus. In a two-by-two design, object exploration training or no training was combined with post-training administration of a memory-enhancing dose of corticosterone or vehicle. Genomic effects were studied by chromatin immunoprecipitation followed by sequencing (ChIP-seq) of GR and pCREB 45 min after training and transcriptome analysis after 3 hr. Corticosterone administration induced differential GR DNA-binding and regulation of target genes within the hippocampus, largely independent of training. Training alone did not result in long-term memory nor did it affect GR or pCREB DNA-binding and gene expression. No strong evidence was found for an interaction between GR and pCREB. Combination of the GR DNA-binding and transcriptome data identified a set of novel, likely direct, GR target genes that are candidate mediators of corticosterone effects on memory consolidation. Cell-specific expression of the identified target genes using single-cell expression data suggests that the effects of corticosterone reflect in part non-neuronal cells. Together, our data identified new GR targets associated with memory consolidation that reflect effects in both neuronal and non-neuronal cells.Development and application of statistical models for medical scientific researc

The input pathways to the circadian clock: from nocturnality to diurnality

Most organisms on Earth anticipate changes between day and night using an internal biological clock. This clock is localized in a small brain area: the suprachiasmatic nucleus (SCN). The SCN generates rhythms of approximately 24 hours (circadian) which are synchronized to light. We investigated how light signals reach the SCN and how the effects of light and behavioural activity on the SCN differ between diurnal and nocturnal species. In this thesis, we show that cone photoreceptors, involved in colour vision, contribute to the light responses of the circadian clock. As observed in fMRI experiments, the human SCN was also sensitive to different colours of light. Furthermore, the SCN of diurnal species was partly inhibited by light, whereas the SCN of nocturnal species was mostly excited. Finally, we observed that behavioural activity activates the SCN of diurnal species, whereas in nocturnal species the SCN is inhibited. We concluded that the input pathways to the circadian clock differ between diurnal and nocturnal species and hypothesize that these differences could contribute to the temporal activity profiles. Together, our findings facilitate the translation of circadian research from nocturnal laboratory animals to diurnal humans, for the benefit of biological rhythms and health in general.</p

Sleep Deprivation Does not Change the Flash Electroretinogram in Wild-type and Opn4(-/-)Gnat1(-/-) Mice

Sleep deprivation reduces the response of neuronal activity in the suprachiasmatic nucleus (SCN) and the phase shift in circadian behaviour to phase shifting light pulses, and thus seems to impair the adaptation of the circadian clock to the external light-dark cycle. The question remains where in the pathway of light input to the SCN the response is reduced. We therefore investigated whether the electroretinogram (ERG) changes after sleep deprivation in wild-type mice and in Opn4(-/-)Gnat1(-/-) mutant mice. We found that the ERG is clearly affected by the Opn4(-/-)Gnat1(-/-) mutations, but that the ERG after sleep deprivation does not differ from the baseline response. The difference between wild-type and mutant is in accordance with the lack of functional rod and melanopsin in the retina of the mutant mice. We conclude that the decrease in light responsiveness of the SCN after sleep deprivation is probably not caused by changes at the retinal level, but rather at the postsynaptic site within the SCN, reflecting affected neurotransmitter signalling.Ophthalmic researc

Inhibitory responses to retinohypothalamic tract stimulation in the circadian clock of the diurnal rodent Rhabdomys pumilio

In both diurnal and nocturnal mammals, the timing of activity is regulated by the central circadian clock of the suprachiasmatic nucleus (SCN). The SCN is synchronized to the external light cycle via the retinohypothalamic tract (RHT). To investigate potential differences in light processing between nocturnal mice and the diurnal rodent Rhabdomys pumilio, we mimicked retinal input by stimulation of the RHT ex vivo. Using Ca2+ imaging, we observed excitations as well as inhibitions of SCN neurons in response to electrical RHT stimulation. In mice, the vast majority of responses were excitatory (85%), whereas in Rhabdomys, the proportion of excitatory and inhibitory responses was similar (51% excitatory, 49% inhibitory). Glutamate blockers AP5 and CNQX blocked the excitatory responses to RHT stimulation but did not abolish the inhibitory responses in mice or Rhabdomys, indicating that the inhibitions were monosynaptically transmitted via the RHT. Simultaneous application of glutamate blockers with the GABA(A) antagonist gabazine blocked all inhibitory responses in mice, but not in Rhabdomys. Collectively, our results indicate that in Rhabdomys, considerably more inhibitory responses to light are present and that these responses are driven directly by the RHT. We propose that this increased proportion of inhibitory input could reflect a difference in the entrainment mechanism employed by diurnal rodents.Circadian clocks in health and diseas

Single cell model for re-entrainment to a shifted light cycle

Our daily 24-h rhythm is synchronized to the external light-dark cycle resulting from the Earth's daily rotation. In the mammalian brain, the suprachiasmatic nucleus (SCN) serves as the master clock and receives light-mediated input via the retinohypothalamic tract. Abrupt changes in the timing of the light-dark cycle (e.g., due to jet lag) cause a phase shift in the circadian rhythms in the SCN. Here, we investigated the effects of a 6-h delay in the light-dark cycle on PERIOD2::LUCIFERASE expression at the single-cell level in mouse SCN organotypic explants. The ensemble pattern in phase shift response obtained from individual neurons in the anterior and central SCN revealed a bimodal distribution; specifically, neurons in the ventrolateral SCN responded with a rapid phase shift, while neurons in the dorsal SCN generally did not respond to the shift in the light-dark cycle. We also stimulated the hypothalamic tract in acute SCN slices to simulate light-mediated input to the SCN; interestingly, we found similarities between the distribution and fraction of rapid shifting neurons (in response to the delay) and neurons that were excited in response to electrical stimulation. These results suggest that a subpopulation of neurons in the ventral SCN that have an excitatory response to light input, shift their clock more readily than dorsal located neurons, and initiate the SCN's entrainment to the new light-dark cycle. Thus, we propose that light-excited neurons in the anterior and central SCN play an important role in the organism's ability to adjust to changes in the external light-dark cycle.Circadian clocks in health and diseas

The photobiology of the human circadian clock

In modern society, the widespread use of artificial light at night disrupts the suprachiasmatic nucleus (SCN), which serves as our central circadian clock. Existing models describe excitatory responses of the SCN to primarily blue light, but direct measures in humans are absent. The combination of state-of-the-art neuroimaging techniques and custom-made MRI compatible light-emitting diode devices allowed to directly measure the light response of the SCN. In contrast to the general expectation, we found that blood oxygen level-dependent (BOLD) functional MRI signals in the SCN were suppressed by light. The suppressions were observed not only in response to narrowband blue light (lambda max: 470 nm) but remarkably, also in response to green (lambda max: 515 nm) and orange (lambda max: 590 nm), but not to violet light (lambda max: 405 nm). The broadband sensitivity of the SCN implies that strategies on light exposure should be revised: enhancement of light levels during daytime is possible with wavelengths other than blue, while during nighttime, all colors are potentially disruptive.Circadian clocks in health and diseas