Thermoregulation in the cold changes depending on the time of day and feeding condition: physiological and anatomical analyses of involved circadian mechanisms

The circadian rhythm of body temperature (T_b) is a well-known phenomenon. However, it is unknown how the circadian system including the suprachiasmatic nucleus (SCN) and clock genes affects thermoregulation. Food deprivation in mice induces a greater reduction of T_b particularly in the light phase. We examined the role of Clock, one of key clock genes and the SCN during induced hypothermia. At 20 °C with fasting, mice increased their metabolic heat production in the dark phase and maintained T_b, whereas in the light phase, heat production was less, resulting in hypothermia. Under these conditions, neuronal activity in the SCN, assessed by cFos expression, increased only in the light phase. However, such differences in thermoregulatory and neural responses between the phases in Clock mutant mice were less marked. The neural network between the SCN and paraventricular nucleus appeared to be important in hypothermia. These findings suggest that the circadian system per se is influenced by both the feeding condition and environmental temperature and that it modulates thermoregulation.     

Compartmentalized expression of light-induced clock genes in the suprachiasmatic nucleus of the diurnal grass rat (Arvicanthis niloticus)

Photic responses of the circadian system are mediated through light-induced clock gene expression in the suprachiasmatic nucleus (SCN). In nocturnal rodents, depending on the timing of light exposure, Per1 and Per2 gene expression shows distinct compartmentalized patterns that correspond to the behavioral responses. Whether the gene- and region-specific induction patterns are unique to nocturnal animals, or are also present in diurnal species is unknown. We explored this question by examining the light-induced Per1 and Per2 gene expression in functionally distinct SCN subregions, using diurnal grass rats Arvicanthis niloticus. Light exposure during nighttime induced Per1 and Per2 expression in the SCN, showing unique spatiotemporal profiles depending on the phase of the light exposure. After a phase delaying light pulse (LP) in the early night, strong Per1 induction was observed in the retinorecipient core region of the SCN, while strong Per2 induction was observed throughout the entire SCN. After a phase advancing LP in the late night, Per1 was first induced in the core and then extended into the whole SCN, accompanied by a weak Per2 induction. This compartmentalized expression pattern is very similar to that observed in nocturnal rodents, suggesting that the same molecular and intercellular pathways underlying acute photic responses are present in both diurnal and nocturnal species. However, after an LP in early subjective day, which induces phase advances in diurnal grass rats, but not in nocturnal rodents, we did not observe any Per1 or Per2 induction in the SCN. This result suggests that in spite of remarkable similarities in the SCN of diurnal and nocturnal rodents, unique mechanisms are involved in mediating the phase shifts of diurnal animals during the subjective day.     

Gene expression in the suprachiasmatic nuclei and the photoperiodic time integration

In mammals, the 24 h-rhythmicity of many physiological events is driven by the circadian clock contained in the suprachiasmatic nuclei (SCN). In the SCN, clock gene expressions produce the rhythmicity and control the expression of clock-controlled genes which play a role in the distribution of daily messages. The daily expression of all these genes is modulated by the duration of the light phase (i.e. photoperiod). The aim of this study was first to determine if these daily changes of expression reflect a real integration of a new photoperiod by the circadian clock or reflect only a passive effect of the light. In this way, we performed a time course of the modifications of gene expression after a transfer of Syrian hamsters from long to short photoperiod (LP and SP). Our results demonstrate that the core of the SCN (clock genes) integrates quickly a new photoperiod which entrains a slow adaptation of the clock-controlled gene expressions and induces a differential daily functioning of an SCN-target tissue, the pineal gland. We next asked the question whether SCN are involved in the photorefractory phase observed in Syrian hamsters exposed to SP for 26 weeks. All genes analyzed present a similar daily expression in SP-refractory and in SP with the exception of Clock. Its particular expression in SP-refractory is different than ones observed in SP or in LP. Thus, Clock seems to play a role in the development of the photorefractory phase, or this physiological state may modify the expression of Clock in the SCN. As a conclusion, it appears that the photoperiodic time measurement involves daily modifications of the molecular functioning of the SCN and that SCN also play a role in the measurement of the duration of the time passed in a short photoperiod.     

PER2 rhythms in the amygdala and bed nucleus of the stria terminalis of the diurnal grass rat (Arvicanthis niloticus)

The suprachiasmatic nucleus (SCN) of the hypothalamus is the central pacemaker that controls circadian rhythms in mammals. In diurnal grass rats (Arvicanthis niloticus), many functional aspects of the SCN are similar to those of nocturnal rodents, making it likely that the difference in the circadian system of diurnal and nocturnal animals lies downstream from the SCN. Rhythms in clock genes expression occur in several brain regions outside the SCN that may function as extra-SCN oscillators. In male grass rats PER1 is expressed in the oval nucleus of the bed nucleus of the stria terminalis (BNST-ov) and in the central and basolateral amygdala (CEA and BLA, respectively); several features of PER1 expression in these regions of the grass rat brain differ substantially from those of nocturnal species. Here we describe PER2 rhythms in the same three brain regions of the grass rat. In the BNST-ov and CEA PER2 expression peaked early in the light period Zeitgeber time (ZT) 2 and was low during the early night, which is the reverse of the pattern of nocturnal rodents. In the BLA, PER2 expression was relatively low for most of the 24-h cycle, but showed an acute elevation late in the light period (ZT10). This pattern is also different from that of nocturnal rodents that show elevated PER2 expression in the mid to late night and into the early day. These results are consistent with the hypothesis that diurnal behavior is associated with a phase change between the SCN and extra-SCN oscillators.     

Differential hypothalamic tyrosine hydroxylase distribution and activation by light in adult mice reared under different light conditions during the suckling period

In mammals, early light experience during a critical period within the first 3 weeks of postnatal development has long-lasting effects on circadian locomotor activity behaviour and neuropeptide expression in the suprachiasmatic nucleus (SCN) of the hypothalamus, site of the principal pacemaker. Dopamine is thought to be involved in the modulation of photic input within the SCN and in tadpoles, the expression of tyrosine hydroxylase (TH), a rate-limiting enzyme in the synthesis of dopamine, in the SCN is altered by previous light history. We thus hypothesised that dopaminergic neurons may be important for the development of the adapted responses to light that we have previously observed. To test this, we raised mice in either constant darkness, 12:12 h light–dark cycles or constant light during the first 3 weeks after birth, and later examined the expression of TH and FOS in the hypothalamus of these mice as adults, both in the dark and after exposure to a light pulse. We found that early light experience affects TH and FOS expression, both baseline levels and in response to a light pulse, in brain areas which are directly connected to the SCN, and are associated with the circadian control of neuroendocrine function. Therefore, our results suggest that the long-lasting alterations induced by early light environment on several hypothalamic nuclei may be relayed through the SCN, and that TH-expressing cells may play a role in conveying/establishing these alterations. These data suggest a role of early light experience in the regulation of future hormonal homeostasis and circadian behaviour.     

Attenuation of circadian light induced phase advances and delays by neuropeptide Y and a neuropeptide Y Y1/Y5 receptor agonist

Circadian rhythms can be synchronised to photic and non-photic stimuli. The circadian clock, anatomically defined as the suprachiasmatic nucleus in mammals, can be phase shifted by light during the night. Non-photic stimuli reset the circadian rhythm during the day. Photic and non-photic stimuli have been shown to interact during the day and night. Precise mechanisms for these complex interactions are unknown. A possible pathway for non-photic resetting of the clock is thought to generate from the intergeniculate leaflet, which conveys information to the suprachiasmatic nucleus (SCN) through the geniculohypothalamic tract and utilises neuropeptide Y (NPY) as its primary neurotransmitter.Interactions between light and NPY were investigated during the early (2 h after activity onset) and late (6 h after activity onset) night in male Syrian hamsters. NPY microinjections into the region of the SCN significantly attenuated light-induced phase delay, during the early subjective night. Phase advances to light were completely inhibited by the administration of NPY during the late night.The precise mechanism by which NPY attenuates or blocks photic phase shifts is unclear, but the NPY Y5 receptor has been implicated in the mediation of this inhibitory effect. The NPY Y1/Y5 receptor agonist, [Leu(31),Pro(34)]NPY, was administered via cannula microinjections following light exposure during the early and late night. [Leu(31),Pro(34)]NPY significantly attenuated phase delays to light during the early night and blocked phase advances during the late night, in a manner similar to NPY.These results show the ability of NPY to attenuate phase shifts to light during the early night and block light-induced phase advances during the late night. Furthermore, this is the first in vivo study implicating the involvement of the NPY Y1/Y5 receptors in the complex interaction of photic and non-photic stimuli during the night. The alteration of photic phase shifts by NPY may influence photic entrainment within the circadian system.     

Circadian and developmental regulation of N-methyl-d-aspartate-receptor 1 mRNA splice variants and N-methyl-d-aspartate-receptor 3 subunit expression within the rat suprachiasmatic nucleus

The circadian rhythms of mammals are generated by the circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Its intrinsic period is entrained to a 24 h cycle by external cues, mainly by light. Light impinging on the SCN at night causes either advancing or delaying phase shifts of the circadian clock. N-methyl-d-aspartate receptors (NMDAR) are the main glutamate receptors mediating the effect of light on the molecular clockwork in the SCN. They are composed of multiple subunits, each with specific characteristics whose mutual interactions strongly determine properties of the receptor. In the brain, the distribution of NMDAR subunits depends on the region and developmental stage. Here, we report the circadian expression of the NMDAR1 subunit in the adult rat SCN and depict its splice variants that may constitute the functional receptor channel in the SCN. During ontogenesis, expression of two of the NMDAR1 subunit splice variants, as well as the NMDAR3A and 3B subunits, exhibits developmental loss around the time of eye opening. Moreover, we demonstrate the spatial and developmental characteristics of the expression of the truncated splice form of NMDAR1 subunit NR1-E in the brain. Our data suggest that specific properties of the NMDAR subunits we describe within the SCN likely influence the photic transduction pathways mediating the clock entrainment. Furthermore, the developmental changes in NMDAR composition may contribute to the gradual postnatal maturation of the entrainment pathways.     

Effect of intermittent fasting on circadian rhythms in mice depends on feeding time

Calorie restriction (CR) resets circadian rhythms and extends life span. Intermittent fasting (IF) also extends life span, but its affect on circadian rhythms has not been studied. To study the effect of IF alongside CR, we imposed IF in FVB/N mice or IF combined with CR using the transgenic FVB/N alphaMUPA mice that, when fed ad libitum, exhibit spontaneously reduced eating and extended life span. Our results show that when food was introduced during the light period, body temperature peak was not disrupted. In contrast, IF caused almost arrhythmicity in clock gene expression in the liver and advanced mPer2 and mClock expression. However, IF restored the amplitudes of clock gene expression under disruptive light condition regardless whether the animals were calorically restricted or not. Unlike daytime feeding, nighttime feeding yielded rhythms similar to those generated during ad libitum feeding. Taken together, our results show that IF can affect circadian rhythms differently depending on the timing of food availability, and suggest that this regimen induces a metabolic state that affects the suprachiasmatic nuclei (SCN) clock.     

The central clock controls the daily rhythm of <Emphasis Type="Italic">Aqp5</Emphasis> expression in salivary glands

Salivary secretion displays day–night variations that are controlled by the circadian clock. The central clock in the suprachiasmatic nucleus (SCN) regulates daily physiological rhythms by prompting peripheral oscillators to adjust to changing environments. Aquaporin 5 (Aqp5) is known to play a key role in salivary secretion, but the association between Aqp5 and the circadian rhythm is poorly understood. The aim of our study was to evaluate whether Aqp5 expression in submandibular glands (SMGs) is driven by the central clock in the SCN or by autonomous oscillations. We observed circadian oscillations in the activity of period circadian protein homolog 2 and luciferase fusion protein (PER2::LUC) in cultured SMGs with periodicity depending on core clock genes. A daily rhythm was detected in the expression profiles of Aqp5 in SMGs in vivo. In cultured SMGs ex vivo, clock genes showed distinct circadian rhythms, whereas Aqp5 expression did not. These data indicate that daily Aqp5 expression in the mouse SMG is driven by the central clock in the SCN.     

Neuropeptide Y,GABA and circadian phase shifts to photic stimuli

Circadian rhythms can be phase shifted by photic and non-photic stimuli. The circadian clock, anatomically defined as the suprachiasmatic nucleus (SCN), can be phase delayed by light during the early subjective night and phase advanced during the late subjective night. Non-photic stimuli reset the clock when presented during the subjective day. A possible pathway for the non-photic resetting of the clock is thought to originate from the intergeniculate leaflet, which conveys information to the SCN through the geniculohypothalamic tract and utilizes among others neuropeptide Y (NPY) and GABA as neurotransmitters. Photic and non-photic stimuli have been shown to interact during the early and late subjective night. Microinjections of NPY or muscimol, a GABA_A receptor agonist, into the region of the SCN can attenuate light-induced phase shifts during the early and late subjective night. The precise mechanism for these interactions is unknown.

In the current study we investigate the involvement of a GABAergic mechanism in the interaction between NPY and light during the early and late subjective night. Microinjections of NPY significantly attenuated light-induced phase delays and inhibited phase advances (P<0.05). The administration of bicuculline during light exposure, before NPY microinjection did not alter the ability of NPY to attenuate light-induced phase delays and block photic phase advances.

These results indicate that NPY attenuates photic phase shifts via a mechanism independent of GABA_A receptor activation. Furthermore it is evident that NPY influences circadian clock function via differing cellular pathways over the course of a circadian cycle.     

Altered photic and non-photic phase shifts in 5-HT(1A) receptor knockout mice

The mammalian circadian clock located in the suprachiasmatic nucleus (SCN) is thought to be modulated by 5-HT. 5-HT is though to inhibit photic phase shifts by inhibiting the release of glutamate from retinal terminals, as well as by decreasing the responsiveness of retinorecipient cells in the SCN. Furthermore, there is also evidence that 5-HT may underlie, in part, non-photic phase shifts of the circadian system. Understanding the mechanism by which 5-HT accomplishes these goals is complicated by the wide variety of 5-HT receptors found in the SCN, the heterogeneous organization of both the circadian clock and the location of 5-HT receptors, and by a lack of sufficiently selective pharmacological agents for the 5-HT receptors of interest. Genetically modified animals engineered to lack a specific 5-HT receptor present an alternative avenue of investigation to understand how 5-HT regulates the circadian system. Here we examine behavioral and molecular responses to both photic and non-photic stimuli in mice lacking the 5-HT(1A) receptor. When compared with wild-type controls, these mice exhibit larger phase advances to a short late-night light pulse and larger delays to long 12 h light pulses that span the whole subjective night. Fos and mPer1 expression in the retinorecipient SCN is significantly attenuated following late-night light pulses in the 5-HT(1A) knockout animals. Finally, non-photic phase shifts to (+/-)-8-hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT) are lost in the knockout animals, while attenuation of the phase shift to the long light pulse due to rebound activity following a wheel lock is unaffected. These findings suggest that the 5-HT(1A) receptor plays an inhibitory role in behavioral phase shifts, a facilitatory role in light-induced gene expression, a necessary role in phase shifts to 8-OH-DPAT, and is not necessary for activity-induced phase advances that oppose photic phase shifts to long light pulses.     

Gastrin releasing peptide and neuropeptide Y exert opposing actions on circadian phase

Microinjection of gastrin releasing peptide (GRP) into the third ventricle or the suprachiasmatic nucleus (SCN) induces circadian phase shifts similar to those produced by light. Administration of GRP during the day does not alter circadian phase. In contrast, neuropeptide Y (NPY) induces phase shifts of circadian rhythms during the day but has little effect when administered at night, similar to the effects of most non-photic stimuli. NPY inhibits the phase shifting effects of light, and GRP is thought to be part of the photic signaling system within the SCN. This experiment was designed to test whether GRP and NPY inhibit each other's effects on circadian phase. Adult male Syrian hamsters equipped with guide cannulas aimed at the SCN were housed in constant darkness until stable free-running rhythms of wheel running activity were apparent. Microinjection of GRP during the early subjective night induced phase delays that were blocked by simultaneous administration of NPY. During the middle of the subjective day, microinjection of NPY caused phase advances that were blocked by simultaneous administration of GRP. These data suggest that GRP and NPY oppose each other's effects on the circadian clock, and that the actions of NPY on the photic phase shifting mechanism in the SCN occur at least in part downstream from retinorecipient cells.     

High frequency of mosaic pathogenic variants in genes causing epilepsy-related neurodevelopmental disorders

PurposeMosaicism probably represents an underreported cause of genetic disorders due to detection challenges during routine molecular diagnostics. The purpose of this study was to evaluate the frequency of mosaicism detected by next-generation sequencing in genes associated with epilepsy-related neurodevelopmental disorders.MethodsWe conducted a retrospective analysis of 893 probands with epilepsy who had a multigene epilepsy panel or whole-exome sequencing performed in a clinical diagnostic laboratory and were positive for a pathogenic or likely pathogenic variant in one of nine genes (CDKL5, GABRA1, GABRG2, GRIN2B, KCNQ2, MECP2, PCDH19, SCN1A, or SCN2A). Parental results were available for 395 of these probands.ResultsMosaicism was most common in the CDKL5, PCDH19, SCN2A, and SCN1A genes. Mosaicism was observed in GABRA1, GABRG2, and GRIN2B, which previously have not been reported to have mosaicism, and also in KCNQ2 and MECP2. Parental mosaicism was observed for pathogenic variants in multiple genes including KCNQ2, MECP2, SCN1A, and SCN2A.ConclusionMosaic pathogenic variants were identified frequently in nine genes associated with various neurological conditions. Given the potential clinical ramifications, our findings suggest that next-generation sequencing diagnostic methods may be utilized when testing these genes in a diagnostic laboratory.