Patrick Vuillez

Photoperiod differentially regulates clock genes’ expression in the suprachiasmatic nucleus of Syrian hamster

The suprachiasmatic nuclei (SCN) contain the master circadian pacemaker in mammals. Generation and maintenance of circadian oscillations involve clock genes which interact to form transcriptional/translational loops and constitute the molecular basis of the clock. There is some evidence that the SCN clock can integrate variations in day length, i.e. photoperiod. However, the effects of photoperiod on clock-gene expression remain largely unknown. We here report the expression pattern of Period (Per) 1, Per2, Per3, Cryptochrome (Cry) 1, Cry2, Bmal1 and Clock genes in the SCN of Syrian hamsters when kept under long (LP) and short (SP) photoperiods. Our data show that photoperiod differentially affects the expression of all clock genes studied. Among the components of the negative limb of the feedback loop, Per1, Per2, Per3, Cry2 but not Cry1 genes show a shortened duration of their peak expression under SP compared with LP. Moreover, mRNA expression of Per1, Per3 and Cry1 are phase advanced in SP compared with LP. Per3 shows an mRNA peak of higher amplitude under SP conditions whereas Per1 and Per2 peak amplitudes are unaffected by photoperiod changes. Bmal1 expression is phase advanced without a change of duration in SP compared with LP. Furthermore, the expression of Clock is rhythmic under SP whereas no rhythm is observed under LP. These results, which provide further evidence that the core clock mechanisms of the SCN integrate photoperiod, are discussed in the context of the existing molecular model.

In Syrian and European hamsters, the duration of sensitive phase to light of the suprachiasmatic nuclei depends on the photoperiod.

Light induction of the expression of Fos protein in the suprachiasmatic nuclei was used to investigate the photosensitive state of the clock in Syrian and European hamster kept under different photoperiods. We observed that the duration of the photosensitive phase is variable and tied to the length of the night. A maximal extension has been determined in both species studied. Finally, a 4 h lengthening of the phase of photosensitivity take approximately 3 weeks, while 3 days only are needed for its shortening.

Systematic presence of GABA-immunoreactivity in the tubero-infundibular and tubero-hypophyseal dopaminergic axonal systems: an ultrastructural immunogold study on several mammals

SummaryImmunoreactivities for tyrosine hydroxylase (TH), gamma-aminobutyric acid (GABA) and, in some cases, glutamic acid decarboxylase (GAD) were detected by light and electron microscopy in axons projecting into the median eminence and pituitary gland of various mammals (rats, mice, guinea pigs, cats, rabbits and hares). Light microscope immunoperoxidase reactions were performed on adjacent semithin sections of plasticembedded samples. In the median eminence external zone, the distributions of the TH- and GAD- or GABA-immunoreactive endings were very similar in the anterior and lateral areas, while medially the GABA-labelled endings predominated. Comparable distribution patterns were found in the various species examined. In the pituitary gland, the distributions of GABA- and TH- immunoreactivities were superimposable in the intermediate lobes of all species examined, except in the rabbit and hare in which both types of innervation were lacking. For electron microscopy, the immunogold procedure was applied to sections of lowicryl-embedded samples; simultaneous detection of GABA- and TH-immunoreactivities was enabled by recto-verso double labelling with gold particles of distinct diameters. In the median eminence, GABA-immunoreactivity occurred systematically in the TH-positive endings, while distinct GABA-positive/TH-negative axons were also detected. In the intermediate lobe, the colocalization of TH- and GABA-immunoreactivities was a constant feature of the axons innervating the melanotrophic cells in all the species examined, except in the Leporidae. The functional significance of this colocalization remains to be determined.

Seasonal variations of clock gene expression in the suprachiasmatic nuclei and pars tuberalis of the European hamster (Cricetus cricetus)

In mammals, day length (photoperiod) is read and encoded in the main circadian clock, the suprachiasmatic nuclei (SCN). In turn, the SCN control the seasonal rhythmicity of various physiological processes, in particular the secretion pattern of the pineal hormone melatonin. This hormone then operates as an essential mediator for the control of seasonal physiological functions on some tissues, especially the pars tuberalis (PT). In the European hamster, both hormonal (melatonin) and behavioral (locomotor activity) rhythms are strongly affected by season, making this species an interesting model to investigate the impact of the seasonal variations of the environment. The direct (on SCN) and indirect (via melatonin on PT) effect of natural short and long photoperiod was investigated on the daily expression of clock genes, these being expressed in both tissues. In the SCN, photoperiod altered the expression of all clock genes studied. In short photoperiod, whereas Clock mRNA levels were reduced, Bmal1 expression became arrhythmic, probably resulting in the observed dramatic reduction in the rhythm of Avp expression. In the PT, Per1 and Rev‐erbα expressions were anchored to dawn in both photoperiods. The daily profiles of Cry1 mRNA were not concordant with the daily variations in plasma melatonin although we confirmed that Cry1 expression is regulated by an acute melatonin injection in the hamster PT. The putative role of such seasonal‐dependent changes in clock gene expression on the control of seasonal functions is discussed.

Photic induction of Fos immunoreactivity in the suprachiasmatic nuclei of the blind mole rat(Spalax ehrenbergi).

Exposure to light during the dark phase entrains c-fos expression in cells of the suprachiasmatic nuclei in the blind mole rat (Spalax ehrenbergi). Labeled cells are mainly located in the ventral region of the nucleus where retinal afferents terminate. Despite severe atrophy of the eye and regression of thalamic and tectal structures, the retinal pathways and central mechanisms involved in photoperiodic perception and circadian regulation appear to be similar to those in other rodents.

Photoreceptor organisation and phenotypic characterization in retinas of two diurnal rodent species: potential use as experimental animal models for human vision research.

To characterize rod and cone distribution and composition in two diurnal mouse-like rodents, retinas from adult Arvicanthis ansorgei and Lemniscomys barbarus were processed for immunohistochemistry using multiple rod- and cone-specific antibodies. Antibodies tested included rhodopsin, cone opsins, pan-arrestin and cone arrestin, recoverin, and cGMP dependent ion channel. In both species, retinas were composed of approximately 33% cones, and most antibodies gave similar staining patterns. Data show these two diurnal rodents possess large numbers of cones, organised in a strict anatomical array. This suggests that diurnal rodents in general possess elevated cone numbers and could constitute valuable models for investigating cone pathophysiology.

Fos-like immunoreactivity in the circadian timing system of calorie-restricted rats fed at dawn: daily rhythms and light pulse-induced changes

Daily rhythms of pineal melatonin, body temperature, and locomotor activity are synchronized to the light-dark cycle (LD) via a circadian clock located in the suprachiasmatic nuclei (SCN). A timed caloric restriction in rats fed at dawn induces phase-advances and further phase-stabilization of these rhythms, suggesting that the circadian clock can integrate conflicting daily photic and non-photic cues. The present study investigated the daily expression of Fos-like immunoreactivity (Fos-ir) and light pulse-induced Fos-ir in the SCN, the intergeniculate leaflet (IGL) and the paraventricular thalamic nucleus (PVT) in calorie-restricted rats fed 2 h after the onset of light and in controls fed ad libitum. A daily rhythm of Fos-ir in the SCN was confirmed in control rats, with a peak approximately 2 h after lights on. At this time point (i.e. just prior to the feeding time), the level of SCN Fos-ir was lowered in calorie-restricted rats. Concomitantly, IGL Fos-ir was higher in calorie-restricted vs. control rats. In response to a light pulse during darkness, Fos-ir induction was found to be specifically (i.e. phase-dependently) lowered in the SCN and IGL of calorie-restricted rats. Observed changes of Fos-ir in the PVT were possibly related to the wake state of the animals. This study shows that repetitive non-photic cues presented in addition to a LD cycle affect the Fos expression in the circadian timing system.

Photoperiod does not act on the suprachiasmatic nucleus photosensitive phase through the endogenous melatonin, in the Syrian hamster

The duration of the sensitive phase to light of the suprachiasmatic nuclei, in terms of Fos protein expression, depends on the photoperiod. In Syrian hamsters, a 4 h lengthening of the photosensitive phase occurs within 3-4 weeks after a transfer from a long to a short photoperiod. The absence of endogenous melatonin following pinealectomy does not prevent the lengthening of the photosensitive phase. Thus, even if the pineal production is able to convey photoperiodic information, it does not feed back on the circadian clock to allow its integration.

Does the intergeniculate leaflet play a role in the integration of the photoperiod by the suprachiasmatic nucleus

The circadian clock located in the suprachiasmatic nuclei (SCN) is influenced by the photoperiod. After the transfer from a long (LP 14:10) to a short photoperiod (SP 10:14), the adjustment of the light sensitivity of the SCN, in terms of Fos expression, takes 25 nights. To examine the contribution of the thalamic intergeniculate leaflet (IGL) and its NPY-immunoreactive projection in the extension of the duration of the photosensitive phase of the SCN, male Syrian hamsters received electrolytic lesions of the IGL. We showed a lower number of Fos-ir cells in the SCN of IGLx hamsters following a light pulse applied 13 h after dark onset, 25 nights after the transfer from LP to SP compared to sham operated hamsters. The present study shows that the integrity of the IGL is necessary to have a complete integration of photoperiodic changes by the SCN. This demonstrates the involvement of the IGL in the integration of photoperiodic information by the SCN.

Intergeniculate leaflets lesion delays but does not prevent the integration of photoperiodic change by the suprachiasmatic nuclei

The duration of the photosensitive phase of the suprachiasmatic nuclei (SCN), as revealed by light-induced Fos protein expression, depends on the photoperiod and is tied to the length of the night. We show here in Syrian hamsters that after a transfer from long to short photoperiod, lengthening of the photosensitive phase of the SCN is significantly delayed but not abolished when the intergeniculate leaflets (IGL) are lesioned. Thus IGL modulate the integration by the SCN of a photoperiodic change.