Cloning and expression of arylalkylamine N-acetyltranferase-2 during early development and metamorphosis in the sole Solea senegalensis.

The arylalkylamine N-acetyltransferase (AANAT) is a key enzyme in the rhythmic production of melatonin. Two Aanats are expressed in teleost fish, one retinal specific, Aanat1, and the other one pineal specific, Aanat2, being the latter the main enzyme responsible of the plasma nocturnal melatonin increase in fish. In anurans melatonin has been involved in metamorphosis through antagonizing thyroid hormone function; however, no available data reports a relationship between melatonin system and metamorphosis in fish. In this study, we have cloned the AANAT2 (SsAanat2) in a flatfish, Solea senegalensis, and studied its sites of expression and developmental expression pattern by in situ hybridization and Real Time PCR. These studies allowed us to demonstrate a specific signal in the pineal gland of sole larvae from 2 days post-fertilization (dpf), which was evident until post-metamorphosis. Immunohistochemical analysis on the hybridized slides showed that the sole pineal Aanat2 expressing cells corresponded to pineal photoreceptor cells. Real Time PCR was performed in animals kept under natural photoperiod and sampled at different stages from 0 to 21 dpf (including pre-, early-, middle- and late-metamorphic stages) and at midlight (ML) and middark (MD) daytimes. Sole Aanat2 expression was higher at MD than at ML from 2 dpf and at most developmental stages analyzed. The highest AANAT2 mRNA abundance was observed at 2 and 4 dpf. A significant 60-fold reduction in Aanat2 expression was seen just before metamorphosis demonstrating, for the first time in a vertebrate species, that the expression of pineal AANAT and thyroid hormones levels exhibit an inverse pattern during metamorphosis.

Daily behavioral rhythmicity and organization of the suprachiasmatic nuclei in the diurnal rodent, Lemniscomys barbarus.

Wheel-running activity was recorded in Lemniscomys barbarus exposed to different lighting conditions. This rodent shows rhythmic locomotor activity under natural twilight-light/dark (LD) as well as squared-LD cycles. A mean of 77% of the activity occurred during the light phase. Under different controlled photoperiods, the quantity of daily locomotor activity was relatively stable except for a lower level in the shortest photoperiod tested (LD 06:18). The duration of the active phase tended to increase with the duration of the light phase, especially in the longer photoperiods. Whatever the lighting conditions, Lemniscomys barbarus started running before lights-on and stopped after lights-off. The phase angle of activity offset relative to lights-off was stable in each squared-photoperiod, whereas the phase angle of activity onset relative to lights-on was significantly the highest under the shortest photoperiods. Recording of activity under constant lighting conditions showed that the daily rhythm of locomotor activity is fundamentally circadian. The endogenous period was slightly<24 h (mean=23.8 h) in permanent darkness and>24 h (mean=24.5 h) in continuous light. Re-entrainment of the locomotor activity rhythm after a 6 h phase advance or delay requires only four days on average. Moreover, the phase-responses curve to a 30 min light pulse (200 lux) in Lemniscomys barbarus kept in constant dark reveals large phase shifts according to circadian times (CT). With CT0 being defined as the onset of daily activity, maximum phase delay and advance shifts were observed at CT11 (Delta Psi=-5.7 h+/-2.3 h) and CT21 (Delta Psi =4.9+/-1.2 h), respectively. Interestingly, the phase-response curve to light did not show any dead zone. Immunohistochemical staining of the suprachiasmatic nuclei indicates that arginine vasopressin-immunoreactive cell bodies and fibers delimited a dorsal subregion that extends laterally and medially. The ventral subregion is rich in vasoactive intestinal peptide-immunoreactive neurones overlapping a smaller area containing gastrin-releasing peptide-expressing cells and receives numerous fibers labeled with neuropeptide Y antibody. The results of this study clearly demonstrate that Lemniscomys barbarus is a diurnal species highly sensitive to the shifting effects of light. Overall, this rodent can be considered a new and interesting model for circadian rhythm neurobiology.