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The daily pattern of locomotor activity of the ruin lizard Podarcis sicula in its natural environment changes from unimodal in spring (with only one activity peak per day) to bimodal in summer (with two well-separated activity peaks per day) and it becomes unimodal again in autumn. In order to establish whether such seasonal changes in pattern might be at least in part controlled by endogenous temporal programs, lizards were collected at different times of the year and immediately after capture their locomotor behavior was tested in the laboratory under constant temperature (29°C) and in darkness. For some individuals tested in the laboratory the locomotor pattern previously expressed in the field was known. Seasonal differences in pattern have been unequivocally found to have an endogenous component, as most lizards in constant conditions retained the locomotor pattern shown in the field during the same season. Besides, in the bimodal lizards the freerunning period of locomotor rhythms (z) was significantly shorter and circadian activity time (~) longer than in the unimodal ones. Altogether the data are compatible with the idea that both the interdependent changes of • and ~ and the changes in locomotor pattern occurring seasonally in the circadian activity rhythms of P. sicula would depend on changes in the phase relationship between mutually coupled oscillators which drive these rhythms.

We have shown previously that in ruin lizards (Podarcis sicula) the ablation of all known photoreceptive structures (lateral eyes, pineal and parietal eye) in the same individual animal does not prevent entrainment of their circadian locomotor rhythms to light. The present study was aimed at identifying the circadian brain photoreceptors mediating entrainment. For this purpose, we looked for opsin expression in the brain by means of immunocytochemistry. Using anti-cone-opsin antiserum CERN 874 we have localized photoreceptors in the periventricular area of hypothalamus, near the third cerebral ventricle. We also cloned a brain opsin cDNA that, on the basis of the deduced amino acid sequence, appears to belong to the RH2 class of cone-opsins. We named the cloned cone-opsin Ps-RH2. To examine whether brain cone-opsins mediate photic entrainment of circadian locomotor rhythms, we performed post-transcriptional inactivation experiments by injecting an expression eukaryotic vector transcribing the antisense cone-opsin Ps-RH2 mRNA in the third cerebral ventricle of pinealectomized-retinectomized lizards previously entrained to a light-dark (LD) cycle. Injections of the antisense construct abolished photic entrainment of circadian locomotor rhythms of pinealectomized-retinectomized lizards to the LD cycle for 6-9 days. CERN 874 completely failed to label cells within the periventricular area of hypothalamus of brains injected with antisense construct. Thus, abolishment of photic entrainment is due to inactivation of endogenous brain cone-opsins mRNA. The present results demonstrate for the first time in a vertebrate that brain cone-opsins are part of a true circadian brain photoreceptor participating in photic entrainment of behavioural rhythms.

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