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Identification of circadian brain photoreceptors mediating photic entrainment of behavioural rhythms in lizards

Authors

  • Massimo Pasqualetti,

    1. Laboratori di Biologia cellulare e dello Sviluppo, Università di Pisa, Via G. Carducci 13, Pisa, Italy
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    • M.P. and C.B. contributed equally to this work.

  • Cristiano Bertolucci,

    1. Dipartimento di Biologia e Centro di Neuroscienze, Università di Ferrara, via L. Borsari 46, I-44100, Ferrara, Italy
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    • M.P. and C.B. contributed equally to this work.

  • Michela Ori,

    1. Laboratori di Biologia cellulare e dello Sviluppo, Università di Pisa, Via G. Carducci 13, Pisa, Italy
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  • Augusto Innocenti,

    1. Dipartimento di Biologia e Centro di Neuroscienze, Università di Ferrara, via L. Borsari 46, I-44100, Ferrara, Italy
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  • Maria C. Magnone,

    1. Dipartimento di Biologia e Centro di Neuroscienze, Università di Ferrara, via L. Borsari 46, I-44100, Ferrara, Italy
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    • *

      Present address: Institut de Biochimie, Université de Fribourg, Ch. du Musée, 5, 1700, Fribourg, Switzerland.

  • Willem J. De Grip,

    1. Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, University of Nijmegen, Nijmegen, the Netherlands
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  • Irma Nardi,

    1. Laboratori di Biologia cellulare e dello Sviluppo, Università di Pisa, Via G. Carducci 13, Pisa, Italy
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  • Augusto Foà

    1. Dipartimento di Biologia e Centro di Neuroscienze, Università di Ferrara, via L. Borsari 46, I-44100, Ferrara, Italy
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: Dr Augusto Foà, as above.
E-mail: foa@unife.it

Abstract

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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