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  • The eye of the blind mole rat, spalax ehrenbergi, a behavioural and molecular analysis

    27 November 2018

    Purpose. To investigate arcadian responses to light and identify and characterise the photopigments within the atrophied non-image forming eye of the blind mole rat Methods. Using mouse specific green cone opsm pnmers, RT-PCR was used to isolate a green-like cone opsm cDNA clone from mole rat ocular RNA. Mole rat specific PCR primers were designed and 3′ and 5′ RACE PCR were earned out to isolate the full coding region of the gteen-like cone opsm By exposing mole rats to differing light/dark (L/D) cycles, and monitoring wheel running activity as an assay of circadian behaviour, the effects of light on photoentrainment were determined 18 adult mole rats were exposed to the following lighting regime, 42 days at 12L/12D cycle (lights on at 7am), 30 days in constant darkness (DD); 30 days at 12L, 12D (lights on at 7am), 45 days 12L/12D cycle with a phase advance of 6 hours (lights on at Ipin) Results. The deduced ammo acid sequence of the mole rat green-like cone opsm shows a high percentage identity with both mouse (86%) and human (85%) green cone opsms The opsm has seven transmembrane domains and all the known structural features required for a functional photopigment These include a putative Schiff base lysine, glutamine countenon, and a paii of cysteines required for disulphide bond formation. Under DD animals showed freerunning rhythms significantly different from 24 hours All animals showed stable entramment to the differing L/D cycles after 5-7 days Enucleated animals failed to entrain to a L/D cycle Conclusions. The subcutaneous location and degenerate nature of the mole rat eye preclude image formation However, blind mole rats with entrain and phase shift their arcadian locomotor rhythms to a L/D cycle. To date only a single green-like cone opsm has been isolated from the mole rat eye Our findings are consistant with previous studies which have implicated green cone and short wave sensitive opsins in rodent photoentrainment We are currently looking for additional opsins in the mole rat eye. Wellcome Trust to RGF none.

  • Localization of opsins in the photoreceptor systems of Anolu. carolinensis

    27 November 2018

    Purpose. Four photorecepior organs -the lateral and parietal eyes, the pineal organ, and the deep brain photoreceptor - are ecogmsed in the lizard Anolis carolinensis, mediating visual and circadian responses to light. Other investigators have identified and sequcnced five different opsins in the Anolis genome. We have studied the expression of these genes in the eye, pinral, and brain. Methods Total RNA was used for the synthesis of cDNA by Supeiscnpt II. Primers were designed lor the specific amplification of rhodopsin, L opsin, rhodopsm-likc M opsin, SI opsin, and S2 opsin, as well as 16S rRNA as a positive control, and used for PCR amplification of the different samples. Results. The different opsins were localized to the dillcrenl photoreceptor organs as follows. PCR pr xlucts were subcloned for sequencing in order to determine specificity: Eye Pineal Brain Rhodopsin + + + L opsin + - - Rhodopsin-like M opsin + - - 51 opsin - + - 52 opsin + + - Conclusions. The detection of rhodopsin mRNA in the rod less Anolis retina confirms immunocytochemical data publ shed by other investigators. The finding of four different photopigments in the eye b consistent with its role in colour vision. Among the circadian photoreceptor systems, we find S opsins in the superficially localized pineal, but not in the deep brain photorecepiors which are unlikely to be exposed to light of these wavelength because of the overlaying tissues. Experiments desiened In confirm our results by RNAse nrntertion assays are in progress.

  • Molecular dissection of the coneless transgenic mouse retina

    27 November 2018

    Purpose. In addition to their role in vision, cone cells are strong candidates for mediating the arcadian responses to light in mammals. Using confess transgenic (Cl) mice (generously donated by J. Nathans and Y. Wang, Johns Hopkins University) consisting of 6.2kb of the human red cone promoter, linked to the coding region of an attenuated diphtheria toxin gene, we have begun a molecular characterization of the retina in order to determine the extent of this genetic lesion, in animals of 45 days of age. Methods. We used Reverse Transcriptase-Polymerase Chain Reaction (RTPCR) to amplify cone-specific messages from retinal cDNA. Various sets of primers designed to amplify cone-specific transcripts were used for PCR amplification. Reanlta. RT-PCR using primers specific for either green cone or UV cone opsins resulted in a product only for the latter, whereas both messages were amplified in the wild type. Further investigation indicates no presence of C-arrestin or a putative green cone-specific gamma phosphodiesterase in the Cl retina. Alpha-2-transduän and alpha phosphodiesterase, which are found in all cone types, were amplified in both wild type and Cl cDNA. Subsequent cloning and sequencing of these products will confirm their identity. Conclusions. No messages specific for green cones could be detected in cDNA from the CI retina. Messages expressed in UV cones were present, although at reduced levels. We intend to extend the analysis to include animals of different ages. Future work will also involve analyzing arcadian responses to light of these animals, and we will correlate this molecular study with the results of the behavioural investigation in order to elucidate exactly how these mammals regulate their biological clock.

  • An in vivo comparison of retinal and pineal melatonin production in C3H mice with normal and degenerate retinas

    27 November 2018

    Purpose. To examine the effects of genetic retinal lesions on the ability of the eyes and pineal to produce melatonin and to regulate melatonin synthesis according to environmental light. Methods. This work compared C3H mice which were either +/+, homozygous for nlavrus alleles or carrying a transgene which selectively destroys cone pholoreceptors ( kindly donated by J Nathans and Y Wang, Johns Hopkins University). Pineals and eyes were dissected from animals at around 80 days of age, homogenised by exposure to ultrasound and assayed for melatonin using a direct radio immunoassay (after Fraser étal 1983. J.CIinical Chem 29:396-397). Tissue was collected at 2 hour intervals over 24 hours, from animals exposed to a 12:12 light:dark cycle. Results. Diurnal rhythms in+/+ pineal and retinal melatonin were observed tinder these conditions. In both eyes and pineals, melatonin content was low during the day. and peaked at night. Typically, maximal melatonin contents were around 400pg/pineal gland and around 50pg/pair of eyes. The diumal profile of melatonin production in the eye and pineal were significantly different in vivo. Pineal melatonin content was enhanced for a brief period during the second half of the night. By contrast, melatonin levels in the eye remained elevated for much of the dark period. Conclusions. There are significant differences in the diurnal profile of melatonin production in the eyes and pineal in viva. We are currently assessing the impact of photoreceptor degeneration on the ability of eyes and pineals to produce melatonin and to inhibit its synthesis on exposure to light.

  • Identifying the photoreceptive inputs to the mammalian circadian system using transgenic and retinally degenerate mice.

    27 November 2018

    The endogenous circadian clock of mammals retains synchrony with the external light:dark cycle through ocular photoreceptors. To date the identity of the photoreceptors responsible for mediating this response is unknown. This review outlines attempts using transgenic mouse models to address this deficit. Mice bearing specific inherited lesions of both rod and cone photoreceptors retain circadian photosensitivity as assessed by photoentrainment of behavioural rhythms and the light-induced suppression of pineal melatonin. These findings indicate that as yet unidentified non-rod, non-cone ocular photoreceptors are capable of contributing to circadian light responses. Nevertheless, the possibility that circadian photosensitivity is the responsibility of multiple photoreceptor classes including both rod/cone and novel photopigments remains. There is some indirect evidence in favour of this hypothesis. A definitive resolution of this issue is likely to employ comparisons of circadian action spectra in wild type and retinally degenerate mice.

  • The putative brain photoperiodic photoreceptors in the vetch aphid, Megoura viciae

    27 November 2018

    In an attempt to identify the brain photoreceptors that mediate the photoperiodic response of the vetch aphid, Megoura viciae, we utilised immunocytochemical techniques and employed 20 antibodies directed against invertebrate and vertebrate opsins and phototransduction proteins. A sub-set of these antibodies (to Drosophila rhodopsin 1: RH1-1; vertebrate cone opsins: COS-1; CERN-874; CERN-933; vertebrate rod opsin: CERN-901; vertebrate arrestin: AB-Arr; vertebrate transducin+arrestin+rhodopsin kinase+cGMP phosphodiesterase: CERN-911; and vertebrate cellular retinoid binding protein: CRALBP) consistently labelled an anterior ventral neuropile region of the protocerebrum. These anatomical findings, coupled with previous localised illumination and micro-lesion studies, provide strong evidence that this region of the aphid brain houses the photoperiodic photoreceptors. The present study also confirms that the medial (Group I) neurosecretory cells are not the photoperiodic photoreceptors.

  • Antibodies against retinal photoreceptor-specific proteins reveal axonal projections from the photosensory pineal organ in teleosts.

    27 November 2018

    With the aid of specific antisera to the retinal proteins S-antigen and alpha-transducin and to the rhodopsin apoprotein opsin, we have labeled various cell populations in the pineal organ, parapineal organ, habenular nucleus, and subcommissural organ in two teleost species: the rainbow trout and the European minnow. Although these proteins are associated with photoreceptor functions, not only photoreceptor cells but also the majority of parenchymal cells in the pineal organ were immunoreactive. Immunoreactive cells with dendrite- and axonlike processes were observed also in the parapineal organ and the habenular nucleus. Furthermore, S-antigen-immunoreactive, long, axonal processes were observed in the pineal organ and could be traced from the pineal organ to the habenular nucleus and to the pretectal area. In the light of recent HRP electron microscopical and immunocytochemical studies we propose (1) that not only the classical pineal photoreceptor cells of poikilothermic vertebrates but also other types of CSF-contacting neurons may be the phylogenetic ancestors of mammalian pinealocytes, and (2) a close interrelationship between the pineal organ and the limbic system, effectuated by the direct projections from pineal photoreceptors/CSF-contacting neurons/pinealocytes to the habenular nucleus, and by displaced "pinealocytelike" elements scattered in the habenular nucleus.

  • Napping facilitates word learning in early lexical development

    27 November 2018

    © 2015 European Sleep Research Society. Little is known about the role that night-time sleep and daytime naps play in early cognitive development. Our aim was to investigate how napping affects word learning in 16-month-olds. Thirty-four typically developing infants were assigned randomly to nap and wake groups. After teaching two novel object-word pairs to infants, we tested their initial performance with an intermodal preferential looking task in which infants are expected to increase their target looking time compared to a distracter after hearing its auditory label. A second test session followed after approximately a 2-h delay. The delay contained sleep for the nap group or no sleep for the wake group. Looking behaviour was measured with an automatic eye-tracker. Vocabulary size was assessed using the Oxford Communicative Development Inventory. A significant interaction between group and session was found in preferential looking towards the target picture. The performance of the nap group increased after the nap, whereas that of the wake group did not change. The gain in performance correlated positively with the expressive vocabulary size in the nap group. These results indicate that daytime napping helps consolidate word learning in infancy.

  • In the happy realms of light

    12 February 2019

    Rods and cones are not the only photoreceptors of the eye. There also exists a population of directly light sensitive ganglion cells that regulate a wide variety of different 'brightness detection' tasks, they may even adjust our mood and sense of well being.