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Effects of Gap Junction Blockers on Circadian Regulation of Gene Expression in Embryonic Retinal Explant Cultures


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EFFECTS OF GAP JUNCTION BLOCKERS ON CIRCADIAN REGULATION OF GENE EXPRESSION IN EMBRYONIC RETINAL EXPLANT CULTURES By YAN ZHANG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Yan Zhang

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To my beloved parents who always provide the strongest supp ort, the greatest encouragement, and the most sincere advice. To all the kind people who have guided, ta ught, and assisted me through twenty-five years of education

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ACKNOWLEDGMENTS First, I would like to thank my mentor, Dr. Susan L. Semple-Rowland, for her inspiring direction and careful training. She is a great educator and a dedicated scientist. I feel very fortunate to work under her supervision and obtained rapid progress in research during my Ph.D. study. Next, I thank my committee members, Dr. Neil Rowland, Dr. Marieta Heaton, and Dr. Barbara Battelle, for their helpful suggestions on my research. I also want to thank all the past and current members of Dr. Semple-Rowlands laboratory for their friendships and assistances. In particular, I thank Dr. Jason Coleman for the help with immunocytochemistry experiment and the provocative discussions on my research and career choice, Daniel Selbst for the assistance in RNA extraction, Gabby Fuchs for the assistance in Cresyl Violet staining of retinal explant cultures, and Miguel Tepedino for his initial teaching of Northern Blot analysis. Finally, I would express my deep appreciation for my parents for their great support and encouragement, without which I would not have been able to successfully complete twenty-five years of education. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF FIGURES ..........................................................................................................vii ABSTRACT .....................................................................................................................viii CHAPTER 1 INTRODUCTION........................................................................................................1 General Features of Circadian Rhythms.......................................................................1 Basic Properties of Circadian Rhythms.................................................................1 Molecular Bases of Circadian Clocks...................................................................2 Light Entrainment of Circadian Clocks.................................................................4 Circadian Clocks in the Central Nervous System (CNS)......................................5 Circadian Rhythms In Retina........................................................................................6 Retinal Functions Regulated by Circadian Clocks................................................6 Cellular Location of Circadian Clocks Within Retina..........................................7 Light Entrainment of Retinal Photoreceptor Clocks.............................................8 Synchronization of Circadian Clocks...........................................................................9 Synchronization of Circadian Clocks in SCN.......................................................9 Expression and Function of Gap Junctions in Retina..........................................11 2 CHARACTERIZATION OF CIRCADIAN OSCILLATOR FUNCTION IN EMBRYONIC RETINA AND RETINAL EXPLANT CULTURES........................14 Introduction.................................................................................................................14 Materials and Methods...............................................................................................16 Preparation of Retinal Explant Cultures..............................................................16 Explant Morphology............................................................................................17 Iodopsin Gene and Protein Expression in Retinal Explant Cultures...................18 Lighting Paradigms.............................................................................................19 Cyclic light...................................................................................................19 Constant dark................................................................................................19 Reversal of the light cycle............................................................................19 RNA Analyses.....................................................................................................20 Results.........................................................................................................................21 Morphology of Developing Retinal Explant Cultures.........................................21 v

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Comparison of Iodopsin Expression Onset in Retinal Explant Cultures and in ovo....................................................................................................................25 Iodopsin Transcript Rhythms..............................................................................27 Cyclic light...................................................................................................27 Constant dark................................................................................................29 Reversal of the light:dark cycle....................................................................31 Discussion...................................................................................................................33 3 GAP JUNCTION BLOCKERS ABOLISH CIRCADIAN RHYTHMS OF GENE EXPRESSION IN RETINAL PHOTORECEPTORS................................................36 Introduction.................................................................................................................36 Methods and Materials...............................................................................................39 Chemicals and Reagents......................................................................................39 Retinal Explant Cultures......................................................................................40 Lighting and Blocker Delivery Paradigms:.........................................................40 12L:12D:......................................................................................................40 12L:12D followed by constant darkness:.....................................................41 RNA Analysis......................................................................................................41 Results.........................................................................................................................42 Iodopsin and AANAT Transcript Rhythms in Untreated Explant Cultures:......42 Iodopsin Transcript Levels in Explant Cultures Treated With ACO For 48 Or 24 hrs Maintained Under A 12L:12D Cycle...............................................45 Iodopsin Transcript Levels in Explant Cultures Treated With ACO For 24hr Or 12hr Followed By Constant Darkness........................................................47 Effects of ACO Treatment On AANAT and GCAP1 Transcript Levels............49 Iodopsin Transcript Levels in Explant Cultures Treated With 18-GA For 48 Or 24 hrs Maintained Under A 12L:12D Cycle...............................................52 Discussion...................................................................................................................55 4 PROSPECTIVE..........................................................................................................59 A Real-Time Monitoring Culture System For Circadian-Regulated Gene Expression..............................................................................................................59 Possible Mechanisms of Light Entrainment in Embryonic Chicken Retinas.............61 REFERENCE LIST...........................................................................................................65 BIOGRAPHICAL SKETCH.............................................................................................75 vi

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LIST OF FIGURES Figure page 2-1 Morphological comparison of chicken embryonic retina and retinal explant cultures.....................................................................................................................22 2-2 Quantification of the morphological integrity of retinal explant cultures................23 2-3 TH immunostaining of explants and embryonic and post-hatch retinas..................25 2-4 Onset of iodopsin expression in retinal explant cultures..........................................26 2-5 Iodopsin transcript rhythms in explant cultures and embryos maintained under 12L:12D and constant dark conditions.....................................................................28 2-6 Iodopsin transcript rhythms in explant cultures and embryos following reversal of the light:dark cycle...............................................................................................31 3-1 Iodopsin mRNA rhythms in retinal explant cultures...............................................43 3-2 AANAT mRNA rhythms in retinal explant cultures...............................................44 3-3 Iodopsin expression in explant cultures treated with ACO for 48 and 24hrs..........46 3-4 Iodopsin expression in explant cultures treated with ACO followed by constant darkness....................................................................................................................48 3-5 AANAT expression in explant cultures treated with ACO for 24hrs......................49 3-6 GCAP-1 expression in explant cultures treated with ACO for 48 or 24 hrs and maintained under 12L:12D conditions.....................................................................51 3-7 Iodopsin expression in explant cultures treated with 18-GA for 48 and 24hrs and maintained under 12L:12D conditions..............................................................53 3-8 Iodopsin expression in explant cultures treated with GA for 48hrs.........................54 4-1 Iodopsin transcript rhythms in GUCY1*B and White Leghorn chicken retinal explant cultures maintained under 12L:12D followed by reversal of the light cycle.........................................................................................................................63 vii

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECTS OF GAP JUNCTION BLOCKERS ON CIRCADIAN REGULATION OF GENE EXPRESSION IN EMBRYONIC RETINAL EXPLANT CULTURES By Yan Zhang May 2004 Chair: Susan L. Semple-Rowland Major Department: Neuroscience The suprachiasmic nucleus (SCN) is comprised of autonomous, single-cell oscillators that work in concert to generate coordinated circadian rhythms. Substantial evidence demonstrates that intercellular gap junction communication plays a role in the coordination of circadian rhythms in SCN. Chicken retina also contains functional circadian oscillators that drive coordinated transcript rhythms of several photoreceptor-specific genes, including iodopsin and Arylalkylamine N-acetyltransferase (AANAT), tryptophan hydroxylase. Based on the studies in SCN, we hypothesized that gap junction communication also plays a role in coordinating transcript rhythms of circadian-regulated genes in retina To test this hypothesis, we first established a chicken embryonic retinal explant culture system in which robust, self-sustaining, and light-entrainable iodopsin transcript rhythms were observed under different lighting conditions. Although iodopsin transcription in retinas of chicken embryos is primarily driven by light, the functional viii

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characteristics of circadian oscillators driving iodopsin transcript rhythms in culture are similar to those found in post-hatch chicken retina, an observation that supports use of the explant cultures in our study. The role that gap junctions play in coordinating rhythms in retinas was examined using two reversible gap junction blockers, 18glycyrrhetnic acid-3-hemisuccinate (ACO) and 18glycyrrhetnic acid (18-GA), which were applied to the explant cultures maintained under different lighting conditions. Both gap junction blockers produced a rapid and persistent reduction of iodopsin and AANAT transcript levels. Following removal of the blockers, the transcript rhythms of both genes reappeared within a 24 hr period. Our data suggest that the change in iodopsin mRNA levels was not due to disruption of the function or the phase of the circadian oscillators driving the iodopsin rhythms. These blockers may either directly uncouple the circadian oscillators from driving transcription of these genes or alter the stability of these transcripts in the photoreceptors cells. ix

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CHAPTER 1 INTRODUCTION General Features of Circadian Rhythms Basic Properties of Circadian Rhythms Circadian rhythms are self-sustained cyclic changes in physiological processes or behavioral functions that have a period of approximately 24 hours (Chang and Reppert 2001). A circadian system is comprised of three components: a core circadian oscillator that acts like a ticking clock to produce self-sustained rhythmic changes, output pathways through which the oscillators regulate physiological and behavioral functions, and input pathways through which the oscillators are synchronized or entrained to environmental time cues (Dunlap 1999). Circadian rhythms are virtually ubiquitous, controlling a myriad of physiological processes in organisms ranging from spore production in fungi, leaf movement in plants, eclosion in insects, rest:activity cycles in animals, and sleep:wake cycles in humans. Although the physiological processes regulated by circadian clocks may vary between organisms, these rhythms share three basic properties. First, the rhythms are self-sustaining and persist or free-run under constant conditions. Second, the rhythms can be entrained to environmental stimuli, light being the dominant and most potent entraining stimulus. Finally, the rhythms are temperature compensated in the sense that the period of the rhythm stays constant over a range of ambient temperatures (Reppert and Weaver 2001). 1

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2 Molecular Bases of Circadian Clocks The molecular mechanism driving core circadian oscillators consists of interlocking transcription-translation feedback loops. This mechanism is best understood in the fruit fly, Drosophila melanogaster, and in the mouse suprachiasmatic nuclei (SCN). In Drosophila, seven genes that encode the proteins involved in these molecular feedback loops have been identified, period (per), timeless (tim), Drosophila clock (dClk), cycle (cyc), double-time (dbt), shaggy (sgg), and vrille (vri) (Blau and Young 1999;Martinek et al. 2001;Young 2000). In one transcription-translation feedback loop, the basic helix-loop-helix (bHLH)PER-ARNT-SIM (PAS) domain-containing transcription factors, dCLK and CYC, form heterodimers that target E-box regulatory cis elements (CACGTG) located in the promoters of the per and tim genes. Binding of the dCLK and CYC heterodimers to the promoters drives the rhythmic transcription of the per and tim genes (Glossop et al. 1999). As the per and tim mRNAs are translated into PER and TIM proteins, TIM and PER proteins begin to accumulate; however, this process is somewhat slowed by the binding of PER proteins to the constitutively expressed kinase DBT, an interaction that leads to the phosphorylation and degradation of PER. PER is stabilized when TIM protein accumulates to levels sufficient to bind the PER/DBT heterodimers. The formation of the TIM-PER-DBT trimeric protein complex allows it to enter the cell nucleus (Price et al. 1998). Contrary to the effect of DBT, which retards the accumulation of PER and the entry of PER and TIM into the nucleus, the phosphorylation of TIM by the glycogen synthase kinase-3, SGG, accelerates PER/TIM heterodimerization and subsequent translocation into the nucleus (Martinek et al. 2001). Once in the nucleus, the PER/TIM/DBT protein complex interacts with dCLK-CYC heterodimers to reduce the activation of per and tim transcription (Darlington et al. 1998). In the other transcription

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3 translation feedback loop, dCLK and CYC heterodimers repress dClk transcription either directly or via intermediate factors. The binding of PER-TIM-DBT heterodimers to dCLK-CYC heterodimers releases dCLK-CYC dependent repression of dClk transcription, thereby allowing separate activator(s) to activate transcription of the dClk gene (Glossop et al. 1999). In mouse SCN, eight clock genes contribute to the autoregulatory feedback loops that define the core oscillator. One transcription-translation feedback loop involves the dynamic regulation of three period genes, designated mper1, and two cryptochrome genes, known as designated mcry1 and mcry2. Rhythmic transcription of the mper and mcry genes is driven by mCLOCK and mouse mBMAL1 heterodimers. Mouse BMAL1 is a homolog of Drosophila CYC. As the mPER and mCRY proteins are translated, they form multimeric complexes that are translocated to the nucleus. In the nucleus, the mCRY component of the multimer acts as a negative regulator by directly interacting with the mCLOCK: BMAL1 heterodimers and inhibiting the transcription of mper and mcry genes (Reppert and Weaver 2001). Mouse PERs, unlike their counterpart in Drosophila, do not play a critical role in transcriptional inhibition. Instead, mPER1 affects the function of the clock at the post-transcriptional level, presumably through protein-protein interactions that affect the stability and nuclear entry of other clock proteins (Bae et al. 2001). mPER2 drives the rhythmic transcription of mbmal1, which exhibits a phase opposite to that of mper and mcry, forming a positive transcriptional loop. Increased availability of BMAL1 presumably promotes the formation of the CLOCK:BMAL1 heterodimers that are required to restart the mper and mcry

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4 transcription cycle (Shearman et al. 2000). At this point, it appears that mPER3 protein is not essential for the maintenance of circadian rhythmicity (Bae et al. 2001). Light Entrainment of Circadian Clocks Many of the circadian clocks that drive rhythmic processes and behaviors are synchronized (entrained) to the daily changes that occur in the levels of ambient light that occur between dawn and dusk. The imposition of an artificial 12 hour light:12 hour dark (12L:12D) cycle on these clocks forces them to run with a period near 24 hours that is in phase with the light/dark cycle. The phase of the clock is defined by the cyclic changes that occur in the levels of the various proteins that comprise the clock over the course of a single 24-hour light/dark cycle. Light entrainment is a dynamic process. A change in the light/dark cycle causes changes in the concentrations of the clock proteins, which in turn produce a shift in the phase of the clock so that it is properly synchronized to the new light/dark cycle. The direction and amplitude of the phase shift of the clock are determined by the magnitude of the change in the concentration of the clock proteins that is induced by the new light cycle (Devlin and Kay 2001). For example, in Drosophila, levels of TIM protein can be directly modulated by light as a result of TIMs interaction with the flavoprotein CRYPTOCHROME (CRY). Light pulses delivered during the dark period trigger the degradation of TIM and reset the phase of the clock to a point in the cycle where the concentration of TIM would normally be low (Young 2000). In mouse SCN, the transcription of mper1 and/or mper2 genes has been shown to be rapidly induced by light pulses delivered during the subjective night, results that suggest that mPER1 and/or mPER2 protein may be involved in mediating light entrainment of the mammalian clock (Albrecht et al. 1997;Okamura et al. 1999;Shearman et al. 1997). The response of the circadian clock to the changes of light stimuli varies over the course of

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5 the day. In general, light pulses delivered in late afternoon or early night delay the phase of the clock, light pulses administered in late night or early morning result in phase advances, and light pulses delivered during the middle of the day are relatively ineffective in inducing changes in the phase of the clock (Rosenwasser and Dwyer 2001). Circadian Clocks in the Central Nervous System (CNS) In mammals, the master circadian "clock" that controls physiological and behavioral rhythms resides in the suprachiasmatic nuclei (SCN) that are located within the anterior hypothalamus. The clock in the SCN is composed of multiple, autonomous, single-cell circadian oscillators that receive information about ambient light levels directly from the eyes via the retinohypothalamic tracts (Green 1998). Synchronization of these oscillators by light permits the SCN to generate a coordinated circadian output that is capable of regulating overt rhythms (Reppert and Weaver 2001). One of the more important rhythms regulated by the SCN is the production of melatonin by the pineal gland. Two types of regulatory signals are transmitted to the pineal gland through efferents from SCN. One is the permissive signal that originates from the circadian clock and restricts melatonin production to the night. The other is the inhibitory signal that is induced by inappropriate light stimuli at night and acutely suppresses nocturnal melatonin production (Gillette and McArthur 1996). Thus, in mammals, the eye, the SCN and the pineal body function in sequence as photoreceptor, master circadian oscillator, and melatonin output organ for maintaining circadian rhythms at the organism level. In avian species, the eye, the pineal gland, and deep brain structures including the hypothalamus and SCN have all been implicated in the regulation of behavioral rhythms, although diversity among avian species is great. In the House sparrow and the Java sparrow, pinealectomy abolishes free-running circadian rhythms under constant

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6 conditions, observations that indicate that the pineal gland is the site of an essential circadian clock in sparrows (Gaston and Menaker 1968). But in Japanese quails, pinealectomy does not have a significant effect on circadian locomotor activity rhythms (Underwood 1994). The eyes (Underwood 1994) and the hypothalamus (Simpsom and Follett 1981) of the Japanese quail have been suggested to be the locations of the major circadian oscillators in this species. The eyes, pineal, and SCN must all be removed to abolish circadian locomotor rhythms in pigeons, a result that indicates that all of these organs are necessary for maintaining circadian rhythmicity in pigeon (Ebihara et al. 1984). Based on these observations, it has been suggested that the circadian system in the avian CNS contains multiple oscillators comprised of the pineal gland, the eyes, and deep brain structures that include the SCN (Oishi et al. 2001). Circadian Rhythms In Retina Retinal Functions Regulated by Circadian Clocks There is now substantial evidence that vertebrate retinas contain circadian clocks and that these clocks play an important role in maintaining the function and health of the retina (Moog 1995). These functions include the synthesis and release of neuromodulators such as melatonin (Cahill et al. 1991;Tosini 2000) and dopamine (Besharse and Iuvone 1992), photoreceptor disc shedding and phagocytosis by the retinal pigment epithelium (RPE) (Nguyen-Legros and Hicks 2000), retinomotor movement (Burnside 2001), gene expression (Pierce et al. 1993; Green and Besharse 1994; Green et al. 1996; Larkin et al. 1999; Chong et al. 2000), and visual sensitivity (Li and Dowling 2000).

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7 Cellular Location of Circadian Clocks Within Retina Currently, the most direct evidence for the presence of functional circadian clocks in a specific retinal cell type comes from studies of melatonin synthesis in reduced Xenopus retina cultures. Melatonin in the retina is produced by the retinal photoreceptor cells and is regulated by circadian clocks located in the retina. By monitoring melatonin release from the photoreceptor cultures under constant dark conditions, Cahill and Besharse (Cahill and Besharse 1992;1993) were able to show that the circadian oscillators controlling melatonin rhythms in Xenopus retina are localized to the photoreceptor cells. The observations that the activities of tryptophan hydroxylase (TPH) and serotonin N-acetyltransferase (AA-NAT), two key enzymes in the melatonin biosynthesis pathway, are both expressed in Xenopus photoreceptors and are also under the control of a circadian oscillator are consistent with the localization of circadian clocks to these cells (Besharse and Iuvone 1983;Green and Besharse 1994;Green et al. 1995). Indirect support for the presence of circadian oscillators in photoreceptor cells comes from studies of low-density cultures of avian retina. Iodopsin is a red-sensitive pigment that is expressed in cone photoreceptors in the retinas of birds (Yoshizawa and Kuwata 1991). The transcription of the iodopsin gene in chicken retina has been shown to be regulated by a circadian clock (Pierce et al. 1993;Larkin et al. 1999). The observations that transcription of the iodopsin gene exhibits a circadian rhythm in low density cultures of both quail (Pierce 1999) and chicken retina (Pierce et al. 1993) suggest that the circadian clocks driving expression of this gene are located in the cone photoreceptor cells. If circadian clocks are present in photoreceptor cells, then the clock genes should be expressed in these cells. In mouse, transcripts encoding mPER1 (Shearman et al.

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8 1997;Sun et al. 1997;Sun et al. 1997;Shearman et al. 1997), mPER2 (Shearman et al. 1997), mPER3 (Zylka et al. 1998), mTIM (Zylka et al. 1998), mClOCK (Gekakis et al. 1998) and BMAL1 (Gekakis et al. 1998) have been isolated from the retina. In situ hybridization analyses of mouse retina have revealed that mclock, mper1 and mbmal1 are co-expressed in retinal photoreceptors, cells within the inner nuclear layer and in the ganglion cell layer (Gekakis et al. 1998). In Xenopus, XClock (Zhu et al. 2000), Xper2 (Zhuang et al. 2000), and three cryptochromes genes (Xcry1, Xcry2a, Xcry2b) (Anderson and Green 2000) have been cloned and have been shown to be expressed in retina. Many of these clock genes are also expressed in the retinas of birds. Transcripts encoding cCLOCK (Larkin et al. 1999;Chong et al. 2000;Larkin et al. 1999), cBMAL1 and cMOP4 (Chong et al. 2000) have been isolated from chicken retina and transcripts encoding qCLOCK, qPER2 and qPER3 have been isolated from quail retina (Yoshimura et al. 2000). Taken together, the results of the studies of the expression of clock genes and melatonin secretion in retina suggest that vertebrate photoreceptor cells contain functional circadian clocks and output pathways. Light Entrainment of Retinal Photoreceptor Clocks Most information about the responses of retinal circadian clocks to light comes from analyses of processes within the retina that are regulated by these clocks. Studies of melatonin and iodopsin synthesis, both of which are produced by photoreceptor cells, suggest that retinal photoreceptors contain functional input pathways that allow entrainment of the oscillators in these cells to light. For example, in their studies of reduced Xenopus retina cultures, Cahill and Besharse (Cahill and Besharse 1992;1993) noted that the phase of the melatonin rhythms in photoreceptor cells could be reset by

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9 light. In chicken retina, the rhythms of iodopsin transcription in cone photoreceptors can be entrained to the external cyclic lighting conditions and the phase of the rhythms can be shifted by 6 hour light pulses delivered during the dark period (Larkin and Semple-Rowland 2001). These observations together with those mentioned above suggest that photoreceptor cells contain a complete circadian system. Synchronization of Circadian Clocks Synchronization of Circadian Clocks in SCN The mammalian SCN contains 20,000 neurons that work in concert to drive the coordinated circadian rhythms of electrical activity (Herzog et al. 1997), gene expression(Panda et al. 2002), glucose metabolism (Schwartz et al. 1983), and behavior (LeSauter and Silver 1998;Herzog et al. 1997). Individual neurons in SCN dissociated cultures exhibit self-sustained rhythms of spontaneous firing activity, suggesting that functional circadian oscillators are present within these single neurons. However, the firing rhythms of individual oscillators exhibit variable periods and different phase relationships with one another under these culture conditions (Welsh et al. 1995). In SCN explant cultures, in which synapses and cellular appositions are largely preserved, the firing rhythms of individual SCN neurons exhibit periods with significantly less variability and are in phase with each other. Furthermore, the range of the periods in explants is almost identical to that observed for behavioral rhythms (Herzog et al. 2001). These observations suggest that intercellular communication is required to generate and maintain coordinated circadian rhythms in SCN. Synapseand gap junction-mediated intercellular communications represent two possible mechanisms that could synchronize populations of autonomous oscillator cells Accumulating indirect evidence suggests that synaptic transmission does not play a

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10 central role in the synchronization of the circadian oscillators within the SCN neurons. For example, circadian glucose metabolism in the SCN is observed before chemical synapses become functional in the SCN (Reppert and Schwartz 1984;Moore and Bernstein 1989). Consistent with this observation are the observations that disruption of synaptic transmission within the SCN using either tetrodotoxin (TTX) (Shibata and Moore 1993) or calcium-free medium (Bouskila and Dudek 1993) do not alter the ability of the SCN neurons to generate synchronized bursts of activity. Gap junction channels provide another means for intercellular communication. Gap junction channel consists of a hemichannel (a connexon) in the membrane of one cell that is paired with a similar hemichannel in another adjoining cell. A hydrophilic pore at the core of the connexon allows the passage of small ions and low molecular weight metabolites (up to 1 kD) between the cells and functions to connect the cells both electrically and metabolically. Each connexon, in turn, is composed of 6 similar protein subunits known as connexins (Cx). The connexins are members of a multigene family. Connexins 26, 32 and 43 are the first members of this family that are identified and are the most abundant isoforms expressed in the developing CNS (Cook and Becker 1995) Gap junction communication has been shown to play a role in the coordination and synchronization of the activity of SCN neurons in vivo. Studies of connectivity of SCN neurons show that SCN neurons are coupled by low resistance pathways (Colwell 2000;Shinohara et al. 2000;Jiang et al. 1997), the permeability of which are modulated by cell activity. Studies using dyes capable of traversing gap junctions show that SCN cells are extensively dye coupled during the day when the cells exhibit synchronous neural activity and are minimally dye coupled during the night when the cells are electrically

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11 silent, a coupling rhythm that is also maintained under constant dark conditions (Colwell 2000). Evidence for a role of gap junctions in the synchronization of the SCN cell oscillators comes from a recent study that shows that the gap junction blockers, octanol and halothane, can reversibly block vasopressin and vasoactive intestinal polypeptide rhythms in SCN slice cultures (Shinohara et al. 2000). Interestingly, there is a growing body of evidence that suggests that gama-aminobutyric acid (GABA) may modify the responses of the SCN neuronal circadian clocks to light and other entraining stimuli by altering cell-cell coupling through gap junctions. GABA, acting through type A receptors, has recently been shown to be capable of phase shifting and synchronizing the oscillators within individual SCN clock cells in vitro (Liu and Reppert 2000) and of modulating the permeability of gap junction channels in SCN slice cultures (Shinohara et al. 2000) Expression and Function of Gap Junctions in Retina The vertebrate retina is a highly laminated assemblage of five major classes of specialized neurons: a vertical pathway connects photoreceptors to bipolar cell to ganglion cells, while horizontal and amacrine cells provide lateral interactions in the outer and inner retina, respectively. Tracer studies have revealed that in addition to these pathways, many types of retinal neurons are interconnected through gap junctions. In addition to the widespread coupling that is observed between identical cell types, heterologous coupling between rods and cones, between amacrine cells and cone bipolar cells, between different types of bipolar cells, between different types of amacrine cells, and between ganglion cells and amacrine cells is also observed (Vaney and Weiler 2000). There is a growing body of evidence that gap junctions are present and functional in developing retinas. Cx43, 26 and 32 are the major connexin isoforms expressed in developing chicken retina. Among them, Cx43 is the first one to be expressed in the early

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12 neuroepithelium of the eyecup, followed by the expression of Cx32 and 26 at E4-4.5 (Becker et al. 1998). As early as E7, fluorescent dye injected into individual retinal ganglion cells spreads into the cells within the ganglion cell layer, the inner nuclear layer, and also to the cells traversing the whole thickness of retina, results that suggest that an extensive network of gap junctions has been formed in chicken retina at this developmental stage (Catsicas et al. 1998). Functional studies have also shown that gap junction communication is involved in the regulation of synchronized spontaneous neural activity that occurs both before (Catsicas et al. 1998) and during (Wong et al. 1998) synaptogenesis in embryonic chicken retina. Neurotransmitters play a major role in regulation of gap junction permeability in inner retina. For example, dopamine and GABA have both been shown to modulate the permeability of gap junctions that exist between amacrine cells in rabbit retina (Hampson et al. 1992) and between horizontal cells in rabbit and turtle retina (Hampson et al. 1994;Piccolino et al. 1982;Piccolino et al. 1984). Modulation of gap junction coupling either through changes in ambient illumination or through light-induced changes in dopamine release have been postulated to play a role in regulating retinal sensitivity (Li and Dowling 2000;Manglapus et al. 1998;Manglapus et al. 1999). Vertebrate photoreceptor cells are also coupled by gap junctions (Gold and Dowling 1979;Raviola and Gilula 1973;Tsukamoto et al. 1992). There is evidence that the strength of junction coupling between photoreceptors can be modulated by light (Yang and Wu 1989) and by dopamine (Krizaj et al. 1998); however, the effectiveness of these stimuli to alter coupling may be species dependent (Schneeweis and Schnapf 1999). While potentially important in processing of light signals for vision (Schneeweis and

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13 Schnapf 1999;Lebedev et al. 1998), changes in gap junction communication may also play a role in the synchronization of photoreceptor circadian oscillators in retina. We have observed that iodopsin rhythms in dispersed cultures of chicken retina are not as robust as those observed in retinal explant cultures, the rhythms in dispersed cells becoming negligible after one week in culture (Semple-Rowland, unpublished observation). This observation also indicates that, as in the case of SCN, gap junction communication may play a role in generation and maintaintence of iodopsin transcript rhythms in retinal explant cultures.

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CHAPTER 2 Note: This work has been published in Molecular Brain Research 114 (2003) 9-19. CHARACTERIZATION OF CIRCADIAN OSCILLATOR FUNCTION IN EMBRYONIC RETINA AND RETINAL EXPLANT CULTURES Introduction The retinas of several vertebrate species contain light entrainable circadian oscillators that regulate 24-hour cyclic changes in retinal function (Besharse and Iuvone 1983;Cahill and Besharse 1991;Tosini and Menaker 1996;Tosini and Menaker 1998). Currently, there is significant interest in identifying the retinal cells that contain these oscillators and understanding how these oscillators are entrained to light. Cahill and Besharse obtained the first evidence that vertebrate photoreceptor cells contain light-entrainable circadian oscillators (Cahill and Besharse 1993). Using a reduced Xenopus eyecup preparation, these investigators obtained support for the hypothesis that the circadian oscillators controlling melatonin release in Xenopus retina are located in the photoreceptors, and that light entrainment of these oscillators does not require input from cells within the inner retina. Direct evidence for the presence of functional oscillators in Xenopus photoreceptor cells has recently been obtained in a study of transgenic Xenopus tadpoles that express a dominant negative form of Clock. Photoreceptor-specific expression of the mutant Clock protein was found to disrupt rhythmic production of melatonin by these cells (Hayasaka et al. 2002). These data and the observations that transcription of the cone-specific iodopsin gene is regulated in a circadian manner in dispersed, low-density cultures of chicken and quail retina (Pierce et 14

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15 al. 1993;Pierce 1999) suggest that it is likely that the retinal photoreceptor cells of several vertebrate species contain autonomous circadian oscillators. The nature of the biochemical cascades that entrain retinal oscillators to light and the extent to which these oscillators influence each other within the context of the intact retina are currently unknown; however, significant progress has recently been made toward understanding light entrainment of oscillators in avian pineal cells. Using immunocytochemical, molecular and biochemical techniques, Fukada and his colleagues have obtained convincing evidence that an opsin-G 11 -mediated signaling pathway is involved in light entrainment of chicken pineal circadian oscillators (Kasahara et al. 2002). They also found that G 11 is expressed in chicken retinal photoreceptors and that it associates with rhodopsin, one of the opsins in retina, in a lightand GTP-dependent manner (Kasahara et al. 2002). Together, these data suggest that an opsin-G 11 signaling pathway may be involved in mediating the phase shifting effects of light on circadian oscillators in chicken retina. Organ culture systems, which have been successfully used to study circadian regulation of melatonin in Xenopus and hamster retina (Cahill and Besharse 1991;Tosini and Menaker 1996), may also prove valuable in studies of the retinal oscillators that drive iodopsin transcription. In this series of experiments, iodopsin mRNA rhythms in embryonic retinal explants maintained under cyclic light, constant dark, and light reversal conditions were compared to those in retinas from age-matched chicken embryos and in post-hatch (< 2 weeks old) chickens. Our data show that embryonic retinas maintained as explant cultures exhibit robust iodopsin rhythms that are driven by light entrainable circadian oscillators. These observations support the use of explant cultures in studies to

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16 investigate mechanisms responsible for generating and maintaining iodopsin transcript rhythms in retina. Materials and Methods Preparation of Retinal Explant Cultures All experimental procedures mentioned in this dissertation were approved by the University of Florida IACUC Committee and were in accordance with the National Institutes of Health guidelines. Fertile White Leghorn chicken eggs obtained from the University of Florida Poultry Sciences Unit were incubated on a 12 hour light: 12 hour dark (12L:12D) cycle in the incubators illuminated by 20 Watt cool white fluorescent bulbs (90 lux). The lights were on at 9:00 AM (Zeitgeber time, ZT0) and off at 9:00 PM (ZT12) EST. Retinal explant cultures were prepared from embryonic day 9 (E9) and E10 chickens during the 12-hour light period. Dissection and preparation of the cultures was carried out according to a method previously described for preparation of neonatal mouse retina cultures (Ogilvie et al. 1999). The eyes were dissected from the embryos and placed in a pool of Dulbeccos modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics (130 U/ml penicillin and 130 g/ml streptomycin). After the sclera, choroid and retinal pigmented epithelial tissues were removed, the remaining structure, consisting of the vitreous body and the retina, was transferred to a 35 mm culture dish that contained a Millicell membrane insert (0.2 m; Millipore, Bedford, MA) filled with media. The retina was gently peeled away from the vitreous body and several small cuts were made around the periphery of the retina to facilitate flattening of the retina photoreceptor side down onto the membrane. The retinal explants were incubated on a 12L:12D cycle at 37C in 5% CO 2 and were fed every two

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17 days. The 12L:12D period beginning the day after the cultures were prepared was designated day 1 in vitro (1 DIV). Explant Morphology Retinal cultures designated for histological analyses were fixed for 1-2 days in 4% paraformaldehyde at 4C. Retinas were left on the Millicell membrane during the fixation step and in some cases remained attached to the membrane throughout the sectioning process. The tissues were infiltrated with 30% sucrose and sectioned (16 m) using a cryostat. Sections were placed onto glass slides and stored at -20C until processed for staining or immunohistochemistry. Cresyl violet stained sections were examined to assess the general morphology of the retinal explants. To determine if there was significant cell loss within the inner nuclear layer (INL) of these cultured retinas, the mean cell density (number of cells per m 2 ) within the INL was determined for three prescribed regions of three different sections from each explant and the average of these values was corrected by multiplying by the mean thickness of the INL layer (m). The resulting values were compared using Kruskal-Wallis ANOVA on Ranks (SigmaStat, Jandel, CA). The percent pyknotic cells in the INL was estimated in 3, 5 and 12 DIV retinal explants by counting the pyknotic nuclei present in three separate prescribed regions of three different sections from each explant and dividing by the mean cell density. To determine if dopaminergic amacrine cells could be detected in the explant cultures, cryosections taken from the retinal explants used in the morphological analyses were immunostained using a monoclonal antibody (1/10,000 dilution in PBS containing 1% goat serum and 0.1% Triton X-100) for tyrosine hydroxylase (TH; Chemicon,

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18 Temecula, CA), a marker for dopaminergic cells, and a goat anti-mouse secondary antibody (1/500 dilution in PBS) tagged with the Alexa-488 fluorophore (Molecular Probes, Eugene, OR). Sections were incubated with the anti-TH primary antibody for 2 hours at room temperature, rinsed three times with PBS, and then incubated with the secondary antibody for 1 hour at room temperature followed by three additional rinses. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes). The stained tissues were viewed using the appropriate fluorescent filter sets and digital images were acquired using a SPOT 2 Enhanced Digital Camera System mounted on a Zeiss Axioplan 2 fluorescence microscope. Iodopsin Gene and Protein Expression in Retinal Explant Cultures To identify the earliest time that iodopsin transcripts could be detected in retinal explant cultures, cultures prepared from E9 embryos and maintained under 12L:12D conditions for 1-6 DIV were collected at ZT 8 (8 hours after lights came on). To compare iodopsin expression onset in cultures and in ovo, retinas from chicken embryos were also collected at ZT 8 from E10 to E16. The retina samples were quickly frozen in liquid nitrogen and stored at -75C until processed for northern blot analyses. Immunohistochemical staining of retinal explants was carried out to examine expression of iodopsin protein in the explants. Tissue sections were blocked using PBS containing 10% goat serum and incubated overnight at 4C with a polyclonal antibody for chicken iodopsin (CERN874; 1/5000 dilution in PBS containing 1.0% BSA and 0.3% Triton X-100) (Geusz et al. 1997). The primary antibody was visualized by incubating the sections with a goat anti-rabbit secondary antibody (1/1000 dilution in PBS) tagged

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19 with the Alexa-594 fluorophore (Molecular Probes) for 1 hour at room temperature. Sections were counterstained with DAPI and imaged as described above. Lighting Paradigms Cyclic light Explant cultures maintained under 12L:12D conditions were collected from 3 DIV to 9 DIV at ZT0 and ZT12. For the in ovo experiments, the retinas of White Leghorn chicken embryos maintained in ovo under 12L:12D conditions were harvested at ZT0 and ZT12 from E17 to E20. In all experiments, sample collection during the dark period was carried out under a low intensity red safe light (15 Watt bulb, Kodak #2 filter). Constant dark Retinal explants were maintained under 12L:12D conditions for the first 5 days in culture and were placed in constant darkness on days 6-7. Cultures were collected on 4 and 5 DIV at ZT0 and ZT12 and on 6 and 7 DIV just after the lights would have been turned on (circadian time 0 CT0) and just after the lights would have been turned off (CT12). For the in ovo experiments, chicken embryos were incubated under 12L:12D conditions through E17 and were then placed in constant dark conditions from E18 to E20. The retinas of E17 embryos were collected at ZT0 and ZT12. The retinas of E18 to E20 embryos were collected at CT0 and CT12. Reversal of the light cycle Retinal explants were maintained on a 12L:12D cycle for 5 DIV. On 6 DIV the light cycle was reversed to a 12D:12L cycle and the cultures remained on this reversed light cycle through 8 DIV. Cultures were collected at ZT0 and ZT12 from 4 to 8 DIV. For the in ovo experiments, chicken embryos were incubated under 12L:12D conditions through E17. On E18, the light cycle was reversed and the embryos remained on this

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20 reversed cycle through E20. The retinas of E17 through E20 embryos were collected at ZT0 and ZT12. RNA Analyses Retinal explants and age-matched embryo retinas were placed in screw top tubes, frozen in liquid nitrogen and stored at -75C until further processing. Total RNA was extracted from the retinas using an RNeasy kit (Qiagen, Valencia, CA). Northern blots were prepared as previously described (Semple-Rowland and van der 1992), each lane containing 8 g total RNA. RNA slot blots were prepared using a BioSlot apparatus (BioRad). The RNA samples, each containing 2g of total RNA from a retinal explant, were diluted with RNase-free water to a final volume of 10 l. The following solutions were then added to each sample; 20 l of 100% formamide, 7l of 37% formaldehyde, and 2ml of 20X SSC (3M NaCl, 0.3M sodium citrate, pH 7.0). The samples were then incubated at 68C for 15 minutes. During the incubation period, the slot blot apparatus was assembled according to the manufacturers instructions and each sample well was rinsed twice with 1 ml 20X SSC. Following the incubation, the denatured RNA samples were cooled on ice, diluted by adding 2 volumes of 20X SSC, and loaded into the sample wells. A gentle vacuum was applied to the apparatus to load the RNA onto a Magnacharge nylon membrane (MSI, Westburough, MA) and to complete the wash steps. Following application of the samples, each well was rinsed twice with 1 ml 20X SSC. The vacuum was kept on for an additional 5 min following the last wash to dry the membrane. The RNA samples were then cross-linked to the blot using UV light (UV Stratalinker, Stragene). The effectiveness of the transfer was examined by staining the blot with methylene blue. Finally, the blot was dried at 37C for 30 minutes and stored at

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21 room temperature until hybridization. The northern and slot blots were prepared in duplicate and were probed consecutively with radiolabeled cDNA probes specific for iodopsin and 18S rRNA that were synthesized as described previously (Larkin et al. 1999). The amount of probe hybridized to the blots was measured using a BioRad Molecular Imager FX system. Iodopsin transcript levels in individual samples were normalized to the amount of 18S rRNA present in that same sample. These values were then expressed relative to the mean iodopsin/18S rRNA value for each blot. Data were analyzed using two-way ANOVA (SigmaStat). Results Morphology of Developing Retinal Explant Cultures The cellular architecture and structural integrity of the retinal explants were examined as a function of time in culture by comparing cresyl violet-stained sections of explants to those of retinas from embryos. The outer nuclear (ONL), inner nuclear (INL), and ganglion cell (GCL) layers of E13 retinas were readily discernable and fairly well organized (Fig.2-1, top panel). From E15 to E18 there was a gradual decrease in the overall thickness of the nuclear layers, a change due in part to a decrease in extracellular space volume and to the rapid growth of the eye that occurs during this period of development. Retinas cultured for 3, 5 and 12 days (Fig.2-1, bottom panel) resembled those obtained from embryos (Fig.2-1, top panel); however, there were two major changes in the structure of the cultured retinas. First, the ganglion cells, which were detectable in 3 DIV cultures, rapidly degenerated and were no longer evident at 5 DIV. Concomitant with the disappearance of the GCL was a reduction in the overall thickness and gradual loss of the inner plexiform layer

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22 (IPL). Second, the cells within the ONL and INL of the explants remained relatively disorganized compared to those present within these layers in retinas in ovo. Figure 2-1. Morphological comparison of chicken embryonic retina and retinal explant cultures. Images of E13, E15 and E18 retinal cross-sections (top panels) show the cellular organization and development of chicken embryonic retinas in ovo. Images of 3 DIV, 5 DIV and 12 DIV retinal cross-sections (middle panels) show the cellular organization and development of chicken retinal explants harvested from E10 embryos. The bottom panel contains magnified images of the INL from explant cultures that show evidence of cell death within the INL (arrows indicate pyknotic nuclei). DIV days in vitro; RPE retinal pigment epithelium; ONL outer nuclear layer; OPL outer plexiform layer; INL inner nuclear layer; IPL inner plexiform layer; GCL ganglion cell layer. Scale bars = 25 m. To determine if loss of the IPL in cultured retinas was accompanied by a significant decrease in the number of cells within the INL, we measured both the cell density and thickness of the INL layers in retinas cultured for 3, 5 and 12 days. The results of these

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23 analyses showed that there was not a significant change in the number of cells within the INL (H = 0.924, df = 2, p = 0.63) even though there were significant changes in the mean densities (H = 21.0, df = 2, p < 0.0001) and widths of the INL layer (H = 16.1, df = 2, p = 0.003) over time (Fig. 2-2A). 0.00.51.01.52.02.53.03.54.0 0123 0.000.050.100.150.200.250.30 020406080 0246810 Mean INL cell density(cell / m2) Mean width INL layer (m) Mean width INL x Mean cell density(cells / m) 3DIV 5DIV 12DIV3DIV 5DIV 12DIVPercent pyknotic cells in INLAB 0.00.51.01.52.02.53.03.54.0 0123 0.000.050.100.150.200.250.30 020406080 0246810 Mean INL cell density(cell / m2) Mean width INL layer (m) Mean width INL x Mean cell density(cells / m) 3DIV 5DIV 12DIV3DIV 5DIV 12DIVPercent pyknotic cells in INLAB Figure 2-2. Quantification of the morphological integrity of retinal explant cultures. (A) Cell densities (yellow bars) and widths (blue bars) of INL layers in 3, 5 and 12 DIV explant cultures. The mean number of cells per m in the INL calculated from the density and width values is also shown in red. (B) Percent pyknotic cells within the INL of 3, 5 and 12 DIV explant cultures. Bars represent mean SE of measures taken from three separate prescribed regions within three different sections of representative 3, 5 and 12 DIV retinal explants. However, there was evidence of cell death within the INL throughout the 12-day culture period. Pyknotic nuclei were detected within this cell layer as early as 3 DIV, the number significantly increasing by 5 DIV and then dramatically falling off by 12 DIV

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24 (Fig. 2-1, bottom panel). This temporal pattern of cell death is similar to what has been reported in analyses of neural cell death in retinas of developing chick (Cook et al. 1998) and quail embryos (Marin-Teva et al. 1999) and suggests that the loss of cells in the INL may reflect normal changes in cell number that accompany development. The majority of the pyknotic nuclei were located in the inner strata of the INL closest to the IPL. The percent of the total number of INL cells that were pyknotic was relatively small, with mean values ranging from 0.09% (12 DIV) to 3.2% (5 DIV) (Fig. 2-2B). Virtually no pyknotic nuclei were detected in the photoreceptor cell layer in any of the explant cultures examined, consistent with previous studies of normal avian retina development (Cook et al. 1998;Marin-Teva et al. 1999). We were also interested in determining if we could detect dopaminergic amacrine cells in our retinal explants because of their known importance in retinal circadian biology (Besharse and Iuvone 1920). Gardino et al. (Gardino et al. 1993) have previously shown that E13 is the earliest time at which amacrine cells expressing the TH phenotype can be detected immunohistochemically in developing chicken retina. We did not detect any TH-positive amacrine cells in cross-sections of the 3, 5, 8 and 12 DIV retinal explants stained with a monoclonal antibody for TH. TH-positive cells exhibiting the morphology of amacrine cells were detected in the inner margin of the INL in E18 and post-hatch retinas using this antibody, but not in E13 retina (Fig. 2-3). The morphology and the location of the TH-positive cells that we observed in E18 and post-hatch retina were the same as previously described (Gardino et al. 1993). Our inability to detect TH-positive cells in E13 retinal sections may have been due to a combination of factors including low levels of expression of TH and the unique spatial

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25 distribution of these cells across the retina at this stage of development (Gardino et al. 1993). ONLOPLINLIPLGCLE18E18 4DPH ONLOPLINLIPLGCLE18E18 4DPH Figure 2-3. TH immunostaining of explants and embryonic and post-hatch retinas. No TH-immunoreactive cells were detected in 3, 5, 8 or 12 DIV retinal explant cultures. Scattered TH-immunoreactive cells exhibiting amacrine cell morphology were identified in E18 and 4 days post-hatch (DPH) retinas along the border between the INL and the IPL. ONL outer nuclear layer; OPL outer plexiform layer; INL inner nuclear layer; IPL inner plexiform layer; GCL ganglion cell layer. Scale bars = 10 m. Comparison of Iodopsin Expression Onset in Retinal Explant Cultures and in ovo Prior to initiating the studies of clock function in the retinal explant cultures I identified the earliest time at which iodopsin gene expression could be detected in these cultures. Northern blot analyses showed that iodopsin transcripts could be detected in E9 explants that had been cultured for 3 days (Fig.2-4A, left panel), a time point approximately equivalent to E12. On the other hand, iodopsin gene expression was first detected by Northern blots on E15 in embryonic chicken retina (Fig.2-4A, right panel). However, it should be noted that recent studies of normal developing chicken retina place the age of onset earlier at approximately E6-E8 when expression is analyzed using RT-PCR (Adler et al. 2001). The acceleration of the onset of iodopsin gene expression that we observed in the explant cultures is consistent with the results of previous studies of

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26 iodopsin gene expression in cultured chicken retina (Belecky-Adams et al. 1999) and has been proposed to occur as a result of the absence of inhibitory factors that normally delay expression of this gene in vivo(Adler et al. 2001). AIod in vitroIod in ovo E 10 11 12 13 14 15 16 Iod18s DIV 1 2 3 4 5 6 Iod18s BAIod in vitroIod in ovo E 10 11 12 13 14 15 16 Iod18s DIV 1 2 3 4 5 6 Iod18sAIod in vitroIod in ovo E 10 11 12 13 14 15 16 Iod18s DIV 1 2 3 4 5 6 Iod18s B Figure 2-4. Onset of iodopsin expression in retinal explant cultures. (A) Northern blot analyses of iodopsin gene expression. Blots were probed consecutively for iodopsin and 18S rRNA. Each lane contained 8g total RNA. The iodopsin mRNA signal was first detected in retinal explants cultured from E9 embryos on 3 DIV, a time roughly equivalent to E12 in ovo. In contrast, iodopsin transcript signal could not be detected in retinas from chicken embryo until E15. (B) Immunohistochemical analyses of iodopsin expression in retinal explants cultured from E10 embryos. Iodopsin immunostaining was not above background in 3 DIV explants but was easily detected in the rudimentary OS of 5 DIV explants. DIV days in vitro; ONL outer nuclear layer; OPL outer plexiform layer; INL inner nuclear layer; IPL inner plexiform layer; GCL ganglion cell layer. Scale bars = 25 m. Iodopsin protein was not detected immunohistochemically in E9 retinas maintained in culture for 3 DIV and, in contrast, was easily observed in the photoreceptor cells of 5

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27 DIV cultures. Iodopsin immunoreactivity was primarily localized to the rudimentary outer segments of the photoreceptor cells (Fig.2-4B). Iodopsin Transcript Rhythms Iodopsin transcript levels in dispersed embryonic retinal cultures and in post-hatch chicken retinas exhibit a circadian rhythm with minimum levels of the transcript at ZT0 and maximum levels at ZT12 (Larkin and Semple-Rowland 2001;Pierce et al. 1993). Analyses of iodopsin transcription in our explant cultures in which we examined transcript levels at ZT0, ZT6, ZT12 and ZT18 revealed that the same dynamics of the rhythm were preserved in explanted retina. Based on these observations, we chose to examine iodopsin transcript levels at ZT0 and ZT12 in our analyses of retinal circadian oscillator function in ovo and in explant culture. Cyclic light Retinal explants. The levels of iodopsin mRNA from 3DIV to 9DIV in retinal explant cultures maintained under 12L:12D conditions exhibited a robust rhythm. On culture day 3, iodopsin mRNA levels were 2.5 fold higher at ZT12 than at ZT0 (Fig.2-5A). From culture day 4 to 7, the total amount of iodopsin transcript increased dramatically compared to that observed on 3DIV, but the relative increase in iodopsin mRNA levels that occurred between ZT0 and ZT12 remained relatively constant from Day 3 to Day 7. On 8 and 9 DIV, iodopsin transcript levels at ZT0 were similar to that on Day 7, but the relative increase of iodopsin mRNA levels from ZT0 to ZT12 was reduced. The differences observed in levels of iodopsin mRNA between ZT0 and ZT12 were significant over the time period examined (F = 31.03, df = 1, p < 0.0001).

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28 0.00.51.01.52.02.53.0 1010666666Relative LevelsIodopsin mRNA / 18S rRNA 0.00.51.01.52.02.5 71571510101414E17 E18 E19 E20 E17 E18 E19 E20 CD E17 E18 E19 E20FB Relative LevelsIodopsin mRNA / 18S rRNA 0.00.51.01.52.02.5 551515711711773333 3DIV 4DIV 5DIV 6DIV 7DIV 8DIV 9DIVA 0.00.51.01.52.02.53.0 44444444B 234567891011Relative LevelsIodopsin mRNA / 18S rRNA 0.00.51.01.52.02.5 E 4DIV 5DIV 6DIV 7DIV 0.00.51.01.52.02.53.04DIV 5DIV 6DIV 7DIV 0.00.51.01.52.02.53.0 1010666666Relative LevelsIodopsin mRNA / 18S rRNA 0.00.51.01.52.02.5 71571510101414E17 E18 E19 E20 E17 E18 E19 E20 CD E17 E18 E19 E20FB Relative LevelsIodopsin mRNA / 18S rRNA 0.00.51.01.52.02.5 551515711711773333 3DIV 4DIV 5DIV 6DIV 7DIV 8DIV 9DIVA 0.00.51.01.52.02.53.0 44444444B 234567891011Relative LevelsIodopsin mRNA / 18S rRNA 0.00.51.01.52.02.5 E 4DIV 5DIV 6DIV 7DIV E 4DIV 5DIV 6DIV 7DIV 0.00.51.01.52.02.53.04DIV 5DIV 6DIV 7DIV Figure 2-5. Iodopsin transcript rhythms in explant cultures and embryos maintained under 12L:12D and constant dark conditions. (A) Relative iodopsin mRNA levels in explant cultures prepared from E9 embryos and examined at 3, 5, 7 and 9 DIV and (B) in retinas of embryos examined at E17 E20. For panels A and B, the explants and embryos were maintained under 12L:12D conditions throughout the experiment. Retinas were analyzed at ZT0 (white bars) and at ZT12 (black

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29 bars). (C) Relative iodopsin mRNA levels in explant cultures maintained for 5 DIV under 12L:12D and then transferred into constant dark conditions on days 6 and 7. (D) Relative iodopsin mRNA levels in embryos raised under 12L:12D until E18 and then transferred into constant dark conditions from E18 to E20. (E) Comparison of explant cyclic light data shown in panel A (red symbols) and the constant dark data shown in panel C (black symbols). (F) Comparison of embryonic cyclic light data shown in panel B (red symbols) and the constant dark data shown in panel D (black symbols). The 12-hour light and dark periods are indicated below each panel using white and black bars, respectively. In panels E and F, plotted data and light cycles are matched by color, red indicating 12-hour dark periods. In panels A D, the group means SE at each time point are shown and the number of retinas in each group is indicated on the bars. In panels E and F, only the mean values are plotted for each group. Retina in ovo. Retinal iodopsin transcripts were first detected in ovo on E15. No significant rhythms were detected on E15 or E16 and the mean values for relative iodopsin levels at these stages were 0.185 and 0.258, respectively (data not shown). The first evidence of a rhythm in iodopsin transcript levels was detected on E17; the transcript levels measured at ZT12 were 1.6 fold higher than those measured at ZT0 (Fig.2-5B). The amplitude of the rhythm increased with developmental age. On E18, iodopsin mRNA levels at ZT12 were 1.8 fold higher than those at ZT0. By E19 and E20, two-fold increases in iodopsin expression were observed over the course of the 12-hour light period. The emergence of the iodopsin transcript rhythm during the late stages of development was paralleled by a steady and significant increase in the total amount of iodopsin transcript, the levels of which appeared to reach a plateau by E20. The differences observed in levels of iodopsin mRNA between ZT0 and ZT12 were significant over the time period from E17 to E20 (F = 97.57, df = 1, p < 0.0001). Constant dark Retinal explants. Under 12L:12D conditions, the iodopsin transcript levels in retinal explant cultures at 4 and 5DIV exhibit robust rhythms. In the absence of light, a

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30 significant rhythm persisted in retinal cultures for 48 hours, the iodopsin levels measured at CT0 being significantly lower than those measured at CT12 on 6 and 7 DIV (F = 9.86, df = 1, p = 0.003) (Fig.2-5C). The relative increase of iodopsin transcript levels observed from CT0 to CT12 under constant dark conditions was 70% of that observed under cyclic light conditions and the temporal characteristics of the iodopsin mRNA rhythm were similar to those observed under cyclic light (Fig.2-5E). These data suggest that the iodopsin rhythms observed in explant cultures under 12L:12D conditions are being driven by functioning retinal circadian oscillators. Retina in ovo. Significant iodopsin transcript rhythms were observed in E17 chicken embryos that had been maintained in ovo under cyclic light conditions. On E18 when the lights were turned off, iodopsin transcript levels at CT12 were 1.3 fold higher than those at CT0 (Fig.2-5D). This increase was half that observed at the corresponding time point in embryos maintained under cyclic light conditions (Fig.2-5B). These incremental increases in transcript levels continued through E19 and E20. At CT12 on E20, the amount of iodopsin transcript in the retinas of embryos maintained in constant dark had reached levels similar to those observed in embryos that had been maintained under cyclic light. Direct comparisons of the cyclic light and constant dark data (Fig.2-5F) revealed that there was no detectable iodopsin mRNA rhythm present in embryos placed in constant darkness. These data suggest that light and developmental mechanisms act synergistically to up-regulate iodopsin transcription in developing embryonic chicken retina, and that in the absence of light, increases in iodopsin transcript levels are predominantly, if not completely, regulated by developmental mechanisms.

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31 Reversal of the light:dark cycle Retinal explants. Reversal of the light cycle on culture day 6 was followed by a 48-hour transition period during which time the iodopsin transcript levels remained at levels intermediate to those observed during this same time period in the explants that had been maintained under cyclic light conditions (Fig.2-6A, B). 0.00.51.01.52.02.53.0 Relative Levels Iodopsin mRNA / 18S rRNA 0.00.51.01.52.02.53.0 1044444410 E17 E18 E19 E20 E17 E18 E19 E20CDRelative Levels Iodopsin mRNA / 18S rRNA 2345678910111213 0.00.51.01.52.0 4DIV 5DIV 6DIV 7DIV 8DIVB 4DIV 5DIV 6DIV 7DIV 8DIV 0.00.51.01.52.0 771515777766ARelative Levels Iodopsin mRNA / 18S rRNARelative Levels Iodopsin mRNA / 18S rRNA 2345678910111213 0.00.51.01.52.0 4DIV 5DIV 6DIV 7DIV 8DIVB 4DIV 5DIV 6DIV 7DIV 8DIV 0.00.51.01.52.0 771515777766A Figure 2-6. Iodopsin transcript rhythms in explant cultures and embryos following reversal of the light:dark cycle. (A) Relative iodopsin mRNA levels in explant cultures prepared from E9 embryos and examined at 4 -8 DIV. Explants were maintained under 12L:12D until 6 DIV when they were transferred to a 12D:12L reversed cycle. (B) Comparison of iodopsin rhythms obtained from explants exposed to a reversed 12D:12L cycle (black symbols) and those from explants maintained on a 12L:12D cycle (red symbols; Fig. 5E). (C) Relative iodopsin mRNA levels in retinas of embryos maintained under 12L:12D until E18 when they were transferred to a 12D:12L reversed cycle. (D) Comparison

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32 of iodopsin rhythms obtained from embryos exposed to a reversed 12D:12L cycle (black symbols) and those obtained from embryos maintained on a 12L:12D cycle (red symbols; Fig 5F). The 12-hour light and dark periods are indicated below each panel using white and black bars, respectively. In panels B and D, plotted data and light cycles are matched by color, red indicating 12-hour dark periods. In panels A and C, the group means SE at each time point are shown and the number of retinas in each group is indicated on the bars. In panels B and D, only the mean values are plotted for each group. Over the course of the first 12-hour dark period of the reversed cycle (6 DIV), iodopsin mRNA levels increased 1.4 fold to levels comparable to those observed in cultures maintained under cyclic light conditions at 6 DIV (Fig.2-6A, B). No further increase in iodopsin mRNA levels was observed in the reversed cycle cultures during the 12-hour light period on 6 DIV. Comparisons of the iodopsin transcription levels in cultures exposed to the reversed light cycle to those in cultures maintained on the 12L:12D cycle at 7 and 8 DIV showed that the oscillators that drive the iodopsin rhythms in the explant cultures are capable of entraining to a new light cycle and suggest that entrainment is completed within 36-48 hours following light reversal (Fig.2-6B). Retina in ovo. During the first 12-hour dark period of the reversed cycle on E18, the increase in iodopsin transcript levels was minimal. By the end of the subsequent 12-hour light period on E18, the levels of iodopsin mRNA had increased 2-fold (Fig.2-6C). Comparisons of the iodopsin rhythms obtained from these embryos and those obtained from embryos maintained on a normal light:dark cycle (Fig.2-6D) show that reversal of the light cycle produces an immediate shift in the iodopsin transcript rhythm. These data, which are consistent with our constant dark in ovo results, support the hypothesis that iodopsin transcript rhythms in ovo are primarily, if not entirely, driven by light.

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33 Discussion Two major conclusions can be drawn from the results of these experiments. First, circadian oscillators regulate iodopsin transcription in embryonic retinal explant cultures and the rhythms that are observed are similar to those observed in post-hatch chicken retinas (Larkin and Semple-Rowland 2001). Second, iodopsin transcript rhythms in the retinas of chicken embryos in ovo are driven by light and not by circadian oscillators. The iodopsin rhythms in retinal explants share several attributes with those measured in post-hatch chicken retina. In both paradigms the rhythms are robust with the peaks of the rhythms occurring around ZT12 and the troughs at ZT0. Importantly, reversal of the light cycle induced similar shifts in the iodopsin transcript rhythms in both explant cultures and in post-hatch retina. Together, these observations show that the essential components for circadian regulation of iodopsin transcription remain intact and functional in retinas maintained in explant cultures. It is generally accepted that the circadian oscillators that drive the iodopsin rhythm in chicken retina are located within the photoreceptor cells. This conclusion is based on the observation that transcription of this cone pigment gene remains rhythmic in dispersed retinal cell cultures (Pierce et al. 1993). Although our analyses of retinal explants do not provide unequivocal evidence that proves that circadian oscillators are located in photoreceptor cells, the results are consistent with this point of view. It is clear from our analyses that neither removal of the retinal pigment epithelium (RPE) nor degeneration of the ganglion cells is sufficient to abolish the generation or entrainment of circadian iodopsin transcription in retinal explants. Thus, neither retinal ganglion cells nor the RPE are essential for maintaining the iodopsin rhythm in chicken retina.

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34 Furthermore, the absence of dopamine-secreting amacrine cells in these explant cultures suggests that dopamine signaling does not play a central role in regulating this rhythm. In addition to questions related to the location of circadian oscillators in retina, it remains to be determined how the oscillators that drive the iodopsin rhythm are entrained to light. If we assume that the oscillators that drive iodopsin transcription are located within the photoreceptor cells, then a logical starting point for the search for the phototransduction cascades that entrain these oscillators to light would focus on biochemical pathways in these cells. We have previously shown that the absence of guanylate cyclase 1 in retinas of GUCY1*B chickens, an enzyme essential for visual phototransduction, significantly delays but does not prevent light entrainment of iodopsin rhythms to a reversal of the light cycle (Larkin and Semple-Rowland 2001). These data show that the visual phototransduction cascade mediated by the G-protein, transducin, does not directly play a role in entrainment of the oscillators that drive the iodopsin rhythm. Recent analyses of chicken pineal and retina provide provocative new evidence that suggests a possible role for a phototransduction pathway mediated by the pertussis toxin-insensitive G-protein, G 11 in light entrainment of circadian oscillators in these tissues (Kasahara et al. 2002). Activation of a G 11 -mediated pathway would be expected to lead to changes in phosphatidylinositol turnover and calcium mobilization, changes that have been documented to occur in photoreceptors of several vertebrate species in response to light stimulation (Ghalayini and Anderson 1984;Hayashi and Amakawa 1985;Millar et al. 1988). Pharmacological manipulation of this cascade in retinal explant cultures may help to determine if, in fact, this cascade is involved in light entrainment of the oscillators that drive the iodopsin rhythm in chicken retina.

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35 We were surprised, in view of our explant data, to find that light is the predominant regulatory signal that drives iodopsin transcript rhythms in the retinas of chicken embryos in ovo. These data, together with those obtained in our previous studies (Larkin and Semple-Rowland 2001), suggest that the transition from light to circadian regulation of iodopsin transcription in chicken retina occurs at or shortly after hatching. The onset of circadian regulation of the activity of serotonin N-acetyltransferase (NAT) in chicken retina, the product of another photoreceptor-specific gene, is also delayed in ovo (Iuvone 1990). However, unlike iodopsin transcription, significant light driven changes in NAT activity do not appear until late in development (E20, just/one day prior to hatching). Our observations and those of other investigators show that the emergence of circadian regulation of iodopsin transcription and NAT activity in chicken retina is accelerated in vitro {Pierce, Sheshberadaran, et al. 1993 PIERCE1993 /id}{Pierce 1999 PIERCE1999 /id}{Ivanova & Iuvone 2003 IVANOVA2003A /id}. The mechanism responsible for this acceleration is unknown. The culture conditions may accelerate the maturation of retinal circadian oscillators in vitro. It is also possible that preparation of the retinas for culture results in the removal of tissues (e.g. RPE) that normally produce regulatory signals that delay the onset of circadian regulation in the intact, developing retina. In conclusion, the results of our analyses show that chicken retinal explant cultures can be used as an experimental paradigm for studies of retinal circadian oscillator function. The structure and organization of many of the retinal cell and synaptic layers remains intact in the explants, allowing studies of entrainment and synchronization of circadian oscillators under various lighting regimens.

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CHAPTER 3 GAP JUNCTION BLOCKERS ABOLISH CIRCADIAN RHYTHMS OF GENE EXPRESSION IN RETINAL PHOTORECEPTORS Introduction Circadian oscillators in vertebrate retina regulate many aspects of retinal function, including the synthesis and release of melatonin (Cahill et al., 1991;Cahill and Besharse, 1991;Tosini, 2000), photoreceptor disk shedding (Nguyen-Legros and Hicks, 2000), retinomotor movement (Burnside, 2001), and gene expression (Pierce et al., 1993;Green and Besharse, 1994;Green et al., 1996;Larkin et al., 1999;Chong et al., 2000;Bernard et al., 1999). The transcription of the genes that encode iodopsin, a red sensitive photopigment that is expressed specifically in the cone photoreceptors of chicken retina, and arylalkylamine N-acetyltransferase (AANAT) have been shown to be regulated by circadian oscillators in vivo (Larkin et al., 1999;Bernard et al., 1999;Chong et al., 2000;Liu et al., 2004) and in dissociated retinal cultures (Pierce et al., 1993). We are interested in understanding the mechanisms that coordinate and maintain photoreceptor transcript rhythms in vertebrate retina. Studies of the SCN provide clues about the mechanisms that may serve to coordinate the activity of populations of autonomous oscillator neurons. Within the intact SCN, 20,000 neurons work in concert to generate coordinated circadian rhythms of electrical activity (Herzog et al., 1997), gene expression (Panda et al., 2002), and glucose metabolism (Schwartz et al., 1983). When examined in dissociated culture, individual SCN neurons exhibit self-sustaining electrical activity rhythms, but the periods and 36

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37 phases of these rhythms are highly variable between neurons (Welsh et al., 1995). In explant culture in which synapses and cellular appositions are largely preserved, the periods and phases of the firing rhythms of individual SCN neurons exhibit significantly less variability between neurons (Herzog et al., 1998). These observations led to the hypothesis that intercellular communication is required to maintain coordinated circadian rhythms in SCN. Synapseand gap junction-mediated intercellular communication represent two possible mechanisms that could coordinate the circadian rhythms generated by populations of autonomous oscillator cells. The observations that circadian rhythms of glucose metabolism appear in the SCN before chemical synapses become functional (Reppert and Schwartz, 1984;Moore and Bernstein, 1989)and that blockade of synaptic transmission using either tetrodotoxin (TTX) (Shibata and Moore, 1993) or calcium-free medium (Bouskila and Dudek, 1993) does not disrupt the ability of SCN neurons to generate synchronized bursts of activity suggest that synaptic transmission is not a central player in the coordination of circadian rhythms in the SCN. Gap junction channels, which allow the passage of small ions, signaling molecules and low molecular weight metabolites between cells, could, on the other hand, serve as mediators of intercellular communication {Goodenough, Goliger, et al. 1996 GOODENOUGH1996 /id}. Gap junction channels are comprised of two paired hemichannels, known as connexons. Each connexon consists of six protein subunits called connexins (Cx) (Cook and Becker, 1995). Gap junction channels form when two connexon hemichannels located on the membranes of adjacent cells couple (Cook and Becker, 1995). Several studies suggest that gap junction communication plays an integral role in maintaining

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38 coordinated rhythms in SCN. Studies using dyes capable of traversing gap junctions show that SCN cells are extensively coupled during the day when the cells exhibit synchronous neural activity and are minimally coupled during the night when the cells are electrically silent, a coupling rhythm that is also maintained under constant dark conditions (Colwell, 2000). The importance of this gap junction coupling in maintaining circadian rhythms in SCN is demonstrated by the observation that the gap junction blockers, octanol and halothane, disrupt the circadian rhythm of vasopressin and vasoactive intestinal polypeptide secretion from SCN slice cultures that is restored upon removal of the blockers (Shinohara et al., 2000). Extensive networks of gap junctions are present in developing chicken retina as early as embryonic day 7 (Becker et al., 1998;Catsicas et al., 1998). Prior to synaptogenesis, these junctions have been shown to play a role in the propagation of transient Ca 2+ waves that spread across the developing chicken retina (Catsicas et al., 1998). We have recently developed a retinal explant culture system that allows the study of circadian regulation of gene transcription in vitro (Zhang et al., 2003). Using this culture system, we have successfully monitored the coordinated, self-sustained, and light-entrainable iodopsin transcript rhythms normally present in chicken retina (Zhang et al., 2003;Larkin et al., 1999). In the present study, we have conducted a series of experiments to determine if, as in SCN, the gap junction network present in chicken retina plays a role in maintaining coordinated iodopsin and AANAT transcript rhythms in this tissue. To test this hypothesis, we have examined the effects of two reversible gap junction blockers, carbenoxolone (ACO) and 18-glycyrrhetinic acids (18-GA), on iodopsin and AANAT transcript rhythms in explant cultures maintained under different lighting conditions.

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39 Both gap junction blockers are the derivatives of glycyrrhetinic acid. ACO has been reported to reversibly block the gap junction channels comprised of Cx26 (Kamermans et al., 2001), Cx32 (Szente et al., 2002), and Cx43 (Goldberg et al., 1996), the three major connexins that are expressed in developing chicken retina (Becker et al., 2002) and hence the three potential targets of ACO in the retinal explant cultures. More importantly, 100M ACO has been shown to effectively block gap junction channels in outer (Kamermans et al., 2001) and inner retina (Sekaran et al., 2003). Another gap junction blocker 18-GA has been shown to induce phosphotase-mediated dephosphorylation of Cx43 and subsequent disassembly of gap junction plaques (Guan et al., 1996). Based on these observations, we hypothesized that application of these blockers would block the gap junction channels and desynchronize the circadian oscillators driving the gene expression rhythms, and the transcript levels of both genes would assume the average of the peak and trough values of their respective intrinsic rhythms (Kunz and Achermann, 2003). Methods and Materials Chemicals and Reagents The culture media for the explants consisted of Dulbecco's modified Eagle's media (DMEM, Gibco # 11995-065) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and antibiotics (130U/ml penicillin, 130 g/ml streptomycin) (Gibco). The gap junction blockers, carbenoxolone (ACO), 18-glycyrrhetinic acids (18-GA), and the chemicallyrelated inactive compound, glycyrrhizic acid (GA), were purchased from Sigma (St. Louis, MO). Stock solutions of carbenoxolone (163 mM) and GA (5 mM) were dissolved in deionized water. The stock solution of 18-GA (100 mM) was

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40 dissolved in dimethyl sulfoxide (DMSO; Sigma). All blockers were used at a final concentration of 100 M in these experiments. Retinal Explant Cultures All experimental procedures were approved by the University of Florida IACUC Committee and were carried out in accordance with the National Institutes of Health guidelines. Fertile White Leghorn chicken eggs (Charles River Laboratories) were incubated on a 12 hour light: 12 hour dark (12L:12D) cycle in incubators illuminated by 20 Watt cool white fluorescent bulbs (90 lux). The lights were turned on at 9:00 AM (Zeitgeber time, ZT0) and shut off at 9:00 PM (ZT12). Retinal explant cultures were prepared from embryonic day 9 (E9) chickens during the 12-hour light period. Dissection and preparation of the cultures were carried out using methods developed in our laboratory (Zhang et al., 2003). During the first 5 days of culture, all explants were incubated on a 12L:12D cycle at 37C in 5% CO2,.and were fed every two days. The 12L:12D period beginning the day after the cultures were prepared was designated day 1 in vitro (1 DIV). Lighting and Blocker Delivery Paradigms: 12L:12D: Retinal explant cultures were maintained on a 12L:12D cycle throughout the experiments. In experiments utilizing gap junction blockers, ACO or 18-GA were added to the media at ZT12 on 4DIV and removed at ZT12 on either 5DIV or 6DIV. Alternatively, ACO was added at ZT0 on 5DIV and removed 24 hrs later. The affects of ACO on AANAT expression were examined by adding ACO at ZT15 on 4DIV and removing it 24 hours later. Control groups included untreated explants or explants treated

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41 with either 0.1% DMSO or 100 M GA. The final percent of DMSO in the control cultures was equivalent to that in cultures treated with 18-GA. DMSO was added at ZT12 on 4DIV and remained on the cultures for either 24 or 48 hrs. GA was added to the cultures at ZT12 on 4DIV and was removed 48 hrs later. Prior to treatment, the cultures were fed every two days. During the treatment period, the media bathing both the experimental and control cultures was replaced every 12 hours. The cultured retinas were collected every 12 hours from 4DIV to 7DIV for analyses of iodopsin expression. In the experiments designed to examine AANAT mRNA levels, the cultured retinas were collected at ZT0 and ZT15 from 4DIV to 7DIV. The retinal cultures were snap frozen in liquid nitrogen, and stored at -75C until further processing. In all experiments, retinas collected during the dark period were processed under a low intensity safe red light (15 Watt bulb, Kodak #2 filter). 12L:12D followed by constant darkness: Retinal explant cultures were maintained under 12L:12D lighting conditions for 5DIV. ACO was added to the media either at ZT12 on 4DIV or at ZT0 on 5DIV and was removed 24 or 12 hours later, respectively. The cultures were kept in constant dark conditions following treatment and were collected every 12 hours from 4DIV to 7DIV. RNA Analysis Total RNA was extracted from the retinal explant cultures using an RNeasy kit (Qiagen, Valencia, CA, USA). The RNA samples, 2 g total RNA per sample, were loaded onto a Magnacharge nylon membrane (MSI, Westburough, MA) and analyzed using a slot blot format as described previously (Zhang et al., 2003). Blots were prepared in duplicate and were probed consecutively with radio-labeled cDNA probes specific for

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42 iodopsin, and/or guanylate cyclase activating protein 1 (GCAP1) and/or AANAT, and 18S rRNA. The iodopsin, GCAP1, and 18S rRNA cDNA fragments used for probes have been described previously (Semple-Rowland and van der, 1992;Zhang et al., 2003). The AANAT probe was generated using a 1.4 kb BamHI fragment of cDNA clone #9A (kindly provided by David Klein) that contained the entire AANAT open reading frame. The 32 P-labeled cDNA probes were generated using a Stripeasy DNA Kit (Ambion the RNA company, Austin, TX, USA). The amount of probe hybridized to the blots was measured using a BioRad Molecular Imager FX system. Levels of iodopsin, GCAP1, and AANAT transcript present in individual samples were normalized to the amount of 18S rRNA present in that same sample. These values were then expressed relative to the mean normalized value for the corresponding gene on each blot. Data were analyzed using two-way ANOVA (SigmaStat). Results Iodopsin and AANAT Transcript Rhythms in Untreated Explant Cultures: Previous studies have shown that iodopsin transcript levels in dispersed embryonic retinal cultures and in post-hatch chicken retinas exhibit a robust rhythm with minimum levels at ZT0 and maximum levels at ZT12 under cyclic light conditions that is maintained in the absence of light (Pierce et al., 1993;Larkin et al., 1999;Larkin and Semple-Rowland, 2001). To examine the temporal regulation of iodopsin transcript rhythms in the retinal explant cultures, iodopsin mRNA levels were examined every 6 hours on 6DIV. The results revealed that the temporal dynamics of the iodopsin rhythm observed in our explant retina cultures were very similar to those previously observed in dispersed retinal cultures and in post-hatch chicken retinas (Fig 3-1A, B). In the retinal explant cultures maintained under 12L:12D conditions, analyses of iodopsin mRNA

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43 levels in selected cultures at ZT0 and ZT12 over a 7-day culture period revealed well-defined rhythms that persisted under constant darkness for at least 48 hrs (Fig 3-1C, D). These results show that functional circadian oscillators regulate iodopsin mRNA rhythms in our explants and that these rhythms can be monitored by measuring transcript levels at ZT0 and ZT12. 0.40.60.81.01.21.41.6 39534 0.40.60.81.01.21.41.6 0.000.450.901.351.802.25 71571510101414 0.000.450.901.351.802.25 ARelative levels of Iodopsin mRNA/18s rRNA ZT0 6 12 18 24Relative levels of Iodopsin mRNA/18s rRNA ZT0 6 12 18 24BRelative levels of Iodopsin mRNA/18s rRNA Relative levels of Iodopsin mRNA/18s rRNA DC Day 4 5 6 7 Day 4 5 6 7 Figure 3-1. Iodopsin mRNA rhythms in retinal explant cultures. (A) Diurnal iodopsin transcript rhythms every 6 hours observed in the retinal explant cultures on 6DIV. (B) Data in panel A are shown in line graph. (C) Iodopsin transcript rhythms observed in the cultures maintained in 12L:12D conditions for 5DIV and were then transferred to constant darkness on 6 DIV. Relative iodopsin mRNA levels were analyzed every 12 hours. (D) Comparison of iodopsin transcript rhythms observed in the cultures maintained under 12L:12D conditions for 7DIV (black lines) to the data shown in panel C (red lines). The 12-hour light and dark periods are indicated below each panel using white and black bars, respectively. In panel D, plotted data and light cycles are matched by color. In panels A and C, the group means SE at each time point are shown and the number of retinas in each group is indicated on the bars. In panels B and D, only the mean values are plotted for each group.

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44 Rhythmic changes in the levels of AANAT mRNA in chicken retina have also been shown to be driven by endogenous retinal circadian oscillators (Bernard et al., 1999;Haque et al., 2002). 0.000.350.701.051.401.75 0.000.350.701.051.401.75 553344444 0.00.51.01.52.0 ARelative levels of AANAT mRNA/18s rRNA 0.00.51.01.52.0 33333333 ZT0 3 6 9 12 15 18 21 24Relative levels of AANAT mRNA/18s rRNA ZT0 3 6 9 12 15 18 21 24BRelative levels of AANAT mRNA/18s rRNA Day 4 5 6 7CRelative levels of AANAT mRNA/18s rRNA D Day 4 5 6 7 Figure 3-2. AANAT mRNA rhythms in retinal explant cultures. (A) Diurnal AANAT transcript rhythms every 3 hours observed in the retinal explant cultures on 6DIV. (B) Data in panel A are shown in line graph. (C) AANAT transcript rhythms observed in the cultures maintained in 12L:12D conditions for 5DIV and were then transferred to constant darkness on 6 DIV. Relative AANAT mRNA levels were analyzed every 12 hours. (D) Comparison of AANAT transcript rhythms observed in the cultures maintained under 12L:12D conditions for 7DIV (black lines) to the data shown in panel C (red lines). The 12-hour light and dark periods are indicated below each panel using white and black bars, respectively. In panel D, plotted data and light cycles are matched by color. In panels A and C, the group means SE at each time point are shown and the number of retinas in each group is indicated on the bars. In panels B and D, only the mean values are plotted for each group.

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45 Analyses of AANAT transcripts every 3 hours in our explant cultures on 6DIV that had been maintained under 12L:12D conditions showed that AANAT mRNA levels are lowest at ZT0 and reach peak levels at ZT15 (Fig 3-2A, B). In the retinal explant cultures maintained under 12L:12D conditions, analyses of AANAT mRNA levels in selected cultures at ZT0 and ZT15 over a 7-day culture period revealed that diurnal rhythms with an amplitude of 1.8-fold in the levels of AANAT mRNA were first detected on 5DIV (Fig 3-2D) and continued in constant dark conditions (Fig 3-2C, D). These results show that functional circadian oscillators in our explants regulate AANAT mRNA rhythms and that these rhythms can be monitored by measuring transcript levels at ZT0 and ZT15. Iodopsin Transcript Levels in Explant Cultures Treated With ACO For 48 Or 24 hrs Maintained Under A 12L:12D Cycle ACO, a derivative of glycyrrhetinic acid that has been shown to reversibly block gap junction communication in vertebrate retina (Pottek et al., 2003;Kamermans et al., 2001), was added to the media of selected cultures at the beginning of the dark period (ZT12) on 4DIV. Analyses of iodopsin mRNA levels at ZT0 on 5DIV, 12 hours after addition of ACO, revealed that the iodopsin mRNA levels in these cultures were similar to those that had been observed in untreated cultures (Fig 3-3A, D). The affects of ACO on iodopsin mRNA levels were first evident in cultures examined at ZT12 on 5DIV. In untreated cultures, iodopsin mRNA levels increase over the course of each 12hr light period, peaking at ZT12 (Fig.3-3D) (Pierce et al., 1993;Larkin et al., 1999). Treatment of cultures with ACO prevented this increase. In treated cultures, the amount of iodopsin mRNA measured at ZT12 on 5DIV was the same as that measured at ZT0 on 5DIV and remained low throughout the 48 hr treatment period. Removal of ACO at ZT12 on 6DIV produced a rapid, two-fold increase in the amount of iodopsin transcript in the cultures

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46 examined at ZT0 on 7DIV. Surprisingly, this increase occurred over the course of a 12-hour dark period, a time during which iodopsin transcript levels normally fall to their lowest values. The amount of iodopsin mRNA in cultures examined at ZT12 on 7DIV did not increase significantly over the values measured at ZT0 on 7DIV. 0.000.450.901.351.802.25 83338333Relative levels of Iodopsin mRNA/18SrRNA 0.000.450.901.351.802.25 0.000.450.901.351.802.25 88333333 0.000.450.901.351.802.25 0.000.450.901.351.802.25 0.000.450.901.351.802.25 88333333 Day 4 5 6 7 Day 4 5 6 7 Relative levels of Iodopsin mRNA/18SrRNARelative levels of Iodopsin mRNA/18SrRNA Day 4 5 6 7 Day 4 5 6 7 Day 4 5 6 7 Day 4 5 6 7 Day 4 5 6 7 Day 4 5 6 7 Day 4 5 6 7 Day 4 5 6 7 Day 4 5 6 7 Day 4 5 6 7 Relative levels of Iodopsin mRNA/18SrRNARelative levels of Iodopsin mRNA/18SrRNARelative levels of Iodopsin mRNA/18SrRNA ACO 48hrsACO 24hrsACO 24hrsACO 48hrsACO 24hrsACO 24hrsABCDEF Figure 3-3. Iodopsin expression in explant cultures treated with ACO for 48 and 24hrs All the explant cultures were maintained under 12L:12D conditions in this series of experiments. (A) Cultures were incubated with 100 M ACO for 48 hrs. ACO was added to the cultures at ZT12 on 4DIV and was removed at ZT12 on 6DIV. (B) Cultures were incubated with 100 M ACO for 24 hrs. ACO was added to the cultures at ZT12 on 4DIV and was removed at ZT12 on 5DIV. (C) Cultures were incubated with 100M ACO for 24 hrs, ACO was added to the cultures at ZT0 on 5DIV and was removed at ZT0 on 6DIV. In panels A to C, retinal explant cultures were analyzed at ZT0 (white bars) and at ZT12 (black bars). (D) Comparison of the data shown in panel A (red line) and the iodopsin transcript rhythms observed in the untreated cultures (black line). (E) Comparison of data shown in panel B (red line) and the iodopsin transcript rhythms observed in the untreated cultures (black line). (F) Comparison of data shown in panel C (red line) and the iodopsin transcript rhythms observed in the untreated cultures (black line). The 12-hour light and

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47 dark periods are indicated below each panel using white and black bars, respectively. The dashed lines in each panel indicate the period of blocker treatment. In panels A C, the group means SE at each time point are shown and the number of retinas in each group is indicated on the bars. In panels D F, only the mean values are plotted for each group. The rapid suppression of iodopsin mRNA levels by ACO was unexpected. To determine if iodopsin mRNA rhythms would reappear following removal of the blocker and to further examine the temporal characteristics of this phenomenon, cultures were treated with ACO for a shorter period of time. ACO was added to cultures at ZT12 on 4DIV or at ZT0 on 5DIV. In both experiments, ACO was removed 24 hours later. In both paradigms, the iodopsin mRNA levels observed 12 hours following addition ACO were comparable to the low levels observed at ZT0 in control cultures. Importantly, within 24 hours of the removal of the blocker, iodopsin transcript rhythms reappeared in the treated cultures that were indistinguishable from those observed in control cultures (Fig. 3-3E, F). Moreover, introduction of ACO at ZT0 on 5DIV completely blocked the increase in iodopsin mRNA that normally occurred over the course of the 12-hour light period in control cultures (Fig 3-3C, F). Together, these data show that the effect of ACO on iodopsin mRNA levels is reversible and occurs within 12 hours of application. Iodopsin Transcript Levels in Explant Cultures Treated With ACO For 24hr Or 12hr Followed By Constant Darkness To determine if there was any evidence that ACO altered the function or phase of the circadian oscillators that drive iodopsin transcript rhythms, iodopsin mRNA levels were measured in cultures that were treated with ACO and were then maintained in constant darkness. As expected from our previous experiments, addition of ACO at the ZT12 on 4DIV or at ZT0 on 5DIV reduced iodopsin transcript levels (Fig 3-4A, B). Importantly, following removal of the blocker, iodopsin mRNA rhythms re-emerged in

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48 the cultures in the absence of the 12L:12D cycle that were congruent with those observed in the untreated cultures (Fig 3-4C, D). 0.000.450.901.351.802.25 0.000.450.901.351.802.25 Day 4 5 6 7 ARelative levels of Iodopsin mRNA/18SrRNAACO 24hrs Day 4 5 6 7 Day 4 5 6 7 Day 4 5 6 7 ACO 12hrsRelative levels of Iodopsin mRNA/18SrRNARelative levels of Iodopsin mRNA/18SrRNARelative levels of Iodopsin mRNA/18SrRNAACO 24hrsACO 12hrsBCD 0.000.450.901.351.802.25 88333333333 0.000.450.901.351.802.25 8833333333 Figure 3-4. Iodopsin expression in explant cultures treated with ACO followed by constant darkness (A) ACO (100 m) was added to the cultures at ZT12 on 4DIV and was removed at ZT12 on 5DIV. (B) ACO (100 m) was added to the cultures at ZT0 on 5DIV and was removed at ZT12 on 5DIV. In panels A and B, retinal explant cultures were analyzed at ZT0 (white bars) and at ZT12 (black bars). (C) Comparison of the data shown in panel A (red line) and the iodopsin transcript rhythms observed in the untreated cultures (black line). (D) Comparison of data shown in panel B (red line) and the iodopsin transcript rhythms observed in the untreated cultures (black line). The 12-hour light and dark periods are indicated below each panel using white and black bars, respectively. The dashed lines in each panel indicate the period of blocker treatment. In panels A and B, the group means SE at each time point are shown and the number of retinas in each group is indicated on the bars. In panels C and D, only the mean values are plotted for each group.

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49 These data suggest that short-term treatment of cultures with ACO does not alter the function or temporal characteristics of the circadian oscillators that drive iodopsin mRNA rhythms in chicken retina. Effects of ACO Treatment On AANAT and GCAP1 Transcript Levels In this series of experiments, we examined the specificity of the effect of ACO on gene transcript levels by examining the affects of ACO on transcript levels of AANAT and GCAP1, genes also expressed in chicken retinal photoreceptors (Chong et al., 2000;Semple-Rowland et al., 1999). 0.000.350.701.051.401.75 0.000.350.701.051.401.75 55337755 Day4 5 6 7 Day4 5 6 7Relative levels of AANAT mRNA/18S rRNA ACO 24 hrsRelative levels of AANAT mRNA/18S rRNA Day4 5 6 7 Day4 5 6 7 ACO 24 hrsAB Figure 3-5. AANAT expression in explant cultures treated with ACO for 24hrs. The explant cultures were maintained under 12L:12D conditions. (A) ACO (100 m) was added to the cultures at ZT15 on 4DIV and removed at ZT15 on 5DIV. Relative AANAT mRNA levels at ZT0 (white bars) and ZT15 (black bars) in the ACO treated cultures were shown. (B) The AANAT expression in ACO treated cultures (red line) was compared to that observed in untreated cultures (black lines). The 12-hour light and dark periods are indicated below each panel using white and black bars, respectively. The dashed lines in each panel indicate the period of blocker treatment. In panel A, the group means SE at each time point are shown and the number of retinas in each group is indicated on the bars. In panel B, only the mean values are plotted for each group. Treatment of the cultures with ACO beginning at ZT15 on 4DIV produced a significant suppression of AANAT mRNA levels in cultures examined at ZT15 on 5DIV, the values of which were similar to those observed at ZT0 in untreated cultures (Fig. 3

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50 5A, B) Within 24 hours of removal of the blocker, the AANAT rhythms paralleled to those observed in the untreated cultures. Day 4 5 6 7 Day 4 5 6 7 0.000.350.701.051.401.75 1111533333 0.000.350.701.051.401.75 Relative levels of GCAP1 mRNA/18S rRNA Relative levels of GCAP1 mRNA/18S rRNA Day 4 5 6 7 Day 4 5 6 7 0.000.350.701.051.401.75 0.000.350.701.051.401.75 1111355333Relative levels of GCAP1 mRNA/18S rRNA Day 4 5 6 7 Day 4 5 6 7 Relative levels of GCAP1 mRNA/18S rRNA ACO 48hrs ACO 48hrs 0.000.350.701.051.401.75 11113553333 0.000.350.701.051.401.75 Relative levels of GCAP1 mRNA/18S rRNA Relative levels of GCAP1 mRNA/18S rRNA ACO 24hrs ACO 24hrs ABCDEF

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51 Figure 3-6. GCAP-1 expression in explant cultures treated with ACO for 48 or 24 hrs and maintained under 12L:12D conditions. (A) GCAP1 expression in the untreated cultures from 4 to 7DIV. (B) The data in panel A are shown in line graph. (C) ACO (100m) was added to the cultures at ZT12 on 4DIV and removed at ZT12 on 6DIV. (D) The data in panel C are shown in line graph. (E) ACO (100 m) was added to the cultures at ZT12 on 4DIV and removed at ZT12 on 5DIV. (F) The data in panel E are shown in line graph. The 12-hour light and dark periods are indicated below each panel using white and black bars, respectively. The dashed lines indicate the period of ACO treatment. In panels A, C and E, the group means SE at each time point are shown and the number of retinas in each group is indicated on the bars. In panels B, D and F, only the mean values are plotted for each group. In explant cultures, GCAP1 mRNA levels gradually increase over the culture period, reaching relatively stable levels by 5DIV (Fig. 3-6A, B). Incubation of cultures with ACO for either 24 (Fig. 3-6E, F) or 48 hours (Fig. 3-6C, D) did not produce any significant changes in GCAP1 mRNA levels in the cultures. Together, the result of our analyses of AANAT and GCAP1 mRNA levels in explants treated with ACO indicate that ACO does not produce a generalized reduction in mRNA levels in retinal photoreceptors and suggest that the action of ACO on transcript levels may be restricted to genes that are regulated by retinal oscillators.

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52 Iodopsin Transcript Levels in Explant Cultures Treated With 18-GA For 48 Or 24 hrs Maintained Under A 12L:12D Cycle Day 4 5 6 7 ADay 4 5 6 7 0.000.450.901.351.802.25 154444154418-GA48hrs Relative levels of iodopsin mRNA/18S rRNA B Day 4 5 6 7 0.000.450.901.351.802.25 1515664444Relative levels of iodopsin mRNA/18S rRNA 18-GA24hrs 0.000.450.901.351.802.25 Relative levels of iodopsin mRNA/18S rRNA 18-GA48hrs 0.000.450.901.351.802.25 Day 4 5 6 7 Relative levels of iodopsin mRNA/18S rRNA 18-GA24hrs CDE Day 4 5 6 7 Day 4 5 6 7 0.1% DMSO 48hrs Relative levels of iodopsin mRNA/18S rRNA 0.000.450.901.351.802.25 F Day 4 5 6 7 Day 4 5 6 7 Relative levels of iodopsin mRNA/18S rRNA 0.1% DMSO 24hrs 0.000.450.901.351.802.25

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53 Figure 3-7. Iodopsin expression in explant cultures treated with 18-GA for 48 and 24hrs and maintained under 12L:12D conditions. (A) 18-GA (100 m) was added to the cultures at ZT12 on 4DIV and was removed at ZT12 on 6DIV. (B) 18-GA (100 m) was added to cultures on ZT12 on 4DIV and was removed on ZT12 on 5DIV. In panels A and B the relative iodopsin mRNA levels at ZT0 and ZT12 are shown in white bars and black bars, respectively. (C) The data shown in panel A (red lines) were compared to iodopsin transcript rhythms observed in untreated cultures (black lines). (D) The data shown in panel B (red line) were compared to iodopsin transcript rhythms observed in untreated cultures (black lines). The dashed lines in each panel indicate the period of 18-GA treatment. In panels A and B, the group means SE at each time point are shown and the number of retinas in each group is indicated on the bars. In panels C and D, only the mean values are plotted for each group. (E) Cultures were treated with 0.1% DMSO for 48 hrs from ZT12 on 4DIV to ZT12 on 6DIV. (F) Cultures were treated with 0.1% DMSO for 24 hrs from ZT12 on 4DIV to ZT12 on 5DIV. In panels E and F iodopsin mRNA levels in DMSO treated cultures at ZT0 (white bar) and ZT12 (hatched bar) were compared to those observed in untreated cultures (black lines). The dashed lines indicate the period of DMSO treatment. Each time point in the bar graphs represents the mean SE iodopsin mRNA levels measured in 3 cultures. For the line graphs, only the mean values are plotted for each group. Treatment of explant cultures with the glycyrrhetinic acid derivative, 18-GA, also significantly reduced iodopsin mRNA levels in the explant cultures maintained under cyclic light conditions. Treatment of cultures with 18-GA for either 48 or 24 hours reduced iodopsin mRNA levels to values significantly below the trough values observed in untreated cultures at ZT0 (Fig. 3-7A, B, C, D). Removal of the blocker produced a rapid 3 to 3.5-fold increase in iodopsin mRNA levels over the course of the first 12 hours following removal of the blocker. In cultures treated with the blocker for 24 hours, iodopsin mRNA rhythms similar to those observed in untreated cultures were re-established within 24 hours of removal of the blocker (Fig. 3-7D). Unlike cultures treated with 18-GA for 24 hours, the levels of iodopsin mRNA measured in cultures 24 hours following 48-hour treatment with 18-GA were not rhythmic and highly variable (Fig. 3-6B). This result is reminiscent of the desynchronized rhythms observed in SCN following

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54 treatment with octanol and halothane (Shinohara et al., 2000) and suggests that longer exposures of cultures to 18-GA might affect the coordination of the retinal oscillators that drive iodopsin rhythm. Treatment of cultures with 0.1% DMSO, the amount equivalent to that applied to the cultures treated with 100 M 18-GA, did not alter iodopsin mRNA rhythms in the retinal cultures (Fig. 3-6C, D). 0.000.450.901.351.802.25 1111553333 Day4 5 6 7 Day4 5 6 7 0.000.450.901.351.802.25 Day4 5 6 7 Day4 5 6 7Relative levels of iodopsin mRNA/18S rRNA Relative levels of iodopsin mRNA/18S rRNA GA48hrs GA48hrs AB Figure 3-8. Iodopsin expression in explant cultures treated with GA for 48hrs. The explant cultures were maintained under 12L:12D conditions in this experiment. (A) GA (100 m) was added to the cultures at ZT12 on 4DIV and removed at ZT12 on 6DIV. Relative iodopsin mRNA levels at ZT0 (white bars) and ZT12 (black bars) in the GA treated cultures were shown. (B) The iodopsin expression in GA treated cultures (red line) was compared to that observed in untreated cultures (black lines). The 12-hour light and dark periods are indicated below each panel using white and black bars, respectively. The dashed lines in each panel indicate the period of GA treatment. In panel A, the group means SE at each time point are shown and the number of retinas in each group is indicated on the bars. In panel B, only the mean values are plotted for each group. In a second control experiment, we examined the specificity of the actions of ACO and 18-GA on the cultures. Treatment of cultures for 48 hrs with 100 M GA, a chemically related inactive compound, did not alter iodopsin mRNA rhythms in the cultures (Fig. 3-8A, B). This result suggests that the actions of ACO and 18-GA on iodopsin transcript levels are specific to these compounds.

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55 Discussion The results of these experiments show that iodopsin and AANAT mRNA levels exhibit coordinated rhythms in cultures maintained under cyclic light conditions and that these rhythms are maintained under constant darkness. In contrast to iodopsin and AANAT, GCAP1 mRNA levels in cultured retinas gradually increase over the course of the culture period and do not exhibit a discernible rhythm. The presence of the iodopsin and AANAT mRNA rhythms in cultures maintained in constant darkness data confirm that circadian oscillators contribute to the regulation of the transcript levels of these genes in our culture paradigm. Addition of 100 M ACO to the cultures rapidly reduced the amount of iodopsin and AANAT mRNA in the cultures, an effect that was maintained as long as the blocker was present. The chemically related gap junction blocker, 18-GA, produced similar effects on iodopsin transcript rhythms in the retinal explant cultures. Within 24 hours of removal of ACO, the iodopsin and AANAT transcript rhythms reappeared in the cultures. ACO did not alter GCAP1 mRNA levels in the cultured retinas, a result that suggests that the actions of this blocker cannot be attributed to general transcription suppression. The hypothesis that we set out to test was that gap junctions play a role in maintaining coordinated iodopsin and AANAT transcript rhythms in retina. We expected that treatment of retina cultures with gap junction blockers would result in a desynchronization of the retinal oscillators that drive these rhythms and a subsequent loss of the iodopsin and AANAT transcript rhythms. The transcript levels of these two genes would assume the average of the peak and trough values of their rhythms in the presence of the blockers. However, the effects of the blockers were unexpected in light of the

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56 results of similar studies that were conducted in SCN slice cultures. The effects of ACO and 18-GA on iodopsin transcript rhythms in retina exhibited two major differences from the effects of two other gap junction blockers, octanol and halothane, on rhythmic AVP and VIP secretion in SCN slice cultures. First, the treatment of ACO or 18-GA suppressed iodopsin transcript levels to the trough or the values significantly lower than the trough of its rhythms observed in the untreated cultures. Hence, unlike the effects of octanol and halothane on AVP and VIP release in SCN cultures (Shinohara et al., 2000), the total amount of iodopsin mRNA generated in retinal photoreceptors over the treatment period is significantly less than that in the untreated cultures (Fig 3-3, 3-4, 3-7C, D). Second, the reduction of iodopsin transcript levels in response to the addition of the blockers occurs within 12 hours (Fig. 3-3C, F). This time period is much shorter than that observed in the study of SCN slice cultures, the exposure of which to octanol or halothane for 42 hours had no observable effects on the circadian rhythms of peptide secretion. The loss of the peptide release circadian rhythms was not observed until the SCN cultures were treated with the blockers for 7 days (Shinohara et al., 2000). Therefore, the effects of the two gap junction blockers on iodopsin transcript rhythms cannot be attributed to desynchronization of the circadian oscillators, which would be expected to take much longer for the subtle phase angle differences among individual oscillators to become significant enough to affect the overt ensemble gene expression rhythm. The reduction in the levels of iodopsin and AANAT transcripts in the presence of the blockers could result from cytotoxcity of the blockers, disruption of gene transcription or elevation of transcript degradation.

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57 Several of our experimental observations argue strongly against the possibility that the suppressing effects of ACO and 18-GA on iodopsin and AANAT transcript levels were due to cytotoxicity of the two gap junction blockers. First, transcription of the photoreceptor-specific gene GCAP1 was not suppressed by ACO. Second, the amount of iodopsin and AANAT transcript in the cultures rapidly recovered to the intermediate or peak levels 12 hours following the removal of the blockers. Finally, circadian rhythms of iodopsin transcript re-appeared within 24 hours after the block was removed, suggesting that retinal circadian oscillators are functional and the explant cultures are physiologically healthy during and after the treatment of ACO. Together, these observations do not support the thesis that the reduction in iodopsin and AANAT mRNA levels that we observed is due to massive cell death. It is possible that treatment of the cultures with the gap junction blockers disrupted gene transcription. This could occur if the gap junction blockers disrupted oscillator function or if they disrupted the coupling mechanism that normally allows the oscillator to drive transcription of these genes. Disruption of circadian oscillator function is unlikely because recovery of iodopsin rhythms following removal of the blockers was rapid (Fig 3-3). If the blockers altered the function of the oscillators driving iodopsin and AANAT transcript rhythms, we would have observed slower recovery of the rhythms following removal of the blockers. More definitively, iodopsin transcript rhythms were maintained in constant darkness after either 24 or 12hr treatment of ACO (Fig 3-4), suggesting that ACO did not disrupt the ability of the circadian oscillators to regulate iodopsin transcription. On the other hand, ACO may uncouple the functional circadian oscillators and iodopsin or AANAT transcription. A recent study of chick dispersed cell

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58 cultures has shown that the Ca 2+ influx stimulates the formation of cAMP, which in turn couples the circadian oscillators and the rhythms of AANAT enzyme activity that bear the similar temporal characteristics to AANAT transcript rhythms (Ivanova and Iuvone, 2003). How does ACO affect Ca 2+ influx to the photoreceptor cells? It has been shown that blockade of gap junction channels by 100M ACO, reduced Ca 2+ influx to photoreceptor cells from horizontal cells (Kamermans et al., 2001). Moreover, 100M ACO can directly reduce the voltage-gated Ca 2+ channel current by 37% in isolated cones, and inhibit the Ca 2+ influx by 57% in retinal slice preparations (Vessey et al., 2004). Therefore, it is possible that the reduction of Ca 2+ influx in presence of ACO results in the lack of stimulation of cAMP, which uncouples the circadian oscillators and the transcription of the output genes, such as AANAT and iodopsin. The exact molecular mechanisms, however, through which the reduced levels of Ca 2+ and/or cAMP suppress iodopsin and AANAT transcription, need to be further investigated. Based the current data, the possibility that the gap junction blockers increase the transcript degradation cannot be ruled out. It has been shown that a rapid turnover protein increases the degradation of AANAT transcript (Greve et al., 1999). Thus, it is also possible that the gap junction blockers enhance the activity of this protein, and lead to the increased degradation of AANAT and/or iodopsin transcript.

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CHAPTER 4 PROSPECTIVE A Real-Time Monitoring Culture System For Circadian-Regulated Gene Expression The major approaches used in this dissertation for studying the transcription of the genes that are expressed in retinal photoreceptors and regulated by circadian oscillators involve collecting significant number of retinal samples at each time point, homogenizing separate populations of the retinal samples, and measuring steady-state RNA levels through standard RNA assays. These typical methods are straightforward and have helped me observe interesting phenomena to prove the hypothesis. However, these methods have limitations for studying the dynamics of clock-regulated gene transcription. First, sample collections every 12hrs were performed in most experiments studying iodopsin and AANAT mRNA rhythms, the time-resolution of the experiments might not be high enough to reveal the complete temporal characteristics of the clock-regulated gene expression. Especially when the explant cultures were subjected to the changes of light cycle or the treatment of gap junction blockers or both, subtle changes in the phase of iodopsin or AANAT rhythms or the immediate transcriptional response of either genes may not be observed in the two-point analyses every light-dark or circadian cycle. Although the problem can be partly overcome by adding more time points during the period of light changes or blocker treatment, this solution makes the typical methods less efficient and more labor-intensive and time-consuming. Moreover, these approaches exclude the possibility to monitor the dynamics of clock-regulated gene expression in individual retinal explant cultures. The iodopsin 59

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60 transcript level shown at each time point in the end results is the ensemble average of iodopsin mRNA levels from the population of retinal cultures collected at that time point. The average value can reliably reflect iodopsin expression levels in individual cultures only if are the cultures in a certain experiment synchronized, which is the case in the experiments in which the cultures are exposed to light. Nonetheless, in the constant dark experiments, when the lights are turned off, the iodopsin mRNA rhythms in individual cultures begin to desynchronize. Therefore, taking the ensemble average of the desynchronized rhythms leads to the apparent reduction in amplitude across the retinal samples collected during the darkness. This could account for the damped amplitude of iodopsin transcript rhythms observed in the constant dark experiments (Fig2-5C, E). Finally, since the typical approaches measure the steady-state mRNA levels, it is difficult to discern if the manipulations act on transcriptional level or on posttranscriptional level. Although it has been shown that circadian oscillators regulate iodopsin expression at the transcriptional level in retinal cultures maintained under different lighting conditions(Pierce et al. 1993), the sites of actions of the gap junction blockers need further investigation. The monitoring of temporal characteristics of iodopsin transcription could be greatly improved and simplified by establishing a retinal culture system that carries a transgene of iodopsin promoter linked to firefly luciferase coding sequence (Iod:luc). The iodopsin promoter can be isolated from chicken genomic DNA library that is available in my current lab. The luciferase reporter has been used successfully for monitoring the transcription of Per1 with high time-resolution in nervous system of both transgenic Drosophila (Brandes et al. 1996;Stanewsky et al. 1997) and transgenic rats(Abe et al.

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61 2002;Yamazaki et al. 2000). The short half-life (about 2 hrs) of luciferase in vertebrate and the automated quantification system for measuring luciferase activity makes it an excellent reporter for real-time monitoring circadian-regulated iodopsin transcription in chicken retina. The modified lentiviral vector can be used as a novel tool to generate transgenic chicken retina. The lentiviral vector has been shown to be able to transduce both retinal progenitor cells and terminally differentiated cells in chicken embryo with high efficiency (>80% cells transduced) (Coleman et al. 2002;Coleman et al. 2003). The packaged lentivirus carrying the Iod:luc transgene will be injected into the neural tubes of chicken embryos at stage 10 to 12 (~embryonic day 2; E2). Following injection, the eggs will be incubated under 12L:12D conditions until E9. The embryonic retinas will be dissected on E9 and cultured with the media supplemented with luciferin under 12L:12D conditions. By continuously measuring the bioluminescence emitted from the retinal explant cultures, changes of iodopsin expression at the transcriptional level in response to the manipulations, such as light and pharmacological agents, can be monitored with high resolution from individual samples over one or two circadian cycles. Possible Mechanisms of Light Entrainment in Embryonic Chicken Retinas The phase of iodopsin transcript rhythms was reversed following the exposure of the retinal explant cultures to the reversed light cycle (Fig2-6A, B). This observation suggests that the circadian oscillators driving iodopsin mRNA rhythms can be entrained to the environmental light changes. Then what are the possible mechanisms underlying the light entrainment in the retinal explant cultures? The recent remarkable progress in the mechanisms responsible for light entrainment of behavioral rhythms in mammals provides clues for this question. A subset of retinal ganglion cells that express a novel photopigment, melanopsin, has been

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62 discovered to be intrinsically photoresponsive (ipRGCs). The dendrites of ipRGCs form extensive reticular networks to maximally detect light irradiance (Hattar et al. 2002;Provencio et al. 2002). The ipRGCs also arborize in the inner plexiform layer, suggesting that these cells receive synaptic input from the classical rod and cone photoreceptors (Belenky et al. 2003;Provencio et al. 2002). It has been show that either rod and cone image-forming system or ipRGCs system is sufficient to detect and transduce photic information through retinohypothalamic tract to the SCN, but neither system is necessary for the light entrainment process. Therefore, classical photoreceptors and ipRGCs are functionally redundant for light entrainment of the behavioral rhythms in mammals (Van Gelder 2003). However, it is notable that by the time the reversal of the light cycle is performed on 6DIV (Fig2-6A, B), the ganglion cells in the retinal explant cultures have degenerated, and are not detectable by cresyl violet staining (Fig2-1). The observations indicate that retinal ganglion cells are not necessary for the light entrainment of the circadian oscillators driving iodopsin transcript rhythms in the explant cultures. On the other hand, circadian oscillator functions in the explant cultures prepared from GUCY1*B embryonic chicken retinas were also characterized. GUCY1*B chicken carries a null mutation in Guanylate Cyclase gene, hence the classical phototransduction pathway is disabled in the retinal photoreceptors. The morphology of *B retinal explant cultures is indistinguishable from that of the cultures prepared from White Leghorn chicken retinas. The ganglion cells also degenerate by 5DIV (data not shown). Interestingly, the iodopsin mRNA rhythms in *B retinal explants exhibit similar dynamics of phase reversal to those observed in Leghorn explant cultures (Fig 4-1). These data suggest that both ganglion

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63 cells and classical phototransduction pathway are not required to entrain the circadian oscillators driving iodopsin transcription in the retinal explant cultures. 0.000.450.901.351.80 559998999899 0.000.450.901.351.80 Day 4 5 6 7 8 Day 4 5 6 7 8 Day 4 5 6 7 8 Relative Levels Iodopsin mRNA / 18S rRNA Relative Levels Iodopsin mRNA / 18S rRNA AB Figure 4-1. Iodopsin transcript rhythms in GUCY1*B and White Leghorn chicken retinal explant cultures maintained under 12L:12D followed by reversal of the light cycle. The explant cultures were prepared from E9 *B and leghorn chicken embryos. The cultures were maintained under 12L:12D until 6 DIV when they were transferred to a 12D:12L reversed cycle. The iodopsin mRNA rhythms from *B explant cultures were analyzed every 12 hours. The iodopsin transcript rhythms from *B retinal explant cultures (red line) were compared to those from Leghorn explant cultures (black line). The 12-hour light and dark periods are indicated below using white and black bars, respectively. In panel A, the group means SE at each time point are shown and the number of retinas in each group is indicated on the bars. In panel B, only the mean values are plotted for each group. Another candidate that might be important for entraining circadian oscillators driving iodopsin rhythms in the retinal explant cultures is Cryptochromes (Crys). Crys are flavin-based photopigments that are first identified as members of photolyase family in plants (Sancar 2000). Although lack of photolyase activity in animals, Crys have been shown to serve as circadian photopigments for light entrainment in both Drosophila (Sancar 2000) and zebrafish (Cermakian et al. 2002). More directly, both chicken Cry1 (Haque et al. 2002) and Cry2 (Bailey et al. 2002) highly express in retinal photoreceptor cells. The dual regulation of chicken Cry1 transcription by circadian oscillators and light

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64 suggests its involvement in the function of circadian oscillator and/or circadian photoreception in the photoreceptors of chicken retina (Haque et al. 2002).

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REFERENCE LIST Abe M, Herzog ED, Yamazaki S, Straume M, Tei H, Sakaki Y, Menaker M, Block GD (2002) Circadian rhythms in isolated brain regions. J Neurosci 22: 350-356. Adler R, Tamres A, Bradford RL, Belecky-Adams TL (2001) Microenvironmental regulation of visual pigment expression in the chick retina. Dev Biol 236: 454-464. Albrecht U, Sun ZS, Eichele G, Lee CC (1997) A differential response of two putative mammalian circadian regulators, mper1 and mper2, to light. Cell 91: 1055-1064. Anderson FE, Green CB (2000) Symphony of rhythms in the Xenopus laevis retina. Microsc Res Tech 50: 360-372. Bae K, Jin X, Maywood ES, Hastings MH, Reppert SM, Weaver DR (2001) Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian clock. Neuron 30: 525-536. Bailey MJ, Chong NW, Xiong J, Cassone VM (2002) Chickens' Cry2: molecular analysis of an avian cryptochrome in retinal and pineal photoreceptors. FEBS Lett 513: 169-174. Becker D, Bonness V, V, Mobbs P (1998) Cell coupling in the retina: patterns and purpose. Cell Biol Int 22: 781-792. Becker DL, Bonness V, Catsicas M, Mobbs P (2002) Changing patterns of ganglion cell coupling and connexin expression during chick retinal development. J Neurobiol 52: 280-293. Belecky-Adams TL, Scheurer D, Adler R (1999) Activin family members in the developing chick retina: expression patterns, protein distribution, and in vitro effects. Dev Biol 210: 107-123. Belenky MA, Smeraski CA, Provencio I, Sollars PJ, Pickard GE (2003) Melanopsin retinal ganglion cells receive bipolar and amacrine cell synapses. J Comp Neurol 460: 380-393. Bernard M, Guerlotte J, Greve P, Grechez-Cassiau A, Iuvone MP, Zatz M, Chong NW, Klein DC, Voisin P (1999) Melatonin synthesis pathway: circadian regulation of the genes encoding the key enzymes in the chicken pineal gland and retina. Reprod Nutr Dev 39: 325-334. 65

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66 Besharse JC, Iuvone PM (1983) Circadian clock in Xenopus eye controlling retinal serotonin N-acetyltransferase. Nature 305: 133-135. Besharse JC, Iuvone PM (1992) Is dopamine a light-adaptive or a dark-adaptive modulator in retina? Neurochem Int 20: 193-199. Blau J, Young MW (1999) Cycling vrille expression is required for a functional Drosophila clock. Cell 99: 661-671. Bouskila Y, Dudek FE (1993) Neuronal synchronization without calcium-dependent synaptic transmission in the hypothalamus. Proc Natl Acad Sci U S A 90: 3207-3210. Brandes C, Plautz JD, Stanewsky R, Jamison CF, Straume M, Wood KV, Kay SA, Hall JC (1996) Novel features of drosophila period Transcription revealed by real-time luciferase reporting. Neuron 16: 687-692. Burnside B (2001) Light and circadian regulation of retinomotor movement. Prog Brain Res 131: 477-485. Cahill GM, Besharse JC (1991) Resetting the circadian clock in cultured Xenopus eyecups: regulation of retinal melatonin rhythms by light and D2 dopamine receptors. J Neurosci 11: 2959-2971. Cahill GM, Besharse JC (1992) Light-sensitive melatonin synthesis by Xenopus photoreceptors after destruction of the inner retina. Vis Neurosci 8: 487-490. Cahill GM, Besharse JC (1993) Circadian clock functions localized in xenopus retinal photoreceptors. Neuron 10: 573-577. Cahill GM, Grace MS, Besharse JC (1991) Rhythmic regulation of retinal melatonin: metabolic pathways, neurochemical mechanisms, and the ocular circadian clock. Cell Mol Neurobiol 11: 529-560. Catsicas M, Bonness V, Becker D, Mobbs P (1998) Spontaneous Ca2+ transients and their transmission in the developing chick retina. Curr Biol 8: 283-286. Cermakian N, Pando MP, Thompson CL, Pinchak AB, Selby CP, Gutierrez L, Wells DE, Cahill GM, Sancar A, Sassone-Corsi P (2002) Light induction of a vertebrate clock gene involves signaling through blue-light receptors and MAP kinases. Curr Biol 12: 844-848. Chang DC, Reppert SM (2001) The circadian clocks of mice and men. Neuron 29: 555-558. Chong NW, Bernard M, Klein DC (2000) Characterization of the chicken serotonin N-acetyltransferase gene. Activation via clock gene heterodimer/E box interaction. J Biol Chem 275: 32991-32998.

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67 Coleman JE, Fuchs GE, Semple-Rowland SL (2002) Analyses of the guanylate cyclase activating protein-1 gene promoter in the developing retina. Invest Ophthalmol Vis Sci 43: 1335-1343. Coleman JE, Huentelman MJ, Kasparov S, Metcalfe BL, Paton JF, Katovich MJ, Semple-Rowland SL, Raizada MK (2003) Efficient large-scale production and concentration of HIV-1-based lentiviral vectors for use in vivo. Physiol Genomics 12: 221-228. Colwell CS (2000) Rhythmic coupling among cells in the suprachiasmatic nucleus. J Neurobiol 43: 379-388. Cook B, Portera-Cailliau C, Adler R (1998) Developmental neuronal death is not a universal phenomenon among cell types in the chick embryo retina. J Comp Neurol 396: 12-19. Cook JE, Becker DL (1995) Gap junctions in the vertebrate retina. Microsc Res Tech 31: 408-419. Darlington TK, Wager-Smith K, Ceriani MF, Staknis D, Gekakis N, Steeves TD, Weitz CJ, Takahashi JS, Kay SA (1998) Closing the circadian loop: CLOCK-induced transcription of its own inhibitors per and tim. Science 280: 1599-1603. Devlin PF, Kay SA (2001) Circadian photoperception. Annu Rev Physiol 63: 677-694. Dunlap JC (1999) Molecular bases for circadian clocks. Cell 96: 271-290. Ebihara S, Uchiyaya k, Oshima I (1984) Circadian Organization in the pigeon, Coluba livia: The role of the pineal organ and the eye. J Comp Physiol 154A: 59-69. Gardino PF, dos Santos RM, Hokoc JN (1993) Histogenesis and topographical distribution of tyrosine hydroxylase immunoreactive amacrine cells in the developing chick retina. Brain Res Dev Brain Res 72: 226-236. Gaston S, Menaker M (1968) Pineal function: the biological clock in the sparrow? Science 160: 1125-1127. Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, King DP, Takahashi JS, Weitz CJ (1998) Role of the CLOCK protein in the mammalian circadian mechanism. Science 280: 1564-1569. Geusz ME, Foster RG, deGrip WJ, Block GD (1997) Opsin-like immunoreactivity in the circadian pacemaker neurons and photoreceptors of the eye of the opisthobranch mollusc Bulla gouldiana. Cell Tissue Res 287: 203-210. Ghalayini A, Anderson RE (1984) Phosphatidylinositol 4,5-bisphosphate: light-mediated breakdown in the vertebrate retina. Biochem Biophys Res Commun 124: 503-506.

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68 Gillette MU, McArthur AJ (1996) Circadian actions of melatonin at the suprachiasmatic nucleus. Behav Brain Res 73: 135-139. Glossop NR, Lyons LC, Hardin PE (1999) Interlocked feedback loops within the Drosophila circadian oscillator. Science 286: 766-768. Gold GH, Dowling JE (1979) Photoreceptor coupling in retina of the toad, Bufo marinus. I. Anatomy. J Neurophysiol 42: 292-310. Goldberg GS, Moreno AP, Bechberger JF, Hearn SS, Shivers RR, MacPhee DJ, Zhang YC, Naus CC (1996) Evidence that disruption of connexon particle arrangements in gap junction plaques is associated with inhibition of gap junctional communication by a glycyrrhetinic acid derivative. Exp Cell Res 222: 48-53. Goodenough DA, Goliger JA, Paul DL (1996) Connexins, connexons, and intercellular communication. Annu Rev Biochem 65: 475-502. Green CB (1998) How cells tell time. Trends Cell Biol 8: 224-230. Green CB, Besharse JC (1994) Tryptophan hydroxylase expression is regulated by a circadian clock in Xenopus laevis retina. J Neurochem 62: 2420-2428. Green CB, Besharse JC, Zatz M (1996) Tryptophan hydroxylase mRNA levels are regulated by the circadian clock, temperature, and cAMP in chick pineal cells. Brain Res 738: 1-7. Green CB, Cahill GM, Besharse JC (1995) Regulation of tryptophan hydroxylase expression by a retinal circadian oscillator in vitro. Brain Res 677: 283-290. Greve P, Alonso-Gomez A, Bernard M, Ma M, Haque R, Klein DC, Iuvone PM (1999) Serotonin N-acetyltransferase mRNA levels in photoreceptor-enriched chicken retinal cell cultures: elevation by cyclic AMP. J Neurochem 73: 1894-1900. Guan X, Wilson S, Schlender KK, Ruch RJ (1996) Gap-junction disassembly and connexin 43 dephosphorylation induced by 18 beta-glycyrrhetinic acid. Mol Carcinog 16: 157-164. Hampson EC, Vaney DI, Weiler R (1992) Dopaminergic modulation of gap junction permeability between amacrine cells in mammalian retina. J Neurosci 12: 4911-4922. Hampson EC, Weiler R, Vaney DI (1994) pH-gated dopaminergic modulation of horizontal cell gap junctions in mammalian retina. Proc R Soc Lond B Biol Sci 255: 67-72. Haque R, Chaurasia SS, Wessel JH, III, Iuvone PM (2002) Dual regulation of cryptochrome 1 mRNA expression in chicken retina by light and circadian oscillators. Neuroreport 13: 2247-2251.

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69 Hattar S, Liao HW, Takao M, Berson DM, Yau KW (2002) Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity. Science 295: 1065-1070. Hayasaka N, LaRue SI, Green CB (2002) In vivo disruption of Xenopus CLOCK in the retinal photoreceptor cells abolishes circadian melatonin rhythmicity without affecting its production levels. J Neurosci 22: 1600-1607. Hayashi F, Amakawa T (1985) Light-mediated breakdown of phosphatidylinositol-4,5-bisphosphate in isolated rod outer segments of frog photoreceptor. Biochem Biophys Res Commun 128: 954-959. Herzog ED, Geusz ME, Khalsa SB, Straume M, Block GD (1997) Circadian rhythms in mouse suprachiasmatic nucleus explants on multimicroelectrode plates. Brain Res 757: 285-290. Herzog ED, Takahashi JS, Block GD (1998) Clock controls circadian period in isolated suprachiasmatic nucleus neurons. Nat Neurosci 1: 708-713. Iuvone PM (1990) Development of melatonin synthesis in chicken retina: regulation of serotonin N-acetyltransferase activity by light, circadian oscillators, and cyclic AMP. J Neurochem 54: 1562-1568. Ivanova TN, Iuvone PM (2003b) Circadian rhythm and photic control of cAMP level in chick retinal cell cultures: a mechanism for coupling the circadian oscillator to the melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase, in photoreceptor cells. Brain Res 991: 96-103. Ivanova TN, Iuvone PM (2003a) Melatonin synthesis in retina: circadian regulation of arylalkylamine N-acetyltransferase activity in cultured photoreceptor cells of embryonic chicken retina. Brain Res 973: 56-63. Jiang ZG, Yang YQ, Allen CN (1997) Tracer and electrical coupling of rat suprachiasmatic nucleus neurons. Neuroscience 77: 1059-1066. Kamermans M, Fahrenfort I, Schultz K, Janssen-Bienhold U, Sjoerdsma T, Weiler R (2001) Hemichannel-mediated inhibition in the outer retina. Science 292: 1178-1180. Kasahara T, Okano T, Haga T, Fukada Y (2002) Opsin-G11-mediated signaling pathway for photic entrainment of the chicken pineal circadian clock. J Neurosci 22: 7321-7325. Krizaj D, Gabriel R, Owen WG, Witkovsky P (1998) Dopamine D2 receptor-mediated modulation of rod-cone coupling in the Xenopus retina. J Comp Neurol 398: 529-538.

PAGE 79

70 Kunz H, Achermann P (2003) Simulation of circadian rhythm generation in the suprachiasmatic nucleus with locally coupled self-sustained oscillators. J Theor Biol 224: 63-78. Larkin P, Baehr W, Semple-Rowland SL (1999) Circadian regulation of iodopsin and clock is altered in the retinal degeneration chicken retina. Brain Res Mol Brain Res 70: 253-263. Larkin P, Semple-Rowland SL (2001) A null mutation in guanylate cyclase-1 alters the temporal dynamics and light entrainment properties of the iodopsin rhythm in cone photoreceptor cells. Brain Res Mol Brain Res 92: 49-57. Lebedev DS, Byzov AL, Govardovskii VI (1998) Photoreceptor coupling and boundary detection. Vision Res 38: 3161-3169. LeSauter J, Silver R (1998) Output signals of the SCN. Chronobiol Int 15: 535-550. Li L, Dowling JE (2000) Effects of dopamine depletion on visual sensitivity of zebrafish. J Neurosci 20: 1893-1903. Liu C, Fukuhara C, Wessel JH, III, Iuvone PM, Tosini G (2004) Localization of Aa-nat mRNA in the rat retina by fluorescence in situ hybridization and laser capture microdissection. Cell Tissue Res 315: 197-201. Liu C, Reppert SM (2000) GABA synchronizes clock cells within the suprachiasmatic circadian clock. Neuron 25: 123-128. Manglapus MK, Iuvone PM, Underwood H, Pierce ME, Barlow RB (1999) Dopamine mediates circadian rhythms of rod-cone dominance in the Japanese quail retina. J Neurosci 19: 4132-4141. Manglapus MK, Uchiyama H, Buelow NF, Barlow RB (1998) Circadian rhythms of rod-cone dominance in the Japanese quail retina. J Neurosci 18: 4775-4784. Marin-Teva JL, Cuadros MA, Calvente R, Almendros A, Navascues J (1999) Naturally occurring cell death and migration of microglial precursors in the quail retina during normal development. J Comp Neurol 412: 255-275. Martinek S, Inonog S, Manoukian AS, Young MW (2001) A role for the segment polarity gene shaggy/GSK-3 in the Drosophila circadian clock. Cell 105: 769-779. Millar FA, Fisher SC, Muir CA, Edwards E, Hawthorne JN (1988) Polyphosphoinositide hydrolysis in response to light stimulation of rat and chick retina and retinal rod outer segments. Biochim Biophys Acta 970: 205-211. Moog R (1995) [Chronobiological health of the circadian system: examples for disorders of the sleep-waking rhythm]. Wien Med Wochenschr 145: 452-453.

PAGE 80

71 Moore RY, Bernstein ME (1989) Synaptogenesis in the rat suprachiasmatic nucleus demonstrated by electron microscopy and synapsin I immunoreactivity. J Neurosci 9: 2151-2162. Nguyen-Legros J, Hicks D (2000) Renewal of photoreceptor outer segments and their phagocytosis by the retinal pigment epithelium. Int Rev Cytol 196: 245-313. Ogilvie JM, Speck JD, Lett JM, Fleming TT (1999) A reliable method for organ culture of neonatal mouse retina with long-term survival. J Neurosci Methods 87: 57-65. Oishi T, Yamao M, Kondo C, Haida Y, Masuda A, Tamotsu S (2001) Multiphotoreceptor and multioscillator system in avian circadian organization. Microsc Res Tech 53: 43-47. Okamura H, Miyake S, Sumi Y, Yamaguchi S, Yasui A, Muijtjens M, Hoeijmakers JH, van der Horst GT (1999) Photic induction of mPer1 and mPer2 in cry-deficient mice lacking a biological clock. Science 286: 2531-2534. Panda S, Antoch MP, Miller BH, Su AI, Schook AB, Straume M, Schultz PG, Kay SA, Takahashi JS, Hogenesch JB (2002) Coordinated transcription of key pathways in the mouse by the circadian clock. Cell 109: 307-320. Piccolino M, Neyton J, Gerschenfeld HM (1984) Decrease of gap junction permeability induced by dopamine and cyclic adenosine 3':5'-monophosphate in horizontal cells of turtle retina. J Neurosci 4: 2477-2488. Piccolino M, Neyton J, Witkovsky P, Gerschenfeld HM (1982) gamma-Aminobutyric acid antagonists decrease junctional communication between L-horizontal cells of the retina. Proc Natl Acad Sci U S A 79: 3671-3675. Pierce ME (1999) Circadian organization in quail retina: differential regulation of melatonin synthesis and iodopsin gene expression in vitro. Vis Neurosci 16: 843-848. Pierce ME, Sheshberadaran H, Zhang Z, Fox LE, Applebury ML, Takahashi JS (1993) Circadian regulation of iodopsin gene expression in embryonic photoreceptors in retinal cell culture. Neuron 10: 579-584. Pottek M, Hoppenstedt W, Janssen-Bienhold U, Schultz K, Perlman I, Weiler R (2003) Contribution of connexin26 to electrical feedback inhibition in the turtle retina. J Comp Neurol 466: 468-477. Price JL, Blau J, Rothenfluh A, Abodeely M, Kloss B, Young MW (1998) double-time is a novel Drosophila clock gene that regulates PERIOD protein accumulation. Cell 94: 83-95.

PAGE 81

72 Provencio I, Rollag MD, Castrucci AM (2002) Photoreceptive net in the mammalian retina. This mesh of cells may explain how some blind mice can still tell day from night. Nature 415: 493. Raviola E, Gilula NB (1973) Gap junctions between photoreceptor cells in the vertebrate retina. Proc Natl Acad Sci U S A 70: 1677-1681. Reppert SM, Schwartz WJ (1984) The suprachiasmatic nuclei of the fetal rat: characterization of a functional circadian clock using 14C-labeled deoxyglucose. J Neurosci 4: 1677-1682. Reppert SM, Weaver DR (2001) Molecular analysis of mammalian circadian rhythms. Annu Rev Physiol 63: 647-676. Rosenwasser AM, Dwyer SM (2001) Circadian phase shifting: Relationships between photic and nonphotic phase-response curves. Physiol Behav 73: 175-183. Sancar A (2000) Cryptochrome: the second photoactive pigment in the eye and its role in circadian photoreception. Annu Rev Biochem 69: 31-67. Schneeweis DM, Schnapf JL (1999) The photovoltage of macaque cone photoreceptors: adaptation, noise, and kinetics. J Neurosci 19: 1203-1216. Schwartz WJ, Reppert SM, Eagan SM, Moore-Ede MC (1983) In vivo metabolic activity of the suprachiasmatic nuclei: a comparative study. Brain Res 274: 184-187. Sekaran S, Foster RG, Lucas RJ, Hankins MW (2003) Calcium imaging reveals a network of intrinsically light-sensitive inner-retinal neurons. Curr Biol 13: 1290-1298. Semple-Rowland SL, Larkin P, Bronson JD, Nykamp K, Streit WJ, Baehr W (1999) Characterization of the chicken GCAP gene array and analyses of GCAP1, GCAP2, and GC1 gene expression in normal and rd chicken pineal. Mol Vis 5: 14. Semple-Rowland SL, van der WH (1992) Visinin: biochemical and molecular comparisons in normal and rd chick retina. Biochem Biophys Res Commun 183: 456-461. Shearman LP, Sriram S, Weaver DR, Maywood ES, Chaves I, Zheng B, Kume K, Lee CC, van der Horst GT, Hastings MH, Reppert SM (2000) Interacting molecular loops in the mammalian circadian clock. Science 288: 1013-1019. Shearman LP, Zylka MJ, Weaver DR, Kolakowski LF, Jr., Reppert SM (1997) Two period homologs: circadian expression and photic regulation in the suprachiasmatic nuclei. Neuron 19: 1261-1269.

PAGE 82

73 Shibata S, Moore RY (1993) Tetrodotoxin does not affect circadian rhythms in neuronal activity and metabolism in rodent suprachiasmatic nucleus in vitro. Brain Res 606: 259-266. Shinohara K, Funabashi T, Mitushima D, Kimura F (2000) Effects of gap junction blocker on vasopressin and vasoactive intestinal polypeptide rhythms in the rat suprachiasmatic nucleus in vitro. Neurosci Res 38: 43-47. Simpsom SM, Follett BK (1981) Pineal and Hypothalamic pacemakers: their role in regulating circadian rhythmicity in Japanese quail. J Comp Physiol 144A: 381-389. Stanewsky R, Jamison CF, Plautz JD, Kay SA, Hall JC (1997) Multiple circadian-regulated elements contribute to cycling period gene expression in Drosophila. EMBO J 16: 5006-5018. Sun ZS, Albrecht U, Zhuchenko O, Bailey J, Eichele G, Lee CC (1997) RIGUI, a putative mammalian ortholog of the Drosophila period gene. Cell 90: 1003-1011. Szente M, Gajda Z, Said AK, Hermesz E (2002) Involvement of electrical coupling in the in vivo ictal epileptiform activity induced by 4-aminopyridine in the neocortex. Neuroscience 115: 1067-1078. Tosini G (2000) Melatonin circadian rhythm in the retina of mammals. Chronobiol Int 17: 599-612. Tosini G, Menaker M (1996) Circadian rhythms in cultured mammalian retina. Science 272: 419-421. Tosini G, Menaker M (1998) The clock in the mouse retina: melatonin synthesis and photoreceptor degeneration. Brain Res 789: 221-228. Tsukamoto Y, Masarachia P, Schein SJ, Sterling P (1992) Gap junctions between the pedicles of macaque foveal cones. Vision Res 32: 1809-1815. Underwood H (1994) The circadian rhythm of thermoregulation in Japanese quail. I. Role of the eyes and pineal. J Comp Physiol [A] 175: 639-653. Van Gelder RN (2003) Making (a) sense of non-visual ocular photoreception. Trends Neurosci 26: 458-461. Vaney DI, Weiler R (2000) Gap junctions in the eye: evidence for heteromeric, heterotypic and mixed-homotypic interactions. Brain Res Brain Res Rev 32: 115-120. Vessey JP, Lalonde MR, Mizan HA, Welch NC, Kelly ME, Barnes S (2004) Carbenoxolone inhibition of voltage-gated Ca channels and synaptic transmission in the retina. J Neurophysiol.

PAGE 83

74 Welsh DK, Logothetis DE, Meister M, Reppert SM (1995) Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms. Neuron 14: 697-706. Wong WT, Sanes JR, Wong RO (1998) Developmentally regulated spontaneous activity in the embryonic chick retina. J Neurosci 18: 8839-8852. Yamazaki S, Numano R, Abe M, Hida A, Takahashi R, Ueda M, Block GD, Sakaki Y, Menaker M, Tei H (2000) Resetting central and peripheral circadian oscillators in transgenic rats. Science 288: 682-685. Yang XL, Wu SM (1989) Modulation of rod-cone coupling by light. Science 244: 352-354. Yoshimura T, Suzuki Y, Makino E, Suzuki T, Kuroiwa A, Matsuda Y, Namikawa T, Ebihara S (2000) Molecular analysis of avian circadian clock genes. Brain Res Mol Brain Res 78: 207-215. Yoshizawa T, Kuwata O (1991) Iodopsin, a red-sensitive cone visual pigment in the chicken retina. Photochem Photobiol 54: 1061-1070. Young MW (2000) The tick-tock of the biological clock. Sci Am 282: 64-71. Zhang Y, Coleman JE, Fuchs GE, Semple-Rowland SL (2003) Circadian oscillator function in embryonic retina and retinal explant cultures. Brain Res Mol Brain Res 114: 9-19. Zhu H, LaRue S, Whiteley A, Steeves TD, Takahashi JS, Green CB (2000) The Xenopus clock gene is constitutively expressed in retinal photoreceptors. Brain Res Mol Brain Res 75: 303-308. Zhuang M, Wang Y, Steenhard BM, Besharse JC (2000) Differential regulation of two period genes in the Xenopus eye. Brain Res Mol Brain Res 82: 52-64. Zylka MJ, Shearman LP, Weaver DR, Reppert SM (1998) Three period homologs in mammals: differential light responses in the suprachiasmatic circadian clock and oscillating transcripts outside of brain. Neuron 20: 1103-1110.

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BIOGRAPHICAL SKETCH Yan Zhang was born in Tianjin, the third largest city in China, on Dec 28 th 1972. After receiving the award for the best student in the primary school in Tianjin city for three consecutive years, he entered Nankai high school, one of the best high schools in China. During three years of his junior high school, he ranked first of 265 students in 8 out of 12 comprehensive exams. Consequently, he was admitted to Nankai senior high school with the exemption of the final exam. After another three years of endeavors in that highly competitive environment, he chose to enter Tianjin Medical University to study clinical medicine with the intention of his parents and with his own hope of a better future, although he loved and was good at mathematics at the time of graduating from high school. He did not understand the value of clinical medicine until the last two years of medical training when he did probation in the Department of Internal Medicine and an internship in the Department of Surgery. It was during those two years that he truly realized that a good doctor could relieve suffering and save the lives of patients, thus gaining respect from people. However, at the time he graduated from medical school in 1996, molecular biology had just become the hottest area in China. Additionally, he thought that because he was still very young, his education should not end at the age of 23. He then chose the National Key Laboratory of Hormone and Brain Development in China to pursue the MS degree in molecular endocrinology. During three years of work in the laboratory, he participated in the purification of glutamic acid decarboxylase from 75

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76 human brain and the development of an ELISA system for early detection of Type I diabetes using the purified protein as antigen. His thesis, Cloning and Expression of Human Somatostatin Gene in E. coli., summarized his work in molecular biology. After receiving his MS degree in August 1999, he came to the University of Florida, joined the Department of Neuroscience in the interdisciplinary program in the College of Medicine, and began his PhD study under the supervision of Dr. Susan Semple-Rowland. His research during the Ph.D. study is reflected in this dissertation.


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EFFECTS OF GAP JUNCTION BLOCKERS ON CIRCADIAN REGULATION OF
GENE EXPRESSION IN EMBRYONIC RETINAL EXPLANT CULTURES
















By

YAN ZHANG


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2004

































Copyright 2004

by

Yan Zhang


























To my beloved parents who always provide the strongest support, the greatest
encouragement, and the most sincere advice.
To all the kind people who have guided, taught, and assisted me through twenty-five
years of education















ACKNOWLEDGMENTS

First, I would like to thank my mentor, Dr. Susan L. Semple-Rowland, for her

inspiring direction and careful training. She is a great educator and a dedicated scientist. I

feel very fortunate to work under her supervision and obtained rapid progress in research

during my Ph.D. study. Next, I thank my committee members, Dr. Neil Rowland, Dr.

Marieta Heaton, and Dr. Barbara Battelle, for their helpful suggestions on my research.

I also want to thank all the past and current members of Dr. Semple-Rowland's

laboratory for their friendships and assistance. In particular, I thank Dr. Jason Coleman

for the help with immunocytochemistry experiment and the provocative discussions on

my research and career choice, Daniel Selbst for the assistance in RNA extraction, Gabby

Fuchs for the assistance in Cresyl Violet staining of retinal explant cultures, and Miguel

Tepedino for his initial teaching of Northern Blot analysis.

Finally, I would express my deep appreciation for my parents for their great

support and encouragement, without which I would not have been able to successfully

complete twenty-five years of education.















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ......... .................................................................................... iv

LIST OF FIGURES ......... ..................................... ....... vii

A B S T R A C T .......................................... .................................................. v iii

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

General Features of Circadian Rhythms .............................................. .................
Basic Properties of Circadian Rhythm s....................... ............................... 1
M olecular Bases of Circadian Clocks ....................................... ............... 2
Light Entrainment of Circadian Clocks .............................................................4
Circadian Clocks in the Central Nervous System (CNS)................................
C ircadian R hythm s In R etina ................. ....................................................................6
Retinal Functions Regulated by Circadian Clocks..............................................6
Cellular Location of Circadian Clocks Within Retina ..........................................7
Light Entrainment of Retinal Photoreceptor Clocks .........................................8
Synchronization of Circadian Clocks ............... ............... .................. ............... 9
Synchronization of Circadian Clocks in SCN ...................................................9
Expression and Function of Gap Junctions in Retina................................11

2 CHARACTERIZATION OF CIRCADIAN OSCILLATOR FUNCTION IN
EMBRYONIC RETINA AND RETINAL EXPLANT CULTURES ........................14

Intro du action ...................................... ................................................ 14
M materials and M methods ................................................................. ...................... 16
Preparation of Retinal Explant Cultures............... .............................................16
Explant M orphology ................ ...... .......... .......... ... ....... ......... 17
lodopsin Gene and Protein Expression in Retinal Explant Cultures ................. 18
L fighting P aradigm s .......................... .... ................ ... .... .. .......... 19
Cyclic light .................................... .......................... .... ....... .19
C o n stan t d ark .............................................. ................ 19
R reversal of the light cycle ................................ ......................... ........ 19
RNA Analyses .................................... ........................... ........... 20
R e su lts ................. ... ........ ......... ............. ......................... ................ 2 1
Morphology of Developing Retinal Explant Cultures............... ... ...............21









Comparison of Iodopsin Expression Onset in Retinal Explant Cultures and in
ovo ............... ................ .... .................................. ........... 25
Iodopsin Transcript Rhythm s ........................................ ......................... 27
Cyclic light ........................................................... .... ....... .27
C on stant d ark ........................................ ....... ................ 2 9
Reversal of the light:dark cycle.............................................. .............. 31
D discussion ................................ .. .................................. ........... 33

3 GAP JUNCTION BLOCKERS ABOLISH CIRCADIAN RHYTHMS OF GENE
EXPRESSION IN RETINAL PHOTORECEPTORS.................. ...................36

In tro du ctio n ...................................... ................................................ 3 6
M methods and M materials ............................. .................................. .... ...... ...... 39
C hem icals and R eagents......................................................................... ... ... 39
R etinal Explant Cultures........................................................ ... ............... 40
Lighting and Blocker Delivery Paradigms:........................................... 40
12 L :12 D : ................................................................... .. 4 0
12L:12D followed by constant darkness:..................................... ....41
RN A A nalysis................................................... 41
R results ....... ........... .............. ............... .. ...................... ............... 42
lodopsin and AANAT Transcript Rhythms in Untreated Explant Cultures: ......42
lodopsin Transcript Levels in Explant Cultures Treated With ACO For 48
Or 24 hrs Maintained Under A 12L:12D Cycle............................................45
Iodopsin Transcript Levels in Explant Cultures Treated With ACO For 24hr
Or 12hr Followed By Constant Darkness......................................................47
Effects of ACO Treatment On AANAT and GCAP1 Transcript Levels ..........49
Iodopsin Transcript Levels in Explant Cultures Treated With 18-pGA For 48
Or 24 hrs Maintained Under A 12L:12D Cycle.................................... 52
D isc u ssio n ................................................... .................. ................ 5 5

4 PROSPECTIVE ....... ........ ............... .......... .....................59

A Real-Time Monitoring Culture System For Circadian-Regulated Gene
E x p re ssio n ................................... .... .............. ............. ........ ............... 5 9
Possible Mechanisms of Light Entrainment in Embryonic Chicken Retinas.............61

REFER EN CE LIST ........................ .. ........................ .. .... ........ ........ 65

B IO G R A PH IC A L SK E TCH ..................................................................... ..................75
















LIST OF FIGURES


Figure p

2-1 Morphological comparison of chicken embryonic retina and retinal explant
cultures. .............................................................................22

2-2 Quantification of the morphological integrity of retinal explant cultures ...............23

2-3 TH immunostaining of explants and embryonic and post-hatch retinas..................25

2-4 Onset of iodopsin expression in retinal explant cultures.................................26

2-5 Iodopsin transcript rhythms in explant cultures and embryos maintained under
12L:12D and constant dark conditions.................................. ....................... 28

2-6 Iodopsin transcript rhythms in explant cultures and embryos following reversal
of the light:dark cycle......... ...................................................... ..............

3-1 Iodopsin mRNA rhythms in retinal explant cultures. ............................................43

3-2 AANAT mRNA rhythms in retinal explant cultures. ............................................44

3-3 Iodopsin expression in explant cultures treated with ACO for 48 and 24hrs ..........46

3-4 Iodopsin expression in explant cultures treated with ACO followed by constant
darkness .............................. ......... ... ........................ ................. 48

3-5 AANAT expression in explant cultures treated with ACO for 24hrs ....................49

3-6 GCAP-1 expression in explant cultures treated with ACO for 48 or 24 hrs and
maintained under 12L:12D conditions. ........................................ ............... 51

3-7 Iodopsin expression in explant cultures treated with 18-pGA for 48 and 24hrs
and maintained under 12L:12D conditions.. ..................................................... 53

3-8 Iodopsin expression in explant cultures treated with GA for 48hrs.......................54

4-1 Iodopsin transcript rhythms in GUCY *B and White Leghorn chicken retinal
explant cultures maintained under 12L: 12D followed by reversal of the light
cy cle ............................................................................... 6 3















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

EFFECTS OF GAP JUNCTION BLOCKERS ON CIRCADIAN REGULATION OF
GENE EXPRESSION IN EMBRYONIC RETINAL EXPLANT CULTURES

By

Yan Zhang

May 2004

Chair: Susan L. Semple-Rowland
Major Department: Neuroscience

The suprachiasmic nucleus (SCN) is comprised of autonomous, single-cell

oscillators that work in concert to generate coordinated circadian rhythms. Substantial

evidence demonstrates that intercellular gap junction communication plays a role in the

coordination of circadian rhythms in SCN. Chicken retina also contains functional

circadian oscillators that drive coordinated transcript rhythms of several photoreceptor-

specific genes, including iodopsin and Arylalkylamine N-acetyltransferase (AANAT),

tryptophan hydroxylase. Based on the studies in SCN, we hypothesized that gap junction

communication also plays a role in coordinating transcript rhythms of circadian-regulated

genes in retina

To test this hypothesis, we first established a chicken embryonic retinal explant

culture system in which robust, self-sustaining, and light-entrainable iodopsin transcript

rhythms were observed under different lighting conditions. Although iodopsin

transcription in retinas of chicken embryos is primarily driven by light, the functional










characteristics of circadian oscillators driving iodopsin transcript rhythms in culture are

similar to those found in post-hatch chicken retina, an observation that supports use of the

explant cultures in our study.

The role that gap junctions play in coordinating rhythms in retinas was examined

using two reversible gap junction blockers, 18ca-glycyrrhetnic acid-3-hemisuccinate

(ACO) and 183-glycyrrhetnic acid (183-GA), which were applied to the explant cultures

maintained under different lighting conditions. Both gap junction blockers produced a

rapid and persistent reduction of iodopsin and AANAT transcript levels. Following

removal of the blockers, the transcript rhythms of both genes reappeared within a 24 hr

period. Our data suggest that the change in iodopsin mRNA levels was not due to

disruption of the function or the phase of the circadian oscillators driving the iodopsin

rhythms. These blockers may either directly uncouple the circadian oscillators from

driving transcription of these genes or alter the stability of these transcripts in the

photoreceptors cells.














CHAPTER 1
INTRODUCTION

General Features of Circadian Rhythms

Basic Properties of Circadian Rhythms

Circadian rhythms are self-sustained cyclic changes in physiological processes or

behavioral functions that have a period of approximately 24 hours (Chang and Reppert

2001). A circadian system is comprised of three components: a core circadian oscillator

that acts like a ticking clock to produce self-sustained rhythmic changes, output pathways

through which the oscillators regulate physiological and behavioral functions, and input

pathways through which the oscillators are synchronized or entrained to environmental

time cues (Dunlap 1999). Circadian rhythms are virtually ubiquitous, controlling a

myriad of physiological processes in organisms ranging from spore production in fungi,

leaf movement in plants, eclosion in insects, rest:activity cycles in animals, and

sleep:wake cycles in humans. Although the physiological processes regulated by

circadian clocks may vary between organisms, these rhythms share three basic properties.

First, the rhythms are self-sustaining and persist or free-run under constant conditions.

Second, the rhythms can be entrained to environmental stimuli, light being the dominant

and most potent entraining stimulus. Finally, the rhythms are temperature compensated in

the sense that the period of the rhythm stays constant over a range of ambient

temperatures (Reppert and Weaver 2001).









Molecular Bases of Circadian Clocks

The molecular mechanism driving core circadian oscillators consists of interlocking

transcription-translation feedback loops. This mechanism is best understood in the fruit

fly, Drosophila melanogaster, and in the mouse suprachiasmatic nuclei (SCN). In

Drosophila, seven genes that encode the proteins involved in these molecular feedback

loops have been identified, period (per), timeless (tim), Drosophila clock (dClk), cycle

(cyc), double-time (dbt), shaggy (sgg), and vrille (vri) (Blau and Young 1999;Martinek et

al. 2001;Young 2000). In one transcription-translation feedback loop, the basic helix-

loop-helix (bHLH)-PER-ARNT-SIM (PAS) domain-containing transcription factors,

dCLK and CYC, form heterodimers that target E-box regulatory cis elements (CACGTG)

located in the promoters of the per and tim genes. Binding of the dCLK and CYC

heterodimers to the promoters drives the rhythmic transcription of the per and tim genes

(Glossop et al. 1999). As the per and tim mRNAs are translated into PER and TIM

proteins, TIM and PER proteins begin to accumulate; however, this process is somewhat

slowed by the binding of PER proteins to the constitutively expressed kinase DBT, an

interaction that leads to the phosphorylation and degradation of PER. PER is stabilized

when TIM protein accumulates to levels sufficient to bind the PER/DBT heterodimers.

The formation of the TIM-PER-DBT trimeric protein complex allows it to enter the cell

nucleus (Price et al. 1998). Contrary to the effect of DBT, which retards the accumulation

of PER and the entry of PER and TIM into the nucleus, the phosphorylation of TIM by

the glycogen synthase kinase-3, SGG, accelerates PER/TIM heterodimerization and

subsequent translocation into the nucleus (Martinek et al. 2001). Once in the nucleus, the

PER/TIM/DBT protein complex interacts with dCLK-CYC heterodimers to reduce the

activation of per and tim transcription (Darlington et al. 1998). In the other transcription-









translation feedback loop, dCLK and CYC heterodimers repress dClk transcription either

directly or via intermediate factors. The binding of PER-TIM-DBT heterodimers to

dCLK-CYC heterodimers releases dCLK-CYC dependent repression of dClk

transcription, thereby allowing separate activator(s) to activate transcription of the dClk

gene (Glossop et al. 1999).

In mouse SCN, eight clock genes contribute to the autoregulatory feedback loops

that define the core oscillator. One transcription-translation feedback loop involves the

dynamic regulation of three period genes, designated mperl-3, and two cryptochrome

genes, known as designated mcry] and mcry2. Rhythmic transcription of the mper and

mcry genes is driven by mCLOCK and mouse mBMAL1 heterodimers. Mouse BMAL1

is a homolog of Drosophila CYC. As the mPER and mCRY proteins are translated, they

form multimeric complexes that are translocated to the nucleus. In the nucleus, the

mCRY component of the multimer acts as a negative regulator by directly interacting

with the mCLOCK: BMAL1 heterodimers and inhibiting the transcription of mper and

mcry genes (Reppert and Weaver 2001). Mouse PERs, unlike their counterpart in

Drosophila, do not play a critical role in transcriptional inhibition. Instead, mPER1

affects the function of the clock at the post-transcriptional level, presumably through

protein-protein interactions that affect the stability and nuclear entry of other clock

proteins (Bae et al. 2001). mPER2 drives the rhythmic transcription of mbmall, which

exhibits a phase opposite to that of mper and mcry, forming a positive transcriptional

loop. Increased availability of BMAL1 presumably promotes the formation of the

CLOCK:BMAL1 heterodimers that are required to restart the mper and mcry









transcription cycle (Shearman et al. 2000). At this point, it appears that mPER3 protein is

not essential for the maintenance of circadian rhythmicity (Bae et al. 2001).

Light Entrainment of Circadian Clocks

Many of the circadian clocks that drive rhythmic processes and behaviors are

synchronized (entrained) to the daily changes that occur in the levels of ambient light that

occur between dawn and dusk. The imposition of an artificial 12 hour light: 12 hour dark

(12L: 12D) cycle on these clocks forces them to run with a period near 24 hours that is in

phase with the light/dark cycle. The phase of the clock is defined by the cyclic changes

that occur in the levels of the various proteins that comprise the clock over the course of a

single 24-hour light/dark cycle. Light entrainment is a dynamic process. A change in the

light/dark cycle causes changes in the concentrations of the clock proteins, which in turn

produce a shift in the phase of the clock so that it is properly synchronized to the new

light/dark cycle. The direction and amplitude of the phase shift of the clock are

determined by the magnitude of the change in the concentration of the clock proteins that

is induced by the new light cycle (Devlin and Kay 2001). For example, in Drosophila,

levels of TIM protein can be directly modulated by light as a result of TIM's interaction

with the flavoprotein CRYPTOCHROME (CRY). Light pulses delivered during the dark

period trigger the degradation of TIM and reset the phase of the clock to a point in the

cycle where the concentration of TIM would normally be low (Young 2000). In mouse

SCN, the transcription of mperl and/or mper2 genes has been shown to be rapidly

induced by light pulses delivered during the subjective night, results that suggest that

mPER1 and/or mPER2 protein may be involved in mediating light entrainment of the

mammalian clock (Albrecht et al. 1997;Okamura et al. 1999;Shearman et al. 1997). The

response of the circadian clock to the changes of light stimuli varies over the course of









the day. In general, light pulses delivered in late afternoon or early night delay the phase

of the clock, light pulses administered in late night or early morning result in phase

advances, and light pulses delivered during the middle of the day are relatively

ineffective in inducing changes in the phase of the clock (Rosenwasser and Dwyer 2001).

Circadian Clocks in the Central Nervous System (CNS)

In mammals, the master circadian "clock" that controls physiological and

behavioral rhythms resides in the suprachiasmatic nuclei (SCN) that are located within

the anterior hypothalamus. The clock in the SCN is composed of multiple, autonomous,

single-cell circadian oscillators that receive information about ambient light levels

directly from the eyes via the retinohypothalamic tracts (Green 1998). Synchronization of

these oscillators by light permits the SCN to generate a coordinated circadian output that

is capable of regulating overt rhythms (Reppert and Weaver 2001). One of the more

important rhythms regulated by the SCN is the production of melatonin by the pineal

gland. Two types of regulatory signals are transmitted to the pineal gland through

efferents from SCN. One is the permissive signal that originates from the circadian clock

and restricts melatonin production to the night. The other is the inhibitory signal that is

induced by inappropriate light stimuli at night and acutely suppresses nocturnal

melatonin production (Gillette and McArthur 1996). Thus, in mammals, the eye, the SCN

and the pineal body function in sequence as photoreceptor, master circadian oscillator,

and melatonin output organ for maintaining circadian rhythms at the organism level.

In avian species, the eye, the pineal gland, and deep brain structures including the

hypothalamus and SCN have all been implicated in the regulation of behavioral rhythms,

although diversity among avian species is great. In the House sparrow and the Java

sparrow, pinealectomy abolishes free-running circadian rhythms under constant









conditions, observations that indicate that the pineal gland is the site of an essential

circadian clock in sparrows (Gaston and Menaker 1968). But in Japanese quails,

pinealectomy does not have a significant effect on circadian locomotor activity rhythms

(Underwood 1994). The eyes (Underwood 1994) and the hypothalamus (Simpsom and

Follett 1981) of the Japanese quail have been suggested to be the locations of the major

circadian oscillators in this species. The eyes, pineal, and SCN must all be removed to

abolish circadian locomotor rhythms in pigeons, a result that indicates that all of these

organs are necessary for maintaining circadian rhythmicity in pigeon (Ebihara et al.

1984). Based on these observations, it has been suggested that the circadian system in the

avian CNS contains multiple oscillators comprised of the pineal gland, the eyes, and deep

brain structures that include the SCN (Oishi et al. 2001).

Circadian Rhythms In Retina

Retinal Functions Regulated by Circadian Clocks

There is now substantial evidence that vertebrate retinas contain circadian clocks

and that these clocks play an important role in maintaining the function and health of the

retina (Moog 1995). These functions include the synthesis and release of

neuromodulators such as melatonin (Cahill et al. 1991;Tosini 2000) and dopamine

(Besharse and luvone 1992), photoreceptor disc shedding and phagocytosis by the retinal

pigment epithelium (RPE) (Nguyen-Legros and Hicks 2000), retinomotor movement

(Burnside 2001), gene expression (Pierce et al. 1993; Green and Besharse 1994; Green et

al. 1996; Larkin et al. 1999; Chong et al. 2000), and visual sensitivity (Li and Dowling

2000).









Cellular Location of Circadian Clocks Within Retina

Currently, the most direct evidence for the presence of functional circadian clocks

in a specific retinal cell type comes from studies of melatonin synthesis in reduced

Xenopus retina cultures. Melatonin in the retina is produced by the retinal photoreceptor

cells and is regulated by circadian clocks located in the retina. By monitoring melatonin

release from the photoreceptor cultures under constant dark conditions, Cahill and

Besharse (Cahill and Besharse 1992; 1993) were able to show that the circadian

oscillators controlling melatonin rhythms in Xenopus retina are localized to the

photoreceptor cells. The observations that the activities of tryptophan hydroxylase (TPH)

and serotonin N-acetyltransferase (AA-NAT), two key enzymes in the melatonin

biosynthesis pathway, are both expressed in Xenopus photoreceptors and are also under

the control of a circadian oscillator are consistent with the localization of circadian clocks

to these cells (Besharse and luvone 1983;Green and Besharse 1994;Green et al. 1995).

Indirect support for the presence of circadian oscillators in photoreceptor cells comes

from studies of low-density cultures of avian retina. Iodopsin is a red-sensitive pigment

that is expressed in cone photoreceptors in the retinas of birds (Yoshizawa and Kuwata

1991). The transcription of the iodopsin gene in chicken retina has been shown to be

regulated by a circadian clock (Pierce et al. 1993;Larkin et al. 1999). The observations

that transcription of the iodopsin gene exhibits a circadian rhythm in low density cultures

of both quail (Pierce 1999) and chicken retina (Pierce et al. 1993) suggest that the

circadian clocks driving expression of this gene are located in the cone photoreceptor

cells.

If circadian clocks are present in photoreceptor cells, then the clock genes should

be expressed in these cells. In mouse, transcripts encoding mPER1 (Shearman et al.









1997;Sun et al. 1997;Sun et al. 1997;Shearman et al. 1997), mPER2 (Shearman et al.

1997), mPER3 (Zylka et al. 1998), mTIM (Zylka et al. 1998), mClOCK (Gekakis et al.

1998) and BMAL1 (Gekakis et al. 1998) have been isolated from the retina. In situ

hybridization analyses of mouse retina have revealed that mclock, mperl and mbmall are

co-expressed in retinal photoreceptors, cells within the inner nuclear layer and in the

ganglion cell layer (Gekakis et al. 1998). In Xenopus, XClock (Zhu et al. 2000), Xper2

(Zhuang et al. 2000), and three cryptochromes genes (Xcryl, Xcry2a, Xcry2b) (Anderson

and Green 2000) have been cloned and have been shown to be expressed in retina. Many

of these clock genes are also expressed in the retinas of birds. Transcripts encoding

cCLOCK (Larkin et al. 1999;Chong et al. 2000;Larkin et al. 1999), cBMAL1 and

cMOP4 (Chong et al. 2000) have been isolated from chicken retina and transcripts

encoding qCLOCK, qPER2 and qPER3 have been isolated from quail retina (Yoshimura

et al. 2000).

Taken together, the results of the studies of the expression of clock genes and

melatonin secretion in retina suggest that vertebrate photoreceptor cells contain

functional circadian clocks and output pathways.

Light Entrainment of Retinal Photoreceptor Clocks

Most information about the responses of retinal circadian clocks to light comes

from analyses of processes within the retina that are regulated by these clocks. Studies of

melatonin and iodopsin synthesis, both of which are produced by photoreceptor cells,

suggest that retinal photoreceptors contain functional input pathways that allow

entrainment of the oscillators in these cells to light. For example, in their studies of

reduced Xenopus retina cultures, Cahill and Besharse (Cahill and Besharse 1992; 1993)

noted that the phase of the melatonin rhythms in photoreceptor cells could be reset by










light. In chicken retina, the rhythms of iodopsin transcription in cone photoreceptors can

be entrained to the external cyclic lighting conditions and the phase of the rhythms can be

shifted by 6 hour light pulses delivered during the dark period (Larkin and Semple-

Rowland 2001). These observations together with those mentioned above suggest that

photoreceptor cells contain a complete circadian system.

Synchronization of Circadian Clocks

Synchronization of Circadian Clocks in SCN

The mammalian SCN contains 20,000 neurons that work in concert to drive the

coordinated circadian rhythms of electrical activity (Herzog et al. 1997), gene

expression(Panda et al. 2002), glucose metabolism (Schwartz et al. 1983), and behavior

(LeSauter and Silver 1998;Herzog et al. 1997). Individual neurons in SCN dissociated

cultures exhibit self-sustained rhythms of spontaneous firing activity, suggesting that

functional circadian oscillators are present within these single neurons. However, the

firing rhythms of individual oscillators exhibit variable periods and different phase

relationships with one another under these culture conditions (Welsh et al. 1995). In SCN

explant cultures, in which synapses and cellular appositions are largely preserved, the

firing rhythms of individual SCN neurons exhibit periods with significantly less

variability and are in phase with each other. Furthermore, the range of the periods in

explants is almost identical to that observed for behavioral rhythms (Herzog et al. 2001).

These observations suggest that intercellular communication is required to generate and

maintain coordinated circadian rhythms in SCN.

Synapse- and gap junction-mediated intercellular communications represent two

possible mechanisms that could synchronize populations of autonomous oscillator cells

Accumulating indirect evidence suggests that synaptic transmission does not play a









central role in the synchronization of the circadian oscillators within the SCN neurons.

For example, circadian glucose metabolism in the SCN is observed before chemical

synapses become functional in the SCN (Reppert and Schwartz 1984;Moore and

Bernstein 1989). Consistent with this observation are the observations that disruption of

synaptic transmission within the SCN using either tetrodotoxin (TTX) (Shibata and

Moore 1993) or calcium-free medium (Bouskila and Dudek 1993) do not alter the ability

of the SCN neurons to generate synchronized bursts of activity.

Gap junction channels provide another means for intercellular communication.

Gap junction channel consists of a hemichannel (a connexon) in the membrane of one

cell that is paired with a similar hemichannel in another adjoining cell. A hydrophilic

pore at the core of the connexon allows the passage of small ions and low molecular

weight metabolites (up to 1 kD) between the cells and functions to connect the cells both

electrically and metabolically. Each connexon, in turn, is composed of 6 similar protein

subunits known as connexins (Cx). The connexins are members of a multigene family.

Connexins 26, 32 and 43 are the first members of this family that are identified and are

the most abundant isoforms expressed in the developing CNS (Cook and Becker 1995)

Gap junction communication has been shown to play a role in the coordination and

synchronization of the activity of SCN neurons in vivo. Studies of connectivity of SCN

neurons show that SCN neurons are coupled by low resistance pathways (Colwell

2000;Shinohara et al. 2000;Jiang et al. 1997), the permeability of which are modulated by

cell activity. Studies using dyes capable of traversing gap junctions show that SCN cells

are extensively dye coupled during the day when the cells exhibit synchronous neural

activity and are minimally dye coupled during the night when the cells are electrically









silent, a coupling rhythm that is also maintained under constant dark conditions (Colwell

2000). Evidence for a role of gap junctions in the synchronization of the SCN cell

oscillators comes from a recent study that shows that the gap junction blockers, octanol

and halothane, can reversibly block vasopressin and vasoactive intestinal polypeptide

rhythms in SCN slice cultures (Shinohara et al. 2000). Interestingly, there is a growing

body of evidence that suggests that gama-aminobutyric acid (GABA) may modify the

responses of the SCN neuronal circadian clocks to light and other entraining stimuli by

altering cell-cell coupling through gap junctions. GABA, acting through type A receptors,

has recently been shown to be capable of phase shifting and synchronizing the oscillators

within individual SCN clock cells in vitro (Liu and Reppert 2000) and of modulating the

permeability of gap junction channels in SCN slice cultures (Shinohara et al. 2000)

Expression and Function of Gap Junctions in Retina

The vertebrate retina is a highly laminated assemblage of five major classes of

specialized neurons: a vertical pathway connects photoreceptors to bipolar cell to

ganglion cells, while horizontal and amacrine cells provide lateral interactions in the

outer and inner retina, respectively. Tracer studies have revealed that in addition to these

pathways, many types of retinal neurons are interconnected through gap junctions. In

addition to the widespread coupling that is observed between identical cell types,

heterologous coupling between rods and cones, between amacrine cells and cone bipolar

cells, between different types of bipolar cells, between different types of amacrine cells,

and between ganglion cells and amacrine cells is also observed (Vaney and Weiler 2000).

There is a growing body of evidence that gap junctions are present and functional

in developing retinas. Cx43, 26 and 32 are the major connexin isoforms expressed in

developing chicken retina. Among them, Cx43 is the first one to be expressed in the early









neuroepithelium of the eyecup, followed by the expression of Cx32 and 26 at E4-4.5

(Becker et al. 1998). As early as E7, fluorescent dye injected into individual retinal

ganglion cells spreads into the cells within the ganglion cell layer, the inner nuclear layer,

and also to the cells traversing the whole thickness of retina, results that suggest that an

extensive network of gap junctions has been formed in chicken retina at this

developmental stage (Catsicas et al. 1998). Functional studies have also shown that gap

junction communication is involved in the regulation of synchronized spontaneous neural

activity that occurs both before (Catsicas et al. 1998) and during (Wong et al. 1998)

synaptogenesis in embryonic chicken retina.

Neurotransmitters play a major role in regulation of gap junction permeability in

inner retina. For example, dopamine and GABA have both been shown to modulate the

permeability of gap junctions that exist between amacrine cells in rabbit retina (Hampson

et al. 1992) and between horizontal cells in rabbit and turtle retina (Hampson et al.

1994;Piccolino et al. 1982;Piccolino et al. 1984). Modulation of gap junction coupling

either through changes in ambient illumination or through light-induced changes in

dopamine release have been postulated to play a role in regulating retinal sensitivity (Li

and Dowling 2000;Manglapus et al. 1998;Manglapus et al. 1999).

Vertebrate photoreceptor cells are also coupled by gap junctions (Gold and

Dowling 1979;Raviola and Gilula 1973;Tsukamoto et al. 1992). There is evidence that

the strength of junction coupling between photoreceptors can be modulated by light

(Yang and Wu 1989) and by dopamine (Krizaj et al. 1998); however, the effectiveness of

these stimuli to alter coupling may be species dependent (Schneeweis and Schnapf 1999).

While potentially important in processing of light signals for vision (Schneeweis and









Schnapf 1999;Lebedev et al. 1998), changes in gap junction communication may also

play a role in the synchronization of photoreceptor circadian oscillators in retina. We

have observed that iodopsin rhythms in dispersed cultures of chicken retina are not as

robust as those observed in retinal explant cultures, the rhythms in dispersed cells

becoming negligible after one week in culture (Semple-Rowland, unpublished

observation). This observation also indicates that, as in the case of SCN, gap junction

communication may play a role in generation and maintaintence of iodopsin transcript

rhythms in retinal explant cultures.














CHAPTER 2

Note: This work has been published in Molecular Brain Research 114 (2003) 9-19.

CHARACTERIZATION OF CIRCADIAN OSCILLATOR FUNCTION IN
EMBRYONIC RETINA AND RETINAL EXPLANT CULTURES

Introduction

The retinas of several vertebrate species contain light entrainable circadian

oscillators that regulate 24-hour cyclic changes in retinal function (Besharse and luvone

1983;Cahill and Besharse 1991;Tosini and Menaker 1996;Tosini and Menaker 1998).

Currently, there is significant interest in identifying the retinal cells that contain these

oscillators and understanding how these oscillators are entrained to light.

Cahill and Besharse obtained the first evidence that vertebrate photoreceptor cells

contain light-entrainable circadian oscillators (Cahill and Besharse 1993). Using a

reduced Xenopus eyecup preparation, these investigators obtained support for the

hypothesis that the circadian oscillators controlling melatonin release in Xenopus retina

are located in the photoreceptors, and that light entrainment of these oscillators does not

require input from cells within the inner retina. Direct evidence for the presence of

functional oscillators in Xenopus photoreceptor cells has recently been obtained in a

study of transgenic Xenopus tadpoles that express a dominant negative form of Clock.

Photoreceptor-specific expression of the mutant Clock protein was found to disrupt

rhythmic production of melatonin by these cells (Hayasaka et al. 2002). These data and

the observations that transcription of the cone-specific iodopsin gene is regulated in a

circadian manner in dispersed, low-density cultures of chicken and quail retina (Pierce et









al. 1993;Pierce 1999) suggest that it is likely that the retinal photoreceptor cells of several

vertebrate species contain autonomous circadian oscillators.

The nature of the biochemical cascades that entrain retinal oscillators to light and

the extent to which these oscillators influence each other within the context of the intact

retina are currently unknown; however, significant progress has recently been made

toward understanding light entrainment of oscillators in avian pineal cells. Using

immunocytochemical, molecular and biochemical techniques, Fukada and his colleagues

have obtained convincing evidence that an opsin-Gliot-mediated signaling pathway is

involved in light entrainment of chicken pineal circadian oscillators (Kasahara et al.

2002). They also found that G11 is expressed in chicken retinal photoreceptors and that it

associates with rhodopsin, one of the opsins in retina, in a light- and GTP-dependent

manner (Kasahara et al. 2002). Together, these data suggest that an opsin-G11 signaling

pathway may be involved in mediating the phase shifting effects of light on circadian

oscillators in chicken retina.

Organ culture systems, which have been successfully used to study circadian

regulation of melatonin in Xenopus and hamster retina (Cahill and Besharse 1991;Tosini

and Menaker 1996), may also prove valuable in studies of the retinal oscillators that drive

iodopsin transcription. In this series of experiments, iodopsin mRNA rhythms in

embryonic retinal explants maintained under cyclic light, constant dark, and light reversal

conditions were compared to those in retinas from age-matched chicken embryos and in

post-hatch (< 2 weeks old) chickens. Our data show that embryonic retinas maintained as

explant cultures exhibit robust iodopsin rhythms that are driven by light entrainable

circadian oscillators. These observations support the use of explant cultures in studies to









investigate mechanisms responsible for generating and maintaining iodopsin transcript

rhythms in retina.

Materials and Methods

Preparation of Retinal Explant Cultures

All experimental procedures mentioned in this dissertation were approved by the

University of Florida IACUC Committee and were in accordance with the National

Institutes of Health guidelines. Fertile White Leghorn chicken eggs obtained from the

University of Florida Poultry Sciences Unit were incubated on a 12 hour light: 12 hour

dark (12L: 12D) cycle in the incubators illuminated by 20 Watt cool white fluorescent

bulbs (90 lux). The lights were on at 9:00 AM (Zeitgeber time, ZTO) and off at 9:00 PM

(ZT12) EST. Retinal explant cultures were prepared from embryonic day 9 (E9) and E10

chickens during the 12-hour light period. Dissection and preparation of the cultures was

carried out according to a method previously described for preparation of neonatal mouse

retina cultures (Ogilvie et al. 1999). The eyes were dissected from the embryos and

placed in a pool of Dulbecco's modified Eagle's medium (DMEM) supplemented with

10% fetal bovine serum and antibiotics (130 U/ml penicillin and 130 [tg/ml

streptomycin). After the sclera, choroid and retinal pigmented epithelial tissues were

removed, the remaining structure, consisting of the vitreous body and the retina, was

transferred to a 35 mm culture dish that contained a Millicell membrane insert (0.2 [tm;

Millipore, Bedford, MA) filled with media. The retina was gently peeled away from the

vitreous body and several small cuts were made around the periphery of the retina to

facilitate flattening of the retina photoreceptor side down onto the membrane. The retinal

explants were incubated on a 12L: 12D cycle at 370C in 5% CO2 and were fed every two









days. The 12L:12D period beginning the day after the cultures were prepared was

designated day 1 in vitro (1 DIV).

Explant Morphology

Retinal cultures designated for histological analyses were fixed for 1-2 days in 4%

paraformaldehyde at 40C. Retinas were left on the Millicell membrane during the fixation

step and in some cases remained attached to the membrane throughout the sectioning

process. The tissues were infiltrated with 30% sucrose and sectioned (16 [tm) using a

cryostat. Sections were placed onto glass slides and stored at -200C until processed for

staining or immunohistochemistry.

Cresyl violet stained sections were examined to assess the general morphology of

the retinal explants. To determine if there was significant cell loss within the inner

nuclear layer (INL) of these cultured retinas, the mean cell density (number of cells per

tmn2) within the INL was determined for three prescribed regions of three different

sections from each explant and the average of these values was corrected by multiplying

by the mean thickness of the INL layer ([tm). The resulting values were compared using

Kruskal-Wallis ANOVA on Ranks (SigmaStat, Jandel, CA). The percent pyknotic cells

in the INL was estimated in 3, 5 and 12 DIV retinal explants by counting the pyknotic

nuclei present in three separate prescribed regions of three different sections from each

explant and dividing by the mean cell density.

To determine if dopaminergic amacrine cells could be detected in the explant

cultures, cryosections taken from the retinal explants used in the morphological analyses

were immunostained using a monoclonal antibody (1/10,000 dilution in PBS containing

1% goat serum and 0.1% Triton X-100) for tyrosine hydroxylase (TH; Chemicon,









Temecula, CA), a marker for dopaminergic cells, and a goat anti-mouse secondary

antibody (1/500 dilution in PBS) tagged with the Alexa-488 fluorophore (Molecular

Probes, Eugene, OR). Sections were incubated with the anti-TH primary antibody for 2

hours at room temperature, rinsed three times with PBS, and then incubated with the

secondary antibody for 1 hour at room temperature followed by three additional rinses.

Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI; Molecular

Probes). The stained tissues were viewed using the appropriate fluorescent filter sets and

digital images were acquired using a SPOT 2 Enhanced Digital Camera System mounted

on a Zeiss Axioplan 2 fluorescence microscope.

Iodopsin Gene and Protein Expression in Retinal Explant Cultures

To identify the earliest time that iodopsin transcripts could be detected in retinal

explant cultures, cultures prepared from E9 embryos and maintained under 12L:12D

conditions for 1-6 DIV were collected at ZT 8 (8 hours after lights came on). To compare

iodopsin expression onset in cultures and in ovo, retinas from chicken embryos were also

collected at ZT 8 from E10 to E16. The retina samples were quickly frozen in liquid

nitrogen and stored at -750C until processed for northern blot analyses.

Immunohistochemical staining of retinal explants was carried out to examine

expression of iodopsin protein in the explants. Tissue sections were blocked using PBS

containing 10% goat serum and incubated overnight at 40C with a polyclonal antibody

for chicken iodopsin (CERN874; 1/5000 dilution in PBS containing 1.0% BSA and 0.3%

Triton X-100) (Geusz et al. 1997). The primary antibody was visualized by incubating

the sections with a goat anti-rabbit secondary antibody (1/1000 dilution in PBS) tagged









with the Alexa-594 fluorophore (Molecular Probes) for 1 hour at room temperature.

Sections were counterstained with DAPI and imaged as described above.

Lighting Paradigms

Cyclic light

Explant cultures maintained under 12L:12D conditions were collected from 3 DIV

to 9 DIV at ZTO and ZT12. For the in ovo experiments, the retinas of White Leghorn

chicken embryos maintained in ovo under 12L: 12D conditions were harvested at ZTO and

ZT12 from E17 to E20. In all experiments, sample collection during the dark period was

carried out under a low intensity red safe light (15 Watt bulb, Kodak #2 filter).

Constant dark

Retinal explants were maintained under 12L: 12D conditions for the first 5 days in

culture and were placed in constant darkness on days 6-7. Cultures were collected on 4

and 5 DIV at ZTO and ZT12 and on 6 and 7 DIV just after the lights would have been

turned on (circadian time 0 CTO) and just after the lights would have been turned off

(CT12). For the in ovo experiments, chicken embryos were incubated under 12L:12D

conditions through E17 and were then placed in constant dark conditions from E18 to

E20. The retinas of E17 embryos were collected at ZTO and ZT12. The retinas ofE18 to

E20 embryos were collected at CTO and CT12.

Reversal of the light cycle

Retinal explants were maintained on a 12L: 12D cycle for 5 DIV. On 6 DIV the

light cycle was reversed to a 12D: 12L cycle and the cultures remained on this reversed

light cycle through 8 DIV. Cultures were collected at ZTO and ZT12 from 4 to 8 DIV.

For the in ovo experiments, chicken embryos were incubated under 12L: 12D conditions

through E17. On E18, the light cycle was reversed and the embryos remained on this










reversed cycle through E20. The retinas of E17 through E20 embryos were collected at

ZTO and ZT12.

RNA Analyses

Retinal explants and age-matched embryo retinas were placed in screw top tubes,

frozen in liquid nitrogen and stored at -750C until further processing. Total RNA was

extracted from the retinas using an RNeasy kit (Qiagen, Valencia, CA). Northern blots

were prepared as previously described (Semple-Rowland and van der 1992), each lane

containing 8 tg total RNA. RNA slot blots were prepared using a BioSlot apparatus

(BioRad). The RNA samples, each containing 2[tg of total RNA from a retinal explant,

were diluted with RNase-free water to a final volume of 10 [tl. The following solutions

were then added to each sample; 20 [tl of 100% formamide, 7[tl of 37% formaldehyde,

and 2ml of 20X SSC (3M NaC1, 0.3M sodium citrate, pH 7.0). The samples were then

incubated at 680C for 15 minutes. During the incubation period, the slot blot apparatus

was assembled according to the manufacturers' instructions and each sample well was

rinsed twice with 1 ml 20X SSC. Following the incubation, the denatured RNA samples

were cooled on ice, diluted by adding 2 volumes of 20X SSC, and loaded into the sample

wells. A gentle vacuum was applied to the apparatus to load the RNA onto a

Magnacharge nylon membrane (MSI, Westburough, MA) and to complete the wash

steps. Following application of the samples, each well was rinsed twice with 1 ml 20X

SSC. The vacuum was kept on for an additional 5 min following the last wash to dry the

membrane. The RNA samples were then cross-linked to the blot using UV light (UV

Stratalinker, Stragene). The effectiveness of the transfer was examined by staining the

blot with methylene blue. Finally, the blot was dried at 370C for 30 minutes and stored at









room temperature until hybridization. The northern and slot blots were prepared in

duplicate and were probed consecutively with radiolabeled cDNA probes specific for

iodopsin and 18S rRNA that were synthesized as described previously (Larkin et al.

1999). The amount of probe hybridized to the blots was measured using a BioRad

Molecular Imager FX system. Iodopsin transcript levels in individual samples were

normalized to the amount of 18S rRNA present in that same sample. These values were

then expressed relative to the mean iodopsin/18S rRNA value for each blot. Data were

analyzed using two-way ANOVA (SigmaStat).

Results

Morphology of Developing Retinal Explant Cultures

The cellular architecture and structural integrity of the retinal explants were

examined as a function of time in culture by comparing cresyl violet-stained sections of

explants to those of retinas from embryos.

The outer nuclear (ONL), inner nuclear (INL), and ganglion cell (GCL) layers of

E13 retinas were readily discernable and fairly well organized (Fig.2-1, top panel). From

E15 to El 18 there was a gradual decrease in the overall thickness of the nuclear layers, a

change due in part to a decrease in extracellular space volume and to the rapid growth of

the eye that occurs during this period of development. Retinas cultured for 3, 5 and 12

days (Fig.2-1, bottom panel) resembled those obtained from embryos (Fig.2-1, top

panel); however, there were two major changes in the structure of the cultured retinas.

First, the ganglion cells, which were detectable in 3 DIV cultures, rapidly degenerated

and were no longer evident at 5 DIV. Concomitant with the disappearance of the GCL

was a reduction in the overall thickness and gradual loss of the inner plexiform layer









(IPL). Second, the cells within the ONL and INL of the explants remained relatively

disorganized compared to those present within these layers in retinas in ovo.


E13 E15 E1B

A S -OPL


IPL

. ,AGCL

12 DIV
|' tONL
S I* OPL


3 DIV


*. ~r* r


Figure 2-1. Morphological comparison of chicken embryonic retina and retinal explant
cultures. Images ofE13, E15 and E18 retinal cross-sections (top panels) show
the cellular organization and development of chicken embryonic retinas in
ovo. Images of 3 DIV, 5 DIV and 12 DIV retinal cross-sections (middle
panels) show the cellular organization and development of chicken retinal
explants harvested from E10 embryos. The bottom panel contains magnified
images of the INL from explant cultures that show evidence of cell death
within the INL (arrows indicate pyknotic nuclei). DIV days in vitro; RPE -
retinal pigment epithelium; ONL outer nuclear layer; OPL outer plexiform
layer; INL inner nuclear layer; IPL inner plexiform layer; GCL ganglion
cell layer. Scale bars = 25 Ltm.

To determine if loss of the IPL in cultured retinas was accompanied by a significant

decrease in the number of cells within the INL, we measured both the cell density and

thickness of the INL layers in retinas cultured for 3, 5 and 12 days. The results of these


1 LI










analyses showed that there was not a significant change in the number of cells within the

INL (H = 0.924, df = 2, p = 0.63) even though there were significant changes in the mean

densities (H = 21.0, df= 2,p < 0.0001) and widths of the INL layer (H = 16.1, df = 2, p =

0.003) over time (Fig. 2-2A).


A



C
SN
-E
0-
o


2-


0.30
0.25
0.20
0.15
0.10
0.05
0.00


3DIV


3DIV 5DIV 12DIV


80 K
CD

60 S

40
[-*-


20 D
I-
20 3

-1


CD
10 "

8 a

6 |
0
CD
3 m
2 6

0 a
CD
C<


5DIV 12DIV


Figure 2-2. Quantification of the morphological integrity of retinal explant cultures. (A)
Cell densities (yellow bars) and widths (blue bars) of INL layers in 3, 5 and 12
DIV explant cultures. The mean number of cells per pm in the INL calculated
from the density and width values is also shown in red. (B) Percent pyknotic
cells within the INL of 3, 5 and 12 DIV explant cultures. Bars represent mean
+ SE of measures taken from three separate prescribed regions within three
different sections of representative 3, 5 and 12 DIV retinal explants.

However, there was evidence of cell death within the INL throughout the 12-day

culture period. Pyknotic nuclei were detected within this cell layer as early as 3 DIV, the

number significantly increasing by 5 DIV and then dramatically falling off by 12 DIV









(Fig. 2-1, bottom panel). This temporal pattern of cell death is similar to what has been

reported in analyses of neural cell death in retinas of developing chick (Cook et al. 1998)

and quail embryos (Marin-Teva et al. 1999) and suggests that the loss of cells in the INL

may reflect normal changes in cell number that accompany development. The majority of

the pyknotic nuclei were located in the inner strata of the INL closest to the IPL. The

percent of the total number of INL cells that were pyknotic was relatively small, with

mean values ranging from 0.09% (12 DIV) to 3.2% (5 DIV) (Fig. 2-2B). Virtually no

pyknotic nuclei were detected in the photoreceptor cell layer in any of the explant

cultures examined, consistent with previous studies of normal avian retina development

(Cook et al. 1998;Marin-Teva et al. 1999).

We were also interested in determining if we could detect dopaminergic amacrine

cells in our retinal explants because of their known importance in retinal circadian

biology (Besharse and luvone 1920). Gardino et al. (Gardino et al. 1993) have previously

shown that E13 is the earliest time at which amacrine cells expressing the TH phenotype

can be detected immunohistochemically in developing chicken retina.

We did not detect any TH-positive amacrine cells in cross-sections of the 3, 5, 8

and 12 DIV retinal explants stained with a monoclonal antibody for TH. TH-positive

cells exhibiting the morphology of amacrine cells were detected in the inner margin of

the INL in E18 and post-hatch retinas using this antibody, but not in E13 retina (Fig. 2-3).

The morphology and the location of the TH-positive cells that we observed in E18 and

post-hatch retina were the same as previously described (Gardino et al. 1993). Our

inability to detect TH-positive cells in E13 retinal sections may have been due to a

combination of factors including low levels of expression of TH and the unique spatial









distribution of these cells across the retina at this stage of development (Gardino et al.

1993).


ONL
OPL

INL



IPL


GCL


Figure 2-3. TH immunostaining of explants and embryonic and post-hatch retinas. No
TH-immunoreactive cells were detected in 3, 5, 8 or 12 DIV retinal explant
cultures. Scattered TH-immunoreactive cells exhibiting amacrine cell
morphology were identified in E18 and 4 days post-hatch (DPH) retinas along
the border between the INL and the IPL. ONL outer nuclear layer; OPL -
outer plexiform layer; INL inner nuclear layer; IPL inner plexiform layer;
GCL ganglion cell layer. Scale bars = 10 |tm.

Comparison of Iodopsin Expression Onset in Retinal Explant Cultures and in ovo

Prior to initiating the studies of clock function in the retinal explant cultures I

identified the earliest time at which iodopsin gene expression could be detected in these

cultures. Northern blot analyses showed that iodopsin transcripts could be detected in E9

explants that had been cultured for 3 days (Fig.2-4A, left panel), a time point

approximately equivalent to E12. On the other hand, iodopsin gene expression was first

detected by Northern blots on E15 in embryonic chicken retina (Fig.2-4A, right panel).

However, it should be noted that recent studies of normal developing chicken retina place

the age of onset earlier at approximately E6-E8 when expression is analyzed using RT-

PCR (Adler et al. 2001). The acceleration of the onset of iodopsin gene expression that

we observed in the explant cultures is consistent with the results of previous studies of


4DPH










iodopsin gene expression in cultured chicken retina (Belecky-Adams et al. 1999) and has

been proposed to occur as a result of the absence of inhibitory factors that normally delay

expression of this gene in vivo(Adler et al. 2001).


A Iod in vitro lod in ovo
DIV 1 2 3 4 5 6 E10 11 12 13 14 15 16
lod w. al lod

18s 18s mMe



B


OS
ONL
OPL
INL


OS
ONL
OPL
INL


3pI


Figure 2-4. Onset of iodopsin expression in retinal explant cultures. (A) Northern blot
analyses of iodopsin gene expression. Blots were probed consecutively for
iodopsin and 18S rRNA. Each lane contained 8[tg total RNA. The iodopsin
mRNA signal was first detected in retinal explants cultured from E9 embryos
on 3 DIV, a time roughly equivalent to E12 in ovo. In contrast, iodopsin
transcript signal could not be detected in retinas from chicken embryo until
E15. (B) Immunohistochemical analyses of iodopsin expression in retinal
explants cultured from E10 embryos. Iodopsin immunostaining was not above
background in 3 DIV explants but was easily detected in the rudimentary OS
of 5 DIV explants. DIV days in vitro; ONL outer nuclear layer; OPL -
outer plexiform layer; INL inner nuclear layer; IPL inner plexiform layer;
GCL ganglion cell layer. Scale bars = 25 |tm.

Iodopsin protein was not detected immunohistochemically in E9 retinas maintained

in culture for 3 DIV and, in contrast, was easily observed in the photoreceptor cells of 5


5









DIV cultures. Iodopsin immunoreactivity was primarily localized to the rudimentary

outer segments of the photoreceptor cells (Fig.2-4B).

lodopsin Transcript Rhythms

lodopsin transcript levels in dispersed embryonic retinal cultures and in post-hatch

chicken retinas exhibit a circadian rhythm with minimum levels of the transcript at ZTO

and maximum levels at ZT12 (Larkin and Semple-Rowland 2001;Pierce et al. 1993).

Analyses of iodopsin transcription in our explant cultures in which we examined

transcript levels at ZTO, ZT6, ZT12 and ZT18 revealed that the same dynamics of the

rhythm were preserved in explanted retina. Based on these observations, we chose to

examine iodopsin transcript levels at ZTO and ZT12 in our analyses of retinal circadian

oscillator function in ovo and in explant culture.

Cyclic light

Retinal explants. The levels of iodopsin mRNA from 3DIV to 9DIV in retinal

explant cultures maintained under 12L:12D conditions exhibited a robust rhythm. On

culture day 3, iodopsin mRNA levels were 2.5 fold higher at ZT12 than at ZTO (Fig.2-

5A). From culture day 4 to 7, the total amount of iodopsin transcript increased

dramatically compared to that observed on 3DIV, but the relative increase in iodopsin

mRNA levels that occurred between ZTO and ZT12 remained relatively constant from

Day 3 to Day 7. On 8 and 9 DIV, iodopsin transcript levels at ZTO were similar to that

on Day 7, but the relative increase of iodopsin mRNA levels from ZTO to ZT12 was

reduced. The differences observed in levels of iodopsin mRNA between ZTO and ZT12

were significant over the time period examined (F = 31.03, df = 1, p < 0.0001).























f L7

JJJX


1.5

1.0

0.5

0.0


3DIV 4DIV 5DIV' 6DIV' 7DIV 8DIV'9DIV


E17 E18 E19 E20


<2.5 C 3.0 D

2.0 2.5

> c- 2.0
a'- 1.5
oz 1.5
0 1.0
-- 1.0

r 0.5
Q. 0.5
0 7
0.0 0.0


4DIV 5DIV 6DIV 7DIV


4DIV 5DIV 6DIV 7DIV


E17 E18 E19 E20


3.0 F

2.5

2.0

1.5

1.0

0.5

0.0


E17 E18 E19 E20


Figure 2-5. Iodopsin transcript rhythms in explant cultures and embryos maintained under
12L: 12D and constant dark conditions. (A) Relative iodopsin mRNA levels in
explant cultures prepared from E9 embryos and examined at 3, 5, 7 and 9 DIV
and (B) in retinas of embryos examined at E17 E20. For panels A and B, the
explants and embryos were maintained under 12L: 12D conditions throughout
the experiment. Retinas were analyzed at ZTO (white bars) and at ZT12 (black


< 2.5
Z

m0 2.0



1.0



a
-o
2 0.0


zn CO


<
oz
SE
<. ,-

0
-o
0


2.5 E









bars). (C) Relative iodopsin mRNA levels in explant cultures maintained for 5
DIV under 12L: 12D and then transferred into constant dark conditions on
days 6 and 7. (D) Relative iodopsin mRNA levels in embryos raised under
12L: 12D until E18 and then transferred into constant dark conditions from
E18 to E20. (E) Comparison of explant cyclic light data shown in panel A (red
symbols) and the constant dark data shown in panel C (black symbols). (F)
Comparison of embryonic cyclic light data shown in panel B (red symbols)
and the constant dark data shown in panel D (black symbols). The 12-hour
light and dark periods are indicated below each panel using white and black
bars, respectively. In panels E and F, plotted data and light cycles are matched
by color, red indicating 12-hour dark periods. In panels A D, the group
means + SE at each time point are shown and the number of retinas in each
group is indicated on the bars. In panels E and F, only the mean values are
plotted for each group.

Retina in ovo. Retinal iodopsin transcripts were first detected in ovo on E15. No

significant rhythms were detected on E15 or E16 and the mean values for relative

iodopsin levels at these stages were 0.185 and 0.258, respectively (data not shown). The

first evidence of a rhythm in iodopsin transcript levels was detected on E17; the transcript

levels measured at ZT12 were 1.6 fold higher than those measured at ZTO (Fig.2-5B).

The amplitude of the rhythm increased with developmental age. On E18, iodopsin mRNA

levels at ZT12 were 1.8 fold higher than those at ZTO. By E19 and E20, two-fold

increases in iodopsin expression were observed over the course of the 12-hour light

period. The emergence of the iodopsin transcript rhythm during the late stages of

development was paralleled by a steady and significant increase in the total amount of

iodopsin transcript, the levels of which appeared to reach a plateau by E20. The

differences observed in levels of iodopsin mRNA between ZTO and ZT12 were

significant over the time period from E17 to E20 (F = 97.57, df = 1, p < 0.0001).

Constant dark

Retinal explants. Under 12L: 12D conditions, the iodopsin transcript levels in

retinal explant cultures at 4 and 5DIV exhibit robust rhythms. In the absence of light, a










significant rhythm persisted in retinal cultures for 48 hours, the iodopsin levels measured

at CTO being significantly lower than those measured at CT12 on 6 and 7 DIV (F = 9.86,

df = 1, p = 0.003) (Fig.2-5C). The relative increase of iodopsin transcript levels observed

from CTO to CT12 under constant dark conditions was 70% of that observed under cyclic

light conditions and the temporal characteristics of the iodopsin mRNA rhythm were

similar to those observed under cyclic light (Fig.2-5E). These data suggest that the

iodopsin rhythms observed in explant cultures under 12L:12D conditions are being

driven by functioning retinal circadian oscillators.

Retina in ovo. Significant iodopsin transcript rhythms were observed in E17

chicken embryos that had been maintained in ovo under cyclic light conditions. On E18

when the lights were turned off, iodopsin transcript levels at CT12 were 1.3 fold higher

than those at CTO (Fig.2-5D). This increase was half that observed at the corresponding

time point in embryos maintained under cyclic light conditions (Fig.2-5B). These

incremental increases in transcript levels continued through E19 and E20. At CT12 on

E20, the amount of iodopsin transcript in the retinas of embryos maintained in constant

dark had reached levels similar to those observed in embryos that had been maintained

under cyclic light. Direct comparisons of the cyclic light and constant dark data (Fig.2-

5F) revealed that there was no detectable iodopsin mRNA rhythm present in embryos

placed in constant darkness. These data suggest that light and developmental mechanisms

act synergistically to up-regulate iodopsin transcription in developing embryonic chicken

retina, and that in the absence of light, increases in iodopsin transcript levels are

predominantly, if not completely, regulated by developmental mechanisms.











Reversal of the light:dark cycle

Retinal explants. Reversal of the light cycle on culture day 6 was followed by a

48-hour transition period during which time the iodopsin transcript levels remained at


levels intermediate to those observed during this same time period in the explants that

had been maintained under cyclic light conditions (Fig.2-6A, B).


4DIV 5DIV 6DIV 7DIV 8DIV


4DIV 5DIV 6DIV 7DIV 8DIV


4





E1 I

E17 E18 E19 E20


E17 E18 E19 E20


Figure 2-6. Iodopsin transcript rhythms in explant cultures and embryos following
reversal of the light:dark cycle. (A) Relative iodopsin mRNA levels in explant
cultures prepared from E9 embryos and examined at 4 -8 DIV. Explants were
maintained under 12L: 12D until 6 DIV when they were transferred to a
12D:12L reversed cycle. (B) Comparison of iodopsin rhythms obtained from
explants exposed to a reversed 12D:12L cycle (black symbols) and those from
explants maintained on a 12L: 12D cycle (red symbols; Fig. 5E). (C) Relative
iodopsin mRNA levels in retinas of embryos maintained under 12L:12D until
E18 when they were transferred to a 12D:12L reversed cycle. (D) Comparison


3.0
D


3.0

z 2.5
cnU)
|^ 2.0
)z 1.5


-. 0.5
0.0
0

0.0









of iodopsin rhythms obtained from embryos exposed to a reversed 12D:12L
cycle (black symbols) and those obtained from embryos maintained on a
12L:12D cycle (red symbols; Fig 5F). The 12-hour light and dark periods are
indicated below each panel using white and black bars, respectively. In panels
B and D, plotted data and light cycles are matched by color, red indicating 12-
hour dark periods. In panels A and C, the group means SE at each time point
are shown and the number of retinas in each group is indicated on the bars. In
panels B and D, only the mean values are plotted for each group.

Over the course of the first 12-hour dark period of the reversed cycle (6 DIV), iodopsin

mRNA levels increased 1.4 fold to levels comparable to those observed in cultures

maintained under cyclic light conditions at 6 DIV (Fig.2-6A, B). No further increase in

iodopsin mRNA levels was observed in the reversed cycle cultures during the 12-hour

light period on 6 DIV. Comparisons of the iodopsin transcription levels in cultures

exposed to the reversed light cycle to those in cultures maintained on the 12L: 12D cycle

at 7 and 8 DIV showed that the oscillators that drive the iodopsin rhythms in the explant

cultures are capable of entraining to a new light cycle and suggest that entrainment is

completed within 36-48 hours following light reversal (Fig.2-6B).

Retina in ovo. During the first 12-hour dark period of the reversed cycle on E18,

the increase in iodopsin transcript levels was minimal. By the end of the subsequent 12-

hour light period on E18, the levels of iodopsin mRNA had increased 2-fold (Fig.2-6C).

Comparisons of the iodopsin rhythms obtained from these embryos and those obtained

from embryos maintained on a normal light:dark cycle (Fig.2-6D) show that reversal of

the light cycle produces an immediate shift in the iodopsin transcript rhythm. These data,

which are consistent with our constant dark in ovo results, support the hypothesis that

iodopsin transcript rhythms in ovo are primarily, if not entirely, driven by light.









Discussion

Two major conclusions can be drawn from the results of these experiments. First,

circadian oscillators regulate iodopsin transcription in embryonic retinal explant cultures

and the rhythms that are observed are similar to those observed in post-hatch chicken

retinas (Larkin and Semple-Rowland 2001). Second, iodopsin transcript rhythms in the

retinas of chicken embryos in ovo are driven by light and not by circadian oscillators.

The iodopsin rhythms in retinal explants share several attributes with those

measured in post-hatch chicken retina. In both paradigms the rhythms are robust with the

peaks of the rhythms occurring around ZT12 and the troughs at ZTO. Importantly,

reversal of the light cycle induced similar shifts in the iodopsin transcript rhythms in both

explant cultures and in post-hatch retina. Together, these observations show that the

essential components for circadian regulation of iodopsin transcription remain intact and

functional in retinas maintained in explant cultures.

It is generally accepted that the circadian oscillators that drive the iodopsin rhythm

in chicken retina are located within the photoreceptor cells. This conclusion is based on

the observation that transcription of this cone pigment gene remains rhythmic in

dispersed retinal cell cultures (Pierce et al. 1993). Although our analyses of retinal

explants do not provide unequivocal evidence that proves that circadian oscillators are

located in photoreceptor cells, the results are consistent with this point of view. It is clear

from our analyses that neither removal of the retinal pigment epithelium (RPE) nor

degeneration of the ganglion cells is sufficient to abolish the generation or entrainment of

circadian iodopsin transcription in retinal explants. Thus, neither retinal ganglion cells

nor the RPE are essential for maintaining the iodopsin rhythm in chicken retina.









Furthermore, the absence of dopamine-secreting amacrine cells in these explant cultures

suggests that dopamine signaling does not play a central role in regulating this rhythm.

In addition to questions related to the location of circadian oscillators in retina, it

remains to be determined how the oscillators that drive the iodopsin rhythm are entrained

to light. If we assume that the oscillators that drive iodopsin transcription are located

within the photoreceptor cells, then a logical starting point for the search for the

phototransduction cascades that entrain these oscillators to light would focus on

biochemical pathways in these cells. We have previously shown that the absence of

guanylate cyclase 1 in retinas of GUCY *B chickens, an enzyme essential for visual

phototransduction, significantly delays but does not prevent light entrainment of iodopsin

rhythms to a reversal of the light cycle (Larkin and Semple-Rowland 2001). These data

show that the visual phototransduction cascade mediated by the G-protein, transducin,

does not directly play a role in entrainment of the oscillators that drive the iodopsin

rhythm. Recent analyses of chicken pineal and retina provide provocative new evidence

that suggests a possible role for a phototransduction pathway mediated by the pertussis

toxin-insensitive G-protein, G11, in light entrainment of circadian oscillators in these

tissues (Kasahara et al. 2002). Activation of a G,1-mediated pathway would be expected

to lead to changes in phosphatidylinositol turnover and calcium mobilization, changes

that have been documented to occur in photoreceptors of several vertebrate species in

response to light stimulation (Ghalayini and Anderson 1984;Hayashi and Amakawa

1985;Millar et al. 1988). Pharmacological manipulation of this cascade in retinal explant

cultures may help to determine if, in fact, this cascade is involved in light entrainment of

the oscillators that drive the iodopsin rhythm in chicken retina.









We were surprised, in view of our explant data, to find that light is the predominant

regulatory signal that drives iodopsin transcript rhythms in the retinas of chicken embryos

in ovo. These data, together with those obtained in our previous studies (Larkin and

Semple-Rowland 2001), suggest that the transition from light to circadian regulation of

iodopsin transcription in chicken retina occurs at or shortly after hatching. The onset of

circadian regulation of the activity of serotonin N-acetyltransferase (NAT) in chicken

retina, the product of another photoreceptor-specific gene, is also delayed in ovo (Iuvone

1990). However, unlike iodopsin transcription, significant light driven changes in NAT

activity do not appear until late in development (E20, just/one day prior to hatching). Our

observations and those of other investigators show that the emergence of circadian

regulation of iodopsin transcription and NAT activity in chicken retina is accelerated in

vitro {Pierce, Sheshberadaran, et al. 1993 PIERCE1993 /id}{Pierce 1999 PIERCE1999

/id} {Ivanova & luvone 2003 IVANOVA2003A /id. The mechanism responsible for this

acceleration is unknown. The culture conditions may accelerate the maturation of retinal

circadian oscillators in vitro. It is also possible that preparation of the retinas for culture

results in the removal of tissues (e.g. RPE) that normally produce regulatory signals that

delay the onset of circadian regulation in the intact, developing retina.

In conclusion, the results of our analyses show that chicken retinal explant cultures

can be used as an experimental paradigm for studies of retinal circadian oscillator

function. The structure and organization of many of the retinal cell and synaptic layers

remains intact in the explants, allowing studies of entrainment and synchronization of

circadian oscillators under various lighting regimens.















CHAPTER 3
GAP JUNCTION BLOCKERS ABOLISH CIRCADIAN RHYTHMS OF GENE
EXPRESSION IN RETINAL PHOTORECEPTORS

Introduction

Circadian oscillators in vertebrate retina regulate many aspects of retinal function,

including the synthesis and release of melatonin (Cahill et al., 1991;Cahill and Besharse,

1991;Tosini, 2000), photoreceptor disk shedding (Nguyen-Legros and Hicks, 2000),

retinomotor movement (Burnside, 2001), and gene expression (Pierce et al., 1993;Green

and Besharse, 1994;Green et al., 1996;Larkin et al., 1999;Chong et al., 2000;Bernard et

al., 1999). The transcription of the genes that encode iodopsin, a red sensitive

photopigment that is expressed specifically in the cone photoreceptors of chicken retina,

and arylalkylamine N-acetyltransferase (AANAT) have been shown to be regulated by

circadian oscillators in vivo (Larkin et al., 1999;Bernard et al., 1999;Chong et al.,

2000;Liu et al., 2004) and in dissociated retinal cultures (Pierce et al., 1993). We are

interested in understanding the mechanisms that coordinate and maintain photoreceptor

transcript rhythms in vertebrate retina.

Studies of the SCN provide clues about the mechanisms that may serve to

coordinate the activity of populations of autonomous oscillator neurons. Within the intact

SCN, 20,000 neurons work in concert to generate coordinated circadian rhythms of

electrical activity (Herzog et al., 1997), gene expression (Panda et al., 2002), and glucose

metabolism (Schwartz et al., 1983). When examined in dissociated culture, individual

SCN neurons exhibit self-sustaining electrical activity rhythms, but the periods and









phases of these rhythms are highly variable between neurons (Welsh et al., 1995). In

explant culture in which synapses and cellular appositions are largely preserved, the

periods and phases of the firing rhythms of individual SCN neurons exhibit significantly

less variability between neurons (Herzog et al., 1998). These observations led to the

hypothesis that intercellular communication is required to maintain coordinated circadian

rhythms in SCN.

Synapse- and gap junction-mediated intercellular communication represent two

possible mechanisms that could coordinate the circadian rhythms generated by

populations of autonomous oscillator cells. The observations that circadian rhythms of

glucose metabolism appear in the SCN before chemical synapses become functional

(Reppert and Schwartz, 1984;Moore and Bernstein, 1989)and that blockade of synaptic

transmission using either tetrodotoxin (TTX) (Shibata and Moore, 1993) or calcium-free

medium (Bouskila and Dudek, 1993) does not disrupt the ability of SCN neurons to

generate synchronized bursts of activity suggest that synaptic transmission is not a central

player in the coordination of circadian rhythms in the SCN. Gap junction channels, which

allow the passage of small ions, signaling molecules and low molecular weight

metabolites between cells, could, on the other hand, serve as mediators of intercellular

communication {Goodenough, Goliger, et al. 1996 GOODENOUGH1996 /id}.

Gap junction channels are comprised of two paired hemichannels, known as

connexons. Each connexon consists of six protein subunits called connexins (Cx) (Cook

and Becker, 1995). Gap junction channels form when two connexon hemichannels

located on the membranes of adjacent cells couple (Cook and Becker, 1995). Several

studies suggest that gap junction communication plays an integral role in maintaining









coordinated rhythms in SCN. Studies using dyes capable of traversing gap junctions

show that SCN cells are extensively coupled during the day when the cells exhibit

synchronous neural activity and are minimally coupled during the night when the cells

are electrically silent, a coupling rhythm that is also maintained under constant dark

conditions (Colwell, 2000). The importance of this gap junction coupling in maintaining

circadian rhythms in SCN is demonstrated by the observation that the gap junction

blockers, octanol and halothane, disrupt the circadian rhythm of vasopressin and

vasoactive intestinal polypeptide secretion from SCN slice cultures that is restored upon

removal of the blockers (Shinohara et al., 2000).

Extensive networks of gap junctions are present in developing chicken retina as

early as embryonic day 7 (Becker et al., 1998;Catsicas et al., 1998). Prior to

synaptogenesis, these junctions have been shown to play a role in the propagation of

transient Ca2+ waves that spread across the developing chicken retina (Catsicas et al.,

1998). We have recently developed a retinal explant culture system that allows the study

of circadian regulation of gene transcription in vitro (Zhang et al., 2003). Using this

culture system, we have successfully monitored the coordinated, self-sustained, and light-

entrainable iodopsin transcript rhythms normally present in chicken retina (Zhang et al.,

2003;Larkin et al., 1999). In the present study, we have conducted a series of experiments

to determine if, as in SCN, the gap junction network present in chicken retina plays a role

in maintaining coordinated iodopsin and AANAT transcript rhythms in this tissue. To test

this hypothesis, we have examined the effects of two reversible gap junction blockers,

carbenoxolone (ACO) and 183-glycyrrhetinic acids (18-PGA), on iodopsin and AANAT

transcript rhythms in explant cultures maintained under different lighting conditions.









Both gap junction blockers are the derivatives of glycyrrhetinic acid. ACO has been

reported to reversibly block the gap junction channels comprised of Cx26 (Kamermans et

al., 2001), Cx32 (Szente et al., 2002), and Cx43 (Goldberg et al., 1996), the three major

connexins that are expressed in developing chicken retina (Becker et al., 2002) and hence

the three potential targets of ACO in the retinal explant cultures. More importantly,

100[tM ACO has been shown to effectively block gap junction channels in outer

(Kamermans et al., 2001) and inner retina (Sekaran et al., 2003). Another gap junction

blocker 18-3GA has been shown to induce phosphotase-mediated dephosphorylation of

Cx43 and subsequent disassembly of gap junction plaques (Guan et al., 1996). Based on

these observations, we hypothesized that application of these blockers would block the

gap junction channels and desynchronize the circadian oscillators driving the gene

expression rhythms, and the transcript levels of both genes would assume the average of

the peak and trough values of their respective intrinsic rhythms (Kunz and Achermann,

2003).

Methods and Materials

Chemicals and Reagents

The culture media for the explants consisted of Dulbecco's modified Eagle's media

(DMEM, Gibco # 11995-065) supplemented with 10% fetal bovine serum (FBS)

(Hyclone) and antibiotics (130U/ml penicillin, 130 g/ml streptomycin) (Gibco). The gap

junction blockers, carbenoxolone (ACO), 183-glycyrrhetinic acids (18-PGA), and the

chemically- related inactive compound, glycyrrhizic acid (GA), were purchased from

Sigma (St. Louis, MO). Stock solutions of carbenoxolone (163 mM) and GA (5 mM)

were dissolved in deionized water. The stock solution of 18-pGA (100 mM) was









dissolved in dimethyl sulfoxide (DMSO; Sigma). All blockers were used at a final

concentration of 100 [tM in these experiments.

Retinal Explant Cultures

All experimental procedures were approved by the University of Florida IACUC

Committee and were carried out in accordance with the National Institutes of Health

guidelines. Fertile White Leghorn chicken eggs (Charles River Laboratories) were

incubated on a 12 hour light: 12 hour dark (12L: 12D) cycle in incubators illuminated by

20 Watt cool white fluorescent bulbs (90 lux). The lights were turned on at 9:00 AM

(Zeitgeber time, ZTO) and shut off at 9:00 PM (ZT12). Retinal explant cultures were

prepared from embryonic day 9 (E9) chickens during the 12-hour light period. Dissection

and preparation of the cultures were carried out using methods developed in our

laboratory (Zhang et al., 2003). During the first 5 days of culture, all explants were

incubated on a 12L: 12D cycle at 370C in 5% C02,.and were fed every two days. The

12L: 12D period beginning the day after the cultures were prepared was designated day 1

in vitro (1 DIV).

Lighting and Blocker Delivery Paradigms:

12L:12D:

Retinal explant cultures were maintained on a 12L: 12D cycle throughout the

experiments. In experiments utilizing gap junction blockers, ACO or 18-pGA were added

to the media at ZT12 on 4DIV and removed at ZT12 on either 5DIV or 6DIV.

Alternatively, ACO was added at ZTO on 5DIV and removed 24 hrs later. The affects of

ACO on AANAT expression were examined by adding ACO at ZT15 on 4DIV and

removing it 24 hours later. Control groups included untreated explants or explants treated









with either 0.1% DMSO or 100 [tM GA. The final percent of DMSO in the control

cultures was equivalent to that in cultures treated with 183-GA. DMSO was added at

ZT12 on 4DIV and remained on the cultures for either 24 or 48 hrs. GA was added to the

cultures at ZT12 on 4DIV and was removed 48 hrs later. Prior to treatment, the cultures

were fed every two days. During the treatment period, the media bathing both the

experimental and control cultures was replaced every 12 hours. The cultured retinas were

collected every 12 hours from 4DIV to 7DIV for analyses ofiodopsin expression. In the

experiments designed to examine AANAT mRNA levels, the cultured retinas were

collected at ZTO and ZT15 from 4DIV to 7DIV. The retinal cultures were snap frozen in

liquid nitrogen, and stored at -750C until further processing. In all experiments, retinas

collected during the dark period were processed under a low intensity safe red light (15

Watt bulb, Kodak #2 filter).

12L:12D followed by constant darkness:

Retinal explant cultures were maintained under 12L:12D lighting conditions for

5DIV. ACO was added to the media either at ZT12 on 4DIV or at ZTO on 5DIV and was

removed 24 or 12 hours later, respectively. The cultures were kept in constant dark

conditions following treatment and were collected every 12 hours from 4DIV to 7DIV.

RNA Analysis

Total RNA was extracted from the retinal explant cultures using an RNeasy kit

(Qiagen, Valencia, CA, USA). The RNA samples, 2 |tg total RNA per sample, were

loaded onto a Magnacharge nylon membrane (MSI, Westburough, MA) and analyzed

using a slot blot format as described previously (Zhang et al., 2003). Blots were prepared

in duplicate and were probed consecutively with radio-labeled cDNA probes specific for










iodopsin, and/or guanylate cyclase activating protein 1 (GCAP1) and/or AANAT, and

18S rRNA. The iodopsin, GCAP1, and 18S rRNA cDNA fragments used for probes have

been described previously (Semple-Rowland and van der, 1992;Zhang et al., 2003). The

AANAT probe was generated using a 1.4 kb BamHI fragment of cDNA clone #9A

(kindly provided by David Klein) that contained the entire AANAT open reading frame.

The 32P-labeled cDNA probes were generated using a Stripeasy DNA Kit (Ambion the

RNA company, Austin, TX, USA). The amount of probe hybridized to the blots was

measured using a BioRad Molecular Imager FX system. Levels of iodopsin, GCAP1, and

AANAT transcript present in individual samples were normalized to the amount of 18S

rRNA present in that same sample. These values were then expressed relative to the mean

normalized value for the corresponding gene on each blot. Data were analyzed using two-

way ANOVA (SigmaStat).

Results

lodopsin and AANAT Transcript Rhythms in Untreated Explant Cultures:

Previous studies have shown that iodopsin transcript levels in dispersed embryonic

retinal cultures and in post-hatch chicken retinas exhibit a robust rhythm with minimum

levels at ZTO and maximum levels at ZT12 under cyclic light conditions that is

maintained in the absence of light (Pierce et al., 1993;Larkin et al., 1999;Larkin and

Semple-Rowland, 2001). To examine the temporal regulation of iodopsin transcript

rhythms in the retinal explant cultures, iodopsin mRNA levels were examined every 6

hours on 6DIV. The results revealed that the temporal dynamics of the iodopsin rhythm

observed in our explant retina cultures were very similar to those previously observed in

dispersed retinal cultures and in post-hatch chicken retinas (Fig 3-1A, B). In the retinal

explant cultures maintained under 12L:12D conditions, analyses of iodopsin mRNA







43


levels in selected cultures at ZTO and ZT12 over a 7-day culture period revealed well-


defined rhythms that persisted under constant darkness for at least 48 hrs (Fig 3-1C, D).


These results show that functional circadian oscillators regulate iodopsin mRNA rhythms


in our explants and that these rhythms can be monitored by measuring transcript levels at


ZTO and ZT12.


ZTO 6 12 18 24
ZTO 6 12 18 24


ZTO 6 12 18 24


D
2.25
z
0 1.80

_ 1.35
-E
S 0.90

0.45


I I I I I I I I


L,'Iy I I I I I I I I I
Day 4 5 6 7


Figure 3-1. Iodopsin mRNA rhythms in retinal explant cultures. (A) Diurnal iodopsin
transcript rhythms every 6 hours observed in the retinal explant cultures on
6DIV. (B) Data in panel A are shown in line graph. (C) Iodopsin transcript
rhythms observed in the cultures maintained in 12L:12D conditions for 5DIV
and were then transferred to constant darkness on 6 DIV. Relative iodopsin
mRNA levels were analyzed every 12 hours. (D) Comparison of iodopsin
transcript rhythms observed in the cultures maintained under 12L:12D
conditions for 7DIV (black lines) to the data shown in panel C (red lines). The
12-hour light and dark periods are indicated below each panel using white and
black bars, respectively. In panel D, plotted data and light cycles are matched
by color. In panels A and C, the group means + SE at each time point are
shown and the number of retinas in each group is indicated on the bars. In
panels B and D, only the mean values are plotted for each group.


C
2.25
z
S1.80
Sow
S1.35
-E
0.90

0.45

0.00


I . .







44


Rhythmic changes in the levels of AANAT mRNA in chicken retina have also been

shown to be driven by endogenous retinal circadian oscillators (Bernard et al.,

1999;Haque et al., 2002).


z
S1.5
IQ
-z
w> 1.0


Z 0.5
0

0.0


S 3 6 9 15 18 1
ZTO 3 6 9 12 15 18 21 24


Z
, 1.40
0l
1.05
-!
E 0.70

< 0.35

0.00


ZTO 3 6 9 12 15 18 21 24
ZTO 3 6 9 12 15 18 21 24


I II I I I6
Day 4 5 6 7


ay i
Day 4


5 6


7


Figure 3-2. AANAT mRNA rhythms in retinal explant cultures. (A) Diurnal AANAT
transcript rhythms every 3 hours observed in the retinal explant cultures on
6DIV. (B) Data in panel A are shown in line graph. (C) AANAT transcript
rhythms observed in the cultures maintained in 12L:12D conditions for 5DIV
and were then transferred to constant darkness on 6 DIV. Relative AANAT
mRNA levels were analyzed every 12 hours. (D) Comparison of AANAT
transcript rhythms observed in the cultures maintained under 12L: 12D
conditions for 7DIV (black lines) to the data shown in panel C (red lines). The
12-hour light and dark periods are indicated below each panel using white and
black bars, respectively. In panel D, plotted data and light cycles are matched
by color. In panels A and C, the group means + SE at each time point are
shown and the number of retinas in each group is indicated on the bars. In
panels B and D, only the mean values are plotted for each group.


I ...









Analyses of AANAT transcripts every 3 hours in our explant cultures on 6DIV that

had been maintained under 12L: 12D conditions showed that AANAT mRNA levels are

lowest at ZTO and reach peak levels at ZT15 (Fig 3-2A, B). In the retinal explant cultures

maintained under 12L: 12D conditions, analyses of AANAT mRNA levels in selected

cultures at ZTO and ZT15 over a 7-day culture period revealed that diurnal rhythms with

an amplitude of 1.8-fold in the levels of AANAT mRNA were first detected on 5DIV

(Fig 3-2D) and continued in constant dark conditions (Fig 3-2C, D). These results show

that functional circadian oscillators in our explants regulate AANAT mRNA rhythms and

that these rhythms can be monitored by measuring transcript levels at ZTO and ZT15.

Iodopsin Transcript Levels in Explant Cultures Treated With ACO For 48 Or 24
hrs Maintained Under A 12L:12D Cycle

ACO, a derivative of glycyrrhetinic acid that has been shown to reversibly block

gap junction communication in vertebrate retina (Pottek et al., 2003;Kamermans et al.,

2001), was added to the media of selected cultures at the beginning of the dark period

(ZT12) on 4DIV. Analyses of iodopsin mRNA levels at ZTO on 5DIV, 12 hours after

addition of ACO, revealed that the iodopsin mRNA levels in these cultures were similar

to those that had been observed in untreated cultures (Fig 3-3A, D). The affects of ACO

on iodopsin mRNA levels were first evident in cultures examined at ZT12 on 5DIV. In

untreated cultures, iodopsin mRNA levels increase over the course of each 12hr light

period, peaking at ZT12 (Fig.3-3D) (Pierce et al., 1993;Larkin et al., 1999). Treatment of

cultures with ACO prevented this increase. In treated cultures, the amount of iodopsin

mRNA measured at ZT12 on 5DIV was the same as that measured at ZTO on 5DIV and

remained low throughout the 48 hr treatment period. Removal of ACO at ZT12 on 6DIV

produced a rapid, two-fold increase in the amount of iodopsin transcript in the cultures







46


examined at ZTO on 7DIV. Surprisingly, this increase occurred over the course of a 12-

hour dark period, a time during which iodopsin transcript levels normally fall to their

lowest values. The amount of iodopsin mRNA in cultures examined at ZT12 on 7DIV did

not increase significantly over the values measured at ZTO on 7DIV.

A 225 ACO 48hrs B 225 ACO 24hrs C225 ACO 24hrs
I I < I I < I I
z z z
S180 180 180
In) I I0 I 00 80
> 135 13 135 >Q 135
z Z T QI
E 090 3 090 3 090









D 225 AC48hrs E 22045 rs F 225 ACO 24hrs
-004









000 A ll 000-A 000
I II







Day 4 5 6 7 Day 4 5 6 7 Day 4 5 6 7


Figure 3-3. Iodopsin expression in explant cultures treated with ACO for 48 and 24hrs








series of experiments. (A) Cultures were incubated with 100 M ACO for 48
>y 135 6 1357 Dy 5 6 7 Dy 135













hrs. ACO was added to the cultures at ZT12 on 4DIV and was removed at
ZT12 on 6DIV. (B) Cultures were incubated with 100 [iM ACO for 24 hrs.
ACO was added to the cultures at ZT12 on 4DIV and was removed at ZT12
on 5DIV. (C) Cultures were incubated with 01000 M ACO for 24 hrs, ACO was
added to the cultures at ZTO on 5DIV and was removed at ZTO on 6DIV. In
panels A to C, retinal explant cultures were analyzed at ZTO (white bars) and
at ZT12 (black bars). (D) Comparison of the data shown in panel A (red line)
and the iodopsin transcript rhythms observed in the untreated cultures (black
line). (E) Comparison of data shown in panel B (red line) and the iodopsin
transcript rhythms observed in the untreated cultures (black line). (F)
Comparison of data shown in panel C (red line) and the iodopsin transcript
rhythms observed in the untreated cultures (black line). The 12-hour light and









dark periods are indicated below each panel using white and black bars,
respectively. The dashed lines in each panel indicate the period of blocker
treatment. In panels A C, the group means SE at each time point are
shown and the number of retinas in each group is indicated on the bars. In
panels D F, only the mean values are plotted for each group.

The rapid suppression of iodopsin mRNA levels by ACO was unexpected. To

determine if iodopsin mRNA rhythms would reappear following removal of the blocker

and to further examine the temporal characteristics of this phenomenon, cultures were

treated with ACO for a shorter period of time. ACO was added to cultures at ZT12 on

4DIV or at ZTO on 5DIV. In both experiments, ACO was removed 24 hours later. In both

paradigms, the iodopsin mRNA levels observed 12 hours following addition ACO were

comparable to the low levels observed at ZTO in control cultures. Importantly, within 24

hours of the removal of the blocker, iodopsin transcript rhythms reappeared in the treated

cultures that were indistinguishable from those observed in control cultures (Fig. 3-3E,

F). Moreover, introduction of ACO at ZTO on 5DIV completely blocked the increase in

iodopsin mRNA that normally occurred over the course of the 12-hour light period in

control cultures (Fig 3-3C, F). Together, these data show that the effect of ACO on

iodopsin mRNA levels is reversible and occurs within 12 hours of application.

Iodopsin Transcript Levels in Explant Cultures Treated With ACO For 24hr Or
12hr Followed By Constant Darkness

To determine if there was any evidence that ACO altered the function or phase of

the circadian oscillators that drive iodopsin transcript rhythms, iodopsin mRNA levels

were measured in cultures that were treated with ACO and were then maintained in

constant darkness. As expected from our previous experiments, addition of ACO at the

ZT12 on 4DIV or at ZTO on 5DIV reduced iodopsin transcript levels (Fig 3-4A, B).

Importantly, following removal of the blocker, iodopsin mRNA rhythms re-emerged in







48


the cultures in the absence of the 12L: 12D cycle that were congruent with those observed

in the untreated cultures (Fig 3-4C, D).


2.25
ACO 24hrs
1.80 -
I I
1.35 -



0.45- 3
0. 00I I


D 4
Day 4 5 6 7


2.25-
Z
S1.80-

S1.35-

-S c 0.9-

-| 0.45-


2.25

1.80

1.35

0.90

0.45


ACO 24hrs








I 4
II





Day4 5 6 7


ACO 12hrs





I I
81" ill


D ayI I I I I I I I
Day 4 5 6 7


ACO 12hrs


I I


Day 4 5


6 7


Figure 3-4. Iodopsin expression in explant cultures treated with ACO followed by
constant darkness (A) ACO (100 [tm) was added to the cultures at ZT12 on
4DIV and was removed at ZT12 on 5DIV. (B) ACO (100 pm) was added to
the cultures at ZTO on 5DIV and was removed at ZT12 on 5DIV. In panels A
and B, retinal explant cultures were analyzed at ZTO (white bars) and at ZT12
(black bars). (C) Comparison of the data shown in panel A (red line) and the
iodopsin transcript rhythms observed in the untreated cultures (black line). (D)
Comparison of data shown in panel B (red line) and the iodopsin transcript
rhythms observed in the untreated cultures (black line). The 12-hour light and
dark periods are indicated below each panel using white and black bars,
respectively. The dashed lines in each panel indicate the period of blocker
treatment. In panels A and B, the group means + SE at each time point are
shown and the number of retinas in each group is indicated on the bars. In
panels C and D, only the mean values are plotted for each group.


A
Z



0
C
o


I I










These data suggest that short-term treatment of cultures with ACO does not alter

the function or temporal characteristics of the circadian oscillators that drive iodopsin

mRNA rhythms in chicken retina.

Effects of ACO Treatment On AANAT and GCAP1 Transcript Levels

In this series of experiments, we examined the specificity of the effect of ACO on

gene transcript levels by examining the affects of ACO on transcript levels of AANAT

and GCAP1, genes also expressed in chicken retinal photoreceptors (Chong et al.,

2000;Semple-Rowland et al., 1999).


A
1.75 ACO 24 hrs
z I I
S1.40 I I

1.05 I I
z II 7
E 0.70 3 I
5 I I
z 0.35

0.00


z
1- 1.40

S 1.05
wz
SE 0.70

z 0.35

0.00


Day4I I I I I I I I
Day4 5 6 7


ACO 24 hrs





S I
I I
I I

I I

I I
I I



Day4 5 6 7


Figure 3-5. AANAT expression in explant cultures treated with ACO for 24hrs. The
explant cultures were maintained under 12L:12D conditions. (A) ACO (100
[tm) was added to the cultures at ZT15 on 4DIV and removed at ZT15 on
5DIV. Relative AANAT mRNA levels at ZTO (white bars) and ZT15 (black
bars) in the ACO treated cultures were shown. (B) The AANAT expression in
ACO treated cultures (red line) was compared to that observed in untreated
cultures (black lines). The 12-hour light and dark periods are indicated below
each panel using white and black bars, respectively. The dashed lines in each
panel indicate the period of blocker treatment. In panel A, the group means +
SE at each time point are shown and the number of retinas in each group is
indicated on the bars. In panel B, only the mean values are plotted for each
group.

Treatment of the cultures with ACO beginning at ZT15 on 4DIV produced a

significant suppression of AANAT mRNA levels in cultures examined at ZT15 on 5DIV,

the values of which were similar to those observed at ZTO in untreated cultures (Fig. 3-








50



5A, B) Within 24 hours of removal of the blocker, the AANAT rhythms paralleled to


those observed in the untreated cultures.


A
c
z

o
r
(D <
( Z
C-E


(D


1.75

1.40

1.05

0.70

0.35

0.00


1 3


11


< 1.75
Z
o 1.40

1.05

- E 0.70

< 0.35

0.00


I I I I


I I I I


Day 4 5 6 7




ACO 48hrs
I I
I I












Day 4 5 6 7



ACO 24hrs



I I

3 3
I I
11









I I I I I

Day 4 5 6 7


<
z
rY




SE


0
O
(9


<
z

SC


(D Y
ijE


SE
13_
(D
0
0D


1.75

1.40

1.05

0.70

0.35


ACO 48hrs


I I
I I

I I I I I I


Day 4 5 6 7


ACO 24hrs
I I
I I


Day 4 5 6


Day 4 5 6 7


C
<
z
S
CI)



ciE

O
(3


E







<
O
-E
(3

0
0D


1.75

1.40

1.05

0.70

0.35

0.00-


1.75

1.40

1.05

0.70

0.35

0.00


! . .


7









Figure 3-6. GCAP-1 expression in explant cultures treated with ACO for 48 or 24 hrs and
maintained under 12L:12D conditions. (A) GCAP1 expression in the
untreated cultures from 4 to 7DIV. (B) The data in panel A are shown in line
graph. (C) ACO (100[tm) was added to the cultures at ZT12 on 4DIV and
removed at ZT12 on 6DIV. (D) The data in panel C are shown in line graph.
(E) ACO (100 [tm) was added to the cultures at ZT12 on 4DIV and removed
at ZT12 on 5DIV. (F) The data in panel E are shown in line graph. The 12-
hour light and dark periods are indicated below each panel using white and
black bars, respectively. The dashed lines indicate the period of ACO
treatment. In panels A, C and E, the group means + SE at each time point are
shown and the number of retinas in each group is indicated on the bars. In
panels B, D and F, only the mean values are plotted for each group.

In explant cultures, GCAP1 mRNA levels gradually increase over the culture

period, reaching relatively stable levels by 5DIV (Fig. 3-6A, B). Incubation of cultures

with ACO for either 24 (Fig. 3-6E, F) or 48 hours (Fig. 3-6C, D) did not produce any

significant changes in GCAP1 mRNA levels in the cultures.

Together, the result of our analyses of AANAT and GCAP1 mRNA levels in

explants treated with ACO indicate that ACO does not produce a generalized reduction in

mRNA levels in retinal photoreceptors and suggest that the action of ACO on transcript

levels may be restricted to genes that are regulated by retinal oscillators.











lodopsin Transcript Levels in Explant Cultures
hrs Maintained Under A 12L:12D Cycle


Treated With 18-PGA For 48 Or 24


18p-GA 48hrs
I I
I I
I I
I I
I I


2.25-

1.80

1.35

0.90

0.45

0.00-





2.25 -

1.80 -

1.35 -

0.90-

0.45-

0.00-





2.25-

1.80

1.35

0.90

0.45-

0.00


IDay I I4 5 6I
Day4 5 6


18p-GA 24hrs
I I
I I
I I


I 1I


Day 4 5 6


Day 4 5 6 7


7


Day 4 5 6 7


18p-GA 48hrs

I I













Day 4 5 6 7


I r r r


7


I I I I I I
Day 4 5 6 7









Figure 3-7. lodopsin expression in explant cultures treated with 18-PGA for 48 and 24hrs
and maintained under 12L:12D conditions. (A) 18-PGA (100 [tm) was added
to the cultures at ZT12 on 4DIV and was removed at ZT12 on 6DIV. (B) 18-
3GA (100 [tm) was added to cultures on ZT12 on 4DIV and was removed on
ZT12 on 5DIV. In panels A and B the relative iodopsin mRNA levels at ZTO
and ZT12 are shown in white bars and black bars, respectively. (C) The data
shown in panel A (red lines) were compared to iodopsin transcript rhythms
observed in untreated cultures (black lines). (D) The data shown in panel B
(red line) were compared to iodopsin transcript rhythms observed in untreated
cultures (black lines). The dashed lines in each panel indicate the period of 18-
3GA treatment. In panels A and B, the group means SE at each time point
are shown and the number of retinas in each group is indicated on the bars. In
panels C and D, only the mean values are plotted for each group. (E) Cultures
were treated with 0.1% DMSO for 48 hrs from ZT12 on 4DIV to ZT12 on
6DIV. (F) Cultures were treated with 0.1% DMSO for 24 hrs from ZT12 on
4DIV to ZT12 on 5DIV. In panels E and F iodopsin mRNA levels in DMSO
treated cultures at ZTO (white bar) and ZT12 (hatched bar) were compared to
those observed in untreated cultures (black lines). The dashed lines indicate
the period of DMSO treatment. Each time point in the bar graphs represents
the mean SE iodopsin mRNA levels measured in 3 cultures. For the line
graphs, only the mean values are plotted for each group.

Treatment of explant cultures with the glycyrrhetinic acid derivative, 18-3GA, also

significantly reduced iodopsin mRNA levels in the explant cultures maintained under

cyclic light conditions. Treatment of cultures with 18-pGA for either 48 or 24 hours

reduced iodopsin mRNA levels to values significantly below the trough values observed

in untreated cultures at ZTO (Fig. 3-7A, B, C, D). Removal of the blocker produced a

rapid 3 to 3.5-fold increase in iodopsin mRNA levels over the course of the first 12 hours

following removal of the blocker. In cultures treated with the blocker for 24 hours,

iodopsin mRNA rhythms similar to those observed in untreated cultures were re-

established within 24 hours of removal of the blocker (Fig. 3-7D). Unlike cultures treated

with 18-pGA for 24 hours, the levels of iodopsin mRNA measured in cultures 24 hours

following 48-hour treatment with 18-pGA were not rhythmic and highly variable (Fig. 3-

6B). This result is reminiscent of the desynchronized rhythms observed in SCN following







54


treatment with octanol and halothane (Shinohara et al., 2000) and suggests that longer

exposures of cultures to 18-pGA might affect the coordination of the retinal oscillators

that drive iodopsin rhythm. Treatment of cultures with 0.1% DMSO, the amount

equivalent to that applied to the cultures treated with 100 [LM 18-3GA, did not alter

iodopsin mRNA rhythms in the retinal cultures (Fig. 3-6C, D).


A 2.25 GA 48hrs B 2.25 GA 48hrs
I IL
z I I z
I- 1.80 I 1.80
I I
> 1.35 I I 1.35
-z
E 0.90 I -I E 0.90
3 3

e 0.45 GA (100 m) 0.45
0 11 0
0.00 0.00

Day4 5 6 7 Day4 5 6 7


Figure 3-8. iodopsin expression in explant cultures treated with GA for 48hrs. The
explant cultures were maintained under 12L: 12D conditions in this
experiment. (A) GA (100 [ti) was added to the cultures at ZT12 on 4DIV and
removed at ZT12 on 6DIV. Relative iodopsin mRNA levels at ZTO (white
bars) and ZT12 (black bars) in the GA treated cultures were shown. (B) The
iodopsin expression in GA treated cultures (red line) was compared to that
observed in untreated cultures (black lines). The 12-hour light and dark
periods are indicated below each panel using white and black bars,
respectively. The dashed lines in each panel indicate the period of GA
treatment. In panel A, the group means SE at each time point are shown and
the number of retinas in each group is indicated on the bars. In panel B, only
the mean values are plotted for each group.

In a second control experiment, we examined the specificity of the actions of ACO

and 18-PGA on the cultures. Treatment of cultures for 48 hrs with 100 [LM GA, a

chemically related inactive compound, did not alter iodopsin mRNA rhythms in the

cultures (Fig. 3-8A, B). This result suggests that the actions of ACO and 18-PGA on


iodopsin transcript levels are specific to these compounds.


A, B). This result suggests that the actions of ACO and 18-3GA on


iodopsin transcript levels are specific to these compounds.









Discussion

The results of these experiments show that iodopsin and AANAT mRNA levels

exhibit coordinated rhythms in cultures maintained under cyclic light conditions and that

these rhythms are maintained under constant darkness. In contrast to iodopsin and

AANAT, GCAP1 mRNA levels in cultured retinas gradually increase over the course of

the culture period and do not exhibit a discernible rhythm. The presence of the iodopsin

and AANAT mRNA rhythms in cultures maintained in constant darkness data confirm

that circadian oscillators contribute to the regulation of the transcript levels of these genes

in our culture paradigm. Addition of 100 [LM ACO to the cultures rapidly reduced the

amount of iodopsin and AANAT mRNA in the cultures, an effect that was maintained as

long as the blocker was present. The chemically related gap junction blocker, 18-3GA,

produced similar effects on iodopsin transcript rhythms in the retinal explant cultures.

Within 24 hours of removal of ACO, the iodopsin and AANAT transcript rhythms

reappeared in the cultures. ACO did not alter GCAP1 mRNA levels in the cultured

retinas, a result that suggests that the actions of this blocker cannot be attributed to

general transcription suppression.

The hypothesis that we set out to test was that gap junctions play a role in

maintaining coordinated iodopsin and AANAT transcript rhythms in retina. We expected

that treatment of retina cultures with gap junction blockers would result in a

desynchronization of the retinal oscillators that drive these rhythms and a subsequent loss

of the iodopsin and AANAT transcript rhythms. The transcript levels of these two genes

would assume the average of the peak and trough values of their rhythms in the presence

of the blockers. However, the effects of the blockers were unexpected in light of the









results of similar studies that were conducted in SCN slice cultures. The effects of ACO

and 18-3GA on iodopsin transcript rhythms in retina exhibited two major differences

from the effects of two other gap junction blockers, octanol and halothane, on rhythmic

AVP and VIP secretion in SCN slice cultures. First, the treatment of ACO or 18-pGA

suppressed iodopsin transcript levels to the trough or the values significantly lower than

the trough of its rhythms observed in the untreated cultures. Hence, unlike the effects of

octanol and halothane on AVP and VIP release in SCN cultures (Shinohara et al., 2000),

the total amount of iodopsin mRNA generated in retinal photoreceptors over the

treatment period is significantly less than that in the untreated cultures (Fig 3-3, 3-4, 3-

7C, D). Second, the reduction of iodopsin transcript levels in response to the addition of

the blockers occurs within 12 hours (Fig. 3-3C, F). This time period is much shorter than

that observed in the study of SCN slice cultures, the exposure of which to octanol or

halothane for 42 hours had no observable effects on the circadian rhythms of peptide

secretion. The loss of the peptide release circadian rhythms was not observed until the

SCN cultures were treated with the blockers for 7 days (Shinohara et al., 2000).

Therefore, the effects of the two gap junction blockers on iodopsin transcript rhythms

cannot be attributed to desynchronization of the circadian oscillators, which would be

expected to take much longer for the subtle phase angle differences among individual

oscillators to become significant enough to affect the overt ensemble gene expression

rhythm.

The reduction in the levels of iodopsin and AANAT transcripts in the presence of

the blockers could result from cytotoxcity of the blockers, disruption of gene

transcription or elevation of transcript degradation.









Several of our experimental observations argue strongly against the possibility that

the suppressing effects of ACO and 18-PGA on iodopsin and AANAT transcript levels

were due to cytotoxicity of the two gap junction blockers. First, transcription of the

photoreceptor-specific gene GCAP1 was not suppressed by ACO. Second, the amount of

iodopsin and AANAT transcript in the cultures rapidly recovered to the intermediate or

peak levels 12 hours following the removal of the blockers. Finally, circadian rhythms of

iodopsin transcript re-appeared within 24 hours after the block was removed, suggesting

that retinal circadian oscillators are functional and the explant cultures are

physiologically healthy during and after the treatment of ACO. Together, these

observations do not support the thesis that the reduction in iodopsin and AANAT mRNA

levels that we observed is due to massive cell death.

It is possible that treatment of the cultures with the gap junction blockers disrupted

gene transcription. This could occur if the gap junction blockers disrupted oscillator

function or if they disrupted the coupling mechanism that normally allows the oscillator

to drive transcription of these genes. Disruption of circadian oscillator function is

unlikely because recovery of iodopsin rhythms following removal of the blockers was

rapid (Fig 3-3). If the blockers altered the function of the oscillators driving iodopsin and

AANAT transcript rhythms, we would have observed slower recovery of the rhythms

following removal of the blockers. More definitively, iodopsin transcript rhythms were

maintained in constant darkness after either 24 or 12hr treatment of ACO (Fig 3-4),

suggesting that ACO did not disrupt the ability of the circadian oscillators to regulate

iodopsin transcription. On the other hand, ACO may uncouple the functional circadian

oscillators and iodopsin or AANAT transcription. A recent study of chick dispersed cell









cultures has shown that the Ca2+ influx stimulates the formation of cAMP, which in turn

couples the circadian oscillators and the rhythms of AANAT enzyme activity that bear

the similar temporal characteristics to AANAT transcript rhythms (Ivanova and luvone,

2003). How does ACO affect Ca2+ influx to the photoreceptor cells? It has been shown

that blockade of gap junction channels by 100M ACO, reduced Ca2+ influx to

photoreceptor cells from horizontal cells (Kamermans et al., 2001). Moreover, 100[tM

ACO can directly reduce the voltage-gated Ca2+ channel current by 37% in isolated

cones, and inhibit the Ca2+ influx by 57% in retinal slice preparations (Vessey et al.,

2004). Therefore, it is possible that the reduction of Ca2+ influx in presence of ACO

results in the lack of stimulation of cAMP, which uncouples the circadian oscillators and

the transcription of the output genes, such as AANAT and iodopsin. The exact molecular

mechanisms, however, through which the reduced levels of Ca2+ and/or cAMP suppress

iodopsin and AANAT transcription, need to be further investigated.

Based the current data, the possibility that the gap junction blockers increase the

transcript degradation cannot be ruled out. It has been shown that a rapid turnover protein

increases the degradation of AANAT transcript (Greve et al., 1999). Thus, it is also

possible that the gap junction blockers enhance the activity of this protein, and lead to the

increased degradation of AANAT and/or iodopsin transcript.















CHAPTER 4
PROSPECTIVE

A Real-Time Monitoring Culture System For Circadian-Regulated Gene Expression

The major approaches used in this dissertation for studying the transcription of the

genes that are expressed in retinal photoreceptors and regulated by circadian oscillators

involve collecting significant number of retinal samples at each time point, homogenizing

separate populations of the retinal samples, and measuring steady-state RNA levels

through standard RNA assays. These typical methods are straightforward and have

helped me observe interesting phenomena to prove the hypothesis.

However, these methods have limitations for studying the dynamics of clock-

regulated gene transcription. First, sample collections every 12hrs were performed in

most experiments studying iodopsin and AANAT mRNA rhythms, the time-resolution of

the experiments might not be high enough to reveal the complete temporal characteristics

of the clock-regulated gene expression. Especially when the explant cultures were

subjected to the changes of light cycle or the treatment of gap junction blockers or both,

subtle changes in the phase of iodopsin or AANAT rhythms or the immediate

transcriptional response of either genes may not be observed in the two-point analyses

every light-dark or circadian cycle. Although the problem can be partly overcome by

adding more time points during the period of light changes or blocker treatment, this

solution makes the typical methods less efficient and more labor-intensive and time-

consuming. Moreover, these approaches exclude the possibility to monitor the dynamics

of clock-regulated gene expression in individual retinal explant cultures. The iodopsin









transcript level shown at each time point in the end results is the ensemble average of

iodopsin mRNA levels from the population of retinal cultures collected at that time point.

The average value can reliably reflect iodopsin expression levels in individual cultures

only if are the cultures in a certain experiment synchronized, which is the case in the

experiments in which the cultures are exposed to light. Nonetheless, in the constant dark

experiments, when the lights are turned off, the iodopsin mRNA rhythms in individual

cultures begin to desynchronize. Therefore, taking the ensemble average of the

desynchronized rhythms leads to the apparent reduction in amplitude across the retinal

samples collected during the darkness. This could account for the damped amplitude of

iodopsin transcript rhythms observed in the constant dark experiments (Fig2-5C, E).

Finally, since the typical approaches measure the steady-state mRNA levels, it is difficult

to discern if the manipulations act on transcriptional level or on posttranscriptional level.

Although it has been shown that circadian oscillators regulate iodopsin expression at the

transcriptional level in retinal cultures maintained under different lighting

conditions(Pierce et al. 1993), the sites of actions of the gap junction blockers need

further investigation.

The monitoring of temporal characteristics of iodopsin transcription could be

greatly improved and simplified by establishing a retinal culture system that carries a

transgene of iodopsin promoter linked to firefly luciferase coding sequence (lod.luc). The

iodopsin promoter can be isolated from chicken genomic DNA library that is available in

my current lab. The luciferase reporter has been used successfully for monitoring the

transcription of Perl with high time-resolution in nervous system of both transgenic

Drosophila (Brandes et al. 1996;Stanewsky et al. 1997) and transgenic rats(Abe et al.









2002;Yamazaki et al. 2000). The short half-life (about 2 hrs) of luciferase in vertebrate

and the automated quantification system for measuring luciferase activity makes it an

excellent reporter for real-time monitoring circadian-regulated iodopsin transcription in

chicken retina. The modified lentiviral vector can be used as a novel tool to generate

"transgenic" chicken retina. The lentiviral vector has been shown to be able to transduce

both retinal progenitor cells and terminally differentiated cells in chicken embryo with

high efficiency (>80% cells transduced) (Coleman et al. 2002;Coleman et al. 2003). The

packaged lentivirus carrying the Iod.luc transgene will be injected into the neural tubes of

chicken embryos at stage 10 to 12 (-embryonic day 2; E2). Following injection, the eggs

will be incubated under 12L:12D conditions until E9. The embryonic retinas will be

dissected on E9 and cultured with the media supplemented with luciferin under 12L: 12D

conditions. By continuously measuring the bioluminescence emitted from the retinal

explant cultures, changes of iodopsin expression at the transcriptional level in response to

the manipulations, such as light and pharmacological agents, can be monitored with high

resolution from individual samples over one or two circadian cycles.

Possible Mechanisms of Light Entrainment in Embryonic Chicken Retinas

The phase of iodopsin transcript rhythms was reversed following the exposure of

the retinal explant cultures to the reversed light cycle (Fig2-6A, B). This observation

suggests that the circadian oscillators driving iodopsin mRNA rhythms can be entrained

to the environmental light changes. Then what are the possible mechanisms underlying

the light entrainment in the retinal explant cultures?

The recent remarkable progress in the mechanisms responsible for light

entrainment of behavioral rhythms in mammals provides clues for this question. A subset

of retinal ganglion cells that express a novel photopigment, melanopsin, has been









discovered to be intrinsically photoresponsive (ipRGCs). The dendrites of ipRGCs form

extensive reticular networks to maximally detect light irradiance (Hattar et al.

2002;Provencio et al. 2002). The ipRGCs also arborize in the inner plexiform layer,

suggesting that these cells receive synaptic input from the classical rod and cone

photoreceptors (Belenky et al. 2003;Provencio et al. 2002). It has been show that either

rod and cone image-forming system or ipRGCs system is sufficient to detect and

transduce photic information through retinohypothalamic tract to the SCN, but neither

system is necessary for the light entrainment process. Therefore, classical photoreceptors

and ipRGCs are functionally redundant for light entrainment of the behavioral rhythms in

mammals (Van Gelder 2003).

However, it is notable that by the time the reversal of the light cycle is performed

on 6DIV (Fig2-6A, B), the ganglion cells in the retinal explant cultures have degenerated,

and are not detectable by cresyl violet staining (Fig2-1). The observations indicate that

retinal ganglion cells are not necessary for the light entrainment of the circadian

oscillators driving iodopsin transcript rhythms in the explant cultures. On the other hand,

circadian oscillator functions in the explant cultures prepared from GUCY1*B embryonic

chicken retinas were also characterized. GUCY1*B chicken carries a null mutation in

Guanylate Cyclase-1 gene, hence the classical phototransduction pathway is disabled in

the retinal photoreceptors. The morphology of *B retinal explant cultures is

indistinguishable from that of the cultures prepared from White Leghorn chicken retinas.

The ganglion cells also degenerate by 5DIV (data not shown). Interestingly, the iodopsin

mRNA rhythms in *B retinal explants exhibit similar dynamics of phase reversal to those

observed in Leghorn explant cultures (Fig 4-1). These data suggest that both ganglion










cells and classical phototransduction pathway are not required to entrain the circadian

oscillators driving iodopsin transcription in the retinal explant cultures.

A B
1.80 1.80
Z Z
cw, 1.35 9 m )M 1.35
00 T T
< '<
|z 0.90 gz 0.90
S19
o0.45 09 0.45
o 0
0.00 0.00


Day 4 5 6 7 8 Day 4 5 6 7 8


Figure 4-1. Iodopsin transcript rhythms in GUCY1*B and White Leghorn chicken retinal
explant cultures maintained under 12L: 12D followed by reversal of the light
cycle. The explant cultures were prepared from E9 *B and leghorn chicken
embryos. The cultures were maintained under 12L: 12D until 6 DIV when they
were transferred to a 12D: 12L reversed cycle. The iodopsin mRNA rhythms
from *B explant cultures were analyzed every 12 hours. The iodopsin
transcript rhythms from *B retinal explant cultures (red line) were compared
to those from Leghorn explant cultures (black line). The 12-hour light and
dark periods are indicated below using white and black bars, respectively. In
panel A, the group means SE at each time point are shown and the number
of retinas in each group is indicated on the bars. In panel B, only the mean
values are plotted for each group.

Another candidate that might be important for entraining circadian oscillators

driving iodopsin rhythms in the retinal explant cultures is Cryptochromes (Crys). Crys

are flavin-based photopigments that are first identified as members of photolyase family

in plants (Sancar 2000). Although lack of photolyase activity in animals, Crys have been

shown to serve as circadian photopigments for light entrainment in both Drosophila

(Sancar 2000) and zebrafish (Cermakian et al. 2002). More directly, both chicken Cryl

(Haque et al. 2002) and Cry2 (Bailey et al. 2002) highly express in retinal photoreceptor

cells. The dual regulation of chicken Cryl transcription by circadian oscillators and light


ulation of chicken Cryl transcription by circadian oscillators and light






64


suggests its involvement in the function of circadian oscillator and/or circadian

photoreception in the photoreceptors of chicken retina (Haque et al. 2002).















REFERENCE LIST


Abe M, Herzog ED, Yamazaki S, Straume M, Tei H, Sakaki Y, Menaker M, Block GD
(2002) Circadian rhythms in isolated brain regions. J Neurosci 22: 350-356.

Adler R, Tamres A, Bradford RL, Belecky-Adams TL (2001) Microenvironmental
regulation of visual pigment expression in the chick retina. Dev Biol 236: 454-464.

Albrecht U, Sun ZS, Eichele G, Lee CC (1997) A differential response of two putative
mammalian circadian regulators, mperl and mper2, to light. Cell 91: 1055-1064.

Anderson FE, Green CB (2000) Symphony of rhythms in the Xenopus laevis retina.
Microsc Res Tech 50: 360-372.

Bae K, Jin X, Maywood ES, Hastings MH, Reppert SM, Weaver DR (2001) Differential
functions of mPerl, mPer2, and mPer3 in the SCN circadian clock. Neuron 30:
525-536.

Bailey MJ, Chong NW, Xiong J, Cassone VM (2002) Chickens' Cry2: molecular analysis
of an avian cryptochrome in retinal and pineal photoreceptors. FEBS Lett 513: 169-
174.

Becker D, Bonness V, V, Mobbs P (1998) Cell coupling in the retina: patterns and
purpose. Cell Biol Int 22: 781-792.

Becker DL, Bonness V, Catsicas M, Mobbs P (2002) Changing patterns of ganglion cell
coupling and connexin expression during chick retinal development. J Neurobiol
52: 280-293.

Belecky-Adams TL, Scheurer D, Adler R (1999) Activin family members in the
developing chick retina: expression patterns, protein distribution, and in vitro
effects. Dev Biol 210: 107-123.

Belenky MA, Smeraski CA, Provencio I, Sollars PJ, Pickard GE (2003) Melanopsin
retinal ganglion cells receive bipolar and amacrine cell synapses. J Comp Neurol
460: 380-393.

Bernard M, Guerlotte J, Greve P, Grechez-Cassiau A, luvone MP, Zatz M, Chong NW,
Klein DC, Voisin P (1999) Melatonin synthesis pathway: circadian regulation of
the genes encoding the key enzymes in the chicken pineal gland and retina. Reprod
Nutr Dev 39: 325-334.










Besharse JC, luvone PM (1983) Circadian clock in Xenopus eye controlling retinal
serotonin N-acetyltransferase. Nature 305: 133-135.

Besharse JC, luvone PM (1992) Is dopamine a light-adaptive or a dark-adaptive
modulator in retina? Neurochem Int 20: 193-199.

Blau J, Young MW (1999) Cycling vrille expression is required for a functional
Drosophila clock. Cell 99: 661-671.

Bouskila Y, Dudek FE (1993) Neuronal synchronization without calcium-dependent
synaptic transmission in the hypothalamus. Proc Natl Acad Sci U S A 90: 3207-
3210.

Brandes C, Plautz JD, Stanewsky R, Jamison CF, Straume M, Wood KV, Kay SA, Hall
JC (1996) Novel features of drosophila period Transcription revealed by real-time
luciferase reporting. Neuron 16: 687-692.

Burnside B (2001) Light and circadian regulation of retinomotor movement. Prog Brain
Res 131: 477-485.

Cahill GM, Besharse JC (1991) Resetting the circadian clock in cultured Xenopus
eyecups: regulation of retinal melatonin rhythms by light and D2 dopamine
receptors. JNeurosci 11: 2959-2971.

Cahill GM, Besharse JC (1992) Light-sensitive melatonin synthesis by Xenopus
photoreceptors after destruction of the inner retina. Vis Neurosci 8: 487-490.

Cahill GM, Besharse JC (1993) Circadian clock functions localized in xenopus retinal
photoreceptors. Neuron 10: 573-577.

Cahill GM, Grace MS, Besharse JC (1991) Rhythmic regulation of retinal melatonin:
metabolic pathways, neurochemical mechanisms, and the ocular circadian clock.
Cell Mol Neurobiol 11: 529-560.

Catsicas M, Bonness V, Becker D, Mobbs P (1998) Spontaneous Ca2+ transients and
their transmission in the developing chick retina. Curr Biol 8: 283-286.

Cermakian N, Pando MP, Thompson CL, Pinchak AB, Selby CP, Gutierrez L, Wells DE,
Cahill GM, Sancar A, Sassone-Corsi P (2002) Light induction of a vertebrate clock
gene involves signaling through blue-light receptors and MAP kinases. Curr Biol
12: 844-848.

Chang DC, Reppert SM (2001) The circadian clocks of mice and men. Neuron 29: 555-
558.

Chong NW, Bernard M, Klein DC (2000) Characterization of the chicken serotonin N-
acetyltransferase gene. Activation via clock gene heterodimer/E box interaction. J
Biol Chem 275: 32991-32998.









Coleman JE, Fuchs GE, Semple-Rowland SL (2002) Analyses of the guanylate cyclase
activating protein-i gene promoter in the developing retina. Invest Ophthalmol Vis
Sci 43: 1335-1343.

Coleman JE, Huentelman MJ, Kasparov S, Metcalfe BL, Paton JF, Katovich MJ, Semple-
Rowland SL, Raizada MK (2003) Efficient large-scale production and
concentration of HIV-1-based lentiviral vectors for use in vivo. Physiol Genomics
12: 221-228.

Colwell CS (2000) Rhythmic coupling among cells in the suprachiasmatic nucleus. J
Neurobiol 43: 379-388.

Cook B, Portera-Cailliau C, Adler R (1998) Developmental neuronal death is not a
universal phenomenon among cell types in the chick embryo retina. J Comp Neurol
396: 12-19.

Cook JE, Becker DL (1995) Gap junctions in the vertebrate retina. Microsc Res Tech 31:
408-419.

Darlington TK, Wager-Smith K, Ceriani MF, Staknis D, Gekakis N, Steeves TD, Weitz
CJ, Takahashi JS, Kay SA (1998) Closing the circadian loop: CLOCK-induced
transcription of its own inhibitors per and tim. Science 280: 1599-1603.

Devlin PF, Kay SA (2001) Circadian photoperception. Annu Rev Physiol 63: 677-694.

Dunlap JC (1999) Molecular bases for circadian clocks. Cell 96: 271-290.

Ebihara S, Uchiyaya k, Oshima I (1984) Circadian Organization in the pigeon, Coluba
livia: The role of the pineal organ and the eye. J Comp Physiol 154A: 59-69.

Gardino PF, dos Santos RM, Hokoc JN (1993) Histogenesis and topographical
distribution of tyrosine hydroxylase immunoreactive amacrine cells in the
developing chick retina. Brain Res Dev Brain Res 72: 226-236.

Gaston S, Menaker M (1968) Pineal function: the biological clock in the sparrow?
Science 160: 1125-1127.

Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, King DP, Takahashi JS,
Weitz CJ (1998) Role of the CLOCK protein in the mammalian circadian
mechanism. Science 280: 1564-1569.

Geusz ME, Foster RG, deGrip WJ, Block GD (1997) Opsin-like immunoreactivity in the
circadian pacemaker neurons and photoreceptors of the eye of the opisthobranch
mollusc Bulla gouldiana. Cell Tissue Res 287: 203-210.

Ghalayini A, Anderson RE (1984) Phosphatidylinositol 4,5-bisphosphate: light-mediated
breakdown in the vertebrate retina. Biochem Biophys Res Commun 124: 503-506.









Gillette MU, McArthur AJ (1996) Circadian actions of melatonin at the suprachiasmatic
nucleus. Behav Brain Res 73: 135-139.

Glossop NR, Lyons LC, Hardin PE (1999) Interlocked feedback loops within the
Drosophila circadian oscillator. Science 286: 766-768.

Gold GH, Dowling JE (1979) Photoreceptor coupling in retina of the toad, Bufo marinus.
I. Anatomy. J Neurophysiol 42: 292-310.

Goldberg GS, Moreno AP, Bechberger JF, Hearn SS, Shivers RR, MacPhee DJ, Zhang
YC, Naus CC (1996) Evidence that disruption of connexon particle arrangements
in gap junction plaques is associated with inhibition of gap junctional
communication by a glycyrrhetinic acid derivative. Exp Cell Res 222: 48-53.

Goodenough DA, Goliger JA, Paul DL (1996) Connexins, connexons, and intercellular
communication. Annu Rev Biochem 65: 475-502.

Green CB (1998) How cells tell time. Trends Cell Biol 8: 224-230.

Green CB, Besharse JC (1994) Tryptophan hydroxylase expression is regulated by a
circadian clock in Xenopus laevis retina. J Neurochem 62: 2420-2428.

Green CB, Besharse JC, Zatz M (1996) Tryptophan hydroxylase mRNA levels are
regulated by the circadian clock, temperature, and cAMP in chick pineal cells.
Brain Res 738: 1-7.

Green CB, Cahill GM, Besharse JC (1995) Regulation of tryptophan hydroxylase
expression by a retinal circadian oscillator in vitro. Brain Res 677: 283-290.

Greve P, Alonso-Gomez A, Bernard M, Ma M, Haque R, Klein DC, luvone PM (1999)
Serotonin N-acetyltransferase mRNA levels in photoreceptor-enriched chicken
retinal cell cultures: elevation by cyclic AMP. J Neurochem 73: 1894-1900.

Guan X, Wilson S, Schlender KK, Ruch RJ (1996) Gap-junction disassembly and
connexin 43 dephosphorylation induced by 18 beta-glycyrrhetinic acid. Mol
Carcinog 16: 157-164.

Hampson EC, Vaney DI, Weiler R (1992) Dopaminergic modulation of gap junction
permeability between amacrine cells in mammalian retina. J Neurosci 12: 4911-
4922.

Hampson EC, Weiler R, Vaney DI (1994) pH-gated dopaminergic modulation of
horizontal cell gap junctions in mammalian retina. Proc R Soc Lond B Biol Sci
255: 67-72.

Haque R, Chaurasia SS, Wessel JH, III, luvone PM (2002) Dual regulation of
cryptochrome 1 mRNA expression in chicken retina by light and circadian
oscillators. Neuroreport 13: 2247-2251.









Hattar S, Liao HW, Takao M, Berson DM, Yau KW (2002) Melanopsin-containing
retinal ganglion cells: architecture, projections, and intrinsic photosensitivity.
Science 295: 1065-1070.

Hayasaka N, LaRue SI, Green CB (2002) In vivo disruption of Xenopus CLOCK in the
retinal photoreceptor cells abolishes circadian melatonin rhythmicity without
affecting its production levels. J Neurosci 22: 1600-1607.

Hayashi F, Amakawa T (1985) Light-mediated breakdown of phosphatidylinositol-4,5-
bisphosphate in isolated rod outer segments of frog photoreceptor. Biochem
Biophys Res Commun 128: 954-959.

Herzog ED, Geusz ME, Khalsa SB, Straume M, Block GD (1997) Circadian rhythms in
mouse suprachiasmatic nucleus explants on multimicroelectrode plates. Brain Res
757: 285-290.

Herzog ED, Takahashi JS, Block GD (1998) Clock controls circadian period in isolated
suprachiasmatic nucleus neurons. Nat Neurosci 1: 708-713.

luvone PM (1990) Development of melatonin synthesis in chicken retina: regulation of
serotonin N-acetyltransferase activity by light, circadian oscillators, and cyclic
AMP. J Neurochem 54: 1562-1568.

Ivanova TN, luvone PM (2003b) Circadian rhythm and photic control of cAMP level in
chick retinal cell cultures: a mechanism for coupling the circadian oscillator to the
melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase, in
photoreceptor cells. Brain Res 991: 96-103.

Ivanova TN, luvone PM (2003a) Melatonin synthesis in retina: circadian regulation of
arylalkylamine N-acetyltransferase activity in cultured photoreceptor cells of
embryonic chicken retina. Brain Res 973: 56-63.

Jiang ZG, Yang YQ, Allen CN (1997) Tracer and electrical coupling of rat
suprachiasmatic nucleus neurons. Neuroscience 77: 1059-1066.

Kamermans M, Fahrenfort I, Schultz K, Janssen-Bienhold U, Sjoerdsma T, Weiler R
(2001) Hemichannel-mediated inhibition in the outer retina. Science 292: 1178-
1180.

Kasahara T, Okano T, Haga T, Fukada Y (2002) Opsin-G11-mediated signaling pathway
for photic entrainment of the chicken pineal circadian clock. J Neurosci 22: 7321-
7325.

Krizaj D, Gabriel R, Owen WG, Witkovsky P (1998) Dopamine D2 receptor-mediated
modulation of rod-cone coupling in the Xenopus retina. J Comp Neurol 398: 529-
538.










Kunz H, Achermann P (2003) Simulation of circadian rhythm generation in the
suprachiasmatic nucleus with locally coupled self-sustained oscillators. J Theor
Biol 224: 63-78.

Larkin P, Baehr W, Semple-Rowland SL (1999) Circadian regulation of iodopsin and
clock is altered in the retinal degeneration chicken retina. Brain Res Mol Brain Res
70: 253-263.

Larkin P, Semple-Rowland SL (2001) A null mutation in guanylate cyclase-1 alters the
temporal dynamics and light entrainment properties of the iodopsin rhythm in cone
photoreceptor cells. Brain Res Mol Brain Res 92: 49-57.

Lebedev DS, Byzov AL, Govardovskii VI (1998) Photoreceptor coupling and boundary
detection. Vision Res 38: 3161-3169.

LeSauter J, Silver R (1998) Output signals of the SCN. Chronobiol Int 15: 535-550.

Li L, Dowling JE (2000) Effects of dopamine depletion on visual sensitivity of zebrafish.
JNeurosci 20: 1893-1903.

Liu C, Fukuhara C, Wessel JH, III, luvone PM, Tosini G (2004) Localization of Aa-nat
mRNA in the rat retina by fluorescence in situ hybridization and laser capture
microdissection. Cell Tissue Res 315: 197-201.

Liu C, Reppert SM (2000) GABA synchronizes clock cells within the suprachiasmatic
circadian clock. Neuron 25: 123-128.

Manglapus MK, luvone PM, Underwood H, Pierce ME, Barlow RB (1999) Dopamine
mediates circadian rhythms of rod-cone dominance in the Japanese quail retina. J
Neurosci 19: 4132-4141.

Manglapus MK, Uchiyama H, Buelow NF, Barlow RB (1998) Circadian rhythms of rod-
cone dominance in the Japanese quail retina. J Neurosci 18: 4775-4784.

Marin-Teva JL, Cuadros MA, Calvente R, Almendros A, Navascues J (1999) Naturally
occurring cell death and migration of microglial precursors in the quail retina
during normal development. J Comp Neurol 412: 255-275.

Martinek S, Inonog S, Manoukian AS, Young MW (2001) A role for the segment
polarity gene shaggy/GSK-3 in the Drosophila circadian clock. Cell 105: 769-779.

Millar FA, Fisher SC, Muir CA, Edwards E, Hawthorne JN (1988) Polyphosphoinositide
hydrolysis in response to light stimulation of rat and chick retina and retinal rod
outer segments. Biochim Biophys Acta 970: 205-211.

Moog R (1995) [Chronobiological health of the circadian system: examples for disorders
of the sleep-waking rhythm]. Wien Med Wochenschr 145: 452-453.









Moore RY, Bernstein ME (1989) Synaptogenesis in the rat suprachiasmatic nucleus
demonstrated by electron microscopy and synapsin I immunoreactivity. J Neurosci
9: 2151-2162.

Nguyen-Legros J, Hicks D (2000) Renewal of photoreceptor outer segments and their
phagocytosis by the retinal pigment epithelium. Int Rev Cytol 196: 245-313.

Ogilvie JM, Speck JD, Lett JM, Fleming TT (1999) A reliable method for organ culture
of neonatal mouse retina with long-term survival. J Neurosci Methods 87: 57-65.

Oishi T, Yamao M, Kondo C, Haida Y, Masuda A, Tamotsu S (2001) Multiphotoreceptor
and multioscillator system in avian circadian organization. Microsc Res Tech 53:
43-47.

Okamura H, Miyake S, Sumi Y, Yamaguchi S, Yasui A, Muijtjens M, Hoeijmakers JH,
van der Horst GT (1999) Photic induction of mPerl and mPer2 in cry-deficient
mice lacking a biological clock. Science 286: 2531-2534.

Panda S, Antoch MP, Miller BH, Su AI, Schook AB, Straume M, Schultz PG, Kay SA,
Takahashi JS, Hogenesch JB (2002) Coordinated transcription of key pathways in
the mouse by the circadian clock. Cell 109: 307-320.

Piccolino M, Neyton J, Gerschenfeld HM (1984) Decrease of gap junction permeability
induced by dopamine and cyclic adenosine 3':5'-monophosphate in horizontal cells
of turtle retina. J Neurosci 4: 2477-2488.

Piccolino M, Neyton J, Witkovsky P, Gerschenfeld HM (1982) gamma-Aminobutyric
acid antagonists decrease junctional communication between L-horizontal cells of
the retina. Proc Natl Acad Sci U S A 79: 3671-3675.

Pierce ME (1999) Circadian organization in quail retina: differential regulation of
melatonin synthesis and iodopsin gene expression in vitro. Vis Neurosci 16: 843-
848.

Pierce ME, Sheshberadaran H, Zhang Z, Fox LE, Applebury ML, Takahashi JS (1993)
Circadian regulation of iodopsin gene expression in embryonic photoreceptors in
retinal cell culture. Neuron 10: 579-584.

Pottek M, Hoppenstedt W, Janssen-Bienhold U, Schultz K, Perlman I, Weiler R (2003)
Contribution of connexin26 to electrical feedback inhibition in the turtle retina. J
Comp Neurol 466: 468-477.

Price JL, Blau J, Rothenfluh A, Abodeely M, Kloss B, Young MW (1998) double-time is
a novel Drosophila clock gene that regulates PERIOD protein accumulation. Cell
94: 83-95.










Provencio I, Rollag MD, Castrucci AM (2002) Photoreceptive net in the mammalian
retina. This mesh of cells may explain how some blind mice can still tell day from
night. Nature 415: 493.

Raviola E, Gilula NB (1973) Gap junctions between photoreceptor cells in the vertebrate
retina. Proc Natl Acad Sci U S A 70: 1677-1681.

Reppert SM, Schwartz WJ (1984) The suprachiasmatic nuclei of the fetal rat:
characterization of a functional circadian clock using 14C-labeled deoxyglucose. J
Neurosci 4: 1677-1682.

Reppert SM, Weaver DR (2001) Molecular analysis of mammalian circadian rhythms.
Annu Rev Physiol 63: 647-676.

Rosenwasser AM, Dwyer SM (2001) Circadian phase shifting: Relationships between
photic and nonphotic phase-response curves. Physiol Behav 73: 175-183.

Sancar A (2000) Cryptochrome: the second photoactive pigment in the eye and its role in
circadian photoreception. Annu Rev Biochem 69: 31-67.

Schneeweis DM, Schnapf JL (1999) The photovoltage of macaque cone photoreceptors:
adaptation, noise, and kinetics. J Neurosci 19: 1203-1216.

Schwartz WJ, Reppert SM, Eagan SM, Moore-Ede MC (1983) In vivo metabolic activity
of the suprachiasmatic nuclei: a comparative study. Brain Res 274: 184-187.

Sekaran S, Foster RG, Lucas RJ, Hankins MW (2003) Calcium imaging reveals a
network of intrinsically light-sensitive inner-retinal neurons. Curr Biol 13: 1290-
1298.

Semple-Rowland SL, Larkin P, Bronson JD, Nykamp K, Streit WJ, Baehr W (1999)
Characterization of the chicken GCAP gene array and analyses of GCAP 1,
GCAP2, and GC1 gene expression in normal and rd chicken pineal. Mol Vis 5: 14.

Semple-Rowland SL, van der WH (1992) Visinin: biochemical and molecular
comparisons in normal and rd chick retina. Biochem Biophys Res Commun 183:
456-461.

Shearman LP, Sriram S, Weaver DR, Maywood ES, Chaves I, Zheng B, Kume K, Lee
CC, van der Horst GT, Hastings MH, Reppert SM (2000) Interacting molecular
loops in the mammalian circadian clock. Science 288: 1013-1019.

Shearman LP, Zylka MJ, Weaver DR, Kolakowski LF, Jr., Reppert SM (1997) Two
period homologs: circadian expression and photic regulation in the suprachiasmatic
nuclei. Neuron 19: 1261-1269.









Shibata S, Moore RY (1993) Tetrodotoxin does not affect circadian rhythms in neuronal
activity and metabolism in rodent suprachiasmatic nucleus in vitro. Brain Res 606:
259-266.

Shinohara K, Funabashi T, Mitushima D, Kimura F (2000) Effects of gap junction
blocker on vasopressin and vasoactive intestinal polypeptide rhythms in the rat
suprachiasmatic nucleus in vitro. Neurosci Res 38: 43-47.

Simpsom SM, Follett BK (1981) Pineal and Hypothalamic pacemakers: their role in
regulating circadian rhythmicity in Japanese quail. J Comp Physiol 144A: 381-389.

Stanewsky R, Jamison CF, Plautz JD, Kay SA, Hall JC (1997) Multiple circadian-
regulated elements contribute to cycling period gene expression in Drosophila.
EMBO J 16: 5006-5018.

Sun ZS, Albrecht U, Zhuchenko O, Bailey J, Eichele G, Lee CC (1997) RIGUI, a
putative mammalian ortholog of the Drosophila period gene. Cell 90: 1003-1011.

Szente M, Gajda Z, Said AK, Hermesz E (2002) Involvement of electrical coupling in the
in vivo ictal epileptiform activity induced by 4-aminopyridine in the neocortex.
Neuroscience 115: 1067-1078.

Tosini G (2000) Melatonin circadian rhythm in the retina of mammals. Chronobiol Int
17: 599-612.

Tosini G, Menaker M (1996) Circadian rhythms in cultured mammalian retina. Science
272: 419-421.

Tosini G, Menaker M (1998) The clock in the mouse retina: melatonin synthesis and
photoreceptor degeneration. Brain Res 789: 221-228.

Tsukamoto Y, Masarachia P, Schein SJ, Sterling P (1992) Gap junctions between the
pedicles of macaque foveal cones. Vision Res 32: 1809-1815.

Underwood H (1994) The circadian rhythm of thermoregulation in Japanese quail. I. Role
of the eyes and pineal. J Comp Physiol [A] 175: 639-653.

Van Gelder RN (2003) Making (a) sense of non-visual ocular photoreception. Trends
Neurosci 26: 458-461.

Vaney DI, Weiler R (2000) Gap junctions in the eye: evidence for heteromeric,
heterotypic and mixed-homotypic interactions. Brain Res Brain Res Rev 32: 115-
120.

Vessey JP, Lalonde MR, Mizan HA, Welch NC, Kelly ME, Barnes S (2004)
Carbenoxolone inhibition of voltage-gated Ca channels and synaptic transmission
in the retina. J Neurophysiol.









Welsh DK, Logothetis DE, Meister M, Reppert SM (1995) Individual neurons
dissociated from rat suprachiasmatic nucleus express independently phased
circadian firing rhythms. Neuron 14: 697-706.

Wong WT, Sanes JR, Wong RO (1998) Developmentally regulated spontaneous activity
in the embryonic chick retina. J Neurosci 18: 8839-8852.

Yamazaki S, Numano R, Abe M, Hida A, Takahashi R, Ueda M, Block GD, Sakaki Y,
Menaker M, Tei H (2000) Resetting central and peripheral circadian oscillators in
transgenic rats. Science 288: 682-685.

Yang XL, Wu SM (1989) Modulation of rod-cone coupling by light. Science 244: 352-
354.

Yoshimura T, Suzuki Y, Makino E, Suzuki T, Kuroiwa A, Matsuda Y, Namikawa T,
Ebihara S (2000) Molecular analysis of avian circadian clock genes. Brain Res Mol
Brain Res 78: 207-215.

Yoshizawa T, Kuwata O (1991) Iodopsin, a red-sensitive cone visual pigment in the
chicken retina. Photochem Photobiol 54: 1061-1070.

Young MW (2000) The tick-tock of the biological clock. Sci Am 282: 64-71.

Zhang Y, Coleman JE, Fuchs GE, Semple-Rowland SL (2003) Circadian oscillator
function in embryonic retina and retinal explant cultures. Brain Res Mol Brain Res
114: 9-19.

Zhu H, LaRue S, Whiteley A, Steeves TD, Takahashi JS, Green CB (2000) The Xenopus
clock gene is constitutively expressed in retinal photoreceptors. Brain Res Mol
Brain Res 75: 303-308.

Zhuang M, Wang Y, Steenhard BM, Besharse JC (2000) Differential regulation of two
period genes in the Xenopus eye. Brain Res Mol Brain Res 82: 52-64.

Zylka MJ, Shearman LP, Weaver DR, Reppert SM (1998) Three period homologs in
mammals: differential light responses in the suprachiasmatic circadian clock and
oscillating transcripts outside of brain. Neuron 20: 1103-1110.















BIOGRAPHICAL SKETCH

Yan Zhang was born in Tianjin, the third largest city in China, on Dec 28th, 1972.

After receiving the award for the best student in the primary school in Tianjin city for

three consecutive years, he entered Nankai high school, one of the best high schools in

China. During three years of his junior high school, he ranked first of 265 students in 8

out of 12 comprehensive exams. Consequently, he was admitted to Nankai senior high

school with the exemption of the final exam. After another three years of endeavors in

that highly competitive environment, he chose to enter Tianjin Medical University to

study clinical medicine with the intention of his parents and with his own hope of a better

future, although he loved and was good at mathematics at the time of graduating from

high school.

He did not understand the value of clinical medicine until the last two years of

medical training when he did probation in the Department of Internal Medicine and an

internship in the Department of Surgery. It was during those two years that he truly

realized that a good doctor could relieve suffering and save the lives of patients, thus

gaining respect from people. However, at the time he graduated from medical school in

1996, molecular biology had just become the hottest area in China. Additionally, he

thought that because he was still very young, his education should not end at the age of

23. He then chose the National Key Laboratory of Hormone and Brain Development in

China to pursue the MS degree in molecular endocrinology. During three years of work

in the laboratory, he participated in the purification of glutamic acid decarboxylase from






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human brain and the development of an ELISA system for early detection of Type I

diabetes using the purified protein as antigen. His thesis, "Cloning and Expression of

Human Somatostatin Gene in E. coli.," summarized his work in molecular biology. After

receiving his MS degree in August 1999, he came to the University of Florida, joined the

Department of Neuroscience in the interdisciplinary program in the College of Medicine,

and began his PhD study under the supervision of Dr. Susan Semple-Rowland. His

research during the Ph.D. study is reflected in this dissertation.