In zebrafish, the expression of long-wavelength cone (LC) opsin mRNA fluctuated rhythmically between the day and night. In a 24-h period, expression was high in the afternoon and low in the early morning. This pattern of fluctuation persisted in zebrafish that were kept in constant darkness, suggesting an involvement of circadian clocks. Functional expression of Clock, a circadian clock gene that contributes to the central circadian pacemaker, was found to play an important role in maintaining the circadian rhythms of LC opsin mRNA expression. In zebrafish embryos, in which the translation of Clock was inhibited by anti-Clock morpholinos, the circadian rhythms of LC opsin mRNA expression diminished. CLOCK may regulate the circadian rhythms of LC opsin mRNA expression via cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. In control retinas, the concentration of cAMP was high in the early morning and low in the remainder of the day and night. Inhibition of Clock translation abolished the fluctuation in the concentration of cAMP, thereby diminishing the circadian rhythms of opsin mRNA expression. Transient increase of cAMP concentrations in the early morning (i.e. by treating the embryos with 8-bromo-cAMP) restored the circadian rhythms of LC opsin mRNA expression in morpholino-treated embryos. Together, the data suggest that Clock plays important roles in regulating the circadian rhythms in photoreceptor cells.

Dopamine plays important roles in the regulation of central nervous system (CNS) development and functions. In vertebrates, two families of dopamine receptors, collectively known as dopamine D1 and D2 receptors, have been identified. Recently, dopamine receptors have been targeted by pharmacological and therapeutic studies of neurological disorders, such as Parkinson’s disease. Here, we report a study on the molecular characterization of dopamine D1 receptor in zebrafish (Danio rerio). We cloned the full-length cDNA of a zebrafish dopamine D1 receptor, designated as drd1. The sequence of drd1 shares high homology to the sequences of dopamine D1 receptors in mammalian, amphibian, and other fish species. drd1 is expressed in the CNS. The first drd1 expression was observed at approximately 30 hours postfertilization, at which time the expression was seen in the developing diencephalon and hindbrain. In developing retinas, the expression of drd1 was detected in the inner nuclear layer with the exception of the marginal zones. In adult retinas, drd1 expression was detected in most cell types in the inner and outer nuclear layers as well as ganglion cell layer. Differential expression of drd1 in developing and adult retinas may play various roles in regulating visual system functions. Developmental Dynamics 236:1339–1346, 2007.

Endogenous circadian clocks regulate day-night rhythms of animal behavior and physiology. In zebrafish, the circadian clocks are located in the pineal gland and the retina. In the retina, each photoreceptor is considered a circadian oscillator. A critical question is whether the individual circadian oscillators are synchronized. If so, the mechanism that underlies the synchronization needs to be elucidated. We generated a transgenic zebrafish line that expresses short half-life GFP under the transcriptional control of the rhodopsin promoter. Time-lapse imaging of rhodopsin promoter-driven GFP expression revealed that during 24·h in constant darkness, rhodopsin promoter expression in rod photoreceptor cells fluctuated rhythmically. However, the pattern of fluctuation differed between individual cells. In some cells, peak expression was seen in the subjective early morning, whereas in other cells, peak expression was seen in the afternoon or at night. Light transiently decreased rhodopsin expression, thereby synchronizing the multiphasic circadian oscillation. The application of dopamine or dopamine D2 receptor agonist also synchronized the circadian rhythms of rhodopsin promoter expression. When the D2 receptors were pharmacologically blocked, light exposure produced no effect. This suggests that the synchronization of the circadian rhythms of rhodopsin promoter expression by light is mediated by dopamine D2 receptors. The mechanism that underlies the synchronization probably involves dopamine-mediated Ca2+ signaling pathways. Light, as well as dopamine, lowered Ca2+ influx into the rod cells, thereby resetting rhodopsin promoter expression to the initial phase.

The vertebrate retina receives centrifugal input fromthe brain. Inzebrafish, the major centrifugal input originates in the terminal nerve (TN). TN cell bodies are located in the olfactory bulb and ventral telencephalon. The TN projects axons to the retina where they branch in the inner plexiform layer (IPL) and synapse onto several inner retinal cell types, including dopaminergic interplexiform cells (DA-IPCs). This olfactoretinal centrifugal input plays a role in modulating retinal ganglion cell (RGC) activity, probably via dopamine-mediated Ca2+ signalling pathways. Normally, dopamine inhibits RGC firing by decreasing the inward Ca2+ current. Olfactory stimulation with amino acids decreases dopamine release in the retina, thereby reducing dopaminergic inhibition of RGCs. Thismodel of olfacto-visual integration was directly tested by recording single-unitRGCactivity in response to olfactory stimulation in the presence or absence of dopamine receptor blockers. Stimulation of the olfactory neurones increased RGC activity. However, this effect diminished when the dopamine D1 receptors were pharmacologically blocked. In isolated RGCs, the application of dopamine or a dopamine D1 receptor agonist decreased voltage-activated Ca2+ current and lowered Ca2+ influx. Together, the data suggest that olfactory input has a modulatory effect on RGC firing, and that this effect is mediated by dopamine D1 receptor-coupled Ca2+ signalling pathways.

Here we report three dominant nightblindness mutations in zebrafish: nightblindness e (nbe), nightblindness f (nbf) and nightblindness g (nbg). The mutants were isolated in the F1 generation of N-ethyl-N-nitrosourea (ENU) mutagenized zebrafish using a behavioral assay based on visually mediated escape responses. Subsequently, electroretinographic (ERG) recordings were made, and histological sections were screened for degenerative processes. For each mutant line, correlation analysis between behavioral, ERG and histological parameters was performed, and their relationships were determined by either calculating the Pearson correlation coefficient or by ANOVA. nbe is characterized by severe rod outer segments (ROS) degeneration. The degeneration correlates weakly with behavioral threshold and ERG b-wave amplitude, however, behavioral threshold correlates strongly with ERG b-wave. nbf is characterized by a dual histological pathology: patchy ROS-degeneration and ‘gaps’ homogeneously distributed over the outer nuclei layer (ONL) and between cone outer segments (COS). The correlations between histological pathology and behavioral threshold, and between behavioral threshold and ERG bwave amplitude are obvious, but the correlation between histology and b-wave amplitude is less prominent. nbg is characterized by moderate ROS degeneration and moderate correlation between histology and behavioral threshold. Interestingly, behavioral threshold correlated inversely with ERG b-wave amplitude and threshold. Thus, contrary to what is normally seen in other nightblindness mutants, in nbg, the fish with the lowest behavioral threshold had the smallest b-waves amplitudes and the highest b-wave threshold. In our interpretation, the major impairment in nbe is photoreceptor-specific. In nbf, both photoreceptor degeneration and altered post-photoreceptor signaling are responsible for the behavioral deficit. In nbg, we find hypersensitivity at a post-photoreceptoral level concurrently with behavioral impairment.