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The retina is a complex neural circuit, which processes and transmits visual information from light perceiving photoreceptors to projecting retinal ganglion cells. Much of the computational power of the retina rests on signal integrating interneurons, such as bipolar cells. Commercially available antibodies against bovine and human conventional protein kinase C (PKC) α and -ß are frequently used as markers for retinal ON-bipolar cells in different species, despite the fact that it is not known which bipolar cell subtype(s) they actually label. In zebrafish (Danio rerio) five prkc genes (coding for PKC proteins) have been identified. Their expression has not been systematically determined. While prkcg is not expressed in retinal tissue, the other four prkc (prkcaa, prkcab, prkcba, prkcbb) transcripts were found in different parts of the inner nuclear layer and some as well in the retinal ganglion cell layer. Immunohistochemical analysis in adult zebrafish retina using fluorescent in situ hybridization and PKC antibodies showed an overlapping immunolabeling of ON-bipolar cells that are most likely of the BON s6 and BON s6L or RRod type. However, comparison of transcript expression with immunolabeling, implies that these antibodies are not specific for one single zebrafish conventional PKC, but rather detect a combination of PKC -α and -ß variants.

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Conventional protein kinases-consisting of α, ß, and γ family members-play key roles in numerous signal transduction events. Phylogenetic analysis demonstrated the existence of five prkcs (the genes representing PKCs) in zebrafish, two paralogous forms of prkca and prkcb and one prkcg variant. mRNA expression analysis showed distinct, mainly nervous system specific expression, for all five prkc genes. For prkca and prkcb paralogs prominent expression can be seen in the telencephalon, in diencephalic regions such as the habenula or the optic tectum, in hypothalamic areas and in distinct cerebellar structures. Each transcript is additionally expressed in distinct areas: prkcaa is highly abundant in cranial sensory ganglia and in dorsal neurons of the hindbrain and the spinal cord, prkcab is strongly expressed in additional cerebellar regions, prkcba shows expression in the pectoral fin, the otic vesicle and in the proximal convoluted tubule of the kidney, and prkcbb shows prominent expression in different hypothalamic areas. Expression of prkcg is most striking in the cerebellum. As zebrafish PKCs are expressed in structures that are equivalent to mammals, the zebrafish model is well suited to study evolutionary conserved functions of PKCs in development and disease.

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Cryptochromes (Crys) are light sensing receptors that are present in all eukaryotes. They mainly absorb light in the UV/blue spectrum. The extant Crys consist of two subfamilies, which are descendants of photolyases but are now involved in the regulation of circadian rhythms. So far, knowledge about the evolution, phylogeny, and expression of cry genes is still scarce. The inclusion of cry sequences from a wide range of bilaterian species allowed us to analyze their phylogeny in detail, identifying six major Cry subgroups. Selective gene inactivations and stabilizations in multiple chordate as well as arthropod lineages suggest several sub- and/or neofunctionalization events. An expression study performed in zebrafish, the model organism harboring the largest amount of crys, showed indeed only partially overlapping expression of paralogous mRNA, supporting gene sub- and/or neofunctionalization. Moreover, the daily cry expression in the adult zebrafish retina indicated varying oscillation patterns in different cell types. Our extensive phylogenetic analysis provides for the first time an overview of cry evolutionary history. Although several, especially parasitic or blind species, have lost all cry genes, crustaceans have retained up to three crys, teleosts possess up to seven, and tetrapods up to four crys. The broad and cyclic expression pattern of all cry transcripts in zebrafish retinal layers implies an involvement in retinal circadian processes and supports the hypothesis of several autonomous circadian clocks present in the vertebrate retina.

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Glutamate, the most abundant excitatory neurotransmitter of the central nervous system, modulates synaptic transmission and neuronal excitability via metabotropic glutamate receptors (mGluRs). These receptors are essential components for diverse cognitive functions and they represent potential drug targets for the treatment of a number of neurological and psychiatric disorders. Here we describe the phylogenetic relation and mRNA distribution of zebrafish mGluRs. In comparison to the eight mglurs present in the mammalian genome, we identified 13 different mglur genes in the zebrafish genome. In situ hybridization experiments in zebrafish revealed widespread expression patterns for the different mglurs in the central nervous system, implicating their significance in diverse neuronal functions. Prominent mglur expression is found in the olfactory bulb, the optic tectum, the hypothalamus, the cerebellum, and the retina. We show that expression pattern of paralogs generated by the teleost-specific whole genome duplication is overlapping in some brain regions but complementary in others, suggesting sub- and/or neofunctionalization in the latter. Group I mglurs are similarly expressed in brain areas of both larval and adult zebrafish, suggesting that their functions are comparable during these stages.

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The metabotropic glutamate receptor 6 (mGluR6 or GRM6) belongs to the class III of the metabotropic glutamate receptor family. It is the only known mGluR that mediates direct synaptic transmission in the nervous system and is thought to mediate the ON-response in the ON-pathway of the vertebrate retina. Phylogenetic and gene structure analysis indicated that the zebrafish genome harbours two mglur6 paralogs, mglur6a and mglur6b. Besides expression in the inner nuclear layer and distinct regions in the brain, both mglur6 paralogs are expressed in ganglion cells of the retina, an expression pattern which can also be observed in the downstream effector molecules gnaoa and gnaob. This unexpected expression pattern is consistent with immunohistological labeling using a peptide antibody specific for the mGluR6b paralog. These expression patterns contradict the existing view that mGluR6 is solely located on ON-bipolar cells where it functions in signal transmission. Consistent with expression in ON-bipolar cells, we report a decreased b-wave amplitude in the electroretinogram after morpholino-based downregulation of mGluR6b, showing a function in the ON response. Our data suggest more widespread functions of mGluR6 mediated signaling in the central nervous system, possibly including sign reversing synapses in the inner retina.

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BACKGROUND: Visual acuity, the ability of the visual system to distinguish two separate objects at a given angular distance, is influenced by the optical and neuronal properties of the visual system. Although many factors may contribute, the ultimate limit is photoreceptor spacing. In general, at least one unstimulated photoreceptor flanked by two stimulated ones is needed to perceive two objects as separate. This critical interval is also referred to as the Nyquist frequency and is according to the Shannon sampling theorem the highest spatial frequency where a pattern can be faithfully transmitted. We measured visual acuity in a behavioral experiment and compared the data to the physical limit given by photoreceptor spacing in zebrafish larvae. RESULTS: We determined visual acuity by using the optokinetic response (OKR), reflexive eye movements in response to whole field movements of the visual scene. By altering the spatial frequency we determined the visual acuity at approximately 0.16 cycles/degree (cpd) (minimum separable angle = 3.1 degrees ). On histological sections we measured the retinal magnification factor and the distance between double cones, that are thought to mediate motion perception. These measurements set the physical limit at 0.24 cpd (2.1 degrees ). CONCLUSION: The maximal spatial information as limited by photoreceptor spacing can not be fully utilized in a motion dependent visual behavior, arguing that the larval zebrafish visual system has not matured enough to optimally translate visual information into behavior. Nevertheless behavioral acuity is remarkable close to its maximal value, given the immature state of young zebrafish larvae.