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Marion Haug, Edda Kastenhuber, Colette M. Maurer, Matthias Gesemann, Stephan C. Neuhauss; A Glutamate Transporter and the conserved mGluR6/TrpM1 Pathway mediate the Zebrafish ON-Response. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4313.
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Generation of an ON-signal requires an inhibitory response to glutamate at the first visual synapse. This is thought to be mediated by the metabotropic glutamate receptor 6 (mGluR6) whose activation leads - via an intracellular G-protein coupled second messenger pathway - to the closure of the transient receptor channel 1 (TrpM1). However, data on lower vertebrates suggest an involvement of a glutamate transporter (EAAT) that enables sign inversion via chloride conductance. We use the cone dominant retina of larval zebrafish to unravel the contribution of these signaling pathways to the ON-response.
Zebrafish orthologs for genes involved in the ON-pathway were identified via comparative phylogenetic analysis. RNA in situ hybridization and immunohistochemistry were used to locate the transcripts and proteins of the studied molecules. A morpholino antisense knockdown approach was used for functional analysis with electroretinography as a read-out.
Phylogenetic analysis indicated that the zebrafish genome harbours two mglur6 paralogs, -6a and -6b. Besides expression in the inner nuclear layer and in distinct regions of the brain, both paralogs are expressed in the retinal ganglion cell layer (GCL). Bipolar cells (BCs) co-express both mglur6 paralogs and all genes discussed to be involved in its signaling cascade. With the notable exception of both trpm1 paralogs all these genes are additionally expressed in the GCL.The reduced b-wave of mglur6b knockdown larvae in ERG recordings reflects a diminished photopic ON-response. Double-knockdown of eaat7 and mglur6b had a more severe effect on the cone ON-response but a small remaining b-wave was still visible.
Our results indicate that the zebrafish photopic ON-response relies on both mGluR6b and EAAT7. However, as an ON-signal in double-morphant larvae still persists, the involvement of an additional receptor is likely.The expression of mglur6 paralogs and all downstream effector molecules in BCs implies a conservation of the ON-pathway. Interestingly, mglur6 expression in other retinal layers and in the brain suggests more widespread functions of mGluR6 mediated signaling. The detection of nearly all mglur6 signaling cascade members in the GCL even suggests the exciting possibility that sign reversing synapses are also present in the inner retina.
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