Abstract
In the mouse retina, horizontal cells form an electrically coupled network and provide feedback signals to photoreceptors and feedforward signals to bipolar cells. Thereby, horizontal cells contribute to gain control at the first visual synapse and to the antagonistic organization of bipolar and ganglion cell receptive fields. However, the nature of horizontal cell output remains a matter of debate, just as the exact contribution of horizontal cells to center-surround antagonism. To facilitate studying horizontal cell function, we developed a knockin mouse line which allows ablating genes exclusively in horizontal cells. This knockin line expresses a Cre recombinase under the promoter of connexin57 (Cx57), a gap junction protein only expressed in horizontal cells. Consistently, in Cx57+/Cre mice, Cre recombinase is expressed in almost all horizontal cells (>99%) and no other retinal neurons. To test Cre activity, we crossbred Cx57+/Cre mice with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites (GluA4fl/fl). In GluA4fl/fl:Cx57+/Cre mice, GluA4 immunoreactivity was significantly reduced (∼50%) in the outer retina where horizontal cells receive photoreceptor inputs, confirming the functionality of the Cre/loxP system. Whole-cell patch-clamp recordings from isolated horizontal cell somata showed a reduction of glutamate-induced inward currents by ∼75%, suggesting that the GluA4 subunit plays a major role in mediating photoreceptor inputs. The persistent current in GluA4-deficient cells is mostly driven by AMPA and to a very small extent by kainate receptors as revealed by application of the AMPA receptor antagonist GYKI52466 and concanavalin A, a potentiator of kainate receptor-mediated currents. In summary, the Cx57+/Cre mouse line provides a versatile tool for studying horizontal cell function. GluA4fl/fl:Cx57+/Cre mice, in which horizontal cells receive less excitatory input, can thus be used to analyze the contribution of horizontal cells to retinal processing.
Highlights
Horizontal cells are interneurons in the mammalian retina which receive glutamatergic input from photoreceptors via ionotropic glutamate receptors [1]
The mouse retina only contains a single type of horizontal cell - the axon-bearing B-type [3], which forms axo-axonal and dendro-dendritic networks coupled by the gap junction-forming protein connexin57 (Cx57) [4,5,6]
As described previously [7], we generated a targeting vector pKW-DTR-frt-Cre and replaced part of the Cx57 coding sequence in exon 2 with the frt-flanked coding sequence of DTR-eGFP followed by the coding sequence of Cre recombinase (Fig. 1A)
Summary
Horizontal cells are interneurons in the mammalian retina which receive glutamatergic input from photoreceptors via ionotropic glutamate receptors [1]. To test the suitability of the resulting Cx57-Cre mouse line as a tool for studying horizontal cells, we crossbred it with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites. This led to the deletion of the first two transmembrane regions of the GluA4 subunit [22] exclusively in horizontal cells. Success and specificity of GluA4 ablation were demonstrated using quantitative immunohistochemistry, Western blot, and patch-clamp recordings from isolated horizontal cells
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