Expression of connexin36 in cone pedicles and OFF-cone bipolar cells of the mouse retina.
Transgenic technology, immunocytochemistry, electrophysiology, intracellular injection techniques, and reverse transcription PCR were combined to study the expression of neuronal connexin36 (Cx36) in the outer plexiform layer of the mouse retina. Transgenic animals expressed either a fusion protein of full-length Cx36 with enhanced green fluorescent protein (EGFP) attached at the C terminus or exon 2 of Cx36 was replaced bybeta-galactosidase (beta-gal). In the outer nuclear layer,beta-gal-positive cell bodies, which were confined to the most distal region close to the outer limiting membrane, displayed immunoreactivity against S-cone opsin. Cx36-EGFP puncta colocalized with cone pedicles, which were visualized by intracellular injection. In reverse transcriptase PCR experiments, Cx36 mRNA was never detected in samples of rods harvested from the outer nuclear layer. These results strongly suggest expression of Cx36 in cones but not in rods. In vertical sections, Cx36 expression in the vitreal part of the outer plexiform layer was characterized by a patchy distribution. Immunocytochemistry with antibodies against the neurokinin-3 receptor and the potassium channel HCN4 (hyperpolarization-activated cyclic nucleotide-gated potassium channel) displayed clusters of the Cx36 label on the dendrites of OFF-cone bipolar cells. In horizontal sections, these clusters of Cx36 appeared as round or oval-shaped groups of individual puncta, and they were always aligned with the base of cone pedicles. Double-labeling experiments and single-cell reverse transcriptase PCR ruled out expression of Cx36 in horizontal cells and rod bipolar cells. At light microscopic resolution, we found close association of Cx36-EGFP with the AMPA-type glutamate receptor subunit GluR1 but not with GluR2-GluR4, the kainate receptor subunit GluR5, or the metabotropic glutamate receptor mGluR6.
Feigenspan
Andreas
A
Institute of Biology, University of Oldenburg, D-26111 Oldenburg, Germany. andreas.feigenspan@uni-oldenburg.de
Janssen-Bienhold
Ulrike
U
Hormuzdi
Sheriar
S
Monyer
Hannah
H
Degen
Joachim
J
Söhl
Goran
G
Willecke
Klaus
K
Ammermüller
Josef
J
Weiler
Reto
R
D000818:Animals; D017630:Connexins / Q000096:biosynthesis* / Q000235:genetics; D004594:Electrophysiology; D004596:Electroretinography; D015870:Gene Expression / Q000502:physiology; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections; D023421:Models, Animal; D009474:Neurons / Q000378:metabolism*; D015221:Potassium Channels / Q000096:biosynthesis; D021122:Protein Subunits / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018091:Receptors, AMPA / Q000378:metabolism; D017470:Receptors, Glutamate / Q000378:metabolism; D018042:Receptors, Neurokinin-3 / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013569:Synapses / Q000378:metabolism; D000097004:Gap Junction delta-2 Protein
D017630:Connexins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D015221:Potassium Channels; D021122:Protein Subunits; D012333:RNA, Messenger; D018091:Receptors, AMPA; D017470:Receptors, Glutamate; D018042:Receptors, Neurokinin-3; D011993:Recombinant Fusion Proteins; D000097004:Gap Junction delta-2 Protein