API, not shown) are circled with dotted lines. Arrowheads point to ribbon synapses, arrows demarcate conventional chemical synapses. Scale bar inside a,B: ten mm, C: 5 mm. doi:10.1371/journal.pone.0070373.gsynaptic layers, i.e. the outer (OPL) along with the inner (IPL) plexiform layer (Fig. 1B). In the OPL, the appearance of your photoreceptor ribbon synapses did not differ between wt and Pclo-mutant retina. Within the IPL, the amount of discrete Pclo puncta, representingindividual synapses, was apparently lowered within the Pclo-mutant retina (Fig. 1B). This indicates that, when within the brain with the Pclomutant mouse the majority of Pclo is lost from synapses [18], within the retina only a subset of synapses, primarily inside the IPL, is affected.PLOS 1 | plosone.orgPiccolino at Sensory Ribbon SynapsesFigure four. Localization of full-length Pclo at distinct forms of ribbon synapses. A: Wild-type (+/+) and Pclo-mutant (2/2) retinae stained together with the C-terminally binding Pclo six against full-length Pclo. B: Inner plexiform layer (IPL) of +/+ retina double labeled for full-length Pclo (Pclo six; green) and CtBP2/RIBEYE (magenta). C : Pre-embedding immunoelectron micrographs of a rod photoreceptor (C), cone photoreceptor (D), and rod bipolar cell (rbc) ribbon synapse (E) in the +/+ retina stained with Pclo six. Only amacrine cell synapses (E; asterisk) and in no way ribbon synapses (C ; arrowheads) were stained for full-length Pclo. F: Inner hair cells (ihc) double labeled for full-length Pclo (Pclo 6; green) and CtBP2/RIBEYE (magenta). Nuclei (stained with DAPI, not shown) are circled with dotted lines. Arrowheads point to ribbon synapses, arrows demarcate conventional chemical synapses. ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; GCL: ganglion cell layer. hc: horizontal cell; bc: bipolar cell; ac: amacrine cell. Scale bar within a,B: 20 mm, C-E: 200 nm, F: 5 mm. doi:10.1371/journal.pone.0070373.gAs the majority of chemical synapses inside the OPL are photoreceptor ribbon synapses, Pclo seems to be present at these synapses in the Pclo-mutant retina. To confirm this, we double labeled photoreceptor ribbons with all the Pclo44a antibody (Fig. 1C; green) and an antibody against RIBEYE (Fig. 1C; magenta) demonstrating a full co-localization of the two proteins within the OPL on the Pclo-mutant retina (Fig. 1C; merge). In the IPL of wt retina, co-localized Pclo/RIBEYE puncta representing bipolar cell ribbon synapses, and single Pclo puncta representing traditional amacrine cell synapses [16], could be observed (Fig. 1D). In the IPL of Pclo-mutant retina, single Pclo puncta have been missing, and only Pclo/RIBEYE puncta were detectable (Fig.1630815-44-9 Formula 1D).1257850-83-1 web Extra electron microscopical analysis of photoreceptor and bipolar cellribbon synapses revealed no structural differences in between wt and Pclo-mutant retinae (Fig.PMID:23715856 1E ). These data strongly suggest that photoreceptor and bipolar cell ribbon synapses express a Pclo variant that is not impacted by the exon-14 deletion and as a result differs in the Pclo protein described for the brain [23?5]. In agreement together with the previously published molecular weight for Pclo, the antibody Pclo 44a detected 1 prominent band of .500 kDa on Western blots of wt mouse cortex synaptosomal (P2) fractions (Fig. 1H; lane 1). In P2 fractions of wt retina, Pclo 44a labeling revealed an extra prominent band of ,350 kDa, which can be absent from cortex (Fig. 1H; lanes 1+3; see also [9]). In agreement with the reported loss of Pclo at standard syna.