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Abstract: Pannexins (Panx) constitute a new family of gap junction type proteins. Functional expression in paired Xenopus oocytes indicated that pannexins are capable of forming communicating junctions but also proved to be active in forming of unapposed hemichannels. In the vertebrate brain pannexins have been found in neurons. However, the subcellular cerebral localization of pannexin proteins which could gain first clues on their putative function is essentially unknown. Here we demonstrate by light and electron microscopical immunohistochemistry that Panx1 reveals postsynaptic localization in rodent hippocampal and cortical principal neurons accumulating at postsynaptic densities. The postsynaptic localization was corroborated by co-localization of Panx1 with postsynaptic density protein 95 (PSD-95), a prominent postsynaptic scaffolding protein, in hippocampal neurons expressing tagged versions of these proteins. The asymmetric synaptic distribution of Panx1 suggests that it may function in neurons as non-junctional channels (pannexons) at postsynaptic sites and comprises a novel component of the postsynaptic protein complex. [Copyright &y& Elsevier]

Highlights • Zebrafish Cx52.6 is alternatively spliced in the retina. • The spliced isoform (Cx53.4) is expressed in all subtypes of horizontal cells. • Cx53.4 forms dendro-dendritic and axo-axonal gap junctions between zebrafish horizontal cells. Abstract Retinal horizontal cells (HCs) are inhibitory neurons, which modulate the transmission of light-elicited signals from photoreceptors to bipolar cells in the outer retina. HCs of the same physiological type are extensively coupled via gap junctions. In the zebrafish retina, the population of HCs comprises up to four morphologically distinct subtypes. Four different connexins (Cx52.6, Cx52.7, Cx52.9 and Cx55.5) were detected in these cells with overlapping expression patterns. In this study, we show that Cx52.6 is alternatively spliced in the retina, resulting in an additional isoform, designated as Cx53.4, which differs from the originally described Cx52.6 only by the final C-terminal peptide (12 vs. 4 aa). Further protein sequence alignments revealed that Cx53.4 represents the counterpart of alternatively spliced mouse Cx57 and human Cx62. RT-PCR analyses of mRNA expression in different adult zebrafish tissues showed that Cx53.4 is expressed exclusively in the retina. The localization of Cx53.4 protein within the retina was analyzed using a specific antibody. Immunofluorescence analyses demonstrated that the expression of Cx53.4 is restricted to HCs of all four subtypes. Further, immunoelectron microscopy confirmed the presence of Cx53.4 in gap junctions between HC dendrites and between their axon terminals. [ABSTRACT FROM AUTHOR]

In the vertebrate retina, horizontal cells (HCs) reveal homologous coupling by gap junctions (gj), which are thought to consist of different connexins (Cx). However, recent studies in mouse, rabbit and zebrafish retina indicate that individual HCs express more than one connexin. To provide further insights into the composition of gj connecting HCs and to determine whether HCs express multiple connexins, we examined the molecular identity and distribution of gj between HCs of the carp retina. We have cloned four carp connexins designated Cx49.5, Cx55.5, Cx52.6 and Cx53.8 with a close relationship to connexins previously reported in HCs of mouse, rabbit and zebrafish, respectively. Using in situ hybridization, Cx49.5 expression was detected in different subpopulations of retinal neurons including HCs, whereas the Cx52.6 transcript was localized exclusively in HCs. Using specific antibodies, Cx55.5 and Cx53.8 were detected on dendrites of all four HC subtypes and axon terminals. Immunoelectron microscopy confirmed the presence of Cx55.5 and Cx53.8 in gap junctions between these processes and Cx55.5 was additionally observed in HC dendrites invaginating cone pedicles, suggesting its participation in the modulation of photoreceptor output in the carp retina. Furthermore, using single-cell RT–PCR, all four connexins were detected in different subtypes of HCs, suggesting overlapping expression patterns. Thus, the composition of gj mediating homologous coupling between subtypes of carp HCs appears to be more complex than expected. Moreover, BLAST searches of the preliminary carp genome, using novel sequences as query, suggest that most of the analyzed connexin genes are duplicated in carp. [ABSTRACT FROM AUTHOR]

Abstract: In the retina, chemical and electrical synapses couple neurons into functional networks. New candidates encoding for electrical synapse proteins have recently emerged. In the present study, we determined the localization of the candidate protein pannexin1 (zfPanx1) in the zebrafish retina and studied the functional properties of zfPanx1 exogenously expressed in Neuroblastoma 2a (N2a) cells. zfPanx1 was identified on the surface of horizontal cell dendrites invaginating deeply into the cone pedicle near the glutamate release sites of the cones, providing in vivo evidence for hemichannel formation at that location. This strategic position of zfPanx1 in the photoreceptor synapse could potentially allow modulation of cone output. Using whole cell voltage clamp and excised patch recordings of transfected N2a cells, we demonstrated that zfPanx1 forms voltage-activated hemichannels with a large unitary conductance in vitro. These channels can open at physiological membrane potentials. Functional channels were not formed following mutation of a single amino acid within a conserved protein motif recently shown to be N-glycosylated in rodent Panx1. Together, these findings indicate that zfPanx1 displays properties similar to its mammalian homologues and can potentially play an important role in functions of the outer retina. [Copyright &y& Elsevier]

Abstract: Receptor protein tyrosine phosphatases (RPTPs) appear to coordinate many aspects of neural development, including cell proliferation, migration and differentiation. Here we investigated potential roles of RPTPs in the developing mouse retina. Using a degenerate oligonucleotide-based reverse transcription polymerase chain reaction approach, we identified 11 different RPTPs in the retina at embryonic stage 13 (E13). Subsequently, the expression patterns of RPTPκ, RPTPJ, RPTPRR, RPTPσ, RPTPε and RPTPγ in the retina from embryonic stages to adult were analyzed in detail using quantitative real-time-PCR, in situ hybridization, immunohistochemistry and Western blotting. At E13, all six RPTPs are expressed in actively cycling retinal progenitor cells and postmitotic newborn retinal neurons. With ongoing maturation, RPTPκ, RPTPJ, RPTPRR, RPTPσ, RPTPε and RPTPγ display a different spatiotemporal regulation of mRNAs and proteins in the pre- and postnatal retina. Finally, in adulthood these six RPTPs localize to distinct cellular compartments of multiple retinal neurons. Additional studies in RPTPγ−/− and RPTPβ/ζ−/− (also known as PTPRZ1, RPTPβ or RPTPζ) mice at postnatal stage P1 reveal no apparent differences in retinal laminar organization or in the expression pattern of specific retinal cell-type markers when compared with wild type. However, in RPTPβ/ζ−/− retinas, immunoreactivity of vimentin, a marker of Müller glial cells, is selectively reduced and the morphology of vimentin-immunoreactive radial processes of Müller cells is considerably disturbed. Our results suggest distinct roles of RPTPs in cell proliferation and establishing phenotypes of different retinal cells during retinogenesis as well as later in the maintenance of mature retina. [Copyright &y& Elsevier]