Gap junctions, direct cell-cell connections deemed crucial in facilitating the synchronisation and collection of electrical signals, are widely prevalent in the nervous system. In vertebrates, gap junctions are comprised of two adjacent hemichannels, which in turn, are made up of six connexins (Cx). During neural development, connexins and their gap junctions emerge before chemical synapse formation and are thought to be involved in establishing distinct neural circuits. Despite being the most abundant in mouse retinas, it is unclear to what extent Cx45 and Cx36 are involved in retinal circuit creation, or indeed, how much they contribute to this process in human induced pluripotent stem cell derived retinal organoids (ROs). 

To assess whether developmental parallels can be found in both systems, we quantified the expression patterns of Cx45 and Cx36 in mice retina (postnatal day 8-16) (Hilgen et al, 2022) and ROs at varying differentiation stages (days 40, 90, 150 and 200, respectively). In order to do this, we developed a custom software enabling automatic analysis of multiple anatomical parameters (e.g. size, overlap and near-by location) of all channels within a confocal microscopy stack. Our results show that numerous soma-somatic Cx45 and Cx36 gap junctions exist in ROs, and, both connexins are localised on and near synaptic terminals in both tissues (shown by synaptophysin and vesicular glutamate transporter 1 staining). Expression levels of Cx45 and Cx36 in both plexiform layers of the mouse retina increased until eye opening, which then reduced slightly from this stage. Within ROs, expression patterns of Cx45 showed increasing densities at the latter stages of differentiation (number of analysed sections for d20 = 125, d45 = 67, d90 = 133, d>150 = 214), whereas Cx36 expression was less pronounced than in mice (number of analysed sections for d20 = 112, d45 = 21, d90 = 73, d>150 = 119) 

Heterotypic Cx45/Cx36 gap junctions, which can be found in the rod pathway between ON cone bipolar cells and all amacrine cells, were similarly expressed in both mice and ROs. Within mice, the percentage of Cx45/Cx36 gap junctions was found to be higher before eye opening (number of analysed sections for P8 =54, P10 = 50, P12 =51, P16 = 66), whereas, in ROs, it steadily increased (number of analysed sections for d20 = 24, d45 = 54, d90 = 78, d>150 = 216). In addition to this, our multielectrode array recordings from ROs revealed gap-junction-coupled retinal ganglion cells (RGCs) which is consistent with our findings from both connexins between RGCs in mice (not yet quantified). 

In conclusion, our data suggests that both mice and ROs have very comparable Cx45 and Cx36 expression patterns. In addition to this, we have shown for the first time, ROs have both heterotypic Cx45/Cx36 gap junctions, and functional gap junctions between RGCs. Together, these findings imply that both connexins play a pivotal role during the development of both mouse retina and ROs.