Morphological properties of the axon initial segment‐like process ofAIIamacrine cells in the rat retina

Abstract

Signal processing within the retina is generally mediated by graded potentials, whereas output is conveyed by action potentials transmitted along optic nerve axons. Among retinal neurons, amacrine cells seem to be an exception to this general rule, as several types generate voltage-gated Na + (Na v ) channel-dependent action potentials. The AII, a narrow-field, bistratified axon-less amacrine cell found in mammalian retinas, displays a unique process that resembles an axon initial segment (AIS), with expression of Na v channels colocalized with the cytoskeletal protein ankyrin-G, and generates action potentials. As the role of spiking in AIIs is uncertain, we hypothesized that the morphological properties of the AIS-like process could provide information relevant for its functional importance, including potential pre-and/or postsynaptic connectivity. For morphological analysis, we injected AII amacrine cells in slices with fluorescent dye and immunolabeled the slices for ankyrin-G. Subsequently, this enabled us to reliably identify AII-type processes among ankyrin-Glabeled processes in wholemount retina. We systematically analyzed the laminar localization, spatial orientation, and distribution of the AIS-like processes as a function of retinal eccentricity. In the horizontal plane, the processes displayed no preferred orientation and terminal endings were randomly distributed. In the vertical plane, the processes displayed a horizontal preference, but also ascended and descended into the inner nuclear layer and proximal inner plexiform layer, respectively. These results suggest that the AII amacrine AIS-like process is unlikely to take part in conventional synaptic connections, but may instead be adapted to respond to volume neurotransmission by means of extrasynaptic receptors.

The mosaic of AII amacrine cell bodies in rat retina is indistinguishable from a random distribution

“…The peak density observed for the two retinas in our study (~5200 and ~4900 cells/mm 2 ) is somewhat lower than the peak densities reported for the two retinas analyzed by Wässle et al (1993; 7000 and 7500 cells/mm 2 , respectively). As suggested in a previous study from our laboratory (Liu et al, 2021), the difference could be due to the exact locations sampled, the extent of shrinkage, and age differences. Importantly, whereas the spatial density varies as a function of retinal region and eccentricity, with reduced density toward the periphery, the number of nearest neighbors stays essentially constant across the retina.…”

“…The characteristic thick apical dendrites of AII amacrine cells that descend vertically into the inner plexiform layer cannot be seen in the horizontal images and slabs illustrated in Fig. 1 , but were displayed in Liu et al ( 2021 ). The smaller and even more weakly labeled structures between the labeled cell bodies in Fig.…”

“…The retina was then incubated for four nights (at 4°C) with primary antibody (guinea pig anti-parvalbumin, diluted 1:1000) in antibody incubation solution (with 0.2% Triton X-100). Both retinas were also immunolabeled with an antibody against ankyrin-G, but this was reported in Liu et al ( 2021 ) and will not be further commented on here. After incubation, the retina was washed six times (10 min each) in PBS and incubated overnight (at 4°C) with secondary antibodies diluted 1:1000 in antibody incubation solution (with 0.2% Triton X-100).…”

“…For each image stack, we estimated an optimal value for the user-selectable SNR parameter by repeating the deconvolution for several values of the SNR while keeping all other parameters and settings constant (see Zandt et al, 2017 ). For additional details, see Liu et al ( 2021 ).…”

“…Second, the cell bodies of the widefield amacrines are located slightly more distally in the retina (i.e., closer to the outer plexiform layer) compared to AII amacrines. Third, in contrast to the AIIs which have a thick (parvalbumin-positive) apical dendrite that extends vertically into the inner plexiform layer, the cell bodies of the widefield amacrine cells display a number of (parvalbumin-positive) processes that run laterally for some distance before they descend toward stratum 5 (S5) of the inner plexiform layer (for a detailed description, see Liu et al, 2021 ).…”