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The retinas of many species show regional specialisations that are evident in the differences in the processing of visual input from different parts of the visual field. Regional specialisation is thought to reflect an adaptation to the natural visual environment, optical constraints, and lifestyle of the species. Yet, little is known about regional differences in synaptic circuitry. Here, we were interested in the topographical distribution of connexin-36 (Cx36), the major constituent of electrical synapses in the retina. We compared the retinas of mice, rats, and cats to include species with different patterns of regional specialisations in the analysis. First, we used the density of Prox1-immunoreactive amacrine cells as a marker of any regional specialisation, with higher cell density signifying more central regions. Double-labelling experiments showed that Prox1 is expressed in AII amacrine cells in all three species. Interestingly, large Cx36 plaques were attached to about 8–10% of Prox1-positive amacrine cell somata, suggesting the strong electrical coupling of pairs or small clusters of cell bodies. When analysing the regional changes in the volumetric density of Cx36-immunoreactive plaques, we found a tight correlation with the density of Prox1-expressing amacrine cells in the ON, but not in the OFF sublamina in all three species. The results suggest that the relative contribution of electrical synapses to the ON- and OFF-pathways of the retina changes with retinal location, which may contribute to functional ON/OFF asymmetries across the visual field.

Connexin-36 (Cx36) is the major constituent of mammalian retinal gap junctions positioned in key signal pathways. Here, we examined the laminar and large-scale topographical distribution of Cx36 punctate immunolabels in the retina of the cat, a classical model of the mammalian visual system. Calretinin-immunoreactive (CaR-IR) cell populations served to outline the nuclear and plexiform layers and to stain specific neuronal populations. CaR-IR cells included horizontal cells in the outer retina, numerous amacrine cells, and scattered cells in the ganglion cell layer. Cx36-IR plaques were found among horizontal cell dendrites albeit without systematic colocalization of the two labels. Diffuse Cx36 immunoreactivity was found in the cytoplasm of AII amacrine cells, but no colocalization of Cx36 plaques was observed with either the perikarya or the long varicose dendrites of the CaR-IR non-AII amacrine cells. Cx36 puncta were seen throughout the entire inner plexiform layer showing their highest density in the ON sublamina. The densities of AII amacrine cell bodies and Cx36 plaques in the ON sublamina were strongly correlated across a wide range of eccentricities suggesting their anatomical association. However, the high number of plaques per AII cell suggests that a considerable fraction of Cx36 gap junctions in the ON sublamina is formed by other cell types than AII amacrine cells drawing attention to extensive but less studied electrically coupled networks. Electronic supplementary material The online version of this article (10.1007/s00429-019-01876-y) contains supplementary material, which is available to authorized users.

For decades, a role for the Ca2+-binding protein calmodulin (CaM) in Ca2+-dependent presynaptic modulation of synaptic transmission has been recognized. Here, we investigated the influence of CaM on evoked and spontaneous neurotransmission at rod bipolar (RB) cell→AII amacrine cell synapses in the mouse retina. Our work was motivated by the observations that expression of CaM in RB axon terminals is extremely high and that [Ca2+] in RB terminals normally rises sufficiently to saturate endogenous buffers, making tonic CaM activation likely. Taking advantage of a model in which RBs can be stimulated by expressed channelrhodopsin-2 (ChR2) to avoid dialysis of the presynaptic terminal, we found that inhibition of CaM dramatically decreased evoked release by inhibition of presynaptic Ca channels while at the same time potentiating both Ca2+-dependent and Ca2+-independent spontaneous release. Remarkably, inhibition of myosin light chain kinase (MLCK), but not other CaM-dependent targets, mimicked the effects of CaM inhibition on evoked and spontaneous release. Importantly, initial antagonism of CaM occluded the effect of subsequent inhibition of MLCK on spontaneous release. We conclude that CaM, by acting through MLCK, bidirectionally regulates evoked and spontaneous release at retinal ribbon synapses. Significance Statement Calmodulin (CaM), a Ca2+-binding protein expressed throughout the CNS, functions as a presynaptic modulator of synaptic transmission. We showed that CaM was strongly expressed in the axon terminals of rod bipolar (RB) cells of the mouse retina. Inhibition of CaM decreased evoked release while potentiating spontaneous release at RB→AII amacrine cell synapses; this effect was mimicked by inhibition of myosin light chain kinase (MLCK), a CaM target. Thus, we conclude that CaM, acting through MLCK, bidirectionally regulates evoked and spontaneous release at retinal ribbon synapses.

Dopaminergic amacrine cells (DACs) are among the most well-characterized neurons in the mammalian retina, and their connections to AII amacrine cells have been described in detail. However, the stratification of DAC dendrites differs based on their location in the inner plexiform layer (IPL), raising the question of whether all AII lobules are modulated by dopamine release from DACs. The present study aimed to clarify the relationship between DACs and AII amacrine cells, and to further elucidate the role of dopamine at synapses with AII amacrine cell. In the rabbit retina, DAC dendrites were observed in strata 1, 3, and 5 of the IPL. In stratum 1, most DAC dendritic varicosities—the presumed sites of neurotransmitter release—made contact with the somata and lobular appendages of AII amacrine cells. However, most lobular appendages of AII amacrine cells localized within stratum 2 of the IPL exhibited little contact with DAC varicosities. In addition, double- or triple-labeling experiments revealed that DACs did not express the GABAergic neuronal markers anti-GABA, vesicular GABA transporter, or glutamic acid decarboxylase. These findings suggest that the lobular appendages of AII amacrine cells are involved in at least two different circuits. We speculate that the circuit associated with stratum 1 of the IPL is modulated by DACs, while that associated with stratum 2 is modulated by unknown amacrine cells expressing a different neuroactive substance. Our findings further indicate that DACs in the rabbit retina do not use GABA as a neurotransmitter, in contrast to those in other mammals., Graphical Abstract

Exocytotic release of neurotransmitter can be quantified by electrophysiological recording from postsynaptic neurons. Alternatively, fusion of synaptic vesicles with the cell membrane can be measured as increased capacitance by recording directly from a presynaptic neuron. The “Sine + DC” technique is based on recording from an unbranched cell, represented by an electrically equivalent RC-circuit. It is challenging to extend such measurements to branching neurons where exocytosis occurs at a distance from a somatic recording electrode. The AII amacrine is an important inhibitory interneuron of the mammalian retina and there is evidence that exocytosis at presynaptic lobular dendrites increases the capacitance. Here, we combined electrophysiological recording and computer simulations with realistic compartmental models to explore capacitance measurements of rat AII amacrine cells. First, we verified the ability of the “Sine + DC” technique to detect depolarizationevoked exocytosis in physiological recordings. Next, we used compartmental modeling to demonstrate that capacitance measurements can detect increased membrane surface area at lobular dendrites. However, the accuracy declines for lobular dendrites located further from the soma due to frequency-dependent signal attenuation. For sine wave frequencies ≥1 kHz, the magnitude of the total releasable pool of synaptic vesicles will be significantly underestimated. Reducing the sine wave frequency increases overall accuracy, but when the frequency is sufficiently low that exocytosis can be detected with high accuracy from all lobular dendrites (~100 Hz), strong electrical coupling between AII amacrines compromises the measurements. These results need to be taken into account in studies with capacitance measurements from these and other electrically coupled neurons. publishedVersion

It has been shown in rd1 and rd10 models of photoreceptor degeneration (PD) that inner retinal neurons display spontaneous and rhythmic activities. Furthermore, the rhythmic activity has been shown to require the gap junction protein connexin 36, which is likely located in AII amacrine cells (AII-ACs). In the present study, an autosomal dominant PD model called rhoΔCTA, whose rods overexpress a C-terminally truncated mutant rhodopsin and degenerate with a rate similar to that of rd1, was used to investigate the generality and mechanisms of heightened inner retinal activity following PD. To fluorescently identify cholinergic starburst amacrine cells (SACs), the rhoΔCTA mouse was introduced into a combined ChAT-IRES-Cre and Ai9 background. In this mouse, we observed large amplitude excitatory postsynaptic currents (EPSCs) oscillations and non-rhythmic inhibitory postsynaptic currents (IPSCs) in both ON- and OFF-SACs. The IPSCs were more noticeable in OFF- than in ON-SACs. Similar to reported retinal ganglion cell (RGC) oscillation in rd1 mice, EPSC oscillation was synaptically driven by glutamate and sensitive to blockade of NaV channels and gap junctions. These data suggest that akin to rd1 mice, AII-AC is a prominent oscillator in rhoΔCTA mice. Surprisingly, OFF-SAC but not ON-SAC EPSC oscillation could readily be enhanced by GABAergic blockade. More importantly, weakening the AII-AC gap junction network by activating retinal dopamine receptors abolished oscillations in ON-SACs but not in OFF-SACs. Furthermore, the latter persisted in the presence of flupirtine, an M-type potassium channel activator recently reported to dampen intrinsic AII-AC bursting. These data suggest the existence of a novel oscillation mechanism in mice with PD.