In many parts of the central nervous system, including the retina, it is unclear whether cholinergic transmission is mediated by rapid, point-to-point synaptic mechanisms, or slower, broad-scale ‘non-synaptic’ mechanisms. Here, we characterized the ultrastructural features of cholinergic connections between direction-selective starburst amacrine cells and downstream ganglion cells in an existing serial electron microscopy data set, as well as their functional properties using electrophysiology and two-photon acetylcholine (ACh) imaging. Correlative results demonstrate that a ‘tripartite’ structure facilitates a ‘multi-directed’ form of transmission, in which ACh released from a single vesicle rapidly (~1 ms) co-activates receptors expressed in multiple neurons located within ~1 µm of the release site. Cholinergic signals are direction-selective at a local, but not global scale, and facilitate the transfer of information from starburst to ganglion cell dendrites. These results suggest a distinct operational framework for cholinergic signaling that bears the hallmarks of synaptic and non-synaptic forms of transmission.

Much of the computational power of the retina derives from the activity of amacrine cells, a large and diverse group of GABAergic and glycinergic inhibitory interneurons. Here, we identify an ON-type orientation-selective, wide-field, polyaxonal amacrine cell (PAC) in the rabbit retina and demonstrate how its orientation selectivity arises from the structure of the dendritic arbor and the pattern of excitatory and inhibitory inputs. Excitation from ON bipolar cells and inhibition arising from the OFF pathway converge to generate a quasi-linear integration of visual signals in the receptive field center. This serves to suppress responses to high spatial frequencies, thereby improving sensitivity to larger objects and enhancing orientation selectivity. Inhibition also regulates the magnitude and time course of excitatory inputs to this PAC through serial inhibitory connections onto the presynaptic terminals of ON bipolar cells. This presynaptic inhibition is driven by graded potentials within local microcircuits, similar in extent to the size of single bipolar cell receptive fields. Additional presynaptic inhibition is generated by spiking amacrine cells on a larger spatial scale covering several hundred microns. The orientation selectivity of this PAC may be a substrate for the inhibition that mediates orientation selectivity in some types of ganglion cells.

Recent studies indicate that the precise timing and location of excitation and inhibition (E/I) within active dendritic trees can significantly impact neuronal function. How synaptic inputs are functionally organized at the subcellular level in intact circuits remains unclear. To address this issue, we took advantage of the retinal direction-selective ganglion cell circuit, where directionally tuned inhibition is known to shape non-directional excitatory signals. We combined two-photon calcium imaging with genetic, pharmacological, and single-cell ablation methods to examine the extent to which inhibition ‘vetoes’ excitation at the level of individual dendrites of direction-selective ganglion cells. We demonstrate that inhibition shapes direction selectivity independently within small dendritic segments (<10µm) with remarkable accuracy. The data suggest that the parallel processing schemes proposed for direction encoding could be more fine-grained than previously envisioned.

The asymmetric summation of kinetically distinct glutamate inputs across the dendrites of retinal 'starburst' amacrine cells is one of the several mechanisms that have been proposed to underlie their direction-selective properties, but experimentally verifying input kinetics has been a challenge. Here, we used two-photon glutamate sensor (iGluSnFR) imaging to directly measure the input kinetics across individual starburst dendrites. We found that signals measured from proximal dendrites were relatively sustained compared to those measured from distal dendrites. These differences were observed across a range of stimulus sizes and appeared to be shaped mainly by excitatory rather than inhibitory network interactions. Temporal deconvolution analysis suggests that the steady-state vesicle release rate was ~ 3 times larger at proximal sites compared to distal sites. Using a connectomics-inspired computational model, we demonstrate that input kinetics play an important role in shaping direction selectivity at low stimulus velocities. Together, these results provide direct support for the 'space-time wiring' model for direction selectivity.

Motion sensitivity requires the comparison of neural responses activated by nearby points in visual space. In this issue of Neuron, Manookin et al. (2018) find that in the primate retina, such comparisons are already manifest in second-order retinal bipolar cells, relying on lateral excitation mediated by gap junctions.