Synaptic inputs to broad thorny ganglion cells in macaque retina

Abstract

In primates, broad thorny retinal ganglion cells are highly sensitive to small, moving stimuli. They have tortuous, fine dendrites with many short, spine-like branches that occupy three contiguous strata in the middle of the inner plexiform layer. The neural circuits that generate their responses to moving stimuli are not well-understood, and that was the goal of this study. A connectome from central macaque retina was generated by serial block-face scanning electron microscopy, a broad thorny cell was reconstructed, and its synaptic inputs were analyzed. It received fewer than 2% of its inputs from both ON and OFF types of bipolar cells; the vast majority of its inputs were from amacrine cells. The presynaptic amacrine cells were reconstructed, and seven types were identified based on their characteristic morphology. Two types of narrow-field cells, knotty bistratified Type 1 and wavy multistratified Type 2, were identified. Two types of medium-field amacrine cells, ON starburst and spiny, were also presynaptic to the broad thorny cell. Three types of wide-field amacrine cells, wiry Type 2, stellate wavy, and semilunar Type 2, also made synapses onto the broad thorny cell. Physiological experiments using a macaque retinal preparation in vitro confirmed that broad thorny cells received robust excitatory input from both the ON and the OFF pathways. Given the paucity of bipolar cell inputs, it is likely that amacrine cells provided much of the excitatory input, in addition to inhibitory input.

Keywords: RRID: SCR_001622; RRID: SCR_017350; RRID:SCR_003584; RRID:SCR_005986; connectomics; electron microscopy; interneuron; motion sensitivity; primate; vision.

FIGURE 1

Broad thorny ganglion cell 103 and the approximate boundaries of the connectome (white) are shown as they would appear in a flat mount preparation viewed from the scleral side of the retina. The cell is colored according to depth in the inner plexiform layer (IPL), with red as the inner nuclear layer and violet as the ganglion cell layer; the dendrites stratify broadly in the three middle strata. Some dendrites on the left were truncated when they reached the edge of the connectome. Note the varicose axon at the lower left (arrowhead). Scale bar = 100 μm [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

Two bipolar cell axon terminals (green) make ribbon synapses (arrowheads) onto dendrites of broad thorny ganglion cell 103 (yellow). The synapses are shown at low (a) and somewhat higher (b) magnification. In this and in all subsequent figures, the markers are located in the presynaptic cells. Synapses like these provided less than 2% of the input to this ganglion cell. Scale bars = 500 nm [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 3

It was common to observe two (a) or three (b) amacrine cell dendrites (orange) converging onto spines of ganglion cell 103 (yellow) and making synapses (arrowheads). Scale bar = 500 nm [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 4

(a) Distribution of synapses from bipolar cell axons (green) onto broad thorny ganglion cell 103. Inputs from ON bipolar cells (light green) are more numerous than those from OFF bipolar cells (dark green). Note that only 15 of more than 1000 synapses originated from bipolar cells and that the distribution appeared to be random. (b) Inputs from amacrine cell axons and dendrites (red) were far more common. The box indicates the area shown at higher magnification in panel (c) Note that there are four clusters of synapses (arrowheads) onto cell 103. Inputs from wide-field amacrine cells are shown in panel (d). These also appear to be randomly distributed. Panels a, b, and d scale bars = 50 μm; panel c scale bar = 5 μm [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 5

Bipolar cell inputs to broad thorny ganglion cell 103 as they would appear in a flat mount preparation. Thirteen bipolar cells made one synapse each, and one, a giant bipolar cell, made two synapses. The depth code in this and subsequent figures is the same as in Figure 1. Scale bar = 100 μm [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 6

Representative presynaptic bipolar cells as they would appear in vertical sections. Synapses onto cell 103 are indicated with white circles. (a) Diffuse bipolar cell 40761 ramifying at 60–70% depth. (b) Diffuse bipolar cell 41145 ramifying at 35–45%. (c) Diffuse bipolar cell 42040 ramifying at 90–100% depth, (d). Diffuse bipolar cell 42569 ramifying at 70–80%, (e) giant bipolar cell 30586 axon terminal ramifying at 50–60% depth. Scale bar = 10 μm [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 7

Knotty bistratified Type 1 amacrine cell 43016 was one type of narrow-field amacrine cell presynaptic to broad thorny ganglion cell 103 (white circle). It is shown as it would appear in flat mount and in a vertical section. Note that the soma is in the inner nuclear layer and that the dendrites ramify in two distinct strata near the center of the inner plexiform layer (IPL). The inset color scheme in this and subsequent figures is the same as in Figure 3. Arrowheads in the electron micrographs represent synaptic specializations in this and subsequent figures. Scale bars = 10 μm; inset scale bar = 500 nm [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 8

Wavy multistratified Type 2 amacrine cell 40879 was another type of narrow-field amacrine cell presynaptic to broad thorny ganglion cell 103. It is shown as it would appear in flat mount and in a vertical section. It made five synapses onto cell 103. The soma was in the inner nuclear layer, and the dendritic arbor was larger in diameter than that of the knotty bistratified cell illustrated in Figure 7. Scale bars = 10 μm; inset scale bar = 500 nm [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 9

Spiny amacrine cell 40654 was a medium-field amacrine cell that made five synapses (white circles) onto broad thorny ganglion cell 103. Note that the soma is in the inner nuclear layer and the dendrites have small spines and mainly ramify in the center of the inner plexiform layer (IPL). Scale bar = 10 μm; inset scale bar = 500 nm [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 10

ON starburst amacrine cell 3111 was another medium-field amacrine cell presynaptic to ganglion cell 103. Although it was not completely reconstructed, it was classified based on its thin, tortuous dendrites ramifying at 65–70% depth in the inner plexiform layer (IPL) that were enlarged at their distal ends. Note that the soma is located in the ganglion cell layer. Scale bar = 10 μm; inset scale bar = 500 nm [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 11

Wiry Type 2 amacrine cell 182 was a wide-field amacrine cell that made four synapses (white circles) onto broad thorny ganglion cell 103. Note that the soma was located in the ganglion cell layer and that the long, unbranched distal dendrites ramified at 60% depth in the inner plexiform layer (IPL). Scale bar = 10 μm; inset scale bar = 500 nm [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 12

Semilunar Type 2 amacrine cell 4781 was another wide-field amacrine cell presynaptic to broad thorny ganglion cell 103. The soma was located in the inner plexiform layer (IPL) and the dendrites were larger in diameter than those of the wiry Type 2 cells illustrated in Figure 11. Thinner axons (arrowheads) arose from both the soma and the distal tips of the dendrites. Scale bar = 10 μm; inset scale bar = 500 nm [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 13

Stellate wavy amacrine cell 4308 was a third type of wide-field amacrine cell presynaptic to ganglion cell 103. It made three synapses (white circles). The soma was located in the ganglion cell layer, and the dendrites were intermediate in diameter between those of wiry and semilunar cells. Thinner axons (arrowheads) arose from secondary and distal dendrites, and both types of processes ramified between 20% and 30% depth in the inner plexiform layer (IPL), on average. Scale bar = 10 μm; inset scale bar = 500 nm [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 14

OFF responses of broad thorny ganglion cells do not depend on the ON pathway. (a) Whole-cell voltage-clamp responses of a broad thorny cell in control conditions at two different holding potentials to reveal excitatory and inhibitory synaptic currents. The stimulus is indicated above the recording traces. (b) Recording of the cell as above but during application of L-AP4 (5 μM) and LY341495 (7.5 μM) to block the ON pathway [Color figure can be viewed at wileyonlinelibrary.com]