ON cone bipolar cell axonal synapses in the OFF inner plexiform layer of the rabbit retina

Abstract

Analysis of the rabbit retinal connectome RC1 reveals that the division between the ON and the OFF inner plexiform layer (IPL) is not structurally absolute. ON cone bipolar cells make noncanonical axonal synapses onto specific targets and receive amacrine cell synapses in the nominal OFF layer, creating novel motifs, including inhibitory crossover networks. Automated transmission electron microscopic imaging, molecular tagging, tracing, and rendering of ~400 bipolar cells reveals axonal ribbons in 36% of ON cone bipolar cells, throughout the OFF IPL. The targets include γ-aminobutyrate (GABA)-positive amacrine cells (γACs), glycine-positive amacrine cells (GACs), and ganglion cells. Most ON cone bipolar cell axonal contacts target GACs driven by OFF cone bipolar cells, forming new architectures for generating ON-OFF amacrine cells. Many of these ON-OFF GACs target ON cone bipolar cell axons, ON γACs, and/or ON-OFF ganglion cells, representing widespread mechanisms for OFF to ON crossover inhibition. Other targets include OFF γACs presynaptic to OFF bipolar cells, forming γAC-mediated crossover motifs. ON cone bipolar cell axonal ribbons drive bistratified ON-OFF ganglion cells in the OFF layer and provide ON drive to polarity-appropriate targets such as bistratified diving ganglion cells (bsdGCs). The targeting precision of ON cone bipolar cell axonal synapses shows that this drive incidence is necessarily a joint distribution of cone bipolar cell axonal frequency and target cell trajectories through a given volume of the OFF layer. Such joint distribution sampling is likely common when targets are sparser than sources and when sources are coupled, as are ON cone bipolar cells.

Figure 1. RC1 overview

A. The RC1 volume with its top section (001) beginning in mid-INL and ending in the GCL at section 371, shown in a mirror image below. RC1 is a short cylinder ≈ 250 μm in diameter and ≈ 30 μm high containing 341 TEM sections and 11 intercalated CMP sections. The cylinder is capped at top and bottom with 10-section CMP series allowing molecular segmentation. TEM section 001 is a near-horizontal plane section through the INL visualized with GABA.glycine.glutamate → red.green.blue transparency mapping and a dark gold alpha channel (ANDed taurine + glutamine channels) described in Anderson et al. 2011a. Similarly TEM section 371 is a near-horizontal plane section through the GCL visualized with GABA.AGB.glutamate → red.green.blue transparency mapping. B. Representative cells contained in RC1 are rendered in 3D onto the volume. Many complete copies of small cells exist (tens to hundreds) such as rod bipolar cells (cells 1,2) and A_II ACs (cell 3). A few semi-complete copies (5-10) of medium-diameter cell classes have their somas and much of their arbors within RC1, but extend outside it, such as interstitial γACs (cell 4) and A_I amacrine cells (cell 5). Finally, RC1 contains many processes from partial cells: large cells such as wide-field amacrine cells or OFF α ganglion cells (cell 6) with somas outside the volume and often fully traversing it.

Figure 2. A subset of ON cone bipolar cells make en passant & branched axonal ribbons

A. Vertically oriented renderings of 53 CBbs (neutral and warm colors) with axonal ribbons in the OFF IPL plotted against 48 CBas (cool colors). Cone bipolar cell color corresponds to depth of IPL stratification as follows: CBa1, sage; CBa2, green; CBb3, tan; CBb3-4, dark mustard; CBb4, silver; CBb5, mustard; CBb6, bright red; wide-field cone bipolar cell, deep red. Arrows, somas of CBbs referenced in B-P. Scale bar, 25 μm. B-K. CBbs indicated in A are confirmed as glycine-positive (B-F, TEM of CBb somas; G-K, glycine-positive labeling of corresponding somas in B-F). Scale bars, 5 μm. L-P. TEM of gap junctions between CBbs indicated in A and A_II ACs. White arrows delineate gap junctions; A-II, A_II amacrine cell; WF BC, wide field bipolar cell; scale bars, 0.5 μm. A, B, G, L. CBb3 1637 rendering (A), TEM of soma (B), corresponding glycine-positive signature (G), and indirect A_II AC coupling via a gap junction with CBb3-4 1724 (L, left subpanel) which is couple to A_II AC 514 (L, right subpanel). A, C, H, M. CBb4 593 rendering (A), TEM of soma (C), corresponding glycine-positive signature (H), and gap junction with A_II AC 3679 (M). A, D, I, N. CBb5w 6156 rendering (A), TEM of soma (D), corresponding glycine-positive signature (I), and gap junction with A_II AC 476 (N). A, E, J, O. CBb6 4570 rendering (A), TEM of soma (E), corresponding glycine-positive signature (J), and gap junction with A_II AC 3257 (O). A, F, K, P. Wide-field cone bipolar cell 5283 rendering (A), TEM of soma (F), corresponding glycine-positive signature (K), and gap junction with A_II AC 3679 (P). Q. CBb4 485 (silver) and CBb5w 180 (copper) form en passant axonal ribbon synapses (circles) among CBa1 and CBa2 arbors. Scale bar, 5 μm. R. Wide-field cone bipolar cell 16026 (red) forms branched axonal ribbon synapses (circle) among CBa1 and CBa2 arbors. Scale bar, 5 μm.

Figure 3. All major classes of ON cone bipolar cells possess axonal ribbons, stereogram

The five CBbs highlighted in figure 2 are displayed in isolation for clarity. Varied numbers of axonal ribbons across CBb classes span the IPL. Cone bipolar cell color corresponds to depth of IPL stratification. Specific cone bipolar cell colors as follows: CBb3, tan; CBb4, silver; CBb5w, copper; CBb6, bright red (left); wide-field cone bipolar cell, deep red (right). Note the class-specific arborization thickness, pattern of varicosities, and axonal arbor diameters. Spatial relationships are preserved. Scale bar,10 μm.

Figure 4. CBb versus rod bipolar cell axonal ribbon depths

The distribution of 160 axonal ribbons in 54 CBbs and 63 ribbons in 63 of 104 rod bipolar cells in RC1. Ribbon positions are measured relative to the sublamina a/b border, defined as the proximal face of the nearest A_II amacrine cell lobule. CBb axonal ribbons are distributed throughout sublamina a. Rod bipolar cell axonal ribbons are excluded from 80% of sublamina a. ACL, amacrine cell layer; Rod BC, rod bipolar cell.

Figure 5. Rod bipolar cell axonal ribbons cannot drive M1 ipRGCs

All 63 rod bipolar cells (ghosts) with axonal ribbons in RC1 are displayed against ipRGC 12208 (sand). Note that all ribbon synapses (bright green dots), including the axonal ribbons, are too proximal in the IPL to form synapses with the ipRGC. Scale bar, 20 μm.

Figure 6. Ganglion cell axonal ribbon targets

A-D. Renderings of five CBb classes forming axonal ribbons onto multiple ganglion cell classes, vertical orientation. Circles indicate location of synapses shown in E-L. Scale bars (A-B), 25 μm; scale bars (C-D), 20 μm. E-L. TEM of synapses indicated in A-D. White arrows indicate synapse directionality. GC, ganglion cell; WF BC, wide field bipolar cell; AC, amacrine cell; r, ribbons; c, cistern; pcd, post-citernal density; scale bars, 0.5 μm;. A,E. CBb4 3116 (left cell of the silver pair that intersect ganglion cell 15796 (red)) forms an axonal single-ribbon dyad with bsdGC 15796 and an unknown cell. CBb4 3116 participates in a chain of seven coupled CBb3s (tan) and CBb4s (silver). The bsdGC 15796 dendritic target of the axonal ribbon abruptly ascends to the OFF IPL where it receives the input before returning to the ON IPL distally (far right of panel A). B,F. CBb5 400 (mustard) forms an axonal multi-ribbon dyad with ON-OFF ganglion cell 5118 (red) and an unknown cell. C,G,H. CBb5w 6156 (copper) and wide-field cone bipolar cell 5283 (red) converge an axonal single-ribbon monad and axonal multi-ribbon monad, respectively, onto ipRGC 12208 (off white). Note the omega figure in the right subpanel of panel G. Wide-field cone bipolar cell 5283 forms an axonal cistern onto γAC 20537 (not shown in C, see Fig. 8, B-C) in the same plane of section as the four-ribbon axonal monad onto ipRGC 12208. D,I,J,K,L. CBb6 353 (red, left cell) and CBb6 447 (red, right cell) both form multi-ribbon axonal dyads (I,J) onto OFF-layer monostratified ganglion cell 21779 (silver) and another amacrine cell, amacrine cell 22210 (not shown inD for clarity,I) and amacrine cell 32273 (upper bright green cell in D, D inset,J), respectively. Amacrine cell 32273 creates both feedback (J, right subpanel) and feedforward (K) inhibition motifs via conventional synapses onto CBb6 447 and ganglion cell 21779, respectively. CBb6 447 also forms a single-ribbon axonal dyad in the ON IPL onto multistratified ganglion cell process 34336 (beige in D, D inset,L left subpanel) and amacrine cell 34337 (lower bright green cell in D, D inset,L left subpanel). Amacrine cell 34337 forms a conventional synapse onto ganglion cell 34336 (L right subpanel), thus completing a feedforward inhibition motif.

Figure 7. GAC axonal ribbon targets

A-D. Renderings of CBbs targeting both mono- and multi-stratified GACs with axonal ribbons, vertical orientation. Circles indicate locations of synapses shown in E-H. Scale bars, 10 μm. E-H. TEM of axonal synapses at locations indicated A-D. White arrows indicate synapse directionality. r, ribbons; scale bars, 0.5 μm. I-L. TEM of GAC somas. Scale bars, 5 μm. M-P. Glycine-positive signatures of the corresponding GAC somas in I-L. Scale bars, 5 μm. A, E. CBb6 4570 (red) forms a single-ribbon monadic reciprocal synapse with GAC 906 (patina). B,F. CBb5w 309 (copper) forms a single-ribbon monadic synapse onto GAC 310 (patina). C,G. CBb5w 6997 (copper) forms a single-ribbon axonal monad with GAC 5507. The ribbon is very light, but possesses the characteristic halo of clear vesicles, and both pre- and post-synaptic densities are visible. D,H. CBb5w 6156 (copper) forms a single-ribbon axonal monad with GAC 5575 (patina).

Figure 8. γAC axonal ribbon targets & axonal cisterns

A-C. Renderings of axonal-ribbon driven γACs mediating within- and cross-channel, divergent and convergent, inhibitory networks, vertical orientation (A-B), horizontal orientation (C). Arrows, locations of γ+ signatures shown in D-E; circles, locations of synapses shown in F-K; scale bars, 20 μm. D-E. TEM of γACs in A-C with corresponding γ+ signatures. Scale bar (D), 5 μm; scale bar (E), 0.5 μm. F-K. TEM of synapses indicated in A-C. White arrows indicate synapse directionality. AC, amacrine cell; WF BC, wide field bipolar cell; r, ribbons; c, cistern; pcd, post-cisternal density; scale bars, 0.5 μm. A. CBb5 5562 (mustard, left) forms an axonal single ribbon monad onto multistratified γAC 5294 (silver, F). γAC 5294 forms a conventional synapse (A inset, G) onto CBb5 5645 (mustard, right), thus completing an axonal ribbon-mediated within channel inhibition motif. 5294’s soma is γ+ (D). B. A chain of five CBbs converge and diverge axonal ribbon and cistern contacts onto common γAC and ganglion cell targets, vertical orientation. CBb6 5536 (red, right) provides divergent input to amacrine cell 19571process (silver) and wide-field γAC 20537(silver) with an axonal ribbon dyad (H) at locations indicated in C insets. Wide-field γAC 20537 is γ+ (E). Amacrine cell 19571 cannot be confirmed as γ+, but is glycine negative, and participates in nested feedback with γAC 20537 (H, right subpanel). CBb5 176 (mustard) and wide-field cone bipolar cell 5283 (deep red, center) converge axonal cistern contacts onto γAC 20537 (K, Fig. 6 H, respectively). In the same plane of section wide-field cone bipolar cell 5283 drives ipRGC 12208 with a four-ribbon axonal monad (Fig. 6 H). This ipRGC receives convergent axonal ribbon input from CBb5w 6156 (copper, Fig. 5 G). C. Horizontal view of B. Scale bar, 20 μm. (Left inset): Rotated and zoomed-in vertical view of the circled area in the main panel (some cells removed for clarity). CBb6 5536 (red) and wide-field cone bipolar cell 16026 (sand) provide convergent, branched axonal ribbon input to γAC 20537 (H left subpanel & I, respectively). This view looks down the length of γAC 20537 (silver) between wide-field cone bipolar cell 16026 in the right foreground and CBb6 5536 in the left background. Wide-field cone bipolar cell 5283 (red, right) can be seen close to wide-field cone bipolar cell 16026. Scale bar, 2.5 μm. (Right inset): Rotated and zoomed-in vertical view of CBb > γAC ≥ CBa crossover inhibition. CBb6 5536 (red) provides a branched axonal ribbon dyad onto amacrine cell 19571 (H, left subpanel). amacrine cell 19571 forms a conventional synapse (J) onto CBa2 5539 (green) nearby, thus completing the crossover inhibition motif. Scale bar, 5 μm.

Figure 9. Novel network topologies construct an ON-OFF GAC & underlie glycine-mediated within- and crosschannel inhibition

A-D. Vertically orientated enderings of ON-OFF GAC construction and CBa > GAC ≥ CBb crossover inhibition (A), and CBb > GAC ≥ ganglion cell within- and crosschannel (crossover) inhibition motifs (B-D). Circles, location of synapses shown in E-J; scale bar (A), 10 μm; scale bar (A inset), 5 μm; scale bar (B-C), 20 μm; scale bar (D), 10 μm. E-J. TEM of synapses indicated by circles in A-D. White arrows indicate synapse directionality; GC,ganglion cell; scale bars, 0.5 μm. A,E-F. Axonal ribbon topologies employed for construction of a mono-stratified, ON-OFF GAC and CBa > GAC ≥ CBb crossover inhibition motifs. (A Inset): Rotated and zoomed in horizontal view of CBa2 424 (green), CBa2 478 (sage), GAC 906 (silver), and CBb6 4570 (red). CBa2 424 and CBa2 478 converge a single-ribbon monad and single ribbon dyad onto GAC 906 (E & F, respectively). GAC 906 forms a conventional synapse onto CBb6 4570 (red), reciprocal to an axonal ribbon (Fig. 6 E). B,G-J. Parallel CBb > GAC ≥ ON-OFF ganglion cell crosschannel inhibition, and divergent within- (CBb > GAC ≥ bsdGC) and crosschannel (CBb > GAC ≥ ON-OFF ganglion cell) inhibition. CBb6 4570 (red) drives GAC 906 (green) at the axonal synapse described in A. GAC 906 forms a conventional synapse onto mono-stratified ON-OFF ganglion cell 18693 (off-white, G). CBb5w 6156 (copper) drives narrow-field multi-stratified GAC 5575 (patina) with an axonal ribbon (Fig. 6 H). GAC 5575 forms conventional synapses onto mono-stratified ON-OFF ganglion cell 18693 at two locations (H,I). The above two synaptic chains thus form parallel CBb > GAC ≥ ON-OFF ganglion cell motifs that converge onto the same ganglion cell target. GAC 5575 also forms a conventional synapse onto bsdGC 15796 (sand, J), thereby creating divergent inhibitory motifs from CBb5w 6156 to two distinct classes of ganglion cell. C,H-J. Rotated zoom-in and isolation of divergent inhibition shown in B. Multi-stratified, narrow-field GAC 5575 (patina) receives axonal ribbon input from CBb5w 6156 (copper) at an OFF-layer branch (Fig. 7 H, not circled for anatomical clarity), and forms conventional synapses with ganglion cell 18693 (off-white,H-I) and bsdGC 15796 (sand, J). D,H-J. Zoom-in of GAC 5575 divergent inhibition in B-C for anatomical clarity and detail, better appreciation of network topologies, and synapse locations.

Figure 10. CBb axon tangency to potential targets without axonal ribbon synapses

A-B. Renderings of CBbs with contact, but not synapses onto ganglion cells, vertical orientation. Circles, locations of synapses shown in C-D; scale bars, 20 μm. C-D. TEM of synapses indicated by circles in A-B. White arrows indicate synapse directionality; GC, ganglion cell; scale bars, 0.5 μm. A,C. CBb3 5513’s (copper) axon is tangent (adjacent with no intervening muller glia) to OFF ganglion cell 13858 (sand), yet does not form a synapse. B,D.CBb4 3116 (silver) forms an axonal ribbon dyad onto bsdGC 15796 (D, Fig. 6 A & E) and an unknown target (D), whereas CBb4 4569 (dark mustard) does not form an axonal ribbon onto the same unknown target despite being tangent to it. Incidently, CBb4 3116 and CBb4 4569 are gap junctionally coupled (data not shown).

Figure 11. Explanation of the interaction between sparse network topologies and joint distributions

A. An array of bipolar cell axons (white) traverses the image plane of the retina. In the top field, a cell class with high coverage is shown in different colors for every instance of the class. Each bipolar cell axon is contacted several times for an average contact of 2.4. B. Two different classes of ganglion cells (yellow, blue) form part of their tiling by sampling from the bipolar cell array. Most bipolar cells are missed, for an average outflow contact of 0.375, which is meaningless. Six circled bipolar cells are contacted by the ganglion cells (none twice), and the ganglion cells are errorless in contacting encountered bipolar cells. As ganglion cells are not space filling cells, further inputs would be superfluous.

Figure 12. Axonal ribbon motifs summary semi-schematic

Wiring diagram for axonal ribbon motifs discovered across all cone bipolar cell classes in RC1 collapsed onto one representative cell. Spatial distributions of axonal ribbons have been preserved as best as possible to represent actual axonal ribbon locations. The axonal branch in sublamina 2 and the bifurcated descending axon are included for completeness, though both occur in a minority of cone bipolar cells. Note that in addition to abundant axonal ribbon output, cone bipolar descending axons are frequently postsynaptic to amacrine cell inputs. S1-S6, IPL sublaminae 1-6; orange arrows, excitatory ribbon synapses; green flathead arrows, inhibitory GAC- or γAC-mediated synapses; GC, ganglion cell.