Submission journal: Nature

Submitted MS & Supporting Materials: ↓ *.pdf 6.7 Mb

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Cover image

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Cover of Retinal Connectome dataset RC1 slice 001 visualized by TEM and CMP. This is a horizontal TEM plane through the retinal inner nuclear layer visualized with GABA.glycine.glutamate > rgb transparency mapping (neurons) and a dark gold taurine alpha channel for glia. It is composed of over 1000 individual TEM images automatically captured as described in Anderson et al. 2009 (PLoS Biol 7, e1000074) on a JEOL JEM 1400 using a Gatan USCAN 4000 camera. The center of a canonical field 250 μm in diameter was identified in each grid using SerialEM (D. N. Mastronarde 2006 , J Struct Biol 152: 32), and captured as an array ofimage tiles at 950-1100 tiles/section and 2.18 nm/pixel resolution. The capture took 5 months (now 3 months with improved capture algorithms, over 341,000 individual captures and requires 16 terabytes of active storage. Each image has 15% area overlap with its neighbors. Image mosaics and volumes were generated using the NCR Toolset (http://www.sci.utah.edu/download/ncrtoolset). SerialEM metadata position information used by the NCR Toolset application ir-translate produces precise initial image mosaics that are refined by ir-refine-grid to correct for image aberrations. Slice-to-slice TEM image registration is automated by ir-stos-brute, ir-stos-grid. CMP-to-TEM registrations are operator-guided with ir-tweak.

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Figure 1

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Figure 1. RC1 datasets visualized by TEM, CMP and excitation mapping. (A) Section 001. A horizontal TEM plane through the inner nuclear layer visualized with GABA.glycine.glutamate > rgb transparency mapping (neurons) and a dark gold taurine alpha channel for glia. (B) Section 030. A slice 2.5 μm deeper in the layer, with GABA.glycine > magenta.green mapping. (C) A higher resolution view of the inset in panel B. Three glycinergic AII ACs are circled. (D) Section 001 visualized with an additional yellow alpha channel from the in vivo AGB signal. (E) A higher resolution view of the outlined patch in panels A and D, highlighting MCs (black arrowheads) and microglia (outlined) in the gold channel. Six different neurons are marked by index numbers (172, 173, 175 OFF cone BCs; 174 AC; 176 ON cone BC; 594 HC. (F) The yellow alpha channel displays the AGB excitation mapping signal across cell classes. MCs (black arrowheads) show no signal. Microglia (outlined) have responses as strong as neurons. The circular fields in A, B, D are 243 μm in diameter and each represents over 1000 individual TEM tiles at 2.18 nm resolution. The inset boxes in A, B, D are 60 μm wide panels enlarged in C, E, F.

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Figure 2

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Figure 2. The mammalian scotopic network. (A) Viking renderings of connected AII ACs (C476, C514, C2610), rod (B518), OFF cone (C478) and ON cone (C1724) BCs. The shaded bands show the laminations of the BC terminal arbors. Scale, 20 μm horizontal, 5 μm vertical. (B) A ribbon synapse (r) from rod BC B518 onto AII AC C476 and γ rod AC C4942 (orange). Terminals C4941, C4942, C4943 and C5006 are from different γ rod ACs presynaptic (arrows) to B518. (C) Synapses (arrows) from AII AC C514 to OFF cone BC C478 at varicosities and fine connecting processes (inset, six slices away). (D) Synapses (arrows) from AII AC C514 and γ AC C5285 to OFF γ+ GC C5150. (E) Heterocellular coupling (between black arrows) of ON cone BC C1724 and AII AC C514. Inset: Re-imaging at film resolution validates the apposition as a gap junction. Inset width 169 nm. (F) Homocellular coupling zones bracketed by black arrows among three AII AC (C476, C514, C3679). Image width 1181 nm. (G) AII AC C514 as a nexus for over a dozen different contacts. B-F scale, 500 nm.

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Figure 3

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Figure 3. Novel network motifs. (A) 3D renderings of ON cone BCs (C166, C177, C180, C483) and γ AC C5303 postsynaptic to C166 and C177 at nanoribbon synapses and presynaptic at C483 which co-stratifies with ON starburst AC C4890. Circles indicate corresponding TEM images. XY Scale bar 20 μm. The right panel is rotated, showing that C180 does not contact C5303. (B) Nanoribbons (r) from ON cone bipolar C180, targeting small neurites immediately after the axon splits. (C) A series through the axon of ON cone BC C177 making a nanoribbon (r) contact onto γ AC C5303. (D) Nanoribbon synapses from C166 onto C5303. C166 receives a vetting synapse (arrow). (E) A nanoribbon from the axon of ON cone BC C461. (F) A vetting synapse from a γ AC onto the axon of ON cone BC C593. (G) A vetting synapse from a γ AC onto the axon of OFF cone BC C224. (H) A cistern contact from the axon of ON cone BC C168 onto a dark neuronal AC process. (I) A tabulation of nanoribbon (black circles) and vetting (white circles) synapses along the axons of 105 reconstructed BCs. Each line indicates the length of the axon from its point of entry to its terminal expansion level in the IPL. Each circle indicates the absolute location on identified types of BCs.

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Figure 4

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Figure 4. Non-neuronal structures viewed in Viking. (A) A binucleate microglial cell of the IPL (C5016) with extensive lateral processes adjacent to rod BCs B519 and B5017. The boxed region is enlarged in panel B. Image width, 18.5 μm. (B) Insertion of a microglial process from C5016 to within 150 nm of the synaptic ribbon (r) vesicle fusion zone of rod BC B5017. C5016 is also directly adjacent to A(II) AC C514 and, in later sections, a nearby γ rod amacrine cell. Image width, 2.7 μm. (C) A section through the endfeet of two MCs (outlined) at the vitreal border of the retina. The endfeet contain a core filament-rich matrix and a large organelle of organized smooth endoplasmic reticulum (OSER). The arrow is 9 μm long. The boxed region is enlarged in panel D. Image width, 23.3 μm. (D) The transitional zone between the MC core matrix and the OSER organelle is bounded by stacks of smooth endoplasmic reticulum (SER). Image width, 3794 nm.

Supplemental Figures

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Figure S2	↓ Full res lzw tif 34 Mb	∞ Full res jpg 6.4 Mb	∞ Low res jpg 600 Kb
Figure S3	↓ Mid res lzw tif 9.8 Mb	∞ Full res jpg 2.3 Mb	∞ Low res jpg 824 Kb
Figure S4	↓ Mid res lzw tif 15.8 Mb	∞ Full res jpg 5.6 Mb	∞ Low res jpg 828 Kb

Cover

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