The Fuzzy Logic of Network Connectivity in Mouse Visual Thalamus

Abstract

In an attempt to chart parallel sensory streams passing through the visual thalamus, we acquired a 100-trillion-voxel electron microscopy (EM) dataset and identified cohorts of retinal ganglion cell axons (RGCs) that innervated each of a diverse group of postsynaptic thalamocortical neurons (TCs). Tracing branches of these axons revealed the set of TCs innervated by each RGC cohort. Instead of finding separate sensory pathways, we found a single large network that could not be easily subdivided because individual RGCs innervated different kinds of TCs and different kinds of RGCs co-innervated individual TCs. We did find conspicuous network subdivisions organized on the basis of dendritic rather than neuronal properties. This work argues that, in the thalamus, neural circuits are not based on a canonical set of connections between intrinsically different neuronal types but, rather, may arise by experience-based mixing of different kinds of inputs onto individual postsynaptic cells.

Figure 1. A 0.07 mm³ volume of LGN was imaged in the electron microscope and a network of >400 neurons reconstructed

(A) Coronal section of LGN (Movie S1). Colors indicate seed Cells A–D and Cell E. Double arrow in this and subsequent images show the superficial (S), medial (M), or rostral (R) directions. (B) Reconstructions of all the cells used for network analysis. (C) Transverse view of the seed cells (A–D) and Cell E with labeled presynaptic RGC boutons. Bouton color indicates which RGC axon gave rise to it. The presynaptic bouton fraction refers to the number of traced presynaptic RGC boutons relative to the total number of RGC boutons identified on that TC. Inset shows the different sizes of boutons on three seed cells: A’s are large, B’s are small and D’s are very large. See also Figure S1.

Figure 2. The structure and synapses of each traced RGC axon innervating the four seed cells

(A) The RGC axon segments (each a separate color) that gave rise to all (212) boutons innervating seed TC A (white). Bar graph (also for panel B–D): The total number of boutons each axon formed on the seed cell (black) and the total number of boutons each axon formed elsewhere in the volume (gray). Most axons provided only a minority of their synaptic boutons to the seed cell. (B) RGC axon segments innervating Cell B. (C) RGC axon segments innervating Cell C. (D) RGC axon segments innervating Cell D. See also Figure S2.

Figure 3. Evidence for both pre- and postsynaptic influences on synaptic structure

(A) A TC (yellow) is innervated by large boutons from RGC axons that also innervate Cell A (red) and by small boutons from RGC axons that also innervate Cell B (green, in circles). (B) RGCs innervating Cell A form large boutons on three groups of TCs. On Cell A, left: 201 boutons, mean diameter = 1.8 μm, 95% range = 1.0–2.5 μm; On other TCs, middle: 343 boutons, mean = 1.9 μm, 95% range = 1.0–3.0 μm; On mixed TCs, right: 246 boutons, mean = 2.0 μm, 95% range = 1.0–3.2 μm) (C) RGCs innervating Cell B form small boutons on three groups of TCs. On Cell B, left: N = 137, mean = 1.2 μm, 95% range = 0.5–1.8 μm, P < 0.001 (compared to panel B); On other TCs, middle: 359 boutons, mean = 1.1 μm, 95% range = 0.52 – 1.7 μm, P < 0.001 (compared to panel B); On mixed TCs, right: 99 boutons, mean = 1.1 μm, range = 0.5 – 1.7 μm, P < 0.001 (compared to panel B). (D) Synaptic features of RGCs that innervate Cell A (left side) and Cell B (right side). Red bars = boutons on TCs that only share RGC input with Cell A, Green bars = boutons on TCs linked to Cell B, and yellow bars are boutons on TCs linked to both Cells A and B. (E) Perforated RGC bouton (highlighted) containing multiple internal TC dendritic spines (blue, arrows). (F) A plot of the size and internal spine number for each RGC bouton innervating Cells A–E. TCs exhibit different numbers of perforated boutons. (G) Most RGC axons linked to seed Cells A–E form both non-perforated and perforated boutons in the volume. (H) The same axons that form only one perforated bouton total on Cell A (of 212, red dot), form many perforated boutons on other TCs. (I) Example axon that forms boutons on both Cell A (red boutons) and Cell E (blue boutons), but that only forms perforated boutons (green circles) on Cell E. Insets show structure of perforated (top) and unperforated (bottom) boutons. See also Figure S3.

Figure 4. A Spring force connectivity map reveals a synaptically interconnected LGN network with both spatial and non-spatial organization

(A) Spring model of synaptically connected (lines) RGCs (triangles) and TCs (circles) forms clusters that correspond to their seed cell association. The color coded legend identifies RGCs based on seed cell innervation. The color of each TC reflects the seed cells with which the TC shares RGC input. Spring strength (indicated by line thickness) reflects the number of boutons connecting each RGC to each TC. Seed Cells (A–D) are excluded from influencing the cell distribution (springs set to 0). See also Figure S4. (B) Segregation of RGCs and TCs with large average bouton diameters (red) from small bouton diameters (blue). Cells with fewer than 5 identified boutons are gray. (C) Non-random distribution of perforated boutons. Cells with fewer than 10 identified boutons are gray. (D) Relative positions and dendritic overlap of seed Cells A–D (stereo pair for crossed eye viewing; see also Movie S4). (E) Non-overlap of the TCs linked to Cell C (cyan) and D (purple). Colored circles indicate seed cell positions (also in panel F). (F) Spatial overlap of TCs linked to Cell A (red), Cell B (green) and both A and B (yellow). (G) Network erosion dendrogram (seed cells excluded) used to identify network substructure (see also Movie S3). Colors indicate seed cell association (left, see Figure 4A for color scheme) or the first six erosion groupings (right, see Movie S3 second 14). (H) Spring force diagram colored according to the erosion groupings from panel G (right side). (I) Relative positions of TCs and RGCs (see key) divided into the six groups shown in panel H. The green group has the widest spatial distribution of any subnetwork and overlaps with several other subnetworks. (J) Spatial mixing and segregation of erosion groupings (color from panel H) of synaptically related TCs. See also Figure S5. (K) Spatial mixing and segregation of erosion groupings (color from panel H) of synaptically related RGCs. (L) Spatial mixing and segregation of synapses within and between erosion groupings (color from panel H) of RGCs and TCs. Left: synapses between RGCs and TCs from the same group. Middle and right: greater mixing of synapses formed between RGCs and TCs from different groups (colored according to the RGC-middle or TC-right). White cross indicates center of traced volume.

Figure 5. TCs with different dendritic arbor shapes found throughout the network

(A) Example of a biconical dendritic arbor (Cell 134). Inset = biconical arbor icon. (B) Example of a symmetric dendritic arbor (Cell 130). Inset = symmetric arbor icon. (C) Different arbor shapes are found in the same subnetworks of the dendrogram. Left side = erosion group membership (colors from Figure 4H) of morphologically characterized TCs. Right side = shape of TCs based on Sholl analysis. Key colors indicate biconical, flattened or radially symmetric arbors. Cells 134 and 130 from panel A and B (arrows) are adjacent in the dendrogram. For 39 TCs, correlation coefficient (r) between similarity of shape and connectivity = 0.15. For randomly assigned cell shape, r = 0, 95% range = −0.09 – 0.10, P = 0.008, adjusted for multiple tests. (D) Plot of TC arbor shape with TCs colored according to erosion grouping (except the four seed cells labeled cyan). (E) Spring force diagram with morphologically characterized TCs colored according to shape (as in panel C). See also Figure S6.

Figure 6. Different subnetworks of RGC axons innervate different parts of individual TC dendritic arbors

(A) Spring force distribution of the three groups of RGCs that innervate Cell A (colors from Figure 4H). (B) Non-uniform dendritic distribution of different groups of RGC synapses (colored as panel A and Figure 4H) on Cell A. (C) Axon arbors (colored as panel A) of the different RGC groupings that generate the bouton distribution in panel B. (D) Network erosion breaking the axons innervating Cell B into five groups. Circles indicate the colors used for these groups in panel E, F and G. See movie S3 second 18. (E) Erosion dendrogram showing the five RGC subgroups linked to seed Cell B. Dotted line indicates the stage of erosion that defines the five groups in panel D. (F) Spring force distribution of the five clusters of RGCs that innervate Cell B. (G) The five groups of RGCs (panel D) innervate largely non-overlapping glomeruli within Cell B’s dendritic arbor. Each color represents a set of axons (orange = 2 axons, green = 4, yellow = 6, purple = 9, and blue = 11, see panel F). The arrow points to the glomerulus shown in panel J. (H) The RGCs of each of the five groups are distributed over a large area relative to their innervation of Cell B (Movie S5). (I) The TCs belonging to each group are distributed over a large area relative to the bouton clusters in panel G. (J) RGC fascicles that jump from one dendrite to another help explain the subneuronal network organization shown in panel G. Many RGC axons forming a synaptic glomerulus on Cell B (the magenta dendrite of seed Cell B) travel together to a nearby dendrite of a different TC (cyan dendrite of Cell 203). (K) A local inhibitory neuron (red) mirrors RGC innervation (green) of two glomeruli on different TCs (magenta and cyan, see panel J). See also Figure S7.

Figure 7. Summary of synaptic and network motifs in this region of the LGN

(A) Four synaptic configurations generated by two RGC types in the traced LGN network. (B) Variations in the properties of RGCs (small or large synaptic boutons) and TCs (“B” = biconical arbor, “S” = symmetric arbor) produce many distinct TC input patterns (eight combinations shown here). All of these input patterns were found within the same interconnected network. Synaptic configurations (from left to right) refer to shaft glomerular, large bouton with external spine, and perforated bouton synapses. Please note that we find considerably more axons converging on each TC than the 1–2 shown here.