Convergence among Non-Sister Dendritic Branches: An Activity-Controlled Mean to Strengthen Network Connectivity

The manner by which axons distribute synaptic connections along dendrites remains a fundamental unresolved issue in neuronal development and physiology. We found in vitro and in vivo indications that dendrites determine the density, location and strength of their synaptic inputs by controlling the distance of their branches from those of their neighbors. Such control occurs through collective branch convergence, a behavior promoted by AMPA and NMDA glutamate receptor activity. At hubs of convergence sites, the incidence of axo-dendritic contacts as well as clustering levels, pre- and post-synaptic protein content and secretion capacity of synaptic connections are higher than found elsewhere. This coupling between synaptic distribution and the pattern of dendritic overlapping results in ‘Economical Small World Network’, a network configuration that enables single axons to innervate multiple and remote dendrites using short wiring lengths. Thus, activity-mediated regulation of the proximity among dendritic branches serves to pattern and strengthen neuronal connectivity

Dendritic branch intersections are structurally regulated targets for efficient axonal wiring and synaptic clustering.

Synaptic clustering on dendritic branches enhances plasticity, input integration and neuronal firing. However, the mechanisms guiding axons to cluster synapses at appropriate sites along dendritic branches are poorly understood. We searched for such a mechanism by investigating the structural overlap between dendritic branches and axons in a simplified model of neuronal networks--the hippocampal cell culture. Using newly developed software, we converted images of meshes of overlapping axonal and dendrites into topological maps of intersections, enabling quantitative study of overlapping neuritic geometry at the resolution of single dendritic branch-to-branch and axon-to-branch crossings. Among dendro-dendritic crossing configurations, it was revealed that the orientations through which dendritic branches cross is a regulated attribute. While crossing angle distribution among branches thinner than 1 µm appeared to be random, dendritic branches 1 µm or wider showed a preference for crossing each other at angle ranges of either 50°-70° or 80°-90°. It was then found that the dendro-dendritic crossings themselves, as well as their selective angles, both affected the path of axonal growth. Axons displayed 4 fold stronger tendency to traverse within 2 µm of dendro-dendritic intersections than at farther distances, probably to minimize wiring length. Moreover, almost 70% of the 50°-70° dendro-denritic crossings were traversed by axons from the obtuse angle's zone, whereas only 15% traversed through the acute angle's zone. By contrast, axons showed no orientation restriction when traversing 80°-90° crossings. When such traverse behavior was repeated by many axons, they converged in the vicinity of dendro-dendritic intersections, thereby clustering their synaptic connections. Thus, the vicinity of dendritic branch-to-branch crossings appears to be a regulated structure used by axons as a target for efficient wiring and as a preferred site for synaptic clustering. This synaptic clustering mechanism may enhance synaptic co-activity and plasticity

Reconstruction of dendritic networks and sub-networks by CCM.

<p>(A) An area in a relatively dense one week old hippocampal culture (A1, MAP2) has ordered (yellow ellipse in (A2)) and less ordered (red circle in (A2)) regions. (A3) Within the ordered area, an even more highly organized region was found (green square) and used for analysis in (B). (B) Dendritic network reconstruction using CCM. (B1) High magnification of the boxed area in (A3). (B2) Reconstruction of (B1). (B3) Connectivity map of (B2). (C) Reconstruction of a sub-network of >1 micron caliber branches. (C1) Reconstructed “thick” branches network. Few thick branches adjacent to the cell bodies were excluded due to deletion of their crossings (located within the blue rectangles). (C2) The connectivity map of the “thick” (red) and fine (green) branches. Yellow arrows show that a large portion of the thin dendrites are at the edges of growing branches. (C3) The “thick” branches sub-network’s connectivity. (D) Comparison between CCM (B3) and the manual reconstructions (D2). (D1) The efficiency of junction and connecting segments detection by CCM compared to that of manual analysis (100%). Shown is the mean ± SEM. Scale (in C3): A - 60 µm; B–D - 15 µm.</p

Axons cluster their synapses near dendro-dendritic crossings by converging predominantly onto these sites.

<p>Green = dendrites (MAP2); red = active synapses (FM1-43). (A) A non-90° dendritic intersection contacted by 7 axons (yellow arrows), most of them traversing through the obtuse angles zones. The axons cluster their active terminals (blue arrow) near the intersection (white arrow). (B) A 90° dendritic intersection traversed by axons from three different zones (yellow arrows), associated with a cluster of active axonal terminals (blue arrow). (C) Size distribution of synaptic clusters located at dendritic intersection vicinities. (D) Synaptic clusters on two dendritic intersections linked by multiple axons (arrows). (E) Same regions as (D) showing an inverted image of dendritic clusters and connecting axons (arrows). (F) A network with active synapses localized to dendritic intersections (arrows). Most clusters are linked through bundles of multiple axons(examples are indicated by pink arrows). Scale: A, B - 10 µm; D - F20 µm.</p

Neighboring dendrites preferentially generate contacts among their branches.

<p>Pictures are inverted images of hippocampal neurons grown in culture for 5 days and labeled with anti-MAP2 antibody. (A) An isolated neuron with no physical contacts with other neurons made a single contact between its own branches (arrow). (B) Within a neuronal assembly, dendritic branches turn (arrows) to contact branches of neighboring dendrites (red spots). (C, D) A dense culture in which all neurons orient dendritic branches toward a major overlapping area (circled) where heavy crossing takes place (D, red spots). (E) Dendrite-dendrite contact density is significantly higher among grouped rather than isolated neurons. Shown is the mean ± SEM. Scale: 20 µm.</p

Axons traverse dendrites preferably near dendritic intersections.

<p>(A–C): The same field seen as merge (A), dendrites (MAP2) (B) and axons (C). Axons traverse predominantly near dendritic intersections (blue arrows) and rarely at non-intersecting regions along dendrites (white arrowhead). Most non-intersecting shafts of dendritic branches are not traversed but rather fasciculated by axons (yellow arrows). (D) Higher magnification of the central region of (A) shows traverse events near (blue arrows) and at a distance (yellow arrow) from dendritic intersections. (E) Further magnification of the left region of (D): Blue scanning circles (4 µm diameter) used for quantification of traverse frequency. (F) Traverse frequency was considered as the number of axons located within the circles. Scale: A–C - 30 µm; D - 13 µm; E - 3 µm.</p

Dendritic branches cross through preferable angles.

<p>(A, B) The procedure for analyzing dendritic crossing angles. A mesh of crossing dendritic branches (A) is reconstructed in (B) based on the intersections location (red spots) and their connections (black lines). Acute and right angles between crossing and bifurcating branches are collected. (C, D) Analysis of the field shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082083#pone-0082083-g003" target="_blank">figure 3D</a>. (C) Crossing angle distribution of combined fine/thick branches. (D) Distribution of angles among >1 micron caliber branches. (E, F) A dendritic network (E) with similar preference of crossing angles ranges among “thick” branches as in (D) but of higher incidence (F). (G) Length distribution of fine (blue bars) and “thick” (red bars) intersections from the field in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082083#pone-0082083-g003" target="_blank">figure 3D</a>. (H) Frequency of crossing angle range selection among 15 dendritic networks. (I) An astrocytic network. (J) Intersection angle distribution of (I). Note the 40°–50° range preference, which is absent from the dendritic networks (H). Scale (under H): A –4 µm; F, I –20 µm.</p

CCM procedure for generating connectivity maps.

<p>(A) Basic principles of identification of dendritic branch intersections and their connections. Dendritic intersections (A1) are identified based on their greater width compared to the dendritic segments composing them (A2, labeled in A3). The intersections are marked (A4), centralized (arrow in A5) and connected by a line (A6, arrow in A7). (B) General description of CCM operation. (B1) Starting image – grayscale, inverted. (B2) Contrast optimization. (B3) Background whitening. (B4) A scanning box with centralized pixel (arrow). (B5) Identified junction area (red) with estimated “intersection center pixels” (black squires). (B6) Areas at and near intersections are removed (colored in blue). (B7) second iteration on an updated image (using varying threshold for intersections) reveals thinner intersections (arrows). (B8) A third iteration generated after removal of the B7 new intersections and using a new threshold revealed additional and even thinner intersections (arrows). (B9) Generation of lines connecting the intersections, according to the neuritic map. Scale: A - 3.5 µm; B - 12 µm.</p

Axons preferentially traverse heavily intersecting dendritic branches.

<p>Green = dendrites (MAP2); Red = axons (NFM). (A) Axons tend to fasciculate (yellow arrow) with dendritic branches at areas of low dendritic branch-to-branch crossing frequency, but prefer to traverse (blue arrows) regions with high rates of dendritic crossing. (B) Two axons defsciculate (blue arrow) from a thick, rarely-crossing dendritic branch to traverse several highly intersected dendritic branches (arrow colors specific to individual axons). (C) Non-crossing dendritic branches (C1) and axons (C2) interact (C3), forming only few axon-dendrite contacts (arrow colors specific to individual axons). (D) When crossing dendritic branches (D1) and axons (D2) interact, the axons heavily traverse dendritic branches (D3), forming axo-dendritic contacts involving multiple axons (D4). (E) A dendritic hub-like organization. The center of the hub (white circle) is composed of heavily crossing branches whereas branches located at the hub’s periphery (blue and yellow marks) have fewer axonal crossing events. (F) Magnification of the yellow rectangle region in (E) shows alignment of axons with rarely crossing dendritic branches and low frequency of axo-dendritic contacts. (G) Magnification of the white circled hub’s center in (E) showing high frequency of dendro-dendritic and axo-dendritic contacts. Scale (black line underneath (E): B - 15 µm; C, D - 6 µm; E - 30 µm; F, G - 10 µm.</p