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BR A IN RE S EA RCH 1 2 51 ( 20 0 9 ) 3 0 –41 a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s Research Report Contacts among non-sister dendritic branches at bifurcations shape neighboring dendrites and pattern their synaptic inputs Joshua Cove a , Pablo Blinder a,1 , Danny Baranes a,b,⁎ a b Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva, 84105, Israel Ariel University Center of Samaria, Ariel, 44837, Israel A R T I C LE I N FO AB S T R A C T Article history: The size and shape of neuronal dendritic arbors affect the number and pattern of synaptic Accepted 1 November 2008 inputs, as well as the complexity and function of brain circuits. However, the means by Available online 19 November 2008 which different dendritic arbors take their final shape and how these shapes are associated with distinct synaptic patterns is still largely unknown. Dendritic ramification is influenced Keywords: by dendrite-dendrite interactions that stabilize specific branching directions and ensure Dendrite morphology appropriate synaptic contacts. Yet, it is not clear by which mechanism these contacts are Synaptic connection allocated. We found that stable dendro-dendritic contacts occur preferentially between non- Synaptic strength sister dendritic branches at sites of bifurcations, and that this process is promoted by Synaptic distribution synaptic activity. Moreover, these contacts are associated with synaptic connections of Dendro-dendritic contact higher density, higher level of synaptophysin, NR1, GluR2 subunits of glutamate receptors and elevated secretion capability than synaptic connections found on contacts made by non-bifurcating branches or along non-contacting parts of the dendrites. Thus, in cultured neurons, stabilization of hetero-neuronal dendro-dendritic contacts at bifurcations is a new mean to pattern and associate morphogenesis and synaptic input distribution in neighboring dendritic trees. © 2008 Elsevier B.V. All rights reserved. Dendrite arborization patterns are critical determinants of neural circuit formation and function (Spruston, 2008). The shape of dendritic trees can influence the type and location of inputs a neuron is able to receive, and influences how these inputs are integrated (Segev and London, 2000; Gulledge et al., 2005). The mechanisms that underlie these influences are not clear, but are likely to be found within the context of dendritic morphogenesis. Dendritic arbor development is a complex, multi-step process that includes initiation of branch growth, outgrowth and guidance, branching and synapse formation, and stabilization (Kossel et al., 1997; Wu et al., 1999; PorteraCailliau et al., 2003; Williams and Truman, 2004; Scott and Luo, 2001). Moreover, dendritic morphogenesis is a highly dynamic process, characterized by extension and retraction of branches, followed by stabilization and growth. Thus, selective stabilization or destabilization of branches probably shapes dendritic arbors. However, the means by which different dendritic arbors take their final shape is still unknown. The size and shape of a dendritic arbor are influenced largely by the combined actions of intrinsic signals and guidance cues. In addition, several observations support a role for neuronal activity in regulating dendrite morphology (McAllister, 2000). For example, pharmacological blockade of synaptic activity in vitro and in vivo ⁎ Corresponding author. Department of Life Sciences Ben Gurion University of the Negev, Beer Sheva, 84105, Israel. Fax: +972 8 647 9011. E-mail address: [email protected] (D. Baranes). 1 Current address: Physics Department, University of California San Diego, La Jolla 92093, USA. 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.11.028 BR A IN RE S E A RCH 1 2 51 ( 20 0 9 ) 3 0 –4 1 31 Fig. 1 – BDI — a novel and ubiquitous structure of dendro-dendritic contact: All images are of MAP2 labeled 12DIV cultures. (A) A contact between two elementary dendritic structural units, segments and bifurcations frequently occurs at the bifurcation site, forming the BDI structure. To be considered part of a contact structure, each dendrite must be distinct and not arrive at the contact structure by fasciculation. BDI definition includes only dendritic segments and bifurcations intersecting precisely at the bifurcation point. (B) Contact dendrites form BDIs (arrows) frequently. (C) A magnification of the area boxed in (B) showing a single dendrites involved in several BDIs. Scale bar (shown above panel C): A — 40 μm; B — 25 μm; C — 5 μm. (McAllister et al., 1996; Rajan and Cline, 1998; Redmond et al., 2002; Portera-Cailliau et al., 2003) and loss of sensory input in both the lateral geniculate nucleus and the visual cortex (Coleman and Riesen, 1968; and Wiesel and Hubel, 1963) leads to deficits in dendritic growth. The commonly held view is that general dendritic structural features result from genetic instructions, whereas fine connectivity details is regulated by functional activity (Portera-Cailliau et al., 2003). A finer tuning of dendritic morphogenesis in vivo occurs through stabilization of dendritic branches through dendrite-dendrite interactions (Komiyama and Luo, 2006). This mechanism has a profound influence on determining the size and shape of the dendritic tree by enabling growing branches to select specific directions. Dendro-dendritic contacts also allow individual cells to refine dendritic targeting (Zhu and Luo, 2004) to their appropriate area and ensures appropriate synaptic contacts. But how are decisions to stabilize dendro-denritic contacts made? One concern is to avoid self contact. For example, the Down syndrome-related cell adhesion molecule (Dscam) genes mediate self-avoidance by generating thousands of different Dscam isoforms (Schmucker et al., 2000). Matched Dscam isoforms on sister Drosophila second-order olfactory PN dendritic branches promote repulsive interactions (Zhu et al., 2006), making non-sisters dendritic branches preferable for contact. Yet, it is not clear whether such non-self recognition-based contact occurs spontaneously, or they are allocated and stabilized by a dedicated mechanism. We found that non-sister dendritic branches make stable contacts preferably at sites of bifurcations. Formation of such contacts is controlled by synaptic activity and is associated with clusters of synaptic connections of higher density and strength than elsewhere along the dendrites. Thus, dendrodendritic contact at bifurcations is an activity-derived neuronal behavior that shapes dendritic trees and links their branching pattern to that of their synaptic inputs. 32 BR A IN RE S EA RCH 1 2 51 ( 20 0 9 ) 3 0 –41 1. Results 1.1. Abundant dendro-dendritic contact at bifurcations Our first aim was to identify recurrent wiring structures in self-assembled neuronal networks in culture. We focused on dendrites as they were easier to quantify than axons, and attempted to identify characteristics along their structures and their contacts. We first defined two basic structures within individual dendritic arbors: i. Dendritic segments – sections spanning between two branch points or between a branch point and dendrite endings; ii. Dendritic bifurcations – sites where a dendritic segment splits into two daughter segments (Fig. 1A). We found that these two structures frequently interacts exactly at the point of bifurcation (Fig. 1A). We termed such contacts as Bifurcation-Dendrite Intersections (BDIs). Intersections between two or three dendritic bifurcations were not analyzed here, since they were extremely rare (less than 1 occurrence per 20 cells). In all BR A IN RE S E A RCH 1 2 51 ( 20 0 9 ) 3 0 –4 1 33 Fig. 3 – High synaptic density and strength at BDIs: (A, B) Representative clustering of synaptophysin (A) or FM1-43 (B) positive synaptic terminals around a BDI. The circle shows the 3.75 μm radius from the BDI center, which was used for analysis in the following panels. The fluorescence of synaptophysin (Ai) or FM1-43 (Bi) per number of dendritic segments involved in each structure, measured within a 3.75 μm radius from the structure center (for Ai: mean ± SEM, p < 0.001 by the Kruskal Wallis test, **p < 0.01 by the Kruskal Wallis multiple comparisons test, n = 14 images from 3 cultures, with 5 dendrites and bifurcations and at least 3 BDIs per image. For Bi: mean ± STD, *p < 0.05 by one way ANOVA and the Bonferroni post hoc test, n = 10 experiments, with one image from each, containing at least 3 of each structure). The number of synaptic terminals as measured by synaptophysin imunnofluorescence (Aii) or FM1-43 staining (Bii) and the density of terminals per axonal length within the same area (for Aii: mean ± SEM, same n as in (Ai); for Bii: mean ± STD, same n as in (Bi)). Scale bar: 5 μm. cases, intersections with additional dendrites in fasciculation upon the structure dendrites were discarded from analysis. BDIs were common enough to affect the orientation of dendritic branches and they involved many of the neurons' dendritic branches (Fig. 1B). Moreover, single dendrites were often involved in several BDIs (Fig. 1C). 1.2. BDIs formation and stability are under regulation It is questionable whether BDIs arise from random intersections between dendrites or from directed growth. We addressed this by comparing the frequency of BDIs, of bifurcations, and of intersections between two dendritic segments (DDIs) in culture to that found in simulations of random distributed neurons. The simulations randomly positioned and rotated images of single GFP expressing neurons, to generate the same density of cells as seen in culture. To justify the suitability of this simulation, we first verified that the same degree of detail of dendritic arbors arose from GFP fluorescence as from MAP2 immunostaining. This was done by comparing the number and density of dendritic bifurcations visible in each visualization method. We found 27 bifurcations per cell with 0.79 ± 0.24 bifurcations per 100 μm of dendrite in MAP2 stained cultures, and 38 bifurcations per cell Fig. 2 – BDIs are frequent, non-random, stable and activity regulated structures: Analysis was performed either on 12DIV cultures immunostained for MAP2, or on phase-contrast time-lapse series of images. Treatments were administered for the final 48 h before immunostaining. (A) Neuronal cultures exhibit significantly higher level of BDIs per dendritic length compared to that found in simulations of random dendritic distribution (mean ± STD, ***p < 0.001 by t-test for each structure, n (culture/simulation) = 79/16 BDIs and 158/229 bifurcations in 7 images from 3 cultures and in 6 simulated images). (B) Representative phase contrast images from part of a time lapse experiment showing: (B1) a stable BDIs, (B2) BDI disassembly. Left panel — a dendrite (red arrow) is close to a bifurcation site of a second dendrite (green arrowhead); middle pane; - the two dendrites merge at the bifurcation site, forming a BDI (yellow arrow); right panel — two dendrites separate. Note that the curvature of the dendrites and the bifurcation angle are different than those in the left panel. (C) The average longevity of those BDIs which were present on the first day of imaging (mean ± SEM, n = 71) (D) BDIs maintain constant numbers per cell, with the rates of formation and dissolution offsetting. Presented are the number of newly formed BDIs, the number of those that disappear since the previous day, and the total number per day per image. n = 117 BDIs (in 2 fields). (E) Pharmacological inhibition of synaptic activity reduces BDI density (mean ± STD, *p < 0.05 by one way ANOVA and the Bonferroni post hoc test. n = 55–69 BDIs, 225–365 DDIs and 158–191 bifurcations in 7–12 images from 3 cultures per treatment). 34 BR A IN RE S EA RCH 1 2 51 ( 20 0 9 ) 3 0 –41 with 0.60 ± 0.07 bifurcations per 100 μm of dendrite in the simulations (p > 0.05, t-test, n = 158 bifurcations in 7 images from 3 cultures and 229 bifurcations from 6 simulated images). Thus the simulation faithfully reproduced the MAP2 immunostained dendritic arbors. In 12 days old cultures, we found 2.82 ± 0.66 DDIs per 100 μm of dendrite, and 0.37 ± 0.1 BDIs per 100 μm of dendrite, (32% of all bifurcations), (Fig. 2A) (79 BDIs, and 171 DDIs in 7 images from 3 cultures). By contrast, there were significantly fewer BDIs in the simulation (mean ± STD, p < 0.001 by t-test for each Fig. 4 – Synapses at BDIs are enriched with postsynaptic NR1 and GluR2: 12DIV cultures immunostained for MAP2 in green and NR1 (A–C) or GluR2 (D) in red shows the highest concentration of these glutamate receptors at BDIs compared to other regions along the dendrites. (E) The fluorescence, per number of dendritic segments, measured within a 3.75 μm radius from the structure center, (mean ± STD, (p < 0.001 for NR1, p = 0.005 for GluR2) by the Kruskall Wallis test and ***p < 0.001 by the Kruskall Wallis multiple comparisons test, n = 20 (NR1), = 15 (GluR2) images with 5 dendrites, bifurcations and DDIs and at least 3 BDIs per image, from 3 cultures). (F) The number of NR1 and GluR2 puncta within the 3.75 μm radius (blue line) and their density per 10 μm of dendrite (mean ± STD, same n as in (E)). Scale bar (shown in C): A, B = 10 μm; C, D = 5 μm. 35 BR A IN RE S E A RCH 1 2 51 ( 20 0 9 ) 3 0 –4 1 contact structure, n (simulation) = 16 BDIs, and 367 DDIs from 6 simulated images). Thus, DDIs, BDIs were ubiquitous and occurred more often than expected from random dendrodendritic contacts. Moreover, with a total of 1000 μm dendrite on average per cell, this means that virtually all neurons form at least one BDI. Do dendrites adhere to each other at BDIs? We performed time lapse experiments over 7 days, beginning on day 19, which revealed BDIs to be stable for up to at least a week (Fig. 2B1). Moreover, we found that disassembly of BDIs results in changes in the structure of the dendrites involved in the BDI (Fig. 2B2). BDIs had a median longevity of 3–4 days, with around 25% remaining stable for a week or more (Fig. 2C, mean ± SEM). Their rate of formation and dissolution offset each other at 6 ± 1.7 a day per image (approximately 440 μm × 350 μm), to generate a steady-state of 35 ± 3 BDIs per image (Fig. 2D, mean, n = 118 BDIs (from 2 fields) taking 1 image per day, for 7 days). Thus, dendrites seem to form stable contacts at BDIs. Another way to verify that BDIs formation is non-random would be if it shows that such process is under regulation. We examined whether the frequency of BDIs varied following manipulations of synaptic activity (Fig. 2E). The frequency (number per 100 μm dendrite) of bifurcations and DDIs was unchanged under all conditions. The frequency of BDIs was reduced following 24 h application of 10 μM CNQX, of 1 μM TTX, 10 μM CNQX or 50 μM APV (mean ± STD, p = 0.025, 0.028 and 0.017 respectively by one way ANOVA and the Bonferroni post hoc test, compared to the control. n = 55-69 BDIs, 225–365 DDIs and 158–191 bifurcations in 7–12 images per treatment from 3 cultures). In conclusion, either the formation or the stability of BDIs are increased by synaptic activity. 1.3. Increased synaptic density and strength at BDIs Based on the logic described in the introduction, we were prompted to check if BDIs affect the development of synaptic connections. We first examined the density and synaptophysin expression level of axonal varicosities within and outside BDIs. Synaptophysin positive puncta were clearly clustered around BDIs, resulting in a patchy distribution clustered around them and had higher fluorescence values than those outside the BDIs (Fig. 3A). To quantify these results, the total fluorescence of synaptophysin puncta within a 3.75 μm radius circle around the center of BDIs and DDIs was measured and normalized by the number of dendritic segments composing the contact (shown in Fig. 3A, left panel). The radius of 3.75 μm was selected as sufficient to include the combined length of a spine and bouton, and thence reflect enrichment of synapses around the structures, but not including other synapses further away. BDIs had significantly higher fluorescence levels per segment compared to dendritic segments (13 fold), bifurcations (3 folds) (Fig. 3Ai). The number of varicosities in a 3.75 μm radius around the center of BDIs was also higher than elsewhere (Fig. 3Aii) (mean ± SEM, same n = 14 as in Fig. 3Ai). The number of segments per structure was considered as follows: dendrite = 2, bifurcation = 3, DDI = 4, BDI = 5 (linear regression: y = 4.8 × − 2.1, R2 = 0.93, p < 0.001), as did also the density of varicosities per axonal length within the same area (linear regression: y = 0.4 × +1.7, R2 = 0.48, p = 0.01). We then imaged active presynaptic terminals with FM1-43 to determine whether the increase in synaptophysin puncta at BDIs reflected an increase in functional axonal terminals (Fig. 3B). Significantly higher FM1-43 fluorescence per dendrite was measured at BDIs than at dendrites or bifurcations (Fig. 3Bi). In addition, the density of FM1-43 positive terminals per axonal length was the highest at BDIs (linear regression: y = 2.4 × –1.5, R2 = 0.91, p < 0.001) (Fig. 3Bii, mean ± STD, same n = 10 as for Fig. 3Bi). We conclude that axonal terminals had a higher density and secretion capability at BDIs than at dendritic segments and bifurcations. 1.4. Dendritic contact structures are enriched with post-synaptic receptors We then turned to perform the same comparative analysis on the post synaptic sites present in the various structures. Clusters of higher content of the NMDA receptor subunit NR1 and the AMPA receptor subunit GluR2 were found in BDIs, compared to the rest of the dendrite (Figs. 4A–E). The number of clusters of NR1 and GluR2 per structure (Fig. 4F blue traces, mean ± STD) was also higher at BDIs than elsewhere (linear regressions, green traces: GluR2, y = 4.7 × − 1.5, R2 = 0.88, p < 0.001; NR1, y = 3.7 × −0.7, R2 = 0.92, p < 0.001). These results suggest that synapses located at BDIs had higher NR1 and GluR2 densities than synapses located elsewhere. As shown in Table 1, we found the degree of synaptic enrichment in BDIs to depend on synaptic activity through NMDA receptors in presynaptic terminals, but not in the postsynaptic site. 1.5. Synaptic enrichment at BDIs arises from synaptogenesis and not from neurite growth The increased number and fluorescence of synaptic terminals at BDIs could result either from enhanced synaptogenesis in axons located near this structure, or from increased Table 1 – Clustering and enrichment of synaptic terminals at BDIs depends on synaptic activity Sph GluR2 NR1 Control TTX CNQX APV Control TTX CNQX APV Control TTX CNQX APV Den Bif DDI BDI 0.09 ± 0.02 0.11 ± 0.03 0.06 ± 0.01 0.07 ± 0.02 0.1 ± 0.02 0.16 ± 0.02 0.16 ± 0.03 0.11 ± 0.06 0.13 ± 0.03 0.13 ± 0.03 0.11 ± 0.02 0.17 ± 0.03 0.23 ± 0.05 0.25 ± 0.04 0.22 ± 0.05 0.20 ± 0.05 0.2 ± 0.04 0.36 ± 0.06 0.31 ± 0.05 0.24 ± 0.06 0.2 ± 0.03 0.18 ± 0.04 0.23 ± 0.04 0.26 ± 0.05 0.14 ± 0.03 0.11 ± 0.03 0.15 ± 0.03 0.09 ± 0.02 0.07 ± 0.03 0.12 ± 0.03 0.19 ± 0.04 0.15 ± 0.05 0.15 ± 0.02 0.11 ± 0.04 0.16 ± 0.03 0.14 ± 0.03 0.36 ± 0.07 0.25 ± 0.05⁎ 0.35 ± 0.07 0.19 ± 0.05⁎ 0.28 ± 0.06 0.28 ± 0.05 0.38 ± 0.08 0.35 ± 0.13 0.31 ± 0.04 0.29 ± 0.06 0.33 ± 0.07 0.25 ± 0.06 The values presented are the fluorescence intensity per dendrite, in arbitrary units, within a circle 3.75 μm in radius, centered on the structure. Shown are mean ± SEM, ⁎p < 0.05 by one way ANOVA and the Bonferroni post hoc test, comparing the same structure over different treatments, n = 14 (Sph), 15 (GluR2) and 20 (NR1) images with 5 dendrites, bifurcations and DDIs and at least 3 BDIs in each, from 3 cultures. 36 BR A IN RE S EA RCH 1 2 51 ( 20 0 9 ) 3 0 –41 convergence of axons into the BDI vicinity. To distinguish between these two possibilities, we quantified the number and lengths of axonal segments within the same 3.75 μm radius from the center of the BDIs (Fig. 5A). Both the number of axons (Fig. 5B) and their lengths (Fig. 5C) were unchanged among all structures checked (mean ± SEM, p > 0.05 by one way ANOVA, n = 45 dendrites, 41 bifurcations, 48 DDIs, 14 BDIs in 3 images from 3 cultures). Thus, the increased number and fluorescence of synaptic terminals at BDIs probably resulted from local increase in synaptogenesis. 1.6. BDIs and its synaptic enrichment occur preferentially among dendritic segments of different cells BDIs are composed of dendrites originating in two different neurons. This was shown either by trasfecting cells with the green fluorescent protein (GFP) and followed by their immunofluoresence labeling with an antibody against GluR2 (Figs. 2A–C), or by staining with anti-MAP2 (Fig. 2B). We compared how often BDIs appear within the dendritic arbor of single neurons (GFP) or between dendritic arbors of different neurons visualized by MAP immunolabeling (Fig. 6B). We found that while the density of dendritic bifurcations (serving as a control) was not different, the occurrence of BDIs between dendritic branches of different neurons was significantly higher than within single dendritic trees (12-fold and 11-fold higher for DDIs and BDIs, respectively, mean ± STD, p < 0.001). Thus, dendritic contact structures occur almost exclusively between dendrites from different neurons. We then compared (Fig. 6C) the synaptic enrichment in each of the studied structures based on the number of cells involved; taking only the cases in which the identity of the Fig. 5 – Axons do not converge into BDIs. 12DIV cultures were immunostained for MAP2 and the axonal marker Neurofilament M. (A) A representative BDI with its surrounding axons. The green arrow points on an axon in fasciculation with one of the BDI's dendritic segments. The red arrow points on a non-fasciculating axon. (B) The average number of fasciculating and non-fasciculating axons found within a 3.75 μm radius from BDIs center, and the average number of dendritic segments fasciculated upon per BDI (mean ± SEM, p > 0.05 by one way ANOVA, n = 45 dendrites, 41 bifurcations, 48 DDIs and 14 BDIs in 3 images from 3 cultures). (C) The average lengths of the axons reported in (B) within the 3.75 μm radius (mean ± SEM, p > 0.05 by one way ANOVA, same n as in (B)). Scale bar: 2 μm for left panel, 4 μm for the right panels. BR A IN RE S E A RCH 1 2 51 ( 20 0 9 ) 3 0 –4 1 37 Fig. 6 – BDIs formation and their synaptic enrichment occur preferentially among dendrites of different cells: 12DIV cultures were immunostained for MAP2 and either synaptophysin, GluR2 or NR1. All fluorescence measurements were performed within a circle 3.75 μm in radius from the structure center. (A–C) Cultures were transfected with the green fluorescent protein to visualize dendritic trees of single cells (A, red arrows). This labeling outlined also axons (white arrowheads). The staining with anti-GluR2 revealed the dendritic arborization of all neurons in the filed (B). The yellow arrow indicates a BDI (arrowheads indicate two GFP-negative segments emerging from the bifurcation). The merger of (A) and (B) showed that the BDIs originated from a green segment of red bifurcation. (D) Contact structures could be comprised of dendrites from a single (yellow arrows) neuron or from two neurons (yellow and green arrows), or be considered unknown (arrowhead). (E) The vast majority of DDIs and BDIs involve dendrites from different neurons, with a small minority involving dendrites from the same neuron (mean ± STD, ***p < 0.001 by the Mann Whitney U test, n = 50 BDIs, 171 DDIs and 109 bifurcations in 4 images from 4 cultures for the multiple cells sample and 4BDIs, 37 DDIs and 229 bifurcations in 7 GFP expressing cells from 4 independent transfections for the single cell sample). (F) The synaptic enrichment per dendritic segment increases when DDIs and BDIs are hetero-neuronal structure (mean ± SEM, *p < 0.05, **p < 0.01 by the Wilcoxon signed ranks test, Sph: n = 10 pairs of DDIs and 8 pairs of BDIs), p = 0.036. GluR2: n = 13 pairs of DDIs and 16 pairs of BDIs, p = 0.01. NR1: n = 10 pairs of DDIs and 6 pairs of BDIs, p = 0.022). Both structures of a pair were always taken from the same image. Scale bar: 20 μm. neurons participating in each intersection was undisputedly clear (see Fig. 6A for example). We found significant increase in the fluorescence of BDIs composed from two dendritic trees than from only one, with all 3 synaptic markers, (mean ± SEM). Thus, neurons employ a mechanism of non-self recognition to increased synaptogenesis at dendro-dendritic contacts. 38 2. BR A IN RE S EA RCH 1 2 51 ( 20 0 9 ) 3 0 –41 Discussion Our results suggest that BDIs are likely to play a significant role in shaping dendritic trees and the pattern of the synaptic inputs. This role is evident from BDIs high frequency and stability, their non-random mechanism of formation their hetero-neuronal nature, and their association with enhanced synaptic formation and strength (see a summary in Fig. 7). 2.1. BDIs may associate between neighboring dendritic tree in terms of morphogenesis and synaptic patterning Our work demonstrates that interactions of a dendritic tree with its dendritic neighbors are non-random (Figs. 2A and E) and therefore should be included when attempting to model or explain dendritic trees morphogenesis. Our results imply that the pattern of branching in a dendritic tree is related to the pattern of contacts that this tree makes with adjacent trees of other neurons (see an example in Fig. 1B). A broader consequence of such a relation is that structural modification of a particular tree, due to growth or retraction, may be propagated to other trees and alter their structure via generation and disassembly of BDIs. Hence, the dynamic ramification of single and network of dendrites can be better understood by considering the pattern of their branching and hetero-neuronal contacts. A similar conclusion can be drawn for the direct correlation between hetero-neuronal contacts and distribution of synaptic contacts and strength (Fig. 6). Due to the clustering and Fig. 7 – Hetero-neuronal BDIs shape the architecture and synaptic map of cultured neuronal networks. (A) When formed between dendrites of different neurons, BDIs are stabilized, leading to synaptic synaptogenesis and synaptic strengthening. (B) Dendro-dendritic intersections cause a patchy distribution of synaptic connections and synaptic strength along the dendritic arbor. strengthening of synapses at BDIs, the patchiness and strength distribution of synaptic connections along a dendritic tree are sensitive to the pattern of BDIs. Since BDIs are composed of dendritic branches from two different neurons, formation or disappearance of BDIs from a single dendritic tree may affect its synaptic distribution but also those that were in contact with him. We have previously reported on another mean for dendritic trees to contact and affect synaptic distribution, by directing their processes toward intersections among other branches (Cove et al., 2006). By difference than BDIs, contact through intersections is not related to the pattern of dendritic branching. We postulate that the architecture of overlapping dendrites and their synaptic maps are an outcome of directed dendrodendritic contacts at both bifurcations and intersections. 2.2. Possible physiological role of synaptic clustering at BDIs Compared to more separated synaptic connections, the increased proximity and secretion levels among the strong clustered synaptic connections at BDIs may reduce the amplitude and increase the frequency of their firing (Liu and Tsien, 1995), promote their summation (Polsky et al., 2004), and increase dendritic activation coupling through glutamate spillover (Grebenyuk et al., 2004; Sargent et al., 2005), perhaps leading to synchronized activity of the participating neurons. 2.3. Possible activity-dependent and -independent mechanisms for BDI formation A mechanism based on heterogeneous adhesion molecule interactions (Hilschmann et al., 2001) could be employed for the hetero-neuronal contact at BDIs. Protocadherins for instance, are variable enough to allow recognition of individual neurons, provided these indeed take advantage of the genetic heterogeneity of these proteins (Hilschmann et al., 2001). Similarly, cadherins (Hirano et al., 2003) and integrins (Clegg et al., 2003) may contribute to the formation of sufficient diversity in the complement of adhesion molecules displayed by each neuron to confer self vs. target recognition at dendro-dendritic contacts. As mentioned in the introduction, the Dscam molecules which were recently implicated in self-avoidance in dendrites (Schmucker et al., 2000; Zhu et al., 2006) could serve for hetero-neuronal BDIs formation as well. BDIs may also be formed by a growth of a dendritic branch toward dendritic bifurcations due to gradients of attractants secreted at the bifurcation. The localized secretion may be possible via the accumulation of Golgi outposts at the branchpoints (Sytnyk et al., 2002; Horton et al., 2005). BDI densities are down-regulated upon blockade of AMPA receptors (Fig. 2E). However, the blockade caused only a partial inhibition of their formation and not a complete arrest. Moreover, we detected BDIs in 3 day old cultures, prior to the onset of synaptic activity (not shown), indicating that synaptic activity through AMPA receptors plays a regulatory, rather than inductive role in their formation. These results suggest that the initial morphology of the neuronal network in culture develops independently of synaptic activity; yet synaptic activity modifies the architecture of the network by stabilizing dendritic branches at BDIs. BR A IN RE S E A RCH 1 2 51 ( 20 0 9 ) 3 0 –4 1 Taken together the high frequency of hetero-neuronal contact at BDIs and its promotion by synaptic activity and the synaptic enrichment at these sites, we suggest that BDIs serve as a novel mechanism to associate dendritic architectures with synaptic activity. Being up-regulated by synaptic activity and associated with enrichment in synaptic density and strength, the association of dendritic branches with other dendrites at bifurcation sites affects the conversion of synaptic information into a map of synaptic connections and synaptic strength distributions. Accordingly, when neuronal network activity increases, the network architecture becomes more enriched with BDIs, leading to an increase in synaptic clustering and strength at these sites. This structuremediated, activity-dependent synaptic strengthening may serve as a novel structural-based mechanism of plasticity and thereby be involved in the mechanism of learning and memory. 3. Experimental procedures 3.1. Cell culture blocked with 3% normal goat serum. The cells were then incubated overnight at 4 °C with mouse anti-microtubule associated protein 2 (MAP2, 1 μg/ml, monoclonal, Sigma, Oakville, Ontario, Canada); rabbit anti-synaptophysin (0.5 μg/ml, polyclonal, DAKO, Mississauga, Ontario, Canada), rabbit anti-NR1 (1:600, polyclonal, Chemicon), anti-GluR1 (1:400, polyclonal, Chemicon). Cells were then washed and incubated for 1 h at room temperature with secondary antibodies conjugated to Alexa-488 or Cy3 (2 μg/ml) (Molecular Probes, Eugene, OR, USA), washed again and mounted on slides. 3.4. 3.2. Transfection of cells with GFP Cultures were transfected at 5–10 days in vitro as described (Kohrmann et al., 1999). Briefly, cells were washed and incubated for 45 min at 37° with warm MEM (Sigma) with 0.5% glucose. Each coverslip was then incubated for 30–40 min at 37° with 80 μl of DNA solution (5 μg DNA (pIRES2-EGFP, Clontech), 250 μl of 250 mM CaCl2 and 250 μl of BBS [in mM: NaCl 280, Na2HPO4 1.5, BES 50, pH 7.1]).until formation of heavy precipitate. Finally, cells were washed twice with warm HBS (in mM: NaCl 135, KCl 4, Na2HPO4 1, CaCl2 2, MgCl2 1, Glucose 10, Hepes 20, pH 7.35) and twice with warm MEM, then returned to their original growth medium. Cells were imaged 5–8 days after transfection. 3.3. Immunocytochemistry Cells were stained as described (Baranes et al., 1998). Briefly, cells were fixed for 10 min at room temperature with 4% paraformaldehyde (PFA), permeabilized with 0.25% Triton, and Light microscopy Imaging was performed on a Zeiss Axiovert 200M microscope. Objectives used were (all from Zeiss): Plan-Neofluar 20X/0.5 and Plan-Apochrome 63X/1.4. Images were captured with a 12MHz CCD camera (SensiCam, PCO, Kolheim, Germany) and an SK3 motorized stage (Marzhauser, Germany). Acquisition and analysis and image processing were performed with commercial software (Metamorph, Universal Imaging, USA, and PhotoShop 7.0, Adobe Systems Inc.). 3.5. Hippocampal Dentate Gyrus-CA3 were dissected from brains of P1-P4 Sprague Dawley rat pups, as described previously (Baranes et al., 1996). Briefly, the tissue was treated for 30 min at 37 °C with 0.25% trypsin (Sigma, type XI); dissociated gently and plated at a concentration of 2 × 105 cells/ml (hippocampus) onto 12 mm glass cover slips coated with poly-D-lysine (Sigma, 20 μg/ml) and laminin (Collaborative Research, 10 μg/ml). Cells were plated in MEM (Sigma) containing 10% heat inactivated normal goat serum, 1% L-glutamine and 0.8% D-glucose. One day after plating, cells were transferred to serum-free medium containing 45% MEM, 40% DMEM, 10% F12, 0.25% (w/v) BSA, 1% DiPorzio supplement, 0.34% D-glucose, 0.5% B27 supplement, 0.25% L-glutamine, 0.01% kinurenic acid, 0.01% of mixed 70% uridine and 30% fluoro-deoxy-uridine. For manipulation of synaptic activity, 1 μM TTX, 10 μM CNQX or 50 μM APV were applied for the final 24 h prior to immunostaining. 39 Simulation of random cell contact Simulations were performed using custom made Matlab based software, as described (Cove et al., 2006). Random cell contact was simulated by superpositioning images of single cells, as visualized in GFP expressing neurons, in random positions and orientations. Average cell densities in the simulation were set as equal to the average in corresponding images of cultures to which they were to be compared. The simulations were created from a data bank of 40 images of GFP expressing cells. 3.6. Definition and Quantification of BDIs The frequency of BDIs in hippocampal cultures were determined using 63X images of neurons immunostained for MAP2. BDIs were defined when a dendrite shared a common pixel with the bifurcation point of another dendrite (Fig. 1A). Cell bodies were identified by their MAP2 fluorescence. Only dendrites that reached a DDI or bifurcation directly without fasciculation were scored. Also, axons were morphologically excluded from the GFP images. For time lapse experiments, one phase contrast image was acquired per day. The coordinates of these images were saved to be returned to successively using a motorized stage. BDIs were identified in a post-hoc manner; their identity to those from previous days was determined by their position and the surrounding topology of the network. 3.7. Fluorescence measurements and FM1-43 labeling All fluorescence measurements were performed within a circle, 3.75 μm in radius, centered on the measured structure. This radius was chosen as sufficient to include the length of a dendritic spine and axonal bouton, without including fluorescence from synapses further away from the structure center. Uptake and secretion of FM1-43 were monitored as described by (Betz and Bewick, 1992). Briefly, 12-17DIV cultures were exposed for 30 s to 15 μM FM1-43 (Molecular Probes) in 40 BR A IN RE S EA RCH 1 2 51 ( 20 0 9 ) 3 0 –41 Tyrode's buffer (in mM: NaCl 119; KCl 5, CaCl2 4, MgCl2, 2; glucose 30, HEPES 20; pH. 7.3), supplemented with 45 mM K+ followed by a 3-min wash with Tyrode's buffer at a rate of 1 ml/min. Two to four randomly selected images per coverslip were acquired. FM1-43 was then secreted in response to 45 mM K+ in Tyrode's buffer for 30 s. followed by a 3-min wash with Tyrode's buffer at a rate of 1 ml/min. Images of the same fields were obtained, and then immunostained with antiMAP2 antibodies. Images acquired after the secretion of FM1-43 were subtracted from those obtained after uptake of FM1-43. The result was considered proportionate to the size of the releasable vesicle pool. These images were analyzed in the same manner as the synaptophysin images described in the previous section. Boutons were estimated by setting a fluorescence threshold within each circle region (as described above). The threshold was used to measure the integrated fluorescence within each object individually, assuming that objects correlated with boutons. To improve the accuracy of the bouton count, objects more than twice the average bouton area were split by dividing them by the average bouton area. 3.8. Statistics All data sets were tested for normal distribution using the Kolmogorov-Smirnov test, in comparison to the Lilliefors distribution, to determine whether to use parametric or nonparametric tests. All tests were two-tailed and examined at a significance level of α= 0.05. 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