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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
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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.
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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
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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).
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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.
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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.
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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.
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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.
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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. Data are presented as mean ±SEM or
STD, as for each case. In Fig. 6C the analysis included only cases
in which a pair of structures were from the same image and were
composed of an undisputedly clear number of neurons. Correspondingly, the Wilcoxon signed ranks test was used for it. p
values for multiple comparison tests including many comparisons are shown as ⁎ for p< 0.05, ⁎⁎ for p <0.01, and ⁎⁎⁎ for p <0.001,
with the appropriate test indicated for each data set in the text.
The details of statistical values are described in the figure
legends.
Acknowledgments
We would like to thank Drs. Elia Abi Jaoude and Myriam
Lafrenier-Roula for performing the initial experiments. This
project was supported by the Canada Foundation of Innovation, and partially by the Horowitz foundation.
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