3
CHAPTER 3
Published in the Journal of Cell Biology
197:327-337, 2012.
63
3
64
Chapter 3
Dendritic position is a major determinant of
presynaptic strength
Arthur PH de Jong1, Sabine K Schmitz1, Ruud FG Toonen1 & Matthijs Verhage1,2
1
Department of Functional Genomics
of Clinical Genetics, Center for Neurogenomics and Cognitive Research,
Neuroscience Campus Amsterdam, VU University and VU Medical Center, Amsterdam, the
Netherlands
2Department
Abstract
Different regulatory principles influence synaptic coupling between neurons, including
positional principles. In dendrites of pyramidal neurons, postsynaptic sensitivity depends
on synapse location, with distal synapses having the highest gain. Here, we investigated
if similar rules exist for presynaptic terminals in mixed networks of pyramidal and dentate
gyrus neurons. Unexpectedly, distal synapses had the lowest staining intensities for
vesicular proteins vGlut, vGAT, Synaptotagmin and VAMP and for many non-vesicular
proteins, including Bassoon, Munc18 and Syntaxin. Concomitantly, distal synapses
displayed less vesicle release upon stimulation. This dependence of presynaptic strength
on dendritic position persisted after chronically blocking action potential firing and
postsynaptic receptors but was markedly reduced on dentate gyrus dendrites compared
to pyramidal dendrites. These data reveal a novel rule, independent of neuronal activity,
that regulates presynaptic strength according to dendritic position, with the strongest
terminals closest to the soma. This gradient is opposite to postsynaptic gradients
observed in pyramidal dendrites and different cell types apply this rule to a different
extent.
65
3
3
Introduction
Presynaptic terminals in the central nervous system, even those formed by a single
neuron, form a highly heterogeneous population in terms of both structure and function.
Ultrastructural analysis of hippocampal synapses revealed a wide range in the number
of synaptic vesicles (40-800 per terminal) and the number of morphologically docked
vesicles (0-15 per terminal) [1-3]. A similar variation was observed using functional
readouts, including synaptic release probability (pr) [4-7] and the number of releaseready vesicles (the readily releasable vesicle pool, RRP) [2,5]. The pr can change rapidly
over time as a result of repetitive stimulation (i.e., short-term plasticity, STP). The
direction of change (facilitation or depression) and the extent of STP depend to a large
extend on the initial pr and the RRP size, and is therefore synapse-specific as well [6,8].
Indeed, presynaptic terminals from a single neuron express different types of STP [9-11].
In addition, each terminal expresses a unique range of presynaptic receptors. Over 70
presynaptic receptors have been described so far, and most of these receptors modulate
pr and STP, via the activation of intracellular signal transduction pathways (see [12]).
These forms of synaptic heterogeneity largely increase the computational power of a
neuronal network, as every terminal acts as an independent information-processing unit
(reviewed in [13,14]).
Figure 3.1 (opposite page). Synapse location determines the amount of vesicles per
terminal. A. Experimental design. A postsynaptic neuron (post, blue) receives presynaptic input
from multiple surrounding presynaptic neurons (pre, grey). The vGlut staining intensity is used
as a measure for the number of vesicles per presynaptic terminal (green). Note that a single
presynaptic neuron may form multiple synapses along the dendritic tree. B. Typical example of
a proximal and distal dendrite stained for vGlut (green) and MAP (blue). Scale bare represents
5 mm. C. Example of an image used for analysis. Scale bare represents 20 mm. D. Analysis of
the image in C. The detected neurites are shown in grey. Neurites from neighbouring neurons
are omitted from the analysis. The dots represent detected synapses, with their vGlut intensity
color coded. The intensity was normalized to the most intense synapse detected in this neuron.
The size of the dots does not represent the surface area of the detected synapse. E. Distance
mask of the neuron in C, used for distance measurements of the detected synapses. Distance
was measured along the MAP staining, and thus takes into account the morphology of the
dendritic tree. F-H. Different methods to plot the results of the analysis in B-D. F. Relationship
between distance from soma and vGlut intensity. Each dot represents an individual synapse,
solid line a linear fit through the data. Inset displays the Spearman correlation coefficient (r)
and significance (p). G. Same data as in F, averaged in bins of 20 mm. Error bars represent SEM.
H. Cumulative histograms of the data in F, in bins of 20 mm. The color of each line indicates
the distance from soma. Legend also applies to panel K and M. P/D is the ratio of proximal
(<50 mm) over distal (last 50 mm). I-K. Results from the complete vGlut data set (n=68 cells,
336.620 synapses). I. Relationship between distance from soma and vGlut intensity. Each dot
represents an individual synapse, solid line a linear fit through the data. J. Same data as in
I, averaged in bins of 20 mm. Error bars are too small to be seen. K. Cumulative histograms
of the data in I. P value is the significance of the P/D ratio (one sample Wilcoxon rank test).
L. Histogram of the slopes of linear line fits through data of individual cells. M. Cumulative
histograms of synapse surface area.
66
B
pre
proximal
proximal
distal
pre
merge
MAP
post
distal
vGlut
A
pre
C
D
E
3
distance (µm)
300
intensity (norm.)
1
0.5
MAP vGlut
G
0.4
0.6
0.4
0.2
0.3
0.2
detection limit
0.1
0
0
100
200
distance from soma (µm)
0
300
I
50
100 150 200 250
distance from soma (µ m)
J
1
0.4
0.2
0
0.3
0.2
detection limit
0.1
0
0
100
200
distance from soma (µm)
300
0
50
100
150
200
distance from soma (µ m)
250
M
cumulative probability
L
# cells
5
0
-15
-5
- 10
slope (AU/µm)
0
0.5
140 >260
P/D = 1.55
n(synapses) = 496
0
0
0.5
vGlut intensity
1
1
0.4
vGlut intensity
0.6
distance (µm):
0
K
0.5
r = -0.34
p < 0.001
0.8
cumulative probability
0.8
0
vGlut intensity
1
r = -0.37
p < 0.001
vGlut ntensity
vGlut intensity
1
H
cumulative probability
F
150
0
0
1
0.5
P/D = 1.26±0.05
p < 0.001
0
0
0.5
normalized vGlut area
67
1
P/D = 1.45 ± 0.04
p <0.001
N=5
n(cells) = 68
n(synapses) = 36620
0.5
0
0
0.5
vGlut intensity
1
Over the recent years it has become increasingly clear that many of these presynaptic
characteristics depend on the dendrite on which the terminal is formed. In hippocampal
neurons in vitro, neighboring synapses have similar pr [6,15], which is thought to be
specific for a dendritic branch [4]. In slices, pr and STP depend on the identity of the
postsynaptic cell [9-11]. Furthermore, the expression of presynaptic receptors depends
on synapse location, both in slices [11,16,17] and in culture [18].
3
The postsynaptic gain, in turn, is largely dependent on the dendritic position of the
synapse. In hippocampal pyramidal cells, distal synapses express an increased number
of AMPA receptors, leading to larger local postsynaptic currents [19,20]. In addition,
synchronous input on distal synapses of hippocampal and cortical pyramidal cells can
be amplified by supralinear summation of excitatory postsynaptic synaptic potentials
(EPSPs) and the initiation of dendritic calcium spikes [21,22]. Dendritic location is therefore
thought to be of crucial importance for the integration of synaptic input (reviewed by
[23]). It can be predicted that presynaptic terminals contribute to this amplification by
the formation of stronger presynaptic inputs at more distal dendritic positions.
In the current study, we investigated the general relationship between presynaptic
strength and the dendritic position of the synapse. Surprisingly, we did not observe the
expected increase in presynaptic strength with increasing distance from the postsynaptic
soma. In contrast, we found that in both inhibitory and excitatory synapses, the number
of synaptic vesicles and the local concentration of presynaptic proteins are highest at
synapses formed close to the soma. This clearly affected vesicle release, since proximal
synapses had a larger RRP. Strikingly, this distance-dependent scaling of presynaptic
strength was insensitive to manipulations of neuronal activity, and was dependent on
the identity of the postsynaptic neuron. These results reveal a novel, cell-wide rule that
determines the strength of presynaptic input along the dendritic tree. We believe that
this will have profound implications for the integration of neuronal activity. Results
The number of synaptic vesicles depends on synapse location
We analyzed the amount of vesicles per presynaptic terminal as a function of its location
on the dendrite relative to the soma. Cultured hippocampal neurons were stained with
antibodies against the vesicular glutamate transporter vGlut (Figure 3.1A-C). We consider
vGlut intensity a reliable measure for the relative amount of synaptic vesicles, since the
number of vGlut copies per vesicle is tightly controlled [24]. Furthermore, only ~1% vGlut
Figure 3.2 (opposite page). Levels of proteins involved in secretion depend on synapse location. A. Example images of hippocampal network cultures stained for dendrite marker MAP,
and presynaptic proteins. Scale bars represent 20 mm. B. Cumulative histograms of the synaptic intensity of proteins involved in vesicle release. Synapses were grouped based on their distance from the postsynaptic soma. All intensities were normalized to the largest value per cell.
P/D is the ratio of proximal (<50 mm) over distal (last 50 mm), p value is the significance of the
P/D ratio. N represents the number of independent experiments. C. Surface area of vGAT and
VAMP puncta. Abbreviations: BSN: Bassoon, M18: Munc18, n.s.: not significant, Syn: Synapsin,
Syt: Synaptotagmin, Syx: Syntaxin.
68
Syx
Syt
Merge MAP
Merge MAP
MAP
Merge
Syn
MAP
Merge
3
P/D = 1.48±0.05
p < 0.001
N=3
n (cells) = 56
n (synapses) = 25082
0.5
0
0
0.5
normalized intensity
1
0
0
0.5
normalized intensity
1
1 Munc18
P/D = 1.50±0.05
p < 0.001
N=2
n (cells) = 30
n (synapses) = 18075
0.5
0
0
0.5
normalized intensity
1
cumulative probability
P/D = 1.23±0.02
p < 0.001
N=5
n (cells) = 130
n (synapses) = 72669
0.5
1 Synapsin
P/D = 1.11±0.03
p < 0.001
N=3
n (cells) = 44
n (synapses) = 20533
0.5
0
0
0.5
normalized intensity
1
1 Syt
P/D = 1.48±0.05
p < 0.001
N=2
n (cells) = 32
n (synapses) = 18156
0.5
0
0
0.5
normalized intensity
1
1
distance (µm):
0.5
P/D = 1.56±0.06
p < 0.001
N=4
n (cells) = 75
n (synapses) = 27922
0
0
0.5
normalized intensity
P/D = 1.20±0.03
p < 0.001
N=5
n (cells) = 130
n (synapses) = 72669
0.5
0
0
0.5
normalized area
140
>280
1
1
VAMP
0
1
1 vGAT
P/D = 1.71±0.1
p < 0.001
N=4
n (cells) = 75
n (synapses) = 27922
0.5
0
0
0.5
normalized area
69
1
cumulative probability
cumulative probability
1
1 Syntaxin
vGAT
C
0.5
normalized intensity
1 VAMP2
cumulative probability
0
cumulative probability
P/D = 1.93±0.22
p < 0.001
N=4
n (cells) = 64
n (synapses) = 33252
0.5
cumulative probability
1 Bassoon
cumulative probability
cumulative probability
cumulative probability
B
cumulative probability
Merge
MAP vGlut
BSN
vGAT
VAMP
Merge MAP
M18
VAMP
VAMP
A
1 Synapsin
P/D = 1.06±0.04
(n.s.)
N=3
n (cells) = 44
n (synapses) = 20533
0.5
0
0
0.5
normalized area
1
is expressed at the cell surface [25]. For every synapse we measured the vGlut intensity
and its distance from the postsynaptic soma along the dendritic marker MAP (Figure 3.1CE). Surprisingly, we found a near perfect gradient of vGlut levels, with proximal synapses
having the highest intensity (Figure 3.1F-K). The median normalized intensity dropped
from 0.50 at the soma to 0.24 at 250 mm from the soma. Note that considerable variation
exists in intensity, even among synapses at similar distance, but that high intensity
synapses are more likely to be found close to the soma (Figure 3.1I-K). However, even
when binned and shown for individual neurons, a clear relation between distance and
intensity of presynaptic staining was observed (Figure 3.1F-H).
3
To test to what extend the distance dependence of staining intensity holds for every
individual neuron, we made linear line fits through the data of each neuron. In 66 of
the 68 cells tested, this fit had a negative slope, indicating that all but two cells showed
decreased vGlut intensity at distal sites (Figure 3.1L). Figure 3.1L also shows that some
heterogeneity probably exists on either side of the median among different cell types
in a mixed culture (Figure 3.1L and see below). To further quantify the effect per cell,
we calculated the ratio of proximal (<50 mm) over distal (last 50 mm) per cell. This P/D
ratio was 1.45 ± 0.04, p < 0.001 (one sample Wilcoxon rank test), again indicating that
individual cells display distance-dependent decline in vGlut intensity. The differences in
surface area of vGlut puncta between proximal and distal terminals were much smaller,
but still significant (median normalized area at the soma: 0.25, at 250 mm: 0.21, Figure
3.1M). It should be noted that, due to the lower vGlut intensity at distal synapses, the area
measurements at distal terminals is less precise. These results suggest that the number
of vesicles per terminal, and to a lesser extend the physical dimensions of the terminal,
depend on the dendritic position of the synapse.
To test if the observed gradients reflect a certain developmental stage rather than a stable
state, we compared sister cultures fixed at 14 or 21 days in vitro (DIV). Despite significant
differences in average synapse area and synapse density, we did not find any difference
in the distance dependency of vGlut intensity of synapse area (Supplemental Figure 3.1).
Thus, the distance dependency of vGlut intensity is stable once mature synapses are
formed.
Protein content of presynaptic terminals depends on synapse location
Next, we tested the distance dependency of the levels of a range of other presynaptic
proteins with immunocytochemistry (Figure 3.2). Synapses were detected with vGlut,
synapsin, vGAT or VAMP, and the detected regions were subsequently used to quantify
the synaptic levels of other proteins. We observed a distance-dependent decline in the
levels of the vesicular GABA transporter vGAT, similar to vGlut (Figure 3.2B). This suggests
that the number of vesicles of excitatory and inhibitory synapses is equally dependent
on synapse location. Furthermore, similar gradients were found for the presynaptic
scaffolding protein Bassoon, and for Munc18, VAMP, Synaptotagmin-1 and Syntaxin,
which are core proteins of the vesicle release machinery [26] (Figure 3.2A and B). Similar
to our results with vGlut stainings (Figure 3.1), we found only a small drop in surface area
at more distal synapses for terminals detected with vGAT, Synapsin or VAMP (Figure 3.2C).
70
A
B
40AP 20Hz
pre
100 AU
1
post
pre: SypHy+
post: alexa filled
2
2s
C
3
3
D
3
1
2
1
0
F
n = 17 cells,
1513 synapses
N=4
60
40
20
0
0
0.5
normalized RRP size
1
cumulative probability
E
number of synapses
SypHy Alexa
1
P/D = 1.55±0.19
p< 0.01
0.5
distance (µm):
0
0
0
80
0.5
normalized RRP size
>160
1
Figure 3.3. RRP size depends on synapse location. A. Experimental design. B. Example
traces of synaptic SypHy fluorescence upon stimulation to release the RRP. C. Example
image of an Alexa-filled neuron receiving synaptic input from a SypHy+ cell. SypHy image
was taken in the presence of 50 mM NH4+. Scale bar represents 20 mm. D. Neurite mask of
the neuron in C (grey) with all SypHy+ synapses formed on this neuron. RRP size colorcoded. Arrows indicate synapses from which the traces in B were obtained. E. Histogram
of RRP size. F. Cumulative histogram of the RRP sizes in E, grouped on synapse location.
71
Hence, the number of vesicles and the abundance of the release machinery in a terminal,
depend on its dendritic location.
RRP size depends on synapse location
Next, we tested whether the correlation between protein levels and synapse position
translates to functional differences in synaptic release properties. We transfected a
fraction of the neurons in the network with SypHy, a fluorescent reporter of synaptic
vesicle release [27]. We made electrophysiological recordings of a pair of neurons, of
which one was SypHy transfected (which we refer to as the presynaptic cell), and one
connected, untransfected cell (referred to as the postsynaptic cell). The postsynaptic cell
DNQX - AP5
TTX
vGlut
MAP
control
500
0 73 68 68
D
*
1
0.15
0.1
0.05
1.5
0.4
1
0.5
0.3
0
**
0
100
200
distance from soma (µm)
0.5
0
0
F
0.5
0.2
area (µm2 )
C
G
slope (AU/µm)
**
P/D ratio
E
normalized intensity
control
DNQX-AP5
TTX
1000
no. synapses /µm
B
vGlut intensity (AU)
Merge
3
A
0
-0.5
-1.0
-1.5
-2.0
Figure 3.4. Neuronal activity does not affect distance-dependent scaling of vGlut intensity.
A. Example images of neurons treated for 7 days with DMSO (control), 10 mM DNQX & 50 mM
AP5 (DNQX-AP5) or 2 mM TTX. Scale bar represents 20 mm. B. Average synaptic vGlut intensity.
C. Synapse density (number of synapses per mm dendrite). D. Surface area of vGlut puncta.
E. Distance dependency of vGlut after DNQX-AP5, TTX or control. Solid lines represent the
median normalized intensity, shaded area the 0.4-0.6 boundaries (See also Supplemental
Figure 3.1). Control: n=73, 34724 synapses; DNQX-AP5: n=68, 36723 synapses; TTX: n=68,
27417 synapses, N=3. * P<0.05, ** P<0.01, compared to control.
72
VAMP
M18
VAMP
GFP
merge
GFP Synapsin BSN
3
merge
merge
GFP
merge
GFP Synapsin RIM
Rab3
A
0
0.5
normalized intensity
1
1 Munc18
P/D = 1.64±0.09
p < 0.001
N=2
n (cells) = 37
n (synapses) = 20860
0.5
0
0
0.5
normalized intensity
1
0.5
0
P/D = 1.51±0.06
p < 0.001
N=4
n (cells) = 64
n (synapses) = 32664
0
0.5
normalized intensity
1
1 Rab3
0.5
0
P/D = 1.38±0.06
p < 0.001
N=2
n (cells) = 43
n (synapses) = 15300
0
0.5
normalized intensity
1
cumulative probability
0
P/D = 1.42±0.04
p < 0.001
N=5
n (cells) = 119
n (synapses) = 51962
1 Synapsin
cumulative probability
0.5
cumulative probability
1 VAMP
cumulative probability
cumulative probability
cumulative probability
B
1 Bassoon
0.5
0
1
P/D = 1.68±0.13
p < 0.001
N=2
n (cells) = 35
n (synapses) = 16847
0
P/D = 1.88±0.27
p < 0.001
N=2
n (cells) = 22
n (synapses) = 11410
0
0.5
normalized intensity
VAMP
0.5
0
P/D = 1.34±0.05
p<0.001
N=5
n (cells) = 119
n (synapses) = 51962
0
0.5
normalized area
1
cumulative probability
cumulative probability
C
1
1 Synapsin
distance (µm):
0.5
0
P/D = 1.37±0.05
p<0.001
N=4
n (cells) = 64
n (synapses) = 32664
0
0.5
normalized area
73
1
1
RIM
0.5
0
0.5
normalized intensity
0
140
>260
1
was filled with Alexa-468 via the recording pipette to observe its morphology (Figure
3.3A and C). To determine the size of the RRP, we stimulated the presynaptic cell with 40
action potentials (AP) at 20 Hz (a stimulation protocol that is routinely used to probe the
RRP [3,5,28]), and measured the increase in fluorescence in every terminal in the field of
view (Figure 3.2B). The cells where then fixed, and analyzed at a confocal microscope to
identify the terminals formed on the postsynaptic cell (Figure 3.3D). The RRP size of these
synapses followed a skewed distribution (Figure 3.3E), in good agreement with previous
studies [5] [6].
3
In line with our data on presynaptic protein content, we found that proximal synapses
had a larger RRP than distal synapses (Figure 3.3F). The median normalized RRP size
ranged from 0.39 for synapses at the soma to 0.19 for synapses formed at >160 mm from
the soma. The P/D ratio per cell was 1.55 ± 0.19 (p < 0.01). Furthermore, application of
50 mM NH4+ (which de-quenches SypHy in unreleased vesicles [29]) confirmed the
relationship between number of vesicles and synapse position that we observed in fixed
neurons (S/D ratio = 1.15 ± 0.18, p < 0.01, Supplemental Figure 3.2). These results indicate
that the increased vesicle number and release machinery abundance translates into
larger releasable vesicle pools in nerve terminals at proximal dendritic positions.
Distance dependency of presynaptic strength is independent of network activity
Previous studies have shown that neuronal activity fine-tunes synaptic strength
[2,5,30,31] and, for synapses onto single dendritic branches, reduces the heterogeneity
in pr [4]. Furthermore, manipulations of network activity alter synaptic protein levels
[5,32]. The distance-dependent scaling of presynaptic strength could be caused by
local differences in dendritic activity, which causes homeostatic adaptations of pr [4].
We therefore tested the effect of blocking AP firing with TTX, or blockage of excitatory
synaptic transmission with DNQX & AP5 on distance-dependent scaling of presynaptic
terminals. TTX or DNQX & AP5 were added at DIV 7, and samples were fixed at DIV 14
(Figure 3.4A). Both treatments had a substantial effect on average synapse size and
vesicle content (Figure 3.4B-D), as observed before [2,4,32], but the correlation between
vGlut intensity and synapse position remained (Figure 3.4E). In line with this observation,
we did not find any significant differences in the P/D ratio per cell (p = 0.81, Figure 3.4F),
or in the slope of the linear line fit per cell (p = 0.94, Figure 3.4G).
To further test the effect of network activity, we measured the distance dependency
of presynaptic protein levels in autaptic islands. These neurons are grown in complete
Figure 3.5 (previous page). Autaptic islands display distance-dependent decline in
presynaptic protein levels. Scale bars represent 20 mm. A. Example images of hippocampal
autaptic cultures stained for synapse marker Synapsin or VAMP and additional synaptic
proteins. GFP expression (delivered with lenti virus) was used as dendritic marker. B. Cumulative
histograms of the synaptic intensity of proteins involved in vesicle release. Synapses were
grouped based on their distance from the postsynaptic soma. All intensities were normalized
to the largest value per cell. P/D is the ratio of proximal (<50 mm) over distal (last 50 mm), p
value is the significance of the P/D ratio. N represents the number of independent experiments.
C. Surface area of synapsin and VAMP puncta. Abbreviations: BSN: Bassoon; M18: Munc18.
74
non DG
A
distal
merge vGlut
MAP
proximal
DG
distal
3
distance (µm):
0
140 >260
P/D = 1.26±0.03
p < 0.001
N=4
n (cells) = 73
n (synapses) = 29550
0.5
0
0
0.4
0.5
normalized intensity
1
1 DG
0.5
0
E
non DG
DG
0.35
0.3
0.25
0
0.5
normalized intensity
**
1
0.5
0
0
100
200
distance from soma (µm)
P/D = 1.15±0.06
p < 0.001
N=4
n (cells) = 77
n (synapses) = 19148
F
1
0
slope (AU/µm)
C
non DG
cumulative probability
1
P/D ratio
D
normalized intensity
B
cumulative probability
merge vGlut
MAP
proximal
-0.5
-1.0
**
-1.5
Figure 3.6. Strongly reduced distance-dependent scaling in DG cells. A. Example images of
proximal and distal synapses in DG and non DG cells. Scale bars represent 5 mm. B. Cumulative
histograms of normalized vGlut intensity of non DG cells. vGlut intensity was normalized to the
largest synapse per cell. P/D is the ratio of proximal (<50 mm) over distal (last 50 mm), p value
is the significance of the P/D ratio. N represents the number of independent experiments. C. As
B, for DG cells. D. Comparison of the histograms in B and C. Solid line represents the median,
shaded area the 0.4 – 0.6 boundaries. E. P/D ratio of vGlut intensity of DG and non-DG cells. F.
Average slopes of linear line fits through data from individual neurons. ** p < 0.01.
75
isolation, and thus are devoid of network activity [33]. Furthermore, since all presynaptic
terminals on an island are formed by the same axon, every synapse is expected to receive
a similar activity pattern. This type of cultures is routinely used to study presynaptic
function (see for instance [6,34,35]). In these autaptic cultures, we found distancedependent gradients for all presynaptic proteins tested, both vesicular and non-vesicular,
in a similar fashion as observed in network cultures (Figure 3.5). Thus, the dependence of
vesicle number and secretion machinery abundance on dendritic position is independent
of synaptic activity or differences in local postsynaptic current amplitude.
3
Protein levels are not correlated in individual synapse
If the local concentration of release machinery proteins is indicative for presynaptic
strength, it could be predicted that their levels are correlated at the level of individual
synapses. We therefore tested whether such a correlation exists within the co-stainings
described above. As can be expected, the levels of vesicular proteins showed a good
correlation: Spearman correlation coefficient (r) of VAMP vs. Synaptotagmin = 0.59; VAMP
vs. Rab3 r = 0.74. Furthermore, Bassoon intensities correlated well with Synapsin (r =
0.63) and vGlut (r = 0.61), while RIM and Synapsin showed weaker correlation (r = 0.46).
However, VAMP levels did almost no correlation with either Munc18 (r = 0.29) or Syntaxin
(r = 0.22). These results show that although these proteins display a distance-dependent
effect on the population level, differences between individual proteins of the release
machinery exist in individual synapses.
Distance-dependent scaling is strongly reduced in dentate gyrus granule cells
Our data suggest that the previously observed increased postsynaptic gain at distal
synapses [19,22] is counterbalanced by weaker presynaptic inputs. Interestingly, it
was shown that dendrites of granule cells in the dentate gyrus (DG cells) integrate
synaptic input independent of synapse location [36]. We hypothesized that if distancedependent scaling of presynaptic strength is important for the integration of synaptic
input, pyramidal and dentate gyrus cells might also scale their synaptic input differently.
Previous work has shown that hippocampal neurons retain their morphological and
functional characteristics in culture [37-39]. DG cells can be identified in culture using
antibodies against the transcription factor Prox1; the remaining cell population (nonDG cells) consists of pyramidal cells from the cornu ammonis and a small fraction of
interneurons [39,40]. We confirmed the specificity of Prox1 by morphological analysis of
DG and non-DG cells. Comparable to in vivo [41-43], DG cells in culture had a smaller
soma and a smaller and less complex dendritic tree (Supplemental Figure 3.3A-H). We
then quantified the vGlut intensity to measure distance-dependent scaling in both cell
populations (Figure 3.6). In non-DG cells, we found a strong reduction of vGlut intensity
in more distal synapses (normalized median intensity at the soma: 0.39, at 260 mm: 0.26),
consistent with our previous experiments (Figure 3.6B). Strikingly, distance-dependent
scaling was strongly reduced in DG cells (normalized intensity at the soma: 0.39, at 260 mm:
0.31; Figure 3.6C-D). Even though proximal synapses were still slightly more intense than
distal synapses (P/D ratio = 1.15 ± 0.06, p <0.001), this was significantly smaller compared
to non-DG cells (p < 0.01, Figure 3.6E). To further test this, we made line fits through the
data of individual neurons. The slope of this fits was significantly smaller in DG cells (p <
0.01, Figure 3.6F), again indicating that distance-dependent scaling is reduced in these
76
cells. The difference between both groups was even more pronounced when comparing
absolute vGlut intensities (median intensity soma vs. distal: 706.6 vs. 409.1 AU (non-DG);
518.1 vs. 690.8 (DG), Supplemental Figure 3.3I-J). These results suggest that populations
of neurons that display different dendritic integration principles also have different rules
for distance-dependent scaling of presynaptic input.
Discussion
It is well established that the number of vesicles, release machinery abundance and pr can
vary widely between presynaptic terminals [3,6,32]. Since the regulation of presynaptic
efficacy is a crucial element in neuronal information processing and memory formation
[14], the strength of presynaptic terminals is expected to be dictated by tight rules. In
cortical and hippocampal pyramidal neurons, the local amplitude and integration rules
of postsynaptic currents are controlled by synapse location on the dendrite, with distal
synapses having the highest gain [19,22]. We predicted a similar rule for the strength of
presynaptic input, to further amplify the signal of distal synapses. Surprisingly, we found
the exact opposite.
Presynaptic strength depends on the location of the synapse
The synaptic levels of the vesicular proteins vGlut, vGAT, Synaptotagmin and VAMP
decreased with increasing distance from the postsynaptic soma (Figure 3.1 & 2), from
which we conclude that distal synapses have less vesicles. If vesicles at distal synapses
would contain fewer copies of these proteins, that might also contribute to this distance
dependent differences in staining intensity. However, this is unlikely, since synaptic
vesicles actively and rapidly mix between different presynaptic terminals via axonal
transport [1,44,45] and single vesicle quantification of vesicle content showed that the
number of vGlut and Synaptotagmin molecules per vesicle shows little variation [24]. In
addition to differences in the number of vesicles per terminal, we found similar distancedependent differences in the local concentration of non-vesicular proteins essential for
synaptic vesicle release (Figure 3.2 and 4).
At the level of individual synapses, we observed moderate correlations in the levels of
various vesicular proteins, and of scaffold proteins and vesicular markers. However, the
levels of other release-machinery proteins did not correlate well. Thus, although the
concentration of these proteins show a distance-dependent effect on the population
level, large differences between individual proteins do occur in single synapses. In most
experiments described here, presynaptic terminals on a given dendrite originate from
several neighboring neurons. As each presynaptic neuron has its independent gene
expression program, the relationship between the expression of different presynaptic
proteins is expected to be different in different subsets of presynaptic terminals.
Furthermore, recent studies showed that in response to neuronal activity, synaptic levels
of specific proteins are regulated, while other proteins are unaffected [32,46]. For these
reasons, the levels of various presynaptic proteins are not expected to show strong
correlations.
The distance-dependent regulation of the number of vesicles and release-machinery
proteins probably account for the distance dependency of the RRP size (Figure 3.3).
77
3
Indeed, alterations in Munc18 and Bassoon levels are known to correlate with changes
in RRP size [5,35]. Since the size of the RRP is a primary determinant of pr and STP [6,47],
these differences in RRP size will have a profound effect on vesicle release during neuronal
activity.
3
The data described above seem in conflict with previous studies, which did not find a
relationship between pr or RRP size and distance from the postsynaptic soma [4,20]. Our
results display a broad heterogeneity in RRP size, vesicle number and protein levels, even
between synapses with a similar distance from the soma (Figure 3.1-3). This variation
will make a distance-dependent effect on presynaptic strength hard to detect using
low throughput methods. In our SypHy experiments we were able to measure the RRP
size of 1513 synapses from 17 cells. With this relatively large number of synapses per cell
we observed a clear distance-dependent decline in RRP size (Figure 3.3). The apparent
discrepancy between previous studies and our results might thus be due to an increased
power of the methods used here. Our results also clearly illustrate that, besides synapse
position, other factors are involved in the regulation of presynaptic strength. The
identity and activity of the presynaptic cell, as well the activation of intracellular signal
transduction pathways are factors known to be involved in the regulation of presynaptic
vesicle release [48,49]. In a recent study, it was found that terminals formed by the distal
part of the axon were stronger compared to proximal synapses, suggesting presynaptic
strength might also depend on the axonal position of a terminal [50]. Taken together, the
observed pr of a given synapse is the result of all the determinants mentioned above, and
cannot be solely attributed to a single factor.
homeostatic
plasticity
proximal
distal
distance-dependent scaling
Figure 3.7. Pre- and postsynaptic strength are determined by several independent
mechanisms. Chronic changes in synaptic activity lead to compensatory adaptations
in synaptic strength (homeostatic plasticity). These mechanisms act locally, at the level
of individual synapses. On top of this, the strength of the pre- and postsynapse is scaled
in a cell-wide manner, based on the synapse’s position on the dendritic tree (distancedependent scaling). Both mechanisms act in parallel, most likely via independent molecular
mechanisms. The distance-dependent scaling is cell-type specific, and may act in conjunction
with the integration rules of the cell’s dendritic tree.
78
Distance-dependent scaling is dictated by the postsynaptic cell
The synapses studied here are formed by axons from several neighboring neurons.
Thus, each axon is capable of determining the location of a synapse on the dendritic
tree, and to set its presynaptic strength accordingly. It is therefore most likely that the
postsynaptic cell dictates the distance-dependent scaling of presynaptic strength,
by indicating the position of the synapse on the dendrite via retrograde messenger
or cell adhesion molecules. In contrast to known forms of homeostatic plasticity
[30,31], distance-dependent scaling is independent of neuronal activity (Figures 4 & 5).
Therefore, this retrograde signaling to tune presynaptic strength is most likely distinct
from the previously documented, activity dependent signaling pathways (reviewed
by [51]). We postulate that in parallel to local homeostatic mechanisms, presynaptic
strength is determined by cell-wide, distance-dependent scaling, which is controlled
by the postsynaptic cell (Figure 3.7). Distance-dependent scaling might act as a global,
default rule, which is stable over time and insensitive to activity in the neuronal network.
Homeostatic mechanisms, in turn, respond to changes in neuronal activity, and tune
individual synapses based on their synaptic use. Together, distance-dependent and
homeostatic mechanisms ensure the proper integration of synaptic input with optimal
use of metabolic resources [13].
Distance-dependent scaling is cell-type specific
Our results indicate that the gradient of presynaptic strength is the opposite of the
previously observed distance-dependent increase in postsynaptic gain in hippocampal
pyramidal cells [19,22]. The location-specific integration rules for synaptic input are
thought to be of high importance for information processing and memory storage in
these cells [23]. Many of these rules, however, are specific for particular cell types (compare
for instance [52] and [19]). Importantly, DG cells were shown to integrate synaptic input
differently compared to hippocampal pyramidal cells [36]. The absence of dendritic
spikes and the strong attenuation of both EPSPs and back-propagating APs result in a
linear summation of synaptic input in DG granule neurons, with similar gain for proximal
and distal synapses. It was estimated that DG cells require input from ~55 synapses
to reach their AP threshold, compared to only 5 synchronous EPSPs in hippocampal
pyramidal cells [36]. A reduction of presynaptic strength as a function of distance would
further reduce the AP firing probability caused by physiological presynaptic activity. By
comparing DG and non-DG neurons we found that, comparable to postsynaptic gain,
the distance-dependent scaling of presynaptic strength was significantly reduced in DG
cells (Figure 3.6 and Supplemental Figure 3.3). The location-specific scaling of presynaptic
terminals thus was different between different populations of neurons. This illustrates
that distance-dependent scaling of presynaptic strength is a cell-type specific property,
and neurons appear to tune the strength of their afferent input in conjunction with the
cell type-specific integration rules of their dendritic tree.
Distance-dependent scaling is expected to regulate complex network performance
Our data reveal a new rule that regulates the strength of presynaptic terminals formed
along the dendritic tree of hippocampal neurons. Since synaptic integration rules are
highly dependent on dendritic location [23], this may be of crucial importance for the
proper integration of synaptic activity. In the intact hippocampus, different regions of
79
3
the dendritic tree receive inputs from different anatomical sources (reviewed in reference
55). Combined with our data, it can be predicted that these inputs differ in presynaptic
strength (and probably also STP), based on their dendritic location. In line with this
prediction, [53] showed that the perforant path (which projects on distal dendrites of CA1
pyramidal cells) display a low pr and paired-pulse facilitation, while Schaffer collaterals,
projecting on proximal dendrites, have a higher pr and show frequency depression (but
also see [54]). Together with the postsynaptic scaling described before, these distancedependent rules imply that the power of these anatomical sources to influence the
activity of a target neuron depend on the position of their terminals on the dendrite
they project on. To what extend distance-dependent scaling affects the strength of other
inputs, and how this influences information processing in the hippocampus, remains an
important open question.
3
80
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Supplemental Figure 3.1 (opposite page). Distance-dependent scaling of total pool size
is independent of neuronal development. A. To compare distance-binned histograms of
different experimental groups, we made profiles of each histogram. For each distance bin we
took the fluorescence intensity that corresponds with a cumulative probability of 0.4, 0.5 (the
median) and 0.6, which is shown in B. B. Profile of the distance-binned histogram in A. Solid
line is the median, shaded area represents the 0.4-0.6 boundaries. C. Average synaptic vGlut
intensity per cell for neurons fixed at DIV14 or DIV21. D. Average synapse area. E. Average
synapse density per cell. F. Average dendrite length per cell. G. Cumulative histograms of vGlut
intensity of cells fixed at DIV14, grouped by distance from postsynaptic soma. Intensity was
normalized to the largest synapse formed on the cell. Legend applies to all panels. H. As G, for
neurons fixed at DIV21. I. Comparison of histograms in G and H. J. Cumulative histograms of
area of synapses in E. K. Cumulative histograms of area of synapses from H. L. Comparison of
histograms from J and K. * p < 0.05, ** p < 0.01.
83
3
B
A
0.5
1
0.8
normalized intensity
cumulative probability
0.9
0.7
0.6
distance (µm):
0.5
0.4
0
140
>260
0.3
0.4
0.3
0.2
0.1
0.2
0.1
D
E
500
0
33
26
1
0.5
0
0.7
0.6
distance (µm):
0 140 >260
P/D = 1.40±0.05
p<0.001
N=2
n (cells) = 33
n (synapses) = 15533
0.5
0
0
0.5
normalized intensity
1
J
cumulative probability
1 DIV 14
0.1
0
0.3
0.2
0.1
0
DIV 14
DIV 14
0.5
0
P/D = 1.25±0.06
p<0.001
0
0.5
normalized area
1
2000
DIV14
1500
DIV21
1000
500
0
I
1 DIV 21
0
0.5
0
DIV14
DIV21
0.4
0.1 0.2 0.3 P/D
0.4= 1.45±0.05
0.5 0.6 0.7 0.8 0.9
p < 0.001
normalized
intensity
0.2
N=2
n (cells) = 26
n (synapses) = 18654
0
0.5
normalized intensity
1
K
1
250
F
0.4
0.2
100
150
200
distance from soma (mm)
**
0.4
0.5
cumulative probability
cumulative probability
0.8
50
H 0.3
G
cumulative probability
1
0.9
cumulative probability
1000
vGlut area ( mm2 )
vGlut intensity (AU)
*
0
dendrite length (µm)
C
0
1
normalized intensity
3
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
normalized intensity
0
1
0
100
200
distance from soma (mm)
0
100
200
distance from soma (mm)
L
1 DIV 21
0.3
0.5
0
P/D = 1.23±0.05
p<0.01
0
0.5
normalized area
84
1
normalized area
0
no. synapses / µm
0
0.2
0.1
0
A
RRP size (AU)
E
400AU
900AP 20Hz
2
3
3
D
20
0
0
0.5
normalized RRP size
number of synapses
2
40
total pool
F
2000 4000 6000
total pool size (AU)
3
60
40
20
0
1
500
400
300
200
100
NH4
10s
1
cumulative probability
100AU
1s
1
number of synapses
C
B
40AP 20Hz
0
0.5
1
normalized total pool size
1
0.5
distance (µm):
0
0
80
>160
0
0.5
1
normalized total pool size
Supplemental Figure 3.2. Total vesicle pool size is related to RRP size and depends
on synapse position. A. Representative traces of SypHy fluorescence from single
synapses stimulated with 40AP at 20Hz to release the RRP. B. Traces from the same
synapses as in A, stimulated with 900AP at 20Hz to release the releasable pool, and
NH4+ to visualize the pool of unreleased vesicles. C. Histogram of the RRP size as
measured from the SypHy fluorescence. D. Total pool size from the same synapses
in C, measured from the response to 900AP + NH4+. E. Typical example of the
relationship between RRP size and total pool size for synapses formed by one neuron.
F. Relationship between distance from postsynaptic soma and the size of the total
vesicle pool. Data from 12 cells (1066 synapses), N = 3.
85
A
B
10
6
4
2
0
D
400
300
***
200
100
73 * 77
0
0.3
no. synapses / µm
F
*
1500
500
G
0.2
0.1
0
0
400
300
***
200
100
0
1
H
0.8
0.6
0.4
0.2
600
400
200
0
J
distance (µm):
0
300
500
0
non DG
0.5
100
200
distance from soma (µm)
0
140 >260
N=4
n (cells) = 73
n (synapses) = 29550
1000 2000 3000
intensity (AU)
cumulative probability
cumulative probability
1
***
1000
0
I
E
0
1
DG
0.5
00
N=4
n (cells) = 77
n (synapses) = 19148
1000 2000 3000
intensity (AU)
intensity (AU)
2
soma area (µm )
3
2000
synapse area ( µm2 )
C
dendrite length (µm)
MAP vGlut Prox1
non DG
DG
8
number of synapses
number of branches
DG
vGlut intensity (AU)
non DG
700
non DG
DG
600
500
400
0
100
200
distance from soma (µ m)
Supplemental Figure 3.3. Dentate gyrus granule cells retain their identity in culture and show
strongly reduced distance-dependent scaling. A. Example images of a dentate gyrus (DG) cell
and non DG cell in culture. Scale bars represent 20 mm. B-H. Morphological characterization of
DG and non DG cells. B. Sholl analysis of dendritic branching. Legend applies to all panels. C.
Surface area of the soma. D. Total dendrite length in the field of view. E. Number of synapses in
the field of view. F. Synapse density (number of synapses per mm dendrite). G. Surface area of
synapses. H. Average vGlut intensity of all synapses. Data was obtained from 73 non DG and
77 DG cells, N = 4. All data is expressed as average, error bars represent SEM. * p < 0.05, *** p <
0.001. I. Cumulative histograms of absolute vGlut intensity of DG (right) and non DG cells (left).
Synapses were grouped by distance from postsynaptic soma in bin of 20 mm. J. Comparison of
the histograms in I.
86