Supplementary Information: Acute imaging
0 hour
1 hour
Supplementary Figure 1. Acute Imaging. Example of
stable spines over 1 hour (e.g. yellow arrowhead), and a
new thin spine that appeared (blue arrow). Scale bar, 5μm.
Acute experiments were performed in anesthetized young adult mice (PND 35-50; n=4).
Images were collected every 15 minutes for 2 hours. Relatively few changes occur over
these time periods (~2.7% / hour, 367 spines; Supplementary Fig. 1 a, b). Therefore daily
imaging captures the majority of the spine dynamics.
Supplementary Information : Immuno-electron microscopy and 3d reconstruction
Imaged GFP-labeled dendrites were localized at the ultrastructural level using preembedding immunocytochemistry. Within 10 minutes of the final in vivo imaging
session, the already anaesthetized mice were transcardially perfused with a solution of
0.2% glutaraldehyde and 2% paraformaldehyde in 0.1M phosphate buffer, pH7.4. One
hour after perfusion, brains were removed and 60m vibratome (Leica VT100) serial
sections were cut tangential to the surface of barrel cortex, approximately parallel to the
optical sections collected during the in vivo imaging. These sections were then
cryoprotected, freeze thawed in liquid nitrogen, then pretreated in 0.3% hydrogen
peroxide in PBS. After blocking in PBS containing 5% goat serum and 0.1% bovine
serum albumin-c (Aurion, Netherlands), they were incubated overnight in primary
antibody (rabbit anti-GFP, Chemicon #AB3080) at 4C, then in biotinylated secondary
antibody (1:500 goat anti-rabbit (F)ab fragment, Jackson Laboratories, USA) for 4 hours
before being incubated in avidin biotin peroxidase complex (ABC Elite, Vector
Laboratories, USA), and finally enhanced in 3, 3’-diaminobenzidine tetrachloride and
0.015% hydrogen peroxide.
Sections were postfixed in 1% osmium tetroxide in cacodylate buffer (0.1M), dehydrated
in a graded series of alcohol followed by propylene oxide and then embedded between
silicon coated glass slides in Epon resin (Fluka). Once cured, the resin embedded sections
were viewed under a light microscope and compared to projections of image stacks
collected in vivo (Fig. 3 a-b). This was done by matching an image of the vascular
pattern, taken immediately after the final in vivo imaging session, with the arrangement
of blood vessels seen in the first few tangential sections. The exact position of particular
dendrites could then be identified in relation to the pattern of vessels on the surface of the
cortex.
A total of 1,534 serial thin sections (silver/gray interference color) were cut from this
region and placed in formvar-coated, single slot grids. Images of labeled dendrites were
recorded at a magnification of 13,500 times using a Phillips CM10 transmission electron
microscope at a filament voltage of 80kV and collected digitally using a CCD camera
(Gatan, USA) (see Supplementary Movie showing sequential serial sections in layer 1).
To visualize the labeled dendrites in 3D, electron micrographs were aligned
consecutively using Photoshop (Adobe) and then the stack of serial images exported to a
Silicon Graphics workstation running the DepthAnalyser module in Imaris Software
(Bitplane AG, Zurich, Switzerland). Labeled elements were outlined on each serial
section and the completed structure exported to 3D Studio Max (Discreet Software, USA)
for color rendering.
Supplementary Information: Electrophysiology
Sharp electrode intracellular recordings were performed on wild type c57/bl6 five week
old male mice (n = 15) essentially as described 34. Chessboard deprived mice had the
appropriate whiskers trimmed to < 1 mm 3-5 days before the acute experiment. Receptive
fields were constructed by measuring sensory stimulation-evoked synaptic potentials
(PSPs). For each whisker 25 stimulus trials were collected with an interstimulus interval
of 4 seconds. Neurons (14/15) were recovered and assigned to a barrel column using
standard histological techniques 34. Recordings from layer 2/3 (12/15) and layer 5 (3/15)
neurons were pooled since the time-course and extent of plasticity is similar in these
layers 5.
Receptive fields were computed using the amplitude of the averaged PSP for each
whisker. Individual responses were median filtered to remove action potentials. Since the
membrane potential fluctuates between up and down states, where the up state
corresponds to massive and synchronous network excitation 23, 34, trials in which the
neuron was in an up-state during stimulation were removed. To define up-states, an allvalue histogram was computed for the membrane potential measurements during each
stimulus trial. The histogram typically had two peaks corresponding to up and down
states. If, during stimulus onset, the membrane potential was within range of the peak at
higher potentials the trace was removed from the average. Whisker-evoked PSPs were
identified as the largest value within a 50 ms poststimulus window. PSP amplitudes
varied greatly between animals (range: 3.3 – 27 mV). Surround 1 and 2 (1SW and 2SW,
respectively) PSPs were defined as the responses to the adjacent whiskers one or two
rows or columns away from the PW.
Supplementary Information: Analysis of spine volumes and spine shapes
Analysis of spine volumes was performed on a subset of spines that were well-separated
from dendrites (n=301, 6 animals). Relative volumes were estimated as the spine
fluorescence divided by the dendritic fluorescence 29. This estimate assumes that GFP is
homogeneously disitributed throughout the cytoplasm and that dendritic shafts do not
vary much in diameter. Using this estimate we find that spine volumes vary by a factor of
> 50, consistent with serial section EM reconstructions where spines in adult
hippocampus vary by ~ 100 fold in volume 30, 42. Stable spines were on average 75 %
larger than transient spines (p < 0.05) (Supplementary Fig. 2a).
Of the spines suitable for volume analysis, stable spines comprised 61% of the
population, consistent with the total spine population (Supplementary Fig. 2b). 77% of
stable spines had a well-defined spine head, distinct from the spine neck. Conversely,
83% of spines that had a well-defined head were stable, and less than 4% were transient
(Supplementary Fig. 2c). We define mushroom spines as spines with a well-defined
spine head that had large spine volumes (within 50% of the largest spine volumes
measured). We estimate that this corresponds to the larger half of mushroom spines
defined using ultrastructural criteria 30. These large mushroom spines were found to be
exceptionally stable, with 93% surviving as long as they were imaged (8-32 days; Fig.
5a,d; Supplementary Fig. 2d, e).
Relative Volume
a
b
c
*
d
Spines with
well-defined heads
All spines
Large
mushroom spines
10
60.5%
5
0
e
Spine Volumes
15
 1 day
2-7 days  8 days
Transient Semi-stable Stable
Day 1
Day 2
Day 3
Day 4
1 day
Day 5
92.9%
82.9%
Day 6
2-7 days
Day 7
Day 8
8 days
Day 29
Legend
Transient
Stable
Supplementary Figure 2. Larger spines are relatively stable. a, Spine head
volumes as a function of spine lifetimes. Stable spines were significantly larger
than transient and semi-stable spines (p < 0.05). b, Breakdown of spine lifetimes
for the subset of spines selected for volume analysis (n = 301). 61% of spines
existed for at least 8 days (stable). c, Breakdown of spine lifetimes for spines
with well-defined spine heads (e.g. red arrow and yellow arrowhead in e; n =
170). 83% of spines with well-defined spine heads were stable. d, Breakdown of
spine lifetimes for large mushroom spines (yellow arrowhead, but not red arrow;
n = 14). All but one mushroom spine existed for as long as imaged. e, Examples
of stable mushroom spines (e.g. yellow arrowhead) and small transient spines
(e.g. blue arrowheads). Scale bar, 5μm.