SUBJECT AREAS:
IMAGING
NEUROSCIENCE
NEUROANATOMY
NEURODEVELOPMENTAL
DISORDERS
The short-time structural plasticity of
dendritic spines is altered in a model of
Rett syndrome
Silvia Landi1,2*, Elena Putignano1,3*, Elena Maria Boggio3, Maurizio Giustetto4, Tommaso Pizzorusso3,5
& Gian Michele Ratto1,2,3
NEST, Scuola Normale Superiore, Pisa, Italy, 2NEST, Institute Nanoscience CNR, Pisa, Italy, 3Institute of Neuroscience CNR, Pisa,
Italy, 4National Institute of Neuroscience-Italy and Department of Anatomy, Pharmacology and Forensic Medicine, University of
Turin, Italy, 5Dipartimento di Psicologia, Università di Firenze- Florence, Italy.
1
Received
18 May 2011
Accepted
4 July 2011
Published
25 July 2011
Correspondence and
requests for materials
The maturation of excitatory transmission comes about through a developmental period in which dendritic
spines are highly motile and their number, form and size are rapidly changing. Surprisingly, although these
processes are crucial for the formation of cortical circuitry, little is known about possible alterations of these
processes in brain disease. By means of acute in vivo 2-photon imaging we show that the dynamic properties
of dendritic spines of layer V cortical neurons are deeply affected in a mouse model of Rett syndrome (RTT)
at a time around P25 when the neuronal phenotype of the disease is still mild. Then, we show that 24h after a
subcutaneous injection of IGF-1 spine dynamics is restored. Our study demonstrates that spine dynamics in
RTT mice is severely impaired early during development and suggest that treatments for RTT should be
started very early in order to reestablish a normal period of spine plasticity.
should be addressed to
G.M.R. (gianmichele.
ratto@sns.it)
* S.L. and E.P.
contributed equally to
the study.
R
ett syndrome (RTT) is a neurodevelopmental disorder characterized by a period of apparently normal
development of 6–18 months followed by regression and onset of a variety of symptoms including motor
abnormalities, mental retardation, epilepsy and anxiety1. Most cases of RTT involve mutations of methylCpG-binding protein 2 (MeCP2), a gene encoding a methylated DNA-binding protein that regulates gene
transcription, mRNA splicing and chromatin structure2. However, how these genetic defects translate into
RTT symptoms is still unknown. It has been proposed that synaptic alterations constitute a main substrate of
the disease symptoms3,4, but only subtle alterations of neuronal density, dendritic arborisation and number of
dendritic spines (the main site of excitatory synapses) were found in both patients5,6 and mouse models in which
MeCP2 has been deleted7. So far, the available studies7 have been performed in fixed tissue thus not allowing the
visualization of the dynamic processes underlying juvenile dendritic spine maturation. These involve the formation and removal of highly motile filopodia that can be stabilized and transformed into more stable mature spines.
These processes can occur in a time scale of minutes and they are altered in response to manipulations that do not
lead to manifest variations of spine density, suggesting that the short-time dynamic regulation of spine structure
plays an important role in shaping connectivity of neural circuits8. To fill this gap, we analyzed dendritic spine
dynamics in somatosensory cortex by 2-photon time lapse imaging in MeCP2 null mice crossed with Thy-GFP
line9,10. At the onset of the disease (3–4 weeks of age11,12) we found deep alterations in the dynamics of dendritic
spines and filopodia. Later on at P40 when the maturation of the connectivity in the somatosensory cortex is
complete, the dynamics of dendritic spines in RTT mice was identical to control, although a reduction in spine
density was observed in mutants. Finally, we found that 24 hrs after a single subcutaneous injection of IGF-1, a
molecule known to pass the blood brain barrier (BBB13) and of proposed therapeutic potential in RTT14–16, spine
motility was completely restored in the cerebral cortex of RTT mice.
Results
We crossed the MeCP2 null-mutants9 with a mouse line expressing green fluorescent protein (GFP) in a sparse
subset of layer V pyramidal neurons (GFP-M line; 10). Initially we analyzed juvenile (P25-26) GFP-MeCP2-KO
mice (RTT mice), at a time that precedes the onset of evident RTT neurological symptoms11,12. We found that the
general architecture of layer V pyramids was normal but the density of dendritic spines and filopodia was reduced
in mutant mice. Also the length of spines was slightly but significantly shorter when compared with controls
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Figure 1 | Morphological alterations of dendritic spines in GFP-MeCP2-KO mice. (A) Confocal micrographs showing GFP-positive dendrites of
layer V pyramidal neurons in the S1 cortex of perfused-fixed control (cnt) and GFP-MeCP2-KO mice (RTT). Filopodia-like spines are indicated with
arrowheads. Scale bar 2.5 mm. B, C, left and center panels) Both spines and filopodia are less dense in the RTT mice (t-test. B: spines, p,0.03; filopodia,
p,0.05; C spines, p50.01; filopodia, p50.02) when evaluated in fixed tissue (B) or in vivo (C). B, C right panels) Cumulative distributions of spine necklength (B, fixed tissue) and of spine length (C, in vivo) revealed a general decrement of spine length in the RTT mice (KS-test, B: p,0.01; C: p,0.001).
Column plots show the mean and the standard error of the mean.
(Fig. 1). These data emerge both from images obtained in fixed tissue
(Fig. 1B) and in vivo (Fig. 1C), thus indicating that the GFP-MeCP2KO mice show a reduction of the density of dendritic spines, as
previously reported for the MeCP2-KO mice7 and that the conditions
of in vivo imaging do not alter the basic morphometric characteristics of dendritic spines. Moreover, these data confirms that synaptic
connectivity in the cortex of RTT mice might be already impaired at
this early developmental stage7. Then, we proceeded to measure the
short time dynamics of dendritic spines in the RTT mice by means of
2-photon imaging through a cranial window. After identification of a
suitable dendrite, we acquired z-stacks of the selected fields at 5 min
interval over a period of 50–60 min (Fig. 2A). Time lapse imaging
revealed a dramatic effect of the MeCP2 deletion on the short time
structural plasticity of spines (see also Supplementary Video V1 and
V2 online). In RTT mice motility of dendritic spines (expressed as
variation of length) was severely reduced (Fig. 2B). The observed
reduction was largely due to a decreased fraction of filopodia and
to their great stability compared to control. In the subset of spines
with a clearly defined head, we measured its volume in each temporal
frame, and the distribution of the fluctuations of the head volume for
both control and RTT mice are shown in Fig. 2C. It is clear that the
spine head volume is far more stable in the cortex of MeCP2 mutants
compared with control mice. In summary, these data demonstrate
that both filopodia and more mature spines are affected by MeCP2
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deletion. Fluctuations of length and volume report two different
functional properties of dendritic spines, being the length correlated
with connectivity and with the spine-dendrite coupling, while the
head volume is correlated to the size of the postsynaptic density and
with synaptic strength17–20. These parameters are uncorrelated in our
sample, as also shown in previous work on normal mice (Fig. 2D; 20).
During normal cortical development, filopodia almost disappear,
spines reach their mature form and structural stability increases: thus
we expected that the differences in motility observed at P25 would
fade away later on as the circuitry stabilizes. Indeed, time lapse
imaging performed in P40 mice showed that at this age spine motility
in control mice is decreased to levels comparable to the MeCP2
null mice (Fig. 3), whereas spine density remains significantly
impaired in MeCP2 mutants. Thus, the deletion of MeCP2 impairs
spine motility during the critical period for cortical plasticity and
synaptogenesis. The impact of MeCP2 loss of function on early
development suggests that any treatment directed at attenuating
RTT condition should be performed during the critical period
for cortical plasticity. IGF-1 is required for the maintenance of
dendritic spines in the adult cerebellum21, and it has been shown
to improve the RTT symptoms in the MeCP2 null mice16. We
wondered whether IGF-1 might also act early during postnatal
development on the short-term dynamics of dendritic spines. Mice
have been treated with a single injection of the long form of IGF-1
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Figure 2 | Dendritic spine dynamics is altered in a model of Rett syndrome at P25-26. (A) Time lapse imaging in a control and a RTT mouse at P25.
Arrowheads points to motile spines and filopodia. Scale bar 5 mm. B, left) Representative data showing the time course of spine length in a control and a
RTT mouse. Right) Cumulative distribution of the average motility showing that in the RTT mice dendritic spines are more stable (cnt, N57; RTT, N55;
KS-test, p50.014). The dots represent the average motility of each filopodia. C, left) Time course of the fluctuations of the spine head volume in a
control and in a RTT mouse. Right) The cumulative distribution of the average fluctuation of the spine head volume shows that in the RTT mice the spine
head is more stable (KS-test, p50.002). (D) Scatter graph showing that there is no correlation between spine motility and fluctuation of spine head
volume in RTT and control mice.
24 h before two-photon imaging (Fig. 4A; 22). The morphometric
analysis of these data evidenced a recovery in spine length and no
effects on spine density after IGF-1 treatment (Fig. 4B). Furthermore,
we observed a complete recovery in dendritic spine dynamics tested
both by the fluctuation of spine length and by the fluctuation of the
volume of the spine head (Fig. 4C, and Supplementary Video V3
online). Although filopodia density showed a small but not significant increment, their stability was drastically modified by IGF-1
treatment. Stability of each filopodia has been quantified by
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computing a coefficient defined as the number of frames in which
each filopodia is visible divided by the total sequence length: thus a
coefficient of 1 means that the filopodia is present during the
entire imaging session. The distribution of filopodia coefficients of
stability (Fig. 4D) shows that in the RTT mice 90% of filopodia are
present during the entire imaging period whereas in controls this
percentage is only 40%. The treatment with IGF-1 strongly increased
filopodia turnover in the RTT mice to 60%, control becoming similar
to that of mice. Interestingly, the IGF-1 treatment did not cause
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Figure 3 | No differences in spine dynamics in older mice (.P40).
(A left) Time lapse imaging in a control and a RTT mouse at P40.
Scale bar 2.5 mm. A right) Spine density is significantly reduced at P40
between control and RTT mice (cnt, N55, KO, N54; t-test, p,0.001)
(B) Cumulative distributions of spine length fluctuations revealed no
differences between control and RTT at P40 (KS, p50.733).
(C) Cumulative distributions of spine head volume fluctuations showed
no differences between control and RTT at P40 (KS, p50.676).
any change in spine motility in control wild type mice, possibly
indicating that IGF-1 availability is not the rate limiting factor of
the processes controlling structural plasticity during normal
development (Fig. 4C).
Discussion
Our experiments have shown that the dynamic properties of dendritic spines in cortical pyramidal neurons are deeply affected by
MeCP2 mutations suggesting that synaptic defects in RTT are even
more complex than what was previously indicated by neuroanatomical studies. Indeed, an anomalous cortical circuitry is established
in the cortex of RTT mice even before the full appearance of the
neurophysiological expression of RTT. The impairment of the cellular mechanisms regulating short-term dynamics of dendritic
spines and filopodia occurs early during the postnatal development,
in a critical window for cortical plasticity and synaptogenesis. Thus,
the alterations in spine development should prevent the normal
formation and maturation of synaptic connectivity. MeCP2 lossof-function mutation has also been associated with several other
neurodevelopmental disorders, i.e. epilepsy, Angelman syndrome,
autism, infantile encephalopathy, juvenile onset of schizophrenia,
Parkinsonism and X-linked mental retardation in both male and
females1. Therefore, impairments in spine short-time structural plasticity could represent a common pathogenic trait in several brain
disease. IGF-1 is a growth factor important for brain development
and plasticity13 widely expressed in the CNS during normal development23 that strongly promotes neuronal cell survival, synaptic
maturation23,24, and functional plasticity in the developing cortex25.
The putative relationship between RTT and IGF-1 is highlighted by
the observations that IGF-1 concentration is lower in the cerebrospinal fluid of autistic children26 and that MeCP2 regulates directly the
expression of the insulin-like growth factor binding protein 3, thus
regulating IGF-1 action27. Indeed, its pharmacological potential is
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enhanced by the ability to pass the BBB (26,13), property not shared
by other trophic factors, such as BDNF28. IGF-1 is capable of crossing
the BBB, both in the tri-peptide form1–3 IGF-129 and in the long
form30–34. Notably, the BBB architecture is well suited to transport
serum IGF-1 into the brain, although the details of this process are
still incomplete. Both brain vessels, choroid plexus epithelium and
perivascular glial end-feet express IGF-1 receptors35, 36, thus IGF-1 is
transferred by endothelial cells into the perivascular space surrounded by glial end-feet able to endocytose IGF-137. Finally,
IGF-1 could be transferred to adjacent neurons through trans-endocytosis or a related process. In conclusion, the established vision is
that IGF-1 can move into the brain through two ways: serum IGF-1
passes into the CSF by physiological oscillations in blood IGF-1
levels32 in a tonic modality even if, serum IGF-1 levels usually remain
very steady. A second proposed mechanism involves the regulated
passage of serum IGF-1 through an activity-dependent mechanism13.
Here, we have shown that IGF-1 acts on dendritic spines early in
development, during the period of enhanced structural plasticity.
Interestingly, the effects of IGF-1 treatment were present already
one day after injection. At this time, we observed no recovery of spine
density, but the increased turnover of filopodia suggests that as the
treatment progresses, more spines could be added to the dendrites16.
The cellular details of IGF-1 action are not completely clarified and
the mechanisms altered in the MeCP2-KO mouse and that are rescued by IGF-1 are still unknown. We can envision two different lines
of actions, that may, to some extent, coexist. 1) IGF-1 acts on synaptic
transmission by converging on the signalling pathways of PI3K/
pAkt/PSD-9538 and MAPK39, similarly to BDNF. Indeed, both
pAkt signalling40 and BDNF expression are impaired in MeCP2 null
mice41 and different mutations in MeCP2 correlates with different
expression levels of BDNF and with a specific severity of RTT in
general42,41. Thus, exogenous IGF-1 might act by vicariating the
reduced signalling of BDNF. Specifically, IGF-1 has been shown to
elevate excitatory postsynaptic currents significantly43: the action of
IGF-1 on excitatory transmission, is strongly implied by the fact that
treating with IGF-1 neurons cultured from patients carrying different MeCP2 mutations, resulted in an increase in glutamatergic synapse number, suggesting that the drug treatment could correct
defective excitatory transmission44.
2) It is also possible that IGF-1 might act more directly on spine
dynamics, by contributing to the regulation of actin. In muscle cells it
has been shown that IGF-1 modulates the activity of N-WASP resulting in enhanced actin filament formation45. Since, N-WASP is also a
crucial regulator of actin during spine formation and plasticity46 the
possibility arises that actin based motility might be directly influenced by IGF-1 availability. In summary, our study has shown that
structural plasticity is impaired in the MeCP2 mouse, at a time in
which cortical circuitry is refined by experience. Then we have shown
that the pharmacological treatment with IGF-1 can recover most of
the deficit of structural plasticity. Our results indicate that time lapse
imaging of the short-time dynamics of dendritic spines can be used as
a powerful screening tool for designing therapeutic strategies targeting pathologies where structural plasticity is impaired. Finally, our
data suggest that treatments should be administered early on during
postnatal development, when cortical plasticity is still enhanced.
Methods
Preparation of the GFP-MeCP21/2 mice. All experimental procedures were
carried out in compliance with the institutional guidelines of the Italian Ministry for
Public Health. The GFP-MeCP2-KO mice were derived from heterozygous
B6.129SF1-MeCP2tm1Jae knock-out females (MeCP21/2)9. Females were originally
crossed to C57BL6 for one generation, followed by breeding amongst offspring of the
same generation with breeder changes, and were maintained on a mixed background
to reduce mortality and to obtain the high numbers of mice required by the
experiments. Age matched littermates were used in all experiments to control
for possible effects of genetic background unrelated to the MeCP2 mutation47.
MeCP2 1/2 females were then crossed with Thy-1 GFP transgenic mice (line M10)
and only mice expressing GFP at cortical level were used for this study. GFP control
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Figure 4 | IGF-1 treatment restores spine dynamics in RTT mice. (A left) Protocol of the experiment. Right) Time lapse imaging in a RTT mouse and in
a RTT mouse treated with IGF-1. Scale bar 2 mm. B left) Cumulative distribution of spine length showed that the treatment with IGF-1 lead to a partial
recovery of the spine length in RTT mice (data replotted from fig. 1 for control and RTT mice for comparison; KS-test, cnt vs RTT-IGF-1, p50.004; RTT
vs RTT-IGF-1, p50.031). B center and right) Both spine density and filopodia densities were not affected by IGF-1 (One-Way ANOVA, post-hoc Tukey
test, p,0.05). C left) Data showing the distributions of the fluctuation of spine length in the specified conditions. In the RTT mice dendritic spines are
more stable than controls (replotted from fig. 2), while spine motility becomes similar to controls after IGF-1 treatment (cnt vs RTT-IGF-1, KS
test,p50.170; RTT vs RTT-IGF-1, p50.010). The dots represent the average motility of each filopodia. IGF-1 treatment per se does not cause any effect in
control mice (cnt vs cnt IGF-1, p50.16); C,center) Distribution of the fluctuations of the spine head volume in the specified conditions. The data
show that after IGF-1 administration RTT spine head fluctuations become normal (KS-test: cnt vs RTT-IGF1, p50.50; RTT vs RTT-IGF1, p50.006).
(D) Cumulative distribution showing that while filopodia in RTT mice are very stable in comparison to filopodia in controls, 24h after IGF-1 treatment
they become less stable, with parameters intermediate between untreated RTTs and controls (KS-test; cnt vs RTT, p50.029; cnt vs RTT-IGF1, p50.469;
RTT vs RTT-IGF1, p50.528).
mice were separated from GFP-MeCP2-KO mice by PCR screening12. We employed
only mice with an expression level of GFP high enough to provide a sufficient quality
of the imaging.
Confocal microscopy on fixed tissue. GFP-MeCP2 null mice (N54) and GFP
control littermates (N54) were anesthetized with an intraperitoneal injection of
chloral hydrate and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer
(PB). Brains were post-fixed overnight in the same fixative solution, washed several
times in 0.1 M PB and then cut coronally into 90-mm sections on a vibratome (Leica
VT 1000S, Germany). Immunolabelling on free-floating sections with an antibody
against GFP (155000, rabbit, Synaptic Systems) was used to enhance GFP
fluorescence signal. Briefly, sections were blocked for 1 hour at room temperature
(RT) using a solution of 3% bovine serum albumin, 0.05% Triton X-100 and 10%
normal goat serum (NGS) in phosphate buffer saline (PBS) and then incubated
overnight at RT with anti-GFP antibody in a PBS solution containing 3% NGS and
0.05% Triton X-100. Sections were then washed in PBS and subsequently incubated
with a fluorescent secondary antibody (Alexa488 goat anti-rabbit; 151000) in a
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solution of 3% NGS, 0.05% Triton X-100 in PBS for 1 hour at RT, rinsed several times
in PBS and finally mounted on glass slides with Dako fluorescent mounting medium.
Sections were viewed with a confocal microscope (Zeiss LSM-5 Pascal, Germany)
equipped with a 403 oil-immersion objective (pinhole: 1.0 Airy unit). At least 10
z-stack images consisting of 10–15 sections (512 3 512 pixels) spaced 0.5 mm apart
were acquired in the layer II–III of the S1 cortex of each animal from distal tertiary
branches (dendritic segment of length in the 35 to 60 mm range) of layer V pyramidal
neurons as in 48. Dendritic segments and spines were analyzed quantitatively using
ImageJ (NIH, USA, public domain) by an observer blind to the experimental
conditions. Measure of spine density was performed on projected z-stack images and
spine number was divided by the length of the dendritic segment to compute the
density expressed as number of spines per micrometer. If dendritic spines were too
packed to clearly separate them from each other, we turned to serial stack images to
delineate individual spines. The measurement of spine neck length was performed on
single-stack images and evaluated by measuring elongated fluorescent structures
from the dendritic shaft to the beginning of the enlargement of the spine head.
Dendritic protrusions showing a thin neck and finishing with no evident head
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enlargement were considered as filopodia. We matched the criteria used for spine
identification in the experiment on fixed tissue and in-vivo.
Two-photon imaging. Pups born from 19 pregnant females were screened to obtain
20 males MeCP2-KO mice. From this population only 5 males MeCP2-KO provided
usable imaging data, because of the high levels of mortality already described postweaning and because of adverse reaction to anesthesia and surgery12. All experiments
have been performed on mice aged P25-P26. Animals were oxygenated, kept warm
and respiratory and cardiac rhythms were monitored during both surgery and
imaging. Since Rett syndrome is frequently associated with respiratory problems and
ipocapnia49, we took extreme care in the control of the anesthesia levels. The study
included only mice that maintained stationary breathing and cardiac rhythm and that
did not display any other experimental anomalies like dendritic blebbing or excessive
edema. Imaging experiments could not be performed blind because Rett mice were
smaller in size than wild type mice as reported by 3 and 12 and furthermore anesthetic
dosage had to be regulated differently in Rett mice. Indeed, we determined that RTT
mice required a smaller dose of averthine than the control littermates: 0.04 ml 3 10g
(RTT) versus 0.2 ml 3 10g (control). A cranial window was implanted at P25-26
according to a protocol described in 8. Briefly, we opened a small craniotomy (about
4 mm of diameter) over the somatosensory cortex (2 mm posterior from bregma,
2 mm lateral from lambda) leaving the dura mater intact. The ECG was picked up by
two tiny copper clips placed on the forelimbs. The signal was fed with shielded coaxial
cables to a differential amplifier (NPI) and then to a custom made threshold
discriminator that converted the heart beat waveform into a TTL signal used for
imaging synchronization. Two-photon imaging was carried out on a dedicated
2-photon microscope (Ultima IV, Prairie Technology, Wisconsin) equipped with a
10 W laser (Coherent) tuned at 890 nm that delivered 30–50 mW at the sample.
Imaging was restricted to the apical dendrites of layer V pyramidal neurons present in
cortical layers II/III (50–150 mm below the cortical surface). Imaging was conducted
for at least 50–60 min at an interval of 5 minutes and was interrupted if the mouse
vital signs degraded. Images were acquired with a water immersion lens (Olympus,
603 NA 0.95) at a resolution of 5123512 pixels at zoom 4, leading to a field of
50.7350.7 mm and a nominal linear resolution of about 0.1 mm/pixel. The stack step
size was 0.75 mm. We selected the dendrites with the highest levels of GFP expression;
moreover, we chose dendrites roughly parallel to the cortical surface to limit the depth
of the z-stacks required to enclose the entire dendrite. The acquisition of each frame
was synchronized to the hearth beat and this procedure strongly reduced the
mechanical artifact produced by the pulsing brain circulation and improved the
alignment of the image stacks.
Image analysis. Analysis of the imaging data was carried out using ImageJ. The
optical sections of each stack were aligned to compensate for mechanical movements
with a custom macro that used the RegStack plug in50. After alignment of the z-stack
obtained at each time point, the length of each spine was measured starting from the
tip along the neck to the center of the dendrite. These measures were performed with a
custom software that allowed to move easily along the z-stack to identify the spine
features on the sections of optimum focus. For each spine we obtained a temporal
series holding the spine length at each time. Usually the motility is described by
computing the mean value of the derivative of the length. Since this measure scales
with the mean length of the dendritic spine and in RTT mice spines are shorter, we
decided to estimate the spine motility as the standard deviation of the series
normalized to the mean spine length. The head volume has been computed at each
temporal point by normalizing the integrated fluorescence (after background
subtraction) from a ROI draw around the spine head with the mean fluorescence of
the nearby dendrite. This normalization compensates for instrumental variations and
also allows to compare measures made on different mice that have different levels of
GFP expression51. The variability of the spine head has been quantified as the
standard deviation of the volume normalized to the mean volume of each spine head.
This analysis has been performed only on the spines characterized by a clearly
defined head.
IGF-1 treatment. Subcutaneous injections of IGF-1 were performed by using IGF-1
(IU100; 1.8 mg/g body weight; Biovision)52–55 in Rett null mice (n59). We performed a
single injection of this factor 24h before imaging. According the concentration used in
22, we injected 6 ml of a solution made by 1 ml of IGF-1 dissolved in saline.
Subcutaneous injections were performed using a catheter with a 20 gauge needle
connected to a Hamilton syringe. Imaging was successful in 6 RTT mice and 5 control
mice.
Statistics. Statistical analysis was performed by Student t-test using Origin 7 software
(OriginLab Co., Northampton, MA) and Sigma Stat 3.0 (SPSS Inc.). Cumulative
frequencies of dendritic spine neck, mean spines length, motility and spine head
fluctuations in KO and WT mice were compared using, first, the normal distribution
Kolmogorov–Smirnov (KS) fitting test and then KS two-sample tests for subsequent
paired comparisons (http://www.physics.csbsju.edu/stats/KS-test.html).
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Acknowledgements
We thank Paolo Orsini for custom software for spine motility analysis. Thanks for
supporting this project to the following grants: FIRB 2003 Laboratorio nazionale sulle
nanotecnologie per genomica e post-genomica (NG-Lab), Telethon Grant GGP07278 to
G.M.R. and GGP09196 to T.P. and M.G., TLS-Toscana Life Sciences ‘Orphan diseases’ to
G.M.R., Firb FUTURO IN RICERCA2008-Miur to S.L. and the E-Rare EuroRett
Consortium to T.P. and M.G. We acknowledge the help of other members of the lab,
specifically of Marco Brondi, Sebastian Sulis Sato, Luisa de Vivo and Mariangela Panniello.
Author contributions
S.L., E.P., T.P., G.M.R. designed the study. E.M.B. and M.G. performed the experiments on
fixed tissue. The in vivo imaging experiments have been performed by S.L. and E.P. under
the supervision of G.M.R. S.L. and G.M.R. prepared the manuscript and all authors
contributed to the final form.
Additional information
Supplementary Information accompanies this paper at http://www.nature.com/
scientificreports
Competing financial interests: Authors have no Competing Financial Interests.
License: This work is licensed under a Creative Commons
Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this
license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/
How to cite this article: Landi, S. et al. The short-time structural plasticity of dendritic
spines is altered in a model of Rett syndrome. Sci. Rep. 1, 45; DOI:10.1038/srep00045 (2011).
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