In Vivo Imaging of a-Synuclein in Mouse Cortex
Demonstrates Stable Expression and Differential
Subcellular Compartment Mobility
Vivek K. Unni1, Tamily A. Weissman2, Edward Rockenstein3, Eliezer Masliah3, Pamela J. McLean1,
Bradley T. Hyman1*
1Alzheimer’s Research Unit, MassGeneral Institute for Neurodegenerative Disease, MGH Harvard Medical School, Charlestown, Massachusetts, United States of America,
2Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, Cambridge, Massachusetts, United States of America, 3Department of
Neurosciences, University of California San Diego, La Jolla, California, United States of America
Background: Regulation of a-synuclein levels within cells is thought to play a critical role in Parkinson’s Disease (PD)
pathogenesis and in other related synucleinopathies. These processes have been studied primarily in reduced preparations,
including cell culture. We now develop methods to measure a-synuclein levels in the living mammalian brain to study in
vivo protein mobility, turnover and degradation with subcellular specificity.
Methodology/Principal Findings: We have developed a system using enhanced Green Fluorescent Protein (GFP)-tagged
human a-synuclein (Syn-GFP) transgenic mice and in vivo multiphoton imaging to measure a-synuclein levels with
subcellular resolution. This new experimental paradigm allows individual Syn-GFP-expressing neurons and presynaptic
terminals to be imaged in the living mouse brain over a period of months. We find that Syn-GFP is stably expressed by
neurons and presynaptic terminals over this time frame and further find that different presynaptic terminals can express
widely differing levels of Syn-GFP. Using the fluorescence recovery after photobleaching (FRAP) technique in vivo we
provide evidence that at least two pools of Syn-GFP exist in terminals with lower levels of mobility than measured
previously. These results demonstrate that multiphoton imaging in Syn-GFP mice is an excellent new strategy for exploring
the biology of a-synuclein and related mechanisms of neurodegeneration.
Conclusions/Significance: In vivo multiphoton imaging in Syn-GFP transgenic mice demonstrates stable a-synuclein
expression and differential subcellular compartment mobility within cortical neurons. This opens new avenues for studying
a-synuclein biology in the living brain and testing new therapeutics for PD and related disorders.
Citation: Unni VK, Weissman TA, Rockenstein E, Masliah E, McLean PJ, et al. (2010) In Vivo Imaging of a-Synuclein in Mouse Cortex Demonstrates Stable
Expression and Differential Subcellular Compartment Mobility. PLoS ONE 5(5): e10589. doi:10.1371/journal.pone.0010589
Editor: Mark R. Cookson, National Institutes of Health, United States of America
Received February 24, 2010; Accepted April 15, 2010; Published May 11, 2010
Copyright: ? 2010 Unni et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health Grants NS038372, NS063963, T32AG000222, T32NS048005, AG18440 and AG022074. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Multiple lines of evidence implicate abnormal regulation and
aggregation of the synaptic protein a-synuclein in the etiology of
Parkinson’s Disease (PD) [1–2]. Because of this there have been
significant efforts to better understand the biology of a-synuclein,
including mechanisms relating to its synthesis [3–6], degradation
[7–10], regulation by other proteins , and function at synapses
[12–15]. To date, largely because of technical reasons, these
studies have been limited to reduced biochemical preparations,
cell culture models and analysis of fixed animal or human tissue. In
contrast, the study of other neurodegenerative diseases like
Alzheimer’s Disease (AD) has recently been advanced by
development of in vivo multiphoton imaging techniques in mouse
models. New insights into the mechanisms of AD involving the
formation of extracellular beta-amyloid plaques [16–18] and
intracellular tau aggregates [19–20] have come from these studies
that can follow individual plaques and tangles in the mouse brain
The study of PD and other related synucleinopathies would
benefit from analogous techniques to study the biology of a-
synuclein in vivo and its role in neurodegeneration. In this study we
detail a new experimental paradigm that allows real-time in vivo
imaging of fluorescently-tagged human a-synuclein in individual
cortical neurons with subcellular resolution over a period of
months. We demonstrate that this system is stable and allows for
detailed measurements of a-synuclein levels in individual cell
bodies and presynaptic terminals. In addition, we use this system
to provide the first in vivo evidence that a-synuclein protein is
differentially mobile within neurons using the fluorescence
recovery after photobleaching (FRAP) technique. To date FRAP
measurements have been described in numerous systems  and
to study a-synuclein in other models [22–23], but to our
knowledge this is the first in vivo extension of the technique to
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mammalian neurons, demonstrating its potential feasibility for
studying a wide range of neuronal proteins in living brain.
Our development of these approaches opens lines of inquiry
that are difficult to address otherwise. For instance, chronic
imaging of individual Syn-GFP expressing cells and presynaptic
terminals allows precise analysis of possible changes in these
structures over time. In addition, measuring a-synuclein mobility
in different subcellular compartments using FRAP can test how its
physical state, ability to bind to partners or other geometrical
constraints vary within the cell. Understanding these processes in
the living brain is of interest since it may lead to new strategies for
developing PD therapies.
Materials and Methods
Male Syn-GFP transgenic mice were mated with BDF1 female
mice by the MGH Center for Comparative Medicine (CCM).
Animals were held in a light-dark cycle, temperature and
humidity-controlled animal vivarium and maintained under ad
libitum food and water diet supplied by the CCM. All experiments
were approved by the Subcommittee on Research Animal Care
(SRAC) at the MGH and every effort was made to minimize the
number of animals used and their suffering.
Animals were deeply anesthetized and perfused with a
transcardiac approach with ice cold phosphate-buffered saline
followed by paraformaldehyde (4%) solution. The brain was
quickly removed and placed in paraformaldehyde (4%) at 4 C for
a minimum of 24 hr. Next 50–200 mm thick floating sections were
cut on a freezing microtome (Microm, HM400). Alpha-synuclein
immunohistochemistry was performed after blocking tissue with
normal goat serum (10%) at room temperature (RT) for 1 hr. Next
sections were stained with a human specific a-synuclein antibody
LB509 (Zymed, 1:100, 4 C for 24 hr) and a Cy3 anti-mouse
secondary antibody (Jackson Immunoresearch, 1:500, RT for
1 hr). All sections were imaged on a Zeiss META laser scanning
In vivo imaging
The general techniques used for making the ‘‘cranial window’’
have been published previously . After placing the cranial
window, isoflurane anesthetized animals were moved to an in vivo
multiphoton imaging set-up (Olympus Fluoview FV1000 MPE
multiphoton microscope microscope with Spectra Physics Mai-Tai
tunable laser source, set to 860 nm) and a Z-series stack from a
volume of cortex taken (encompassing 10–100 Syn-GFP express-
ing neurons, .10,000 Syn-GFP expressing presynaptic terminals).
Images were analyzed with Image J software (NIH). Somatic Syn-
GFP signal from individual neurons was analyzed over time by
creating a region of interest (ROI) outlining the somatic
compartment for each Syn-GFP expressing cell and measuring
the average fluorescence intensity within this ROI. This same ROI
was used to measure average fluorescence intensity from the same
cell at repeated time points. The average intensity (and standard
deviation) from all neurons is plotted as function of time.
Presynaptic terminal Syn-GFP expression was analyzed in Image
J using the Analyze Particles routine (size 0.2–5 mm2, default
automatic thresholding) to measure terminal density. Histograms
of mean terminal density were plotted in Prism 5 (GraphPad).
Fluorescence recovery after photobleaching (FRAP) was per-
formed on individual Syn-GFP expressing terminals by first
acquiring a baseline image at low laser power (power at sample
,5–10 mW), then a ROI was drawn around one terminal and this
region imaged a high laser power (power at sample ,45 mW) for
2–5 sec until there was an approximately 75% decrease in
terminal signal compared to baseline. Then laser power was
returned to the previous low setting and terminals were imaged
repeatedly every 1–2 min. Fractional recovery of fluorescence
signal was calculated as the fraction of recovered signal over the
total bleached signal. FRAP of Syn-GFP expressing cell bodies was
done in a similar manner as terminal bleaching except a larger
ROI was either made around the whole soma or one half of the
soma and a bleaching pulse of ,5 sec used while moving the focus
in the z-direction to bleach the appropriate volume. All animals
were maintained at an internal temperature 32–37 C, as measured
by rectal probe, with a homeothermic blanket (Harvard
In this study we perform multiphoton laser scanning microscopy
and measure a-synuclein levels in the living brain using a
previously described enhanced Green Fluorescent Protein (GFP)-
tagged human a-synuclein (Syn-GFP) transgenic mouse line called
PDNG78 . This initial characterization demonstrated that
fusion of GFP to human a-synuclein’s C-terminus and expression
under the human Platelet Derived Growth Factor promoter lead
to robust expression in a subset of cortical neurons. In addition,
this previous work demonstrated increased transgene expression at
the mRNA level at ,3-fold higher levels compared to that found
in human brain .
Before starting in vivo imaging we used high resolution confocal
imaging in fixed cortical tissue to determine the cellular
localization of Syn-GFP. This analysis demonstrates that Syn-
GFP is detectable in the cell bodies of a sparse subset (,1–3%) of
layer 2/3 cortical neurons and in multiple neuropil puncta
(Fig. 1A). Visual inspection shows that the vast majority (.99%) of
these puncta are contained within axon-like structures and when
paired with previous work showing colocalization of Syn-GFP
puncta with the presynaptic terminal marker synaptophysin and
electron microscopic localization of Syn-GFP at presynaptic
terminals  strongly suggests that most neuropil puncta
represent presynaptic accumulations of Syn-GFP. Confocal
analysis of fixed Syn-GFP tissue also reveals that immunohisto-
chemical staining for human a-synuclein and GFP colocalize as
would be expected for this fusion protein (Fig. 1C–E).
Multiphoton imaging demonstrates that a similar pattern of
Syn-GFP localization in a sparse subset of neuronal cell bodies and
in neuropil puncta can be detected in vivo (Fig. 1B) as in fixed
tissue. In the in vivo case, however, we are unable to visualize
individual axons given the lower signal-to-noise ratio of these
structures. Another difference between the two techniques is the
greater number of neuropil puncta visualized within the plane of
focus in vivo compared to fixed tissue within a unit area (Fig. 1A–B).
This is likely because of the decreased z-axis resolution of in vivo
multiphoton imaging compared to confocal microscopy.
In order to determine if the density of Syn-GFP positive neurons
or presynaptic terminals changes as animals age we measured their
respective densities in layer 2/3 of cortex over time. A loss of Syn-
GFP positive cells or terminals over time could be a manifestation
of a-synuclein-mediated neurodegeneration. In general genetic
models of PD have not shown large amounts of frank cell loss 
but previous studies have not used similar in vivo techniques to
follow cell or synapse number, which may be more sensitive. In
our first cross-sectional study we found that over a period of more
than 1 year the density of Syn-GFP expressing neurons (age 2
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months: 9830+1470 cells/mm3n=6 animals, age 3 months:
10090+1390 cells/mm3n=5 animals, age 14–17 months:
10200+1460 cells/mm3n=5 animals; Fig. 2A) and presynaptic
terminals (age 4 months 4.56+0.876107/mm3n=5 animals, age
9–12 months 4.75+1.166107/mm3n=4 animals; Fig. 2B) did
not change significantly. In addition, to further characterize the
Syn-GFP neuropil puncta we plotted the distribution of mean
fluorescence intensity for each punctum within a field of view in an
animal 4 months old (Fig. 2C, top panel) and this distribution is
positively skewed with a tail towards higher mean puncta
intensities. This indicates that Syn-GFP levels can vary by
several-fold in different presynaptic terminals. Even with the wide
range in Syn-GFP terminal levels detected the general shape of
this distribution also did not vary with age (Fig. 2C). Next we
performed chronic imaging of the same region of cortex over time.
This second longitudinal analysis shows that the pattern of
expression of Syn-GFP in particular neuronal cell bodies is stable
over a period of weeks (Fig. 3A1-2). A total of 42 individual Syn-
GFP positive cell bodies (n=2 animals) were followed within a
total volume of cortex of 46106mm3over 49 days and all 42 cells
present on day 0 were also present on day 49. In addition, no
additional new Syn-GFP positive neurons were detected within
this volume. These results suggest that there is no large scale
neurodegeneration or synapse loss in Syn-GFP expressing neurons
at the ages tested.
The analysis of individual Syn-GFP positive presynaptic
terminals over days to months is more complicated than that for
Syn-GFP positive cell bodies (described above) since the higher
density of terminals makes it difficult to follow the same terminals
over the course of days. This is in part because of the small
differences in imaging conditions that are inherently present from
day to day and the small amount of movement of individual
terminals relative to each other that likely occurs. However, we
have found that if only the high intensity terminals (those
expressing the most Syn-GFP) are selected their density is low
enough to follow individual high intensity terminals over time
(Fig. 3B1-3, C1-4). This analysis of high intensity terminals showed
that individual terminals could be followed over a period of
months and that some terminals were stably present while others
were lost over time (Fig. 3B1-3, C1-4).
Two different ‘‘window’’ techniques have been developed to
perform in vivo multiphoton imaging in the cortex of rodents, each
with its own advantages and disadvantages [27–29]. We tested
whether the ‘‘glass coverslip’’ cranial window approach used in
this study might produce detectible changes in Syn-GFP over time.
Different changes possibly related to the window placement
process itself have been suggested in some model systems [18,30].
In our case, however, repeated imaging of cortical Syn-GFP did
not produce any noticeable changes in either the pattern of
expression (Fig. 4A–C), density of labeled Syn-GFP cell bodies
(day 0 post-window: 10030+1350 cells/mm3n=16 animals, 6
months post-window: 9130+4000 cells/mm3n=3 animals;
4.73+0.886107/mm3n=6 animals, 6 months post-window:
4.49+1.266107/mm3n=3 animals; Fig. 4E). In addition, the
distribution of mean fluorescence intensity for Syn-GFP neuropil
puncta maintained the same general shape with time after window
placement (Fig. 4F).
Together all the data presented above suggest that this
experimental approach provides a powerful new paradigm for
visualizing a-synuclein in the living brain, something difficult to do
with other methods.
In order to better characterize the mobility of a-synuclein
within neurons in our system and compare this to what has been
Figure 1. Syn-GFP is present in neuronal cell bodies and presynaptic terminals. A. Fixed tissue confocal image of Syn-GFP in the cortex
shows one neuronal soma and that the vast majority of neuropil puncta staining is present within axons. Endogenous GFP fluorescence is shown
without antibody labeling. B. In vivo multiphoton image of Syn-GFP in the cortex shows staining in one neuronal soma and multiple neuropil puncta.
Scale bar for A–B 10 mm. C. Fixed tissue confocal image from the cortex of a Syn-GFP animal showing GFP staining in two somata and in the neuropil.
D. Staining in the same section for human a-synuclein. E. Merged image shows colocalization between GFP signal and human a-synuclein, as
expected for this fusion protein. Scale bar for C–E 20 mm.
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reported in the literature for hippocampal neurons in culture 
and C. elegans body wall muscle , we used the fluorescence
recovery after photobleaching (FRAP) technique in vivo to
photobleach Syn-GFP in individual presynaptic terminals and
measure the recovery of this signal over time. Given the positively
skewed distribution of mean terminal Syn-GFP intensities we
observed (Fig. 2C) and previous work from C. elegans suggesting
two different populations of inclusions in body wall muscle we
decided to selectively photobleach two different populations of
presynaptic terminals, high intensity terminals (those in the top
decile of the intensity distribution) and ‘‘normal’’ terminals (those
outside the top decile). After photobleaching both kinds of
terminals we measure a similar rate of recovery of Syn-GFP
signal with a t1/2,2 min (Fig. 5) in both cases. This is slower than
has been reported in dissociated hippocampal cell cultures for
GFP-tagged a-synuclein at presynaptic terminals  and in C.
elegans body wall muscle for yellow fluorescent protein (YFP)-
tagged a-synuclein . This suggests a difference in presynaptic
terminal a-synuclein mobility in cortical neurons in vivo compared
to neuronal culture or in worm body wall muscle, the possible
causes of which are discussed below. In our experiments
fluorescence signal did not recover fully to its pre-photobleaching
baseline and the fractional level of recovery was significantly
different between high intensity and normal terminals (fractional
recovery at 8 min normal intensity terminals: 0.76+0.22, n=15
terminals; high intensity terminals: 0.20+0.12, n=10 terminals; t-
test p,0.0001; n=5 animals). These fractional levels of recovery
are similar to those reported in the two previous studies and can be
related to the fraction of immobile a-synuclein species present at
terminals, in our case ,25% in normal terminals and ,80% in
high intensity terminals.
Next we tested the turnover and mobility of a-synuclein within
photobleached Syn-GFP throughout the entire cell body and then
measured its time course of recovery. The rate of a-synuclein
synthesis within cells is an issue of importance since multiple
Figure 2. Syn-GFP positive cell body and presynaptic terminal density does not vary with age. A. Group data of average Syn-GFP positive
cell body density in layer 2/3 of cortex at different ages demonstrates no difference with age. B. Group data of average Syn-GFP positive presynaptic
terminal density in layer 2/3 of cortex at different ages demonstrates no difference with age. C. Representative histograms of mean terminal intensity
(in arbitrary units) demonstrate a similar shape at three different ages. n=number of animals, error bars=1 SD.
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mechanisms controlling this synthesis have been postulated to play a
role in PD pathogenesis [3–6]. We measure a t1/2of recovery ,1 hr
after photobleaching Syn-GFP throughout the soma (Fig. 6A and C).
Given this slower time course the source of Syn-GFP contributing to
this recovery is not clear since it may represent the synthesis and
maturation of newSyn-GFP molecules,the movement ofunbleached
Syn-GFP located in other regions (e.g. presynaptic terminals) back to
the cell body after the establishment of a concentration difference by
somatic FRAP, or a combination of both. In order to better
characterize the mobility of a-synuclein within neuronal cell bodies
and determine how this movement might play a role in the rate of
Syn-GFP recovery after whole-cell bleaching we photobleached Syn-
GFP in a region encompassing only one half of the soma and
measured its recovery. Somewhat to our surprise, imaging cell bodies
after half the soma had been photobleached (bleaching pulse ,5 sec
duration) demonstrated that Syn-GFP within the entire cellbody had
been greatly reduced to a level equivalent to that seen with whole-cell
bleaching (relative signal post whole-cell bleach: 0.39+0.15, n=20
cells; half-cell bleach: 0.38+0.24, n=3 cells; n=3 animals; Fig. 6B
and C). In addition, the t1/2of recovery after half-cell bleaching was
essentially identical to that seen with whole-cell bleaching (Fig. 6C).
These results strongly suggest that a-synuclein is rapidly mobile
within the somatic compartment since essentially all somatic Syn-
GFP molecules visit the bleached half of the cell during the 5 sec long
bleaching pulse. Given this rapid mobility it is possible that
redistribution of Syn-GFP from unbleached regions back to the
soma plays a role in the recovery of signal after whole-cell bleaching.
Determining the relative contribution of this process (vs. new
avenue for further study.
Several lines of evidence implicate abnormal a-synuclein
regulation and aggregation in the etiology of PD [1–2]. Because
our understanding of a-synuclein function and dysfunction comes
mainly from in vitro studies, in vivo approaches need to be developed
to test implicated mechanisms and potentially reveal new ones that
are relevant to PD pathogenesis in the living mammalian brain.
The in vivo imaging model we have developed measures
fluorescently-labeled a-synuclein in individual expressing neuronal
cell bodies and presynaptic terminals and can follow them serially
over a period of months. It must of course be noted that Syn-GFP
may have different biophysical properties than untagged a-
synuclein, including an alteration in propensity to aggregate.
Recent studies suggest that split fluorescent protein a-synuclein
constructs are able to form oligomeric and larger species [31–32]
but the similarity of these aggregates to those found in PD and
other synucleinopathies is not clear. Although more work needs to
be done to compare fibrillization kinetics and other biophysical
properties of GFP-tagged a-synuclein to the untagged species, the
use of in vivo imaging in Syn-GFP mice represents a substantial
technical advance that can be used to address many questions
relating to a-synuclein biology in a relevant context. Our data
show that in this transgenic line a sparse subset of cortical neurons
expresses Syn-GFP and that the density of expressing neurons does
not change substantially with age. In addition, chronic imaging
reveals that the subset of Syn-GFP expressing cells seems to be
invariant over the course of months, since the rates at which new
neurons start to express Syn-GFP or already expressing ones stop
expressing the transgene (or degenerate) both appear to be
Figure 3. Syn-GFP cell body and high intensity presynaptic terminal expression is relatively stable over time. In vivo multiphoton
images of the same region at 2 different time points (A1: day 0, A2: day 49) show that Syn-GFP cell body expression is stable over weeks. Arrows show
4 cell Syn-GFP positive bodies that are present at both time points. Scale bar for A1-2 10 mm. In vivo multiphoton images of two different regions (B1-
3 and C1-4) repeatedly imaged at different time points show that high intensity Syn-GFP terminal expression can be followed over months. Arrows
show multiple Syn-GFP positive terminals that are present at all the time points and the arrowheads show high intensity terminals that disappeared
over time. Scale bars for B 10 mm and C 7.5 mm.
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Figure 4. Syn-GFP expression pattern, cell body and terminal density do not vary with time post-window placement. In vivo
multiphoton images of Syn-GFP in the cortex at 3 different times post-window placement (A: day 0, B: 3 months, C: 6 months) shows a similar pattern
of staining. Scale bar 10 mm. D. Group data of average Syn-GFP positive cell body density in layer 2/3 of cortex at different times post window
placement demonstrates no significant difference. E. Group data of average Syn-GFP positive presynaptic terminal density in layer 2/3 of cortex at
different times post-window placement demonstrates no significant difference. F. Representative histograms of mean terminal intensity (in arbitrary
units) demonstrate a similar shape at different times post-window placement. n=number of animals, error bars=1 SD.
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negligible at the ages tested. In addition, the placement of the
cranial window itself does not appear to cause detectable changes
in Syn-GFP expression. These results suggest that this model
system would be particularly useful for experiments that follow a-
synuclein expression in individual neurons and synapses over time,
both before and after particular manipulations. One possible
application for these techniques would be to test the role of
different biochemical pathways in determining steady-state a-
synuclein levels in vivo both in the cell body and at the presynaptic
terminal. Pharmacologic or genetic manipulation can first be
targeted to specific processes thought to be important for a-
synuclein regulation and toxicity such as the ubiquitin-proteasome
system, autophagy or a-synuclein phosphorylation. Then Syn-
GFP can be measured in individual cells and terminals to test if
manipulation causes expression pattern changes or cell loss
suggestive of neurodegeneration. This level of sensitivity is difficult
to obtain with other techniques and holds the promise of revealing
important information about a-synuclein biology in a relevant
We have also measured the mobility of a-synuclein at the
presynaptic terminal in vivo. Interestingly, the t1/2 for terminal
photobleaching recovery we measure (,2 min) is slower than that
measured in a photobleaching study of GFP-tagged a-synuclein at
presynaptic terminals in an acute dissociated hippocampal cell
culture system (,10 sec, ), or YFP-tagged a-synuclein in C.
elegans body wall muscle (,10 sec, ). In addition to the
presynaptic terminal, we have also measured the turnover and
mobility of a-synuclein in the somatic compartment of cortical
neurons in vivo. In contrast to the discrepancy between our
presynaptic terminal results and those reported in the literature,
the rapid mobility of Syn-GFP we measure within the soma
(,5 sec) is in good agreement with these previous studies of a-
synuclein mobility. The cause, however, for the difference between
our measured presynaptic terminal mobility and the previous
studies is not clear but several possibilities exist. One possibility
could be that a-synuclein in mouse cortical neurons in vivo has the
same intrinsic mobility as in hippocampal culture  and worm
body wall muscle  but that differences in the geometry of the
axonal tree in vivo cause slower measured mobility. In cell culture
and in worm body wall muscle nearby reservoirs of unbleached a-
synuclein are likely to be much closer to the bleached region than
in the mouse cortex where the axonal tree is more complicated
and presynaptic terminals (reservoirs of unbleached protein)
farther apart. Alternatively, intrinsic mobility may be different in
the different systems because the binding affinity of monomeric a-
synuclein to lipid membranes in the terminal (a key determinant of
Figure 5. Syn-GFP presynaptic terminal signal recovers differently after photobleaching. A. In vivo multiphoton images of the same
region of Syn-GFP positive terminals over time. One normal terminal (marked by the white circle) is photobleached just after t=0. This sequence
shows full recovery of Syn-GFP signal over several minutes. B. Similar time sequence as in panel A but in this case one high intensity terminal (marked
by the white circle) is photobleached just after t=0. This sequence shows partial recovery of Syn-GFP signal. Scale bar A and B 5 mm. C. Group data
plotting normalized fluorescence before and after presynaptic terminal photobleaching (marked by yellow arrow; normal terminals n=15 terminals,
high intensity terminals n=10 terminals; n=5 animals, error bars=1 SD; * represents statistically significant difference at t=8 min, t-test p,0.0001).
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mobility) may be higher in vivo due to changes in the protein itself
(e.g. phosphorylation state), the membrane (lipid composition), or
differences in temperature. Another possibility could be the
presence of a-synuclein in a multimeric or small aggregate form
which would be expected to diffuse less freely. In support of this
concept the presynaptic terminal has recently been suggested to be
the subcellular location where a-synuclein aggregation begins and
microaggregates form . Alpha-synuclein-GFP fusion con-
structs have been used by many groups to study a-synuclein
function in different contexts [22,34–36], but the effect of this
construct, if any, on oligomerization in synaptic terminals in vivo is
unknown. Previous work in this mouse line does demonstrate the
presence of granular cytoplasmic aggregates of Syn-GFP in
lysosomes  but the relationship of these aggregates to those
found in synucleinopathies is not clear. Which of these (or other)
possibilities give rise to this discrepancy between measured a-
synuclein mobility between our data and that reported in the
literature is not certain. Our work, however, does suggests specific
differences in the mobility of a-synuclein in different subcellular
compartments in mouse cortical neurons in vivo as compared to
other systems that will be interesting to analyze in more detail in
Our measurement of the differential fractional recovery of
fluorescence signal after photobleaching in normal terminals
(,75%) and high intensity terminals (,20%) in vivo suggests that
the amount of immobile a-synuclein is different in these two
groups. Literature values reported for GFP, GFP-tagged wild-type
a-synuclein and GFP-tagged a-synuclein bearing the human
disease mutation A30P in presynaptic terminals of cultured
hippocampal neurons are a fractional recovery of ,70–80%
, similar to what we find for normal terminals in vivo. In
contrast, relatively immobile GFP-tagged synapsin had a lower
fractional recovery ,30–40% , which is closer to the
fractional recovery we measure for high intensity terminals. In
worm body wall muscle two different populations of YFP-tagged
a-synuclein, one with a higher level of fractional recovery (,80%)
and another with a lower level (,40%) have been reported .
This result was interpreted as suggesting the presence of two
Figure 6. Syn-GFP somatic signal recovers slowly after whole-cell photobleaching and is rapidly mobile during half-cell
photobleaching. A. In vivo multiphoton images of the same region of Syn-GFP positive cell bodies over time. One soma (marked by the white
circle) is photobleached just after t=0. This sequence shows partial recovery of Syn-GFP signal over 60 min. B. Similar time sequence as in panel A but
in this case half the soma (marked by the white rectangle) is photobleached just after t=0. Scale bar in A 10 mm and in B 20 mm. C. Group data
plotting normalized fluorescence before and after photobleaching (marked by yellow arrow; whole-cell bleach: n=20 cells, half-cell bleach: n=3 cells;
n=3 animals, error bars=1 SD).
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different kinds of inclusions, one with less and one with more Download full-text
immobile a-synuclein protein. We interpret these data similarly,
and suggest that there are at least two pools of Syn-GFP in the
terminals, one of which has substantially less mobility than the
others. Interestingly, however, the high intensity terminal
population that has a lower mobility is the same population that
shows some evidence for terminal loss over time (Fig 3C).
Our work describes a new model system for studying the biology
of a-synuclein in the living brain over a period of months with
single cell and even single synapse resolution. The data
demonstrate the stability of expression of Syn-GFP within neurons
in this transgenic line over time and provide the first in vivo
measurements of a-synuclein mobility within neurons and
terminals. The ability to study a-synuclein with this new level of
specificity holds the promise of revealing important insights into
the pathobiology of Parkinson’s Disease and related synucleino-
We are grateful to Dr. Kishore Kuchibhotla for helpful discussions and
Conceived and designed the experiments: VKU PJM BTH. Performed the
experiments: VKU TAW. Analyzed the data: VKU. Contributed
reagents/materials/analysis tools: ER EM. Wrote the paper: VKU PJM
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In Vivo Imaging of a-Synuclein
PLoS ONE | www.plosone.org9 May 2010 | Volume 5 | Issue 5 | e10589