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Dendritic mitoflash as a putative signal for stabilizing long-term synaptic plasticity

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Mitochondrial flashes (mitoflashes) are recently discovered excitable mitochondrial events in many cell types. Here we investigate their occurrence in the context of structural long-term potentiation (sLTP) at hippocampal synapses. At dendritic spines stimulated by electric pulses, glycine, or targeted glutamate uncaging, induction of sLTP is associated with a phasic occurrence of local, quantized mitochondrial activity in the form of one or a few mitoflashes, over a 30-min window. Low-dose nigericin or photoactivation that elicits mitoflashes stabilizes otherwise short-term spine enlargement into sLTP. Meanwhile, scavengers of reactive oxygen species suppress mitoflashes while blocking sLTP. With targeted photoactivation of mitoflashes, we further show that the stabilization of sLTP is effective within the critical 30-min time-window and a spatial extent of ~2 μm, similar to that of local diffusive reactive oxygen species. These findings indicate a potential signaling role of dendritic mitochondria in synaptic plasticity, and provide new insights into the cellular function of mitoflashes.
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ARTICLE
Dendritic mitoash as a putative signal for
stabilizing long-term synaptic plasticity
Zhong-Xiao Fu1,2,3,4, Xiao Tan2,3,4,5, Huaqiang Fang3,4, Pak-Ming Lau2,5,
Xianhua Wang3,4, Heping Cheng3,4 & Guo-Qiang Bi1,2,6,7
Mitochondrial ashes (mitoashes) are recently discovered excitable mitochondrial events in
many cell types. Here we investigate their occurrence in the context of structural long-term
potentiation (sLTP) at hippocampal synapses. At dendritic spines stimulated by electric
pulses, glycine, or targeted glutamate uncaging, induction of sLTP is associated with
a phasic occurrence of local, quantized mitochondrial activity in the form of one or a few
mitoashes, over a 30-min window. Low-dose nigericin or photoactivation that elicits
mitoashes stabilizes otherwise short-term spine enlargement into sLTP. Meanwhile,
scavengers of reactive oxygen species suppress mitoashes while blocking sLTP. With
targeted photoactivation of mitoashes, we further show that the stabilization of sLTP is
effective within the critical 30-min time-window and a spatial extent of ~2 μm, similar to that
of local diffusive reactive oxygen species. These ndings indicate a potential signaling role of
dendritic mitochondria in synaptic plasticity, and provide new insights into the cellular
function of mitoashes.
DOI: 10.1038/s41467-017-00043-3 OPEN
1Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, 230027, China. 2School of Life
Sciences, University of Science and Technology of China, Hefei, 230027, China. 3State Key Laboratory of Membrane Biology, Institute of Molecular Medicine,
Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China. 4Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute
of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China. 5CAS Key Laboratory of Brain Function and
Disease, University of Science and Technology of China, Hefei, 230027, China. 6CAS Center for Excellence in Brain Science and Intelligence Technology,
University of Science and Technology of China, Hefei, 230027, China. 7Innovation Center for Cell Signaling Network, University of Science and Technology of
China, Hefei, 230027, China. Zhong-Xiao Fu and Xiao Tan contributed equally to this work. Correspondence and requests for materials should be addressed
to X.W. (email: xianhua@pku.edu.cn) or to H.C. (email: chengp@pku.edu.cn) or to G.-Q.B. (email: gqbi@ustc.edu.cn)
NATURE COMMUNICATIONS |8: 31 |DOI: 10.1038/s41467-017-00043-3 |www.nature.com/naturecommunications 1
Synaptic plasticity is regarded as the neuronal basis of
learning and memory13. It involves a series of subcellular
signals and structural changes, including Ca2+ inux,
activation of protein kinases, reorganization of the cytoskeleton,
as well as synthesis and translocation of proteins47. Two distinct
temporal phases of plasticity have been characterized: an early
phase that involves signaling and structural changes within
seconds to minutes after induction, and a late phase that involves
enduring changes lasting for tens of minutes to hours810, yet the
key process linking the two phases are less well dened.
Like many other cellular events, these synaptic processes are
energy-consuming, and thus likely to require the involvement of
mitochondria that play a pivotal role in the cellular and synaptic
energy supply11. More than just a powerhouse, the mitochon-
drion is also emerging as an important signaling organelle that
plays an active role in regulating synaptic Ca2+ signaling12,13.In
addition, mitochondrial dysfunction is known to result in
synaptic plasticity defects and to be pivotal in various neurolo-
gical disorders12,14,15.
Recently, it has been shown that respiring mitochondria exhibit a
dynamicactivityknownasmitochondrial ashor mitoash,
a transient event comprising mitochondrial depolarization,
reactive oxygen species (ROS) production, and alkalization in the
matrix1621.Mitoash activity is closely associated with cellular and
whole-animal metabolic state22, and modulates neural progenitor
cell differentiation and proliferation23 as well as somatic cell
reprogramming24. Acting as a digital reporter of the aging process,
the frequency of mitoash in a young adults can also predict the
lifespan of Caenorhabditis elegans21. Given that mitochondria con-
stitute one of the most abundant organelles in neuronal processes, it
is conceivable that such mitoash events, if occurring at synapses,
may also participate in activity-induced synaptic plasticity.
In the current study, we explore whether and how mitochon-
drial ashes (mitoashes) participate in synaptic plasticity in
cultured hippocampal neurons, a model system that has been
used to identify important cellular mechanisms and computa-
tional rules of synaptic plasticity2528. Specically, we aim at
determining whether activities that lead to functional and
structural changes at the synapse can also cause mitoash
production in dendritic mitochondria, and, if so, whether such
mitoashes can reciprocally impact on the outcome of synaptic
plasticity. Because of the heterogeneity of synaptic plasticity
across individual synapses, we employ various stimulation para-
digms used in previous studies of long-term potentiation (LTP) to
induce lasting morphological enlargement of individual dendritic
spines, a phenomenon termed structural long-term potentiation
(sLTP)2932.Wend that induction of late-phase sLTP at
synaptic spines is associated with a preceding increase in
mitoash frequency occurring in nearby dendritic shafts, whereas
mitoash activity remains unaltered during induction of only
short-term spine enlargement. Articially-eliciting mitoashes
stabilizes short-term spine enlargement and switches it into
enduring sLTP. Furthermore, mitoashes produce local ROS
elevations on the micrometre scale, and ROS scavengers suppress
mitoash activity and impair late-phase sLTP expression,
permitting only short-term spine enlargement. These results
indicate that dendritic mitoashes may be involved in short-term
to long-term conversion of postsynaptic changes, and that may
reect a spatiotemporally specic, two-way communication
between synaptic spines and dendritic mitochondria in hippo-
campal neurons.
Results
Dendritic mitoashes in hippocampal neurons. To explore the
potential involvement of mitoashes in synaptic plasticity, we
used cultured hippocampal neurons33 transfected with mito-
chondrial matrix-targeting circularly-permuted yellow uorescent
protein (mt-cpYFP) as a mitoash biosensor16. In the spiny
dendrites of these neurons, mitochondria of various lengths
occupied dendritic shafts in single le (Fig. 1a and Supplementary
Fig. 1), and in the vast majority of cases, each spine had a
mitochondrion at its dendritic base (>90%) or was within 2 µmof
the nearest mitochondrion (99%) (Supplementary Fig. 1g, h).
These dendritic mitochondria remained largely stationary during
the period of observation, in contrast to frequent movements of
axonal mitochondria, which were much smaller in size (Supple-
mentary Fig. 2). Importantly, dendritic mitochondria underwent
spontaneous mitoash activity: individual events occurred as
sudden increases of mt-cpYFP uorescence intensity, lasted for
tens of seconds, and were conned to single organelles (Fig. 1a, b;
Supplementary Movie 1). Parallel multiparametric measurements
revealed that mt-cpYFP-reported mitoash activity was accom-
panied by alkalinization reported by mt-pHTomato (Supple-
mentary Fig. 3ac). The rising phase of the mitoash was coupled
with a step-increase of mitoSOX signal (Supplementary Fig. 3d),
with mitoSOX irreversibly reacts with various ROS and displays a
relative selectivity for superoxide. Further, concurrent mito-
chondrial depolarization was visualized as a decreasing tetra-
methylrhodamine ethyl ester (TMRE) signal (Supplementary
Fig. 3e). Thus, dendritic mitoashes of hippocampal neurons each
consists of a ROS burst, a transient pH rise, and a reversible
depolarization, and thus reects an electrical and chemical exci-
tation at the single-organelle level, as is the case in other cell
types16,18,34.
Dendritic mitoashes accompanying sLTP at adjacent spines.
To induce synaptic plasticity, we rst used a chemical LTP (cLTP)
induction paradigm25, which involved 5-min exposure to 100 µM
glycine and resulted in a long-term increase in the amplitude of
miniature excitatory postsynaptic currents (Supplementary
Fig. 4). We found that a prominent phasic increase in mitoash
frequency peaked at ~20 min after glycine exposure (Fig. 1ac).
This increase was completely blocked by the N-methyl-D-
aspartate receptor (NMDAR) antagonist D-AP5 (Fig. 1c), indi-
cating that the glycine-induced mitoash activity requires early
Ca2+ inux through NMDARs, as is the case with the induction
of cLTP25 as well as other forms of LTP4,27,35.
Using co-transfection with actin-mCherry and mt-cpYFP, we
were able to monitor the structural changes of dendritic spines
together with mitoash events through the entire process of cLTP
induction. Consistent with previous studies of cLTP and
electrically-induced LTP25,36, signicant long-term morphologi-
cal growth (termed structural LTP or sLTP hereafter) was
observed in a subpopulation of dendritic spines (~30%) after
glycine exposure, as evidenced by an increase of integrated actin-
mCherry uorescence that peaked early and persisted for at least
50 min after glycine stimulation (Fig. 1d). Interestingly, this
subpopulation of spines, but not those that remained stable
(~57%), exhibited higher dendritic mitoash activity during the
1040 min period following glycine exposure (Fig. 1e, f). Careful
examination further revealed that all spines that underwent sLTP
were each coupled with one or a few local mitoashes during this
period (Fig. 1f, g). It was also notable that ~13% spines decreased
signicantly in size following glycine treatment, and mitochon-
dria adjacent to these spines also showed an elevated mitoash
frequency (Fig. 1g). Such spines might have undergone some
form of long-term depression, in part inuenced by sLTP in
neighboring spines through heterosynaptic signaling37. These
results suggest that dendritic mitoashes may occur in the context
of structural plasticity at dendritic spines.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00043-3
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In addition to chemical stimulation, we further employed the
classical high frequency electrical eld stimulation (HFS, 3 × 100
pulses at 100 Hz, 20 s apart) (Supplementary Fig. 5a) to induce
sLTP. We found that after HFS, out of 179 spines examined,
62 (34.6%) spines showed long-term enlargement, 25 (14%)
spines showed only short-term enlargement, and 92 (51.4%)
spines were stable (Fig. 1h and Supplementary Fig. 12a). Again,
only spines underwent long-term enlargement were found to
have increased local dendritic mitoash frequency within a
30-min time window after HFS (Fig. 1i and Supplementary Fig. 5b).
These results indicate that spine sLTP is generally associated with a
phasic increase in local dendritic mitoash frequency.
In order to precisely delineate the relationship between
synaptic plasticity and mitoashes, we then targeted individual
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spines using two-photon photolysis of MNI-caged glutamate
(4-methoxy-7-nitroindolinyl-caged-L-glutamate), similar to the
method used to induce sLTP in hippocampal slices38,39. Three
paradigms of stimulation were created by varying MNI-caged
glutamate concentration and the number of uncaging laser pulses
(Supplementary Fig. 6a). In the high concentration, strong
uncaging (HS) paradigm, a train of 120 two-photon pulses
(720 nm, 2.5 ms, 8 mW) at 2 Hz in the presence of 6-mM MNI-
caged glutamate induced sLTP at targeted spines, but not at
neighboring spines (Fig. 2a, c), in agreement with previous
reports8,40. Kinetic analysis revealed a biphasic change of spine
size, characterized by an early peak followed by a gradual ~16%
decay, before stabilization at an elevated plateau. In a representa-
tive example, simultaneous mitoash imaging detected seven
events occurring in the mitochondrion immediately beneath the
stimulated spine during the 1040 min period after uncaging, but
only one event in a nearby mitochondrion during the same period
(Fig. 2b). Overall, during the 1040 min after glutamate uncaging,
a robust transient increase of mitoash events occurred in the
mitochondria underlying stimulated spines but not in those
beneath nearby, unstimulated spines (Fig. 2e), the latter did not
undergo sLTP (Fig. 2d and Supplementary Fig. 12b). It is
noteworthy that no change in the amplitude or kinetics of
mitoashes was found after uncaging stimulation (Supplementary
Fig. 6bd). This result indicates that spine activity-triggered
mitoashes and spontaneous ones are indistinguishable, consis-
tent with the notion that mitoash, if serving a signaling role,
would mainly operates via frequency-modulation21,22.
With a lower concentration (2 mM) of MNI-caged glutamate,
strong single spine-targeted glutamate uncaging (low-concentra-
tion strong stimulation, LS paradigm) (Supplementary Fig. 6a)
elicited a slowly developing sLTP that exhibited a slow
monophasic enlargement in 49% of spines (Fig. 2g and
Supplementary Fig. 12c). An increase in mitoash frequency
was again seen only in mitochondria next to the enlarged spines
~1040 min after uncaging, but not in those adjacent to stable
spines (Fig. 2h). Moreover, in both the HS and LS paradigms, the
number of mitoash events beneath a stimulated spine appeared
to positively correlate with the degree of spine size enlargement
(Fig. 2f, i). These set of experiments illustrates a tight
spatiotemporal specicity between sLTP at single spines and
mitoash activation in dendritic shafts.
The induction and expression of sLTP at the dendritic spine
involve a complex signaling cascade including the early processes
such as Ca2+ ion inux through NMDA receptors and activation
of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and
small GTPase6,41,42. The blocking effect of D-AP5 already
showed an essential role of Ca2+ inux through NMDARs.
However, since mitoash activation peaked at ~2030 min after
spine stimulation, it appeared unlikely that Ca2+ transients
directly trigger the delayed mitoash response. To evaluate the
role of signaling events downstream of Ca2+, we performed the
same uncaging experiment in the presence of KN93, an inhibitor
of CaMKII that is known to be crucial for LTP in hippocampal
cultures as well as in other systems28,35. Our results showed that
both sLTP and sLTP-linked mitoash activation were blocked by
KN93, but not by the control compound KN92 (Fig. 3a, b).
Similar results were obtained with two other CaMKII inhibitors,
KN62 and autocamtide-2-related inhibitory peptide (AIP) (Fig. 3c, d).
Thus, CaMKII activation as a key step in LTP induction is
required for mitoash activation.
Mitoashes during short- to long-term transition of sLTP. The
HS paradigm-induced sLTP might be considered as a two-step
process, a fast, early-phase enlargement followed by a slowly
developing stabilization into a sustained structural change.
Plasticity-triggered mitoashes were found in experimental
paradigms when sLTP was fully expressed. To evaluate mitoash
response to the early phases of spine plasticity, we used a weak
uncaging paradigm (40 pulses at 2 Hz, HW) (Supplementary
Fig. 6a), which was shown to induce only short-term enlargement
of targeted spines8. Indeed, while spines responded with a ~1.8-
fold peak enlargement, comparable to those in the HS paradigm,
they completely recovered in ~40 min (Fig. 4a, b and Supple-
mentary Fig. 12d), indicating that such spine enlargement failed
to stabilize. Surprisingly, mitochondria at the bases of targeted
spines showed no signicant increase in mitoash frequency
(Fig. 4c), in contrast to their responses in the HS and LS para-
digms. Thus, although one cannot exclude the possibility that
spine plasticity-triggered phasic mitoashes are an epiphenome-
non accompanying synaptic changes, it is worthwhile to evaluate
the potential role of mitoash as a putative signal to stabilize
spine enlargement after initial induction and to support the short-
to long-term transition of sLTP.
One way to directly test our hypothesis is to articially activate
mitoashes within the critical time-window prior to spine
restoration, i.e., 1040 min after initial induction of spine
enlargement. For this purpose, we rst employed the mitoash
activator nigericin, a K+/H+antiporter, which has recently been
shown in muscle cells to enhance mitoash activity without
disturbing the mitochondrial membrane potential when applied
at low concentrations18,43. We found that 50 nM nigericin
rapidly and persistently augmented mitoash frequency among
the whole population of dendritic mitochondria in hippocampal
neurons (Fig. 4c). More importantly, combination of the HW
paradigm of single spine activation with immediate nigericin
application resulted in persistent sLTP alongside mitoash
Fig. 1 Dendritic mitoashes occurring in chemical and electrical LTP induction. aDendritic mitoash activity before and after glycine stimulation. Left:mt-
cpYFP uorescence revealing mitochondria in a dendritic segment. Dashed lines delimit the boundaries of the segment. Scale bar, 10 μm. Middle and right:
kymographs showing discrete mitoashes (marked by arrows) prior to and 2029 min after glycine treatment. bTime-courses of mitoashes shown in a.
cPhasic increase in mitoash frequency after glycine stimulation (100 μM applied at time zero for 5 min). Note that this increase was abolished by D-AP5.
n=1012 neurons from seven batches for each group. Unless otherwise stated, error bars in all gures hereafter report s.e.m. *P<0.05, **P<0.01 (paired
t-test, after glycine vs. baseline). dDendritic segment visualized by actin-mCherry at 0, 30, and 50 min after glycine treatment. Images are maximal
projections of corresponding Z-stacks. White arrowheads mark a spine undergoing sLTP. Scale bar, 2 μm. eMitoash activity in the same dendrite as in d.
Left: morphology of dendritic shaft and spines outlined by dashed lines overlaid on an mt-cpYFP uorescence image. Scale bar, 2 μm. Right: kymograph and
time-course (green) of a mitoash event at the base of the marked spine in d, occurring at ~8 min after glycine stimulation. fRaster plot of mitoash
occurrence (red triangles) beneath 44 spines located on seven dendritic branches of a neuron, bisected into sLTP and stable groups according to size-
changes after glycine stimulation (blue bar). gRelationship between spine size-change and the number of corresponding mitoash events occurring 050
min after glycine treatment. Size-changes were quantied as mean actin-mCherry uorescence intensity at 30, 40, and 50 min, normalized to that prior to
stimulation. Dashed lines mark 2 SD of the uctuation under basal conditions. n=9 neurons from seven batches. hLong-term spine morphological changes
induced by high frequency eld electrical stimulation. n=179 spines in eight neurons from four batches. iMitoash activity in dendritic mitochondria
corresponding to the spines studied in h.*P<0.05, **P<0.01 (paired t-test, after electrical stimulation vs. baseline)
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00043-3
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activation, suggesting that the articially-elicited mitoashes
might indeed contribute to the conversion of otherwise short-
term spine enlargement into sLTP (Fig. 4a, b and Supplementary
Fig. 12d).
Next, we modied a mitochondrial photo-excitation protocol44
(see Methods, and Supplementary Figs. 3fj, 7) to elicit a photo-
activated mitoash (PA-mitoash) at a precisely designated time
and location. In this experimental setting, two trains of
femtosecond laser pulses (80 MHz, 720 nm, 10 mW, 1 ms;
delivered at a 30-s interval) were targeted to a designated
mitochondrion. The success rate of PA-mitoash activation was
~50% and cases of failure were used as experimental controls
(Fig. 4e, f). In contrast to Shi et al. (80 MHz, 810 nm, 1017 mW,
100 ms3s)
44, our laser stimulation by itself did not induce any
detectable changes in local ROS or calcium concentration
(Supplementary Figs. 3j, 7,9), nor did it cause any mitochondrial
swelling or fragmentation regardless of mitoash activation
(Supplementary Fig. 7). These PA-mitoashes, as detected by
multiple reporters including TMRE and mitoSOX, were indis-
tinguishable from spontaneous and nigericin-triggered mito-
ashes by virtues of amplitude and duration (Supplementary
Fig. 3and Supplementary Table 1). Strikingly, when combined
with one or more early PA-mitoashes (at 10 and/or 20 min after
uncaging stimulation) (Fig. 4d), the HW paradigm evoked robust
sLTP (Fig. 4e, f and Supplementary Fig. 12e). In contrast, no such
sLTP was induced if the same laser pulses failed to elicit a
PA-mitoash (Figs. 4e, f and Supplementary Fig. 12e), or if the
PA-mitoash events were elicited late (30 and/or 40 min after
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Fig. 2 Dendritic mitoashes occurring in sLTP induced by single-spine-targeted glutamate uncaging. aSequential view of spine enlargement following the
HStwo-photon uncaging paradigm (720 nm at 8 mW, 120 pulses of 2.5 ms at 2 Hz, with 6 mM MNI-caged L-glutamate. See also Supplementary Fig. 6a).
Red dot (at 0 min) denotes the uncaging spot placed close to a spine. Red and blue arrowheads mark this target spine and a nearby spine, respectively. Scale
bar, 2 μm. bLocal mitoash activity beneath the spines. Top inset: raster plot of mitoashes facing the target spine (red ticks) and a nearby spine (blue tick)
during a 90-min period. Left:White lines outline the dendritic shaft and spines overlaid on the mt-cpYFP image of dendritic mitochondria. Right: kymograph
showing mitoashes between 20 and 30 min after glutamate uncaging. cChanges in actin-mCherry uorescence of the target and nearby spines.
dStatistics of sLTP induced in the HS paradigm. n=51 spines in ten neurons from four batches for each group. eTime-dependent changes of mitoash
frequency corresponding to d(**P<0.01; paired t-test). fRelationship between mitoash number and spine size change. Data from the same experiments
as in dand e.giRelationship between sLTP at individual spines and local mitoash events, as in df, except that the LS paradigm was used (see also
Supplementary Fig. 6a). n=67 for target and nearby spines in 18 neurons from six batches. The target spines were further divided into responding
(open circles, size change >2 SD of basal uctuation, n=33 spines in 15 neurons from six batches) and non-responding groups (open squares,n=34 spines
in 18 neurons from six batches). Note that no change in mitoash frequency was found in non-responding spines (**P<0.01; paired t-test)
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uncaging) when the recovery from the early-phase spine
enlargement was essentially complete (Fig. 4f and Supplementary
Fig. 12e). Thus, dendritic mitoashes evoked within a critical
time-window appear to be effective in stabilizing spine size and
permitting the full expression of sLTP. These results are
consistent with a signaling role for mitoash in the short-term
to long-term transition of sLTP.
Local ROS signaling in a mitoash. We hypothesized that
diffusive messengers might emanate from the ashing mito-
chondria whereby dendritic mitoashes drive the full induction of
sLTP. We therefore measured local, cytosolic ROS (reported by
2',7'-dichlorouorescein (DCF)), calcium (by GCaMP6f), and
ATP (by magnesium green and PercevalHR) concentrations
during mitoashes. Our results showed that mitoashes were
accompanied by stepwise increases of cytosolic DCF signals that
peaked in about 10 s and was spatially-graded over a distance of
about 2 μm (Supplementary Fig. 8), indicating diffusion of
mitoash ROS into the cytosol (Fig. 5a, b). On the other hand, we
detected no appreciable changes in local calcium and ATP con-
centrations during mitoashes (Supplementary Figs. 9,10). This
does not argue against the importance of general ATP production
in sLTP. In fact, inhibiting ATP synthesis with FCCP or other
ETC inhibitors severely impaired spine actin dynamics and sLTP
(Supplementary Fig. 11). However, it is unlikely that mitoashes
support sLTP as burst of ATP energy supply. Because recent
studies have implicated ROS signaling in synaptic plasticity45,46,
we examined possible effects of the ROS scavengers Tiron and
mitoTEMPO in glutamate uncaging-induced sLTP (Fig. 5ce).
Strikingly, both scavengers suppressed the phasic increase in
mitoash frequency in the HS paradigm (Fig. 5e), and effectively
abolished sLTP, leaving only short-term spine enlargement
(Fig. 5d and Supplementary Fig. 12f). These results are consistent
with an involvement of mitoash ROS in sLTP.
Further, using local PA-mitoashes, we examined the spatial
pattern of mitoash signaling in sLTP. For this purpose, we
elicited PA-mitoashes at various distances from selected spines
at 10 and 20 min after HW uncaging stimulation applied to the
spines (Fig. 6ad). We found that the mitoash signaling in terms
of stabilizing spine enlargement was highly conned to within
only a few micrometres. Within this short distance, the farther
away the PA-mitoashes were, the less the spine growth could be
sustained. Quantitatively, the degree of persistent spine enlarge-
ment tted an exponential decay function of the distance between
spine and PA-mitoash, yielding a length constant of ~2 µm
(Fig. 6e), similar to the range of cytosolic diffusion of mitoash
ROS as indicated by DCF signals. Although these observations do
not exclude the possibility that the ROS elevation is an
epiphephenomon of mitochondrial activity, they are consistent
with the scenario that local cytosolic ROS transient in a mitoash
provides a signal for stabilizing structural plasticity at the spine.
Discussion
We present two ndings suggesting a previously unknown
interplay between synaptic plasticity in the form of sLTP and
dynamic activity of dendritic mitochondria in the form of
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**
Time (min)
Control
AIP
KN62
Mitoflashes under a spine
(10 min)
–1
d
Time (min)
Control
AIP
KN62
Normalized spine fluorescence
(
F
/
F
0
)
c
Time (min)
KN93
KN92
KN93-nearby
Mitoflashes under a spine
(10 min)
–1
b
Time (min)
KN93
KN92
KN93-nearby
Normalized spine fluorescence
(
F
/
F
0
)
a
Fig. 3 Dendritic mitoashes occurring in sLTP depends on CaMKII activation. aEffects of the CaMKII inhibitor KN93 (10 μM) and its analog KN92 (10 μM)
on sLTP induced by glutamate uncaging in the LS paradigm (see Supplementary Fig. 6a). Dashed line indicates the presence of KN93 or KN92. n=6
neurons in four batches for the KN92 and KN93 groups. bGlutamate-uncaging-induced mitoash frequency changes corresponding to the data in a.
*P<0.05; **P<0.01; paired t-test vs. baseline. cEffects of the CaMKII inhibitors AIP (10 μM) and KN62 (10 μM) on sLTP induced by glutamate uncaging
in the LS paradigm. n=6 neurons in three batches for AIP groups, n=5 neurons in three batches for KN62 groups, n=7 neurons in three batches for
control groups. Dashed line indicates the presence of AIP or KN62. dGlutamate uncaging-induced mitoash activity was blocked by AIP and KN62.
Data correspond to those in c.*P<0.05; **P<0.01; paired t-test vs. baseline
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00043-3
6NATURE COMMUNICATIONS |8: 31 |DOI: 10.1038/s41467-017-00043-3 |www.nature.com/naturecom munications
mitoashes (Fig. 6f). Firstly, early signaling events during sLTP
induction in a spine cause a phasic increase in mitoash
frequency in adjacent dendritic mitochondria, regardless whether
the induction is through glycine treatment or high-frequency
electrical stimulation or single-spine-targeted focal glutamate
uncaging. This nding provides direct evidence that dendritic
mitochondria can act as an organelle biosensor capable of
decoding the complex cellular signal originated from the spine. It
is clear that key signals for LTP induction, including Ca2+ inux
through NMDARs and subsequent CaMKII activation2,4,6,
participate in this spine-to-mitochondrion signaling. Basal ROS
signal is also involved because ROS scavengers effectively inhibit
the sLTP-linked mitoash activity. Albeit obligatory, the transient
Ca2+ signal and CaMKII activation are unlikely the direct trigger,
because the synaptic activity-linked mitoashes occur within a
broad 30 min window after synaptic stimulation. One possibility
is that downstream processes responsible for spine enlargement,
such as receptor recruitment and cytoskeletal reorganization,
consume more energy and thereby alter metabolic state in nearby
mitochondria. Indeed, it has been shown that, in other types of
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0 102030405060–10–20
0 102030405060–10–20
*
*
##
###
#
#
##
#
Group1
Group2
Group4
Group3
Time (min)
Normalized spine fluorescence
f
10 S
1 min0 min
–10 min 40 min Mito Flash
Success
Failure
e
min
min
min
Late
Early
Control : HW-uncaging
2P-uncaging of glutamate
Photoactivated mitoflash
2P-laser pulse
Glutamate uncaging site
Mitochondrion
Spine
d
Time (min)
Nearby
Control
Nigericin
Mitoflashes under a spine
(10 min)
–1
c
Time (min)
Nearby
Control
Nigericin
Normalized spine fluorescence
b
–10 min 0 min 1 min 60 min
Nigericin Control
a
Fig. 4 Articially-eliciting dendritic mitoashes converts short-term spine enlargement into sLTP. a,bShort-term spine enlargement induced by local
glutamate uncaging using the HW paradigm (see also Supplementary Fig. 6a) and its conversion to sLTP by nigericin (50 nM). Representative examples
and statistics are shown in aand b, respectively. Arrowheads in adenote uncaging target spines and dotted line in bthe period of nigericin application. Note
that nigericin did not alter the magnitude of enlargement at target spines and the size of nearby spines. Red dots (at 0 min) denote the uncaging spots
placed close to spines. Scale bars, 5 μm. For controls, n=51 spines on nine dendrites in six neurons. For nigericin and nearby groups, n=45 spines on eight
dendrites in six neurons. cDendritic mitoash events corresponding to the spines in b. Note that nigericin evoked greater, sustained mitoash activity.
*P<0.05, #P<0.01; paired t-test compared to baseline. dExperimental protocol with photoactivated mitoashes (PA-mitoashes). eRepresentative
examples showing that PA-mitoashes stabilized otherwise short-term spine enlargement into sLTP (upper row), whereas laser pulses per se in failed
attempts were ineffective (lower row). Horizontal arrowheads denote the target mitochondria for photoactivation. Vertical dashed lines mark the timing of
laser pulses. Scale bars, 5 μm. fStatistics. Group 1: at least one PA-mitoash evoked at 10 or 20 min; Group 2: no PA-mitoash evoked; Group 3: at least
one PA-mitoash evoked at 30 or 40 min; Group 4: no laser stimulation. n=13 neurons and ten batches for group 1 (61 spines) and group 2 (53 spines),
and n=5 neurons and ve batches for group 3 (28 spines) and group 4 (30 spines)
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cells, mitoash frequency varies depending on cellular metabolic
state22,47. Alternatively, the delayed mitoash activation might be
triggered by metabolically independent signals occurring late in
the sLTP process.
Secondly, we found that dendritic mitoashes appear to act
reciprocally on the synapse, to stabilize spine enlargement and
switch on the full expression of sLTP. Phasic increase of mitoash
activity is associated with full-edged sLTP, but not when only a
short-term enlargement is expressed, and this is true for all the
chemical, electrical, and photolytic paradigms of synaptic
stimulation. Manipulations that inhibit mitoashes prevent sLTP
without affecting the expression of short-term spine growth,
suggesting that the mitoash-sLTP link is causal and specic.
Conversely, when combined with weak stimulations, articially
activating the mitoash, with either chemical stimulation or
photo-excitation, is sufcient to stabilize otherwise short-term
spine enlargement into long-term sLTP. With precise photo-
activation methods, we demonstrate that this mitochondrion-to-
spine signaling displays high spatiotemporal specicity:
PA-mitoashes are effective in supporting sLTP only if they are
evoked during the early time window after spine stimulation
(<30 min) and within a distance of a few micrometres. These
results suggest that, in addition to being a biosensor, the mito-
chondrion has the potential to act as a signaling organelle and
play a role in the signaling cascade of synaptic plasticity in
hippocampal neurons.
Alternatively, mitochondrial activity may play a permissive role
in sLTP as the mitochondrion in close proximity to a spine
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**
**
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Time (min)
Control
mitoTEMPO
Tiron
Mitoflashes under a spine
(10 min)–1
e
Time (min)
Tiron
mitoTEMPO
Nearby
Control
Normalized spine fluorescence
(F/F0)
d
–10 min 1 min0 min 40 min
Tiron
Mito
TEMPOControl
c
Time (s)
DCF-0 μm
DCF-1 μm
DCF-2 μm
mt-pHTomato
F/F0
b
Merge
mt-pHTomato
1.5–2.5 μm
0.5–1.5 μm
0 μm
DCF
a
Fig. 5 Local cytosolic ROS signaling during mitoashes. a,bRepresentative images showing cytosolic DCF signal for ROS detection (upper panel),
mt-pHTomato signal for mtioash detection (middle panel), and their merged image (bottom panel). Dashed line in the middle panel delimits a spontaneously
ashing mitochondrion, and local DCF signals were measured in regions of interest marked in the upper panel, at different distances from the mitoash.
Scale bar, 5 μm. bTime courses of local cytosolic DCF signals during mitoashes. n=72 events. See also Supplementary Fig. 8.cRepresentative examples
showing inhibitory effects of the ROS scavengers, Tiron and mitoTEMPO, on sLTP induced in the HS paradigm. Red dots (at 0 min) denote the uncaging
spots placed close to spines. Arrowheads denote uncaging target spines. Scale bars, 5 μm. dTime courses of sLTP induction under different conditions.
n=7 neurons and 4 batches for Tiron (45 spines), n=7 neurons and four batches for mitoTEMPO (32 spines), and n=7 neurons and four batches for
control (48 spines) and nearby groups (48 spines). eTime courses of dendritic mitoash frequency corresponding to d. The nearby group is omitted for
clarity. **P<0.01 vs. baseline
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undergoing sLTP are providing the necessary energy required for
this process. In such a scenario, the mitoashes could simply
reect bursts of energy production in response to cellular stress
due to increased energy demand from the changing spine.
However, measurements with both magnesium green and
percevalHR failed to reveal any uctuations in local ATP
concentration during mitoashes. In contrast, mitoashes are
accompanied by transient increases in local ROS, but not calcium,
that spread over a few micrometres, in a spatial pattern closely
resembling that of mitoash regulation of sLTP. Therefore, while
energy provision by the mitochondria is undoubtedly crucial for
long-term synaptic modication, and we cannot fully exclude the
possibility that mitoash is an epiphenomenon during sLTP, it is
possible that the role of mitoashes go beyond bioenergetics.
Specically, we hypothesize that mitoashes generate local ROS
transients to signal for the stabilization of spine sLTP. Indeed,
emerging evidence indicates that ROS can play physiological roles
in various cellular processes such as regulating synaptic trans-
mission48 and modication of the actin cytoskeleton via its
interaction with small GTPases34,49, and ROS may be directly
involved in signaling synaptic plasticity12,42,45. While previous
studies have focused on roles of extracellularly and cytosolically
generated sustained ROS in LTP induction45, our work points to
a new scenario in which bursts of mitochondria-generated ROS
occur locally and timely to support the expression of sLTP in
adjacent spines. Intriguingly, from the LTD-like size reduction
and increase in mitoash occurrence observed in a small portion
of spines following glycine stimulation (Fig. 1g), it appears that
the mitoash and perhaps the resultant ROS transient also
stabilizes structural changes in the negative direction. More
generally, mitoashes produce packets of ROS signal that are
qualitatively distinguishable from basal mitochondrial ROS
produced by constitutive electron leakage of the electron transfer
chain. Whereas sustained elevation of global ROS is often detri-
mental, mitoash-generated ROS can be event-driven, spatially
conned, and promptly extinguished. All these salient features
point to an appealing cellular mechanism for generating brief and
intense ROS pulses, which could in turn activate high-threshold
ROS effectors and pathways locally. Such a mechanism that
exploits the handy mitochondrial ROS machinery and the ROS
signaling toolkits could in principle exist in various cellular
contexts and should be evaluated in future studies.
The induction and action of mitoashes thus may add a novel,
spatiotemporally specic mechanism to the picture of the
complex and dynamic cellular signaling network underlying
synaptic plasticity6,28. As such, tuning mitoash activity might
afford an effective means to regulate structural plasticity at
synapses. Furthermore, given that more than one spine can share
an extended mitochondrion, this spine-mitochondrion commu-
nication might provide a basis for a subtle form of inter-spine
associationwhereby one spine can facilitate sLTP in another.
Such a process could contribute, together with other diffusible
signaling mechanisms (e.g., Ca2+ and ATP)8, to the clustering of
02468101214
0.0
0.5
1.0
1.5
2.0
–2
–0.5
Actin
Ca
2+
CaMKII
ROS
ROS
AMPA receptor
Mitoflash
Ca
2+
Glutamate
NMDA receptor
Spine
Mitochondrion
f
Distance between mito and spine (
μ
m)
Success
Failure
Fitting curve
Fluorescence change in spine
(
Δ
F/F
0
)
e
20 S
1.14.5Failure 0
–10 min 0 min 1 min 40 min Mito Flash
μ
m
a
b
c
d
Fig. 6 Spatial specicity of putative mitoash signaling in sLTP at dendritic
spines. adRepresentative examples of time-dependent spine enlargement
with PA-mitoashes elicited (or attempted) at various distances from the
target spine 10 min after glutamate uncaging in the HW paradigm (See also
Supplementary Fig. 6a). Red dots (at 0 min) denote the uncaging spots
placed close to spines. White arrowheads denote uncaging target spines.
Red and blue arrowheads mark target spines and target mitochondria,
respectively. Vertical dashed lines mark the timing of laser pulses. Scale bars,
5μm. eDegree of long-term change in spine size as a function of the spine-
mitochondrion distance for successful PA-mitoashes (black dots). A
single-exponential curve tting yielded a spatial decay constant of 1.94 μm.
Cases with failed attempts are shown as controls (red dots). n=12 neurons
and ten batches for both the success (53 spines) and failure groups
(34 spines). fSchematic summary of possible role of mitoash signaling in
the structural plasticity of dendritic spines. Activation of postsynaptic
NMDARs causes Ca2+ inux and subsequent CaMKII activation in the
spine, which leads to the generation of a local dendritic mitoash via yet-to-
be-delineated processes. Dendritic mitoashes so activated stabilize spine
enlargement likely via a diffusive ROS signal that targets the cytoskeleton
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NATURE COMMUNICATIONS |8: 31 |DOI: 10.1038/s41467-017-00043-3 |www.nature.com/naturecommunications 9
dendritic spines during learning40,5052. Further studies of the
two-way signaling between synapses and mitochondria are likely
to reveal new mechanistic insights into synaptic plasticity and
mitochondrion-related brain disorders.
Methods
Isolation and culture of primary hippocampal neurons. All procedures were
performed following the guidelines of the Animal Experiments Committees at the
University of Science and Technology of China and Peking University.
Primary cultures were prepared using neurons from embryonic rat
hippocampus33. Briey, both hippocampi of randomly selected E18 embryos
(without distinguishing sex difference) from timed-pregnant rats (Beijing Vital
River Laboratory Animal Technology
Co. Ltd., Beijing, China) were removed and digested with 0.25% trypsin
(Sigma-Aldrich, St. Louis, MO) for 15 min at 37 °C, followed by washing in Hanks
balanced salt solution (HBSS) without Ca2+ and Mg2+ (Thermo Fisher, Waltham,
MA) and gentle pipetting. The dissociated neurons were then plated on poly-L-
lysine (Sigma-Aldrich)-coated glass coverslips in 35-mm petri dishes at 1 × 105
cells ml1and kept in serum-free medium (plating medium: Neurobasal
medium (Thermo Fisher) supplemented with 2% B27 (Thermo Fisher), 0.5 mM
L-glutaMAX (Thermo Fisher), 37.5 mM NaCl (Sigma-Aldrich), and 25 μM
L-glutamic acid (Sigma-Aldrich)) in 5% CO
2
at 37 °C. 4 days after plating, half of
the plating medium was exchanged with new medium (Neurobasal medium
supplemented with 2% B27, 0.5 mM L-glutaMAX, 37.5 mM NaCl). The medium
was then replaced every 4 days.
Plasmid transfection. Cultured neurons were transfected with an mt-cpYFP-
containing plasmid (CaMKII-mt-cpYFP) and a βactin-mCherry-containing
plasmid (CaMKII-βactinmCherry) at 89 DIV (days in vitro) using a calcium
phosphate transfection kit (Thermo Fisher). Briey, neurons cultured on coverslips
were transferred to a new petri dish with pre-warmed transfection medium
(Neurobasal medium supplemented with 2% B27, 0.5 mM L-glutaMAX, 37.5 mM
NaCl, pH 7.4), while the culture medium was preserved in the original dish.
CaMKII-mt-cpYFP (7.5 μg) and CaMKII-βactin-mCherry DNA (2.0 μg) along
with 15.5 μlof2moll
1CaCl
2
were diluted in ddH
2
O (125 μl in total). The DNA
solution was added into 125 μl 2× HBSS followed by slow pipetting and gentle
vortexing with an eighth volume added each time. The mixture was then incubated
for 1520 min at room temperature and added dropwise to the culture dish with
transfection medium. After incubati ng the neurons for 80 min in 5% CO
2
at 37 °C,
an evenly distributed layer of precipitate particles was observed across the neurons
under a ×4 objective. The DNACa2+-phosphate precipitates were dissolved with
freshly-made dissolution medium (feeding medium with extra 20 mM HEPES, pH
6.8) and incubated for 48 min at room temperature. Typically, no precipitate
particles were then observed. The transfected neurons were then transferred back
to their original dishes containing culture medium. The same transfection protocol
is used for all the other plasmids transfection. All Imaging experiments were done
with 1720 DIV neurons, except for Fig. 2gi, which were done with 1315 DIV
neurons.
Electrophysiology and cLTP induction. Whole-cell perforated patch recordings
were performed on 1315 DIV neurons, using amphotericin B for perforation. The
glass micropipettes were made on a PC-10 puller (Narishige, Tokyo, Japan), and
had resistances of 24MΩ. The pipettes were tip-lled with internal solution
[containing (in mM): potassium gluconate 136.5, KCl 17.5, NaCl 9, MgCl
2
1,
HEPES 10, EGTA 0.2, pH 7.20] and then back-lled with internal solution
containing 150 ng ml1amphotericin B. Neurons were perfused with fresh extra-
cellular bath solution (ECS) [containing (in mM): NaCl 150, KCl 3, CaCl
2
3,
HEPES 10, glucose 8, tetrodotoxin 0.0005, strychnine 0.001, and bicuculline
methiodide 0.02, pH 7.30] at a slow rate (0.7 ml min1) throughout recordings.
Miniature synaptic currents were recorded with a patch-clamp amplier
(MultiClamp 700B; Molecular Devices, Sunnyvale, CA) on an inverted microscope
(Carl Zeiss Axio Observer, Oberkochen, Germany) with phase optics. For cLTP
induction, cultured hippocampal neurons were treated with glycine (100 μM) in
ECS for 5 min and then transferred to ECS without glycine. Signals were ltered at
5 kHz and sampled at 10 kHz. Data were analyzed with Igor Pro (WaveMetrics,
Portland, OR). All experiments were performed at room temperature.
Field electrical stimulation for LTP induction. Primary hippocampal neurons
were perfused in extracellular solution containing (in mM): NaCl 150, KCl 3, CaCl
2
3,
HEPES 10, MgCl
2
2, glucose 8, pH 7.30, at the speed of 0.7 ml min1. Electrical
eld stimulation of about 15 V cm1was applied through a pair of platinum wires,
using 5 ms voltage pulses delivered from high current capacity stimulators.
Neurons were stimulated with high frequency stimulation (HFS, 3 X 100 pulses at
100 Hz, 20 s apart) to induce LTP.
Imaging mitoashes and spine morphology. An upright laser-scanning confocal
microscope (Zeiss LSM NLO 710, Oberkochen, Germany) equipped with a water-
immersion objective (WPlan-Apochromat ×20, numerical aperture 1.0, Zeiss) or
an inverted confocal microscope (Zeiss LSM NLO 710) equipped with an
oil-immersion objective (Plan-Apochromat ×40, numerical aperture 1.3, Zeiss) was
used for imaging. Neurons cultured on a coverslip were perfused with extracellular
solution containing (in mM): NaCl 150, KCl 3, CaCl
2
3, HEPES 10, D-glucose 8,
tetrodotoxin 0.0005, pH 7.4) at rate of 1 ml min1in a perfusion chamber.
For cLTP induction, 1 μM strychnine and 20 μM bicuculline methiodide
(Sigma Aldrich) were included in the extracellular solution.
Time-lapse images of mt-cpYFP were captured by excitation at 488 nm
(laser power, 23%) and collecting the emission at 490550 nm. In typical time-
series recordings of mitoashes, 350 frames of 512 × 512 pixels were collected every
10 min at 0.100.15 μm per pixel in bidirectional scanning mode, and the axial
resolution was set to 0.81.0 μm. In glutamate-uncaging experiments, 270 frames
were collected at 38 frames min1from 3 to 10 min after uncaging. For mitoSOX
(Invitrogen) uorescence measurement, the indicator (3 μM) was loaded at 37 °C
for 20 min followed by three washes, and cells were imaged by excitation at 543 nm
and collecting the emission at >585 nm.
We used ImageJ53, ZEN (Carl Zeiss), and MATLAB (MathWorks, Inc.)
software for image analysis. For mitoash detection, we analyzed the changes of
mt-cpYFP uorescence intensity in target dendritic mitochondria with Zeiss ZEN
software. The standard deviation (SD) of F/F
0
for the entire trace was calculated,
where F
0
is the average baseline uorescence intensity. The criteria for a mitoash
event were: (1) peak F/F
0
>3 SD; and (2) full duration at half-maximum >3s.
To analyze their structural plasticity, we measured changes in the intensity of
actin-mCherry uorescence in spines. Briey, 3-D stacks of mCherry images were
acquired by excitation at 543 nm and collecting the emission at >590 nm at a series
of time points. Typically, 910 frames were collected at 0.100.12 μm per pixel in
unidirectional scanning mode at axial intervals of 0.50.65 μm. One 3-D stack of
mCherry images was acquired every 10 min, alternating with the mitoash image
acquisition described above. After X-Y drift correction with Image Stabilizer54,a
time series of the maximal intensity projection was generated, and photobleaching
correction was carried out using Bleach Correction55. Then, the uorescence
intensity of target spines was analyzed using ImageJ. The uorescence intensity at
different time points was normalized to the basal value (average uorescence
intensity before glycine or uncaging stimulation). The analysis of spine size was
blinded to the results of nearby mitoash events with one author analyzing spine
size, and another analyzing mitoash events separately. The dendrites under severe
Z-axis drifts were excluded from analysis.
ATP detection during mitoashes. For simultaneous imaging of local
ATP detection and mitoashes, primary hippocampal neurons were either
co-transfected with CaMKII-mt-pHTomato and CaMKII-PercevalHR or, in cells
transfected with CaMKII-mt-pHTomato only, loaded with Magnesium Green,
AM (ThemoFisher) prior to imaging, at 5 μM for 30 min and then washed for 3 × 5
min. Image acquisition was performed 7 days after transfection, by excitation at
488 and 543 nm and collecting the emission at 500530 and >600 nm, respectively,
for CaMKII-PercevalHR and CaMKII-mt-pHTomato co-imaging; by excitation at
488 and 543 nm and collecting the emission at 500530 nm and >600 nm,
respectively, for Magnesium Green and CaMKII-mt-pHTomato co-imaging.
Dendritic ROS detection. The indicator DCF (Invitrogen, Carlsbad, CA) (5 μM)
was loaded at 37 °C for 10 min followed by 3 × 5 min washes, and image acquisition
was performed by excitation at 488 nm and collecting the emission at >500 nm
when imaged alone or 500530 nm when co-imag ed with mt-pHTomato. T o avoid
mitochondrial enrichment of DCF, image acquisition was performed only within
30 min after washes.
Calcium imaging. Neurons were transfected with CaMKII-GCaMP6f on 89DIV
and used for imaging 1 week later. Image acquisition was performed by excitation
at 488 nm and collecting the emission at >500 nm when imaging alone, or
500530 nm when co-imaged with mt-pHTomato.
Photo-activation of mitoashes. Zeiss Multispot Macro was used to photo-
activate mitoashes. The protocol was modied from a previous method44 after
optimizing the laser wavelength, power, train duration, and number of trains.
Briey, photo-illumination with a 720-nm laser (Ti: sapphire laser, 6901080 nm,
80 MHz, 10 mW, Coherent, Santa Clara, CA) was positioned at a diffraction-
limited spot on a mitochondrion, with train duration of 1 ms, train number of two,
and train interval of 30 s. The probability at which single laser stimulation
successfully induced a mitoash was ~50%. In contrast to the previous work, our
photo-activation protocol did not induce mitochondrial swelling and fragmenta-
tion (Supplementary Fig. 7), or local ROS or calcium change (Supplementary
Figs. 7,9).
MNI-glutamate uncaging with the two-photon method. MNI-glutamate
(Tocris, Bristol, UK) was photolyzed by a 720-nm beam at 8 mW from a Ti:
sapphire laser (Coherent, 6901080 nm). The stimulus duration was 2.5 ms and the
photolysis procedure was controlled using a Zeiss Multispot Macro. We used three
different photolysis paradigms. First, for strong uncaging with a high concentration
of MNI-glutamate (HS-uncaging), 6 mM MNI-glutamate was used, and 120
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episodes of laser illumination at 2 Hz were applied to a target spine. Second, for
weak uncaging with a high concentration of MNI-glutamate (HW-uncaging),
6 mM MNI-glutamate was used, and 40 episodes at 2 Hz were applied. Third, for
strong uncaging with a low concentratio n of MNI-glutamate (LS-uncaging), 2 mM
MNI-glutamate was used, and 120 episodes at 2 Hz were applied.
Statistics. Required sample sizes were estimated based on experience of similar
experiments. The experiments were not randomized. Data are expressed as mean
±s.e.m. The paired t-test was used to determine the statistical differences between
samples at different time points. The MannWhitney test was used to determine
statistical differences between two groups. For paired t-tests, all data were drawn
from normally distributed populations. For all statistical tests, tests were two-sided
and a Pvalue <0.05 was considered statistically signicant.
Data availability. All supporting data are available from the authors upon request.
Received: 24 August 2016 Accepted: 28 April 2017
References
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Acknowledgements
We thank Bin Zhang for help with culture preparation, Lei Qi and Jigui Zhang for
the actin-mCherry plasmid construction, Xin Gong for the mt-pHTomato plasmid
construction, and Drs Mu-ming Poo and IC Bruce for helpful comments on the
manuscript. This work was supported in part by the Strategic Priority Research Program
of the Chinese Academy of Sciences (XDB02050000), the National Basic Research
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00043-3 ARTICLE
NATURE COMMUNICATIONS |8: 31 |DOI: 10.1038/s41467-017-00043-3 |www.nature.com/naturecommunications 11
Program of China (2013CB835100, 2013CB531200 and 2016YFA0500403), and the
National Science Foundation of China (31130067, 31521062).
Author contributions
H.C. and G.-Q.B. conceived the project, and G.-Q.B., H.C., X.W. and P.-M.L. supervised
the project. Z.-X.F. and X.T. designed and implemented the experiments, collected,
processed and analyzed data, and drafted the manuscript. H.F. contributed to early
experiments. G.-Q.B., H.C., X.W., P.-M.L., Z.-X.F. and X.T. wrote the paper. All authors
participated in discussion and critically reviewed the manuscript.
Additional information
Supplementary Information accompanies this paper at doi:10.1038/s41467-017-00043-3.
Competing interests: The authors declare no competing nancial interests.
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00043-3
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Plasticity of neuronal structure, such as the growth and retraction of individual dendritic spines, is thought to support experience-dependent neural circuit remodeling (Bosch and Hayashi, 2012 and Holtmaat and Svoboda, 2009). Indeed, as neural circuits are modified during learning, their optimization and fine-tuning involves the weakening and loss of superfluous synaptic connections. Manipulations leading to experience-dependent plasticity of neuronal circuits also increase the rate of spine shrinkage and elimination (Holtmaat et al., 2006, Tschida and Mooney, 2012, Xu et al., 2009 and Yang et al., 2009). Yet it remains unclear how neural activity drives the selective shrinkage and loss of individual dendritic spines in response to sensory experience.
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