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Report
Exercise Reverses Behavi
oral and SynapticAbnormalities after Maternal InflammationGraphical Abstract
Highlights
d MIA causes behavioral and synaptic abnormalities in the
offspring
d Microglia-dependent synaptic engulfment is impaired byMIA
d Voluntary running in adulthood ameliorates MIA-induced
abnormalities
d Voluntary running stimulates microglia-mediated engulfment
of surplus synapses
Andoh et al., 2019, Cell Reports 27, 2817–2825June 4, 2019 ª 2019 The Author(s).https://doi.org/10.1016/j.celrep.2019.05.015
Authors
Megumi Andoh, Kazuki Shibata,
Kazuki Okamoto, ..., Yuki Miura,
Yuji Ikegaya, Ryuta Koyama
In Brief
Andoh et al. find that maternal immune
activation (MIA) causes autism spectrum
disorder (ASD)-like behaviors and
synaptic surplus in the offspring in mice.
Voluntary running normalizes synaptic
density and ameliorates abnormal
behaviors even after the onset of ASD-like
behaviors, probably by boosting synaptic
engulfment by microglia.
Cell Reports
Report
Exercise Reverses Behavioral and SynapticAbnormalities after Maternal InflammationMegumi Andoh,1,3 Kazuki Shibata,1,3 Kazuki Okamoto,1 Junya Onodera,1 Kohei Morishita,1 Yuki Miura,1 Yuji Ikegaya,1,2
and Ryuta Koyama1,4,*1Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan2Center for Information and Neural Networks, 1-4 Yamadaoka, Suita City, Osaka 565-0871, Japan3These authors contributed equally4Lead Contact
*Correspondence: [email protected]
https://doi.org/10.1016/j.celrep.2019.05.015
SUMMARY
Abnormal behaviors in individuals with neurodeve-lopmental disorders are generally believed to be irre-versible. Here, we show that voluntary wheel runningameliorates the abnormalities in sociability, repeti-tiveness, and anxiety observed in a mouse model ofa neurodevelopmental disorder induced by maternalimmune activation (MIA). Exercise activates a portionof dentate granule cells, normalizing the density ofhippocampal CA3 synapses, which is excessive inthe MIA-affected offspring. The synaptic surplus inthe MIA offspring is induced by deficits in synapseengulfment by microglia, which is normalized byexercise through microglial activation. Finally, che-mogenetically induced activation of granule cellspromotes the engulfment of CA3 synapses. Thus,our study proposes a role of voluntary exercise inthe modulation of behavioral and synaptic abnormal-ities in neurodevelopmental disorders.
INTRODUCTION
Physical exercise has attracted attention as a potential thera-
peutic strategy for abnormal behaviors in neurodevelopmental
disorders such as autism spectrum disorders (ASDs) (Pan,
2010; Anderson-Hanley et al., 2011), but the cellular and molec-
ular mechanisms for how exercise ameliorates the symptoms
remain unrevealed. Previous studies reported that exercise
may help improve cognitive performance in humans and rodents
(Aberg et al., 2009; Gomes da Silva et al., 2012), probably by
enhancing the production of neurotrophic factors and neurogen-
esis (de Almeida et al., 2013; Pereira et al., 2007). However, the
question of whether exercise modifies synaptic connections,
which are fundamental structures for brain function, remains
unanswered.
The frequency of neurodevelopmental disorders is positively
correlated with maternal immune activation (MIA), which is
induced by viral infection during pregnancy (Knuesel et al.,
2014), and several studies have reported that deficits in synap-
CelThis is an open access article und
tic function and structure underlie the pathogenesis of neurode-
velopmental disorders (Koyama and Ikegaya, 2015). Specif-
ically, the disruption of synapse excitatory versus inhibitory
(E/I) balance (Rubenstein and Merzenich, 2003; Yizhar et al.,
2011; Tyzio et al., 2014) and the increased spine or synapse
density (Tang et al., 2014; Jawaid et al., 2018; Andoh et al.,
2016) are shared pathological features between ASD patients
and ASD animal models. These findings motivated us to
examine whether voluntary exercise in adulthood reverses the
behavioral abnormalities and affects synaptic properties in
mouse offspring prenatally subjected to MIA (MIA offspring).
To test this idea, we focused on the hippocampus, because
physical exercise has been suggested to activate neurons in
the hippocampal dentate gyrus (Clark et al., 2011) and sociabil-
ity, impairment of which is a major symptom of ASD, is partly
mediated by the hippocampus (Hitti and Siegelbaum, 2014;
Okuyama et al., 2016).
In the present study, we examined the role of microglia, the
resident immune cells in the brain, in synaptic deficits in MIA
offspring. Microglia continuously survey the brain environment,
monitoring and modulating synaptic structure and function; mi-
croglia prune synapses in the lateral geniculate nucleus (LGN)
and hippocampal CA1 (Schafer et al., 2012; Paolicelli et al.,
2011) during development. We examined whether MIA induces
synaptic deficits by affecting synaptic pruning by microglia,
because MIA has been suggested to affect microglial properties
such as number, morphology, and cytokine expression (Zhu
et al., 2014; Hui et al., 2018; Mattei et al., 2017), possibly leading
to abnormalities in microglial functions, including their phago-
cytic capacities (Giovanoli et al., 2013; Fernandez de Cossıo
et al., 2017).
RESULTS
Exercise in Adulthood Ameliorates MIA-AssociatedBehaviorsWe used offspring from pregnant mice intraperitoneally injected
with the synthetic double-stranded RNA polyinosinic:polycyti-
dylic acid (poly(I:C)), on embryonic days 12.5 and 17.5 to model
MIA induced by viral infection during pregnancy (Malkova et al.,
2012; Naviaux et al., 2013). Consistent with previous reports, we
found that MIA offspring at postnatal day (P) 60 exhibited deficits
l Reports 27, 2817–2825, June 4, 2019 ª 2019 The Author(s). 2817er the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Figure 1. Adult Exercise Ameliorates MIA-Associated Behaviors
(A) Social interaction behavior assessed by the three-chamber test and graphed as a social preference index (percentage of total investigation time spent
interacting with a stranger). n = 40–45 mice.
(B) Repetitive behavior assessed by self-grooming time. n = 31–33 mice.
(C) Anxiety behavior assessed by the novelty-suppressed feeding test. Latency to feed was used as an index of anxiety and is graphed as the fraction of mice that
did not eat over a period of 5 min. n = 12–24 mice.
(D) Representative confocal images of the dentate gyrus and hippocampal CA3 immunostained for NeuN and c-Fos at P60. Mice underwent exercise from P30 to
P60. DG, dentate gyrus.
(E) Density (percentage of control) of c-Fos(+) cells in several brain regions in P60 mice that underwent exercise. n = 4 mice (2–6 regions/mouse). S1, primary
somatosensory cortex; PRh, perirhinal cortex.
Mean ± SEM (A, B, and E). *p < 0.05 and **p < 0.01 (A–C), and **p < 0.01 versus control (E); Dunnett’s test (A and B), Gehan-Breslow test (C), and Student’s t test
(E). Mice from 7 litters in (A), 6 litters in (B), and 4 litters in (C) were used for each condition (control, MIA, and MIA + exercise).
in social interaction using the three-chamber assay (Figure 1A)
and repetitive behaviors by analyzing self-grooming time (Fig-
ure 1B). BecauseMIA is likely associatedwith anxiety in offspring
(Ulmer-Yaniv et al., 2018), we also performed the novelty-sup-
pressed feeding test and found that the MIA offspring displayed
enhanced anxiety (Figure 1C). Next, we examined whether the
MIA-associated behaviors could be reversed by adult voluntary
exercise. For this purpose, we placed a runningwheel in the cage
of MIA offspring from P30 to P60. After a month of voluntary
running exercise, all observed MIA-associated behaviors were
ameliorated (Figures 1A–1C). We confirmed that neither MIA
treatment nor exercise affected basal motility (total distance:
control, 3,621.29 ± 143.69 cm; MIA, 3,472.24 ± 115.58 cm;
MIA + exercise, 3,225.90 ± 163.16 cm; n = 15–20 mice, mean
± SEM, p > 0.05, Dunnett’s test) or appetite (home cage latency
to feed: control, 58.50 ± 7.53 s; MIA, 62.42 ± 6.73 s; MIA + exer-
cise, 59.67 ± 6.56 s; n = 12–24 mice, mean ± SEM, p > 0.05,
Dunnett’s test) of the mice. Altogether, these results suggest
2818 Cell Reports 27, 2817–2825, June 4, 2019
that exercise can attenuate MIA-associated behaviors even in
adulthood.
Exercise in Adulthood Ameliorates Synaptic Defects inMIA OffspringImmunostaining of the immediate-early gene c-Fos demon-
strated that the running exercise preferentially activated the den-
tate granule cells compared to its effect on other brain regions
(Figures 1D and 1E), in agreement with a previous report (Clark
et al., 2011). Therefore, we focused on the properties of the
synapses between the granule cell axons, i.e., the mossy fibers,
and the CA3 pyramidal cells. First, we measured the develop-
mental changes in the density of mossy fiber synapses, which
were defined as puncta that colocalized presynaptic elements
(synaptoporin [SPO], a mossy fiber-specific presynaptic marker)
(Williams et al., 2011) and postsynaptic elements (postsynaptic
density 95 [PSD95]) in the CA3 (Figures 2A and 2B; Figure S1).
The density of mossy fiber synapses decreased from P15 to
Figure 2. Adult Exercise Ameliorates Synaptic Defects in MIA Offspring
(A) P30 hippocampus immunostained for SPO and PSD95.
(B) Upper: representative images of the mossy fiber pathway stratum lucidum (SL) and CA3 stratum pyramidale (SP) at P30. Lower: colocalized signals of SPO
and PSD95 in the boxed areas in the upper images.
(C) Developmental changes in the mossy fiber synapse density. n = 4–9 mice (4–12 regions/mouse).
(D) Mossy fiber synapse density at P60. n = 4–9 mice (10–12 regions/mouse).
(E) mEPSC traces taken from CA3 pyramidal cells from P30–P34 control or MIA offspring.
(legend continued on next page)
Cell Reports 27, 2817–2825, June 4, 2019 2819
P30, probably via developmental synapse elimination, and the
decreased synapse density was maintained until P60 in control
mice. However, the developmental decrease in synapses was
not observed in MIA offspring (Figure 2C), and the synapse
density at P15 was maintained until P60 (Figure 2D). The hippo-
campal neuron density was comparable between control and
MIA offspring (granule cell: control, 14.78 ± 0.34 3 105/mm3;
MIA, 14.75 ± 0.39 3 105/mm3; pyramidal cell: control, 5.63 ±
0.08 3 105/mm3; MIA, 5.77 ± 0.06 3 105/mm3; mean ± SEM,
n = 4 mice per condition, p > 0.05, Student’s t test) and thus
was unlikely to contribute to the increased synapse density in
MIA offspring. The density of inhibitory synapses was not
affected in MIA offspring (Figure S2). Furthermore, we examined
the functional properties of the mossy fiber synapses using
whole-cell patch-clamp recordings from CA3 pyramidal neu-
rons. We measured the miniature excitatory postsynaptic cur-
rents (mEPSCs) derived from the mossy fiber synapses, defined
as the difference in the mEPSCs before and those after applica-
tion of 10 mM DCG-IV, a mGluR2 agonist that specifically blocks
mossy fiber transmission (Mellor andNicoll, 2001).We found that
the mEPSC frequency, but not the amplitude, was higher in MIA
offspring than in control mice (Figures 2E–2I). Finally, we investi-
gated the structural properties of the large mossy fiber boutons,
which include multiple synaptic sites (Rollenhagen et al., 2007),
using Thy1-mGFP mice in which the membrane structure of a
portion of granule cells was labeled with GFP (Figure 2K) (Tao
et al., 2016). We found that the area of each mossy fiber bouton
was greater in the MIA offspring than in the controls (Figure 2L),
but the overall density of mossy fiber boutons was not changed
(Figure 2M). Because the area of mossy fiber boutons and that of
postsynapses in the boutons were positively correlated (Fig-
ure 2N), these results indicate that the density of functional
mossy fiber synapses was increased in adult MIA offspring.
Exercise in Adulthood Stimulates Microglia to PruneSynapses in MIA OffspringNext, we examined whether and how adult exercise affected the
synaptic properties ofMIA offspring. First, we found that theP30–
P60 exercise normalized the increased density of mossy fiber
synapses in MIA offspring to the control level (Figure 2C). More-
over, the elevated frequency ofmEPSCwas reversed by exercise
to the control level (Figure 2J). These results suggested that
exercise attenuates structural and functional abnormalities of
mossy fiber synapses. Next, we examined whether microglia,
the brain immune cells that prune synapses during development
(Schafer et al., 2012; Paolicelli et al., 2011), were involved in the
elimination of mossy fiber synapses. For this purpose, we per-
(F and G) mEPSC frequency (F) and delta frequency (G), i.e., the mossy fiber transm
(1–2 cells/mouse).
(H and I) mEPSC amplitude (H) and delta amplitude (I). n = 12 cells from 8–10 mi
(J) Delta frequency of mEPSC taken from CA3 pyramidal cells from P60–P67 mic
(K) Left: P30 hippocampus of a Thy1-mGFP mouse. Upper right: images of SL a
(L and M) Area (L) and density (M) of mossy fiber boutons. n = 5–6 mice (25–110
(N) Relationship between the area of mossy fiber boutons and the area of Homer
p = 0.029.
Mean±SEM (C, D, F–J, L, andM). *p < 0.05 and **p < 0.01; Tukey’s test after ANOV
correlation coefficient (N). Mice from 3 litters were used for each condition (contro
2820 Cell Reports 27, 2817–2825, June 4, 2019
formed a synapse engulfment assay (Schafer et al., 2012) in the
mossy fiber pathway to detect the volume of synaptic elements
engulfed by microglia in mice, in which microglia are selectively
labeled with GFP (Zhan et al., 2014) (Figure 3A). We confirmed
that no GFP(+) cell in the hippocampal CA3 was immunopositive
for CCR2 (Prinz and Priller., 2010), an infiltrating cell-specific
marker (Figure S3), while GFP(+) cells were stained with an anti-
body for Siglec-H (Zhang et al., 2006; Konishi et al., 2017) and
P2Y12R (Sasaki et al., 2003; Haynes et al., 2006; Mildner et al.,
2017), which were generally used as microglia-specific markers
(Figure S3). Furthermore, GFP(+) cells expressed interleukin-1b
(IL-1b), one of the typical cytokines released from microglia (Fig-
ure S3). We also confirmed that the expression patterns of these
markers were unchanged, even in MIA or minocycline-injected
animals (data not shown). Daily injection of minocycline from
P15 (75 mg/kg subcutaneously [s.c.]), which is frequently used
to suppress microglial activity (Kobayashi et al., 2013),
decreased the volume of CD68-positive lysosomes in microglia
at P18 (Figures S4A andS4B) and suppressed the developmental
decrease in synapse density at P20 (Figure 3B). Furthermore,
PSD95 was engulfed by microglia in the mossy fiber pathway
(Figure 3A; Figures S4C and S4D), and the engulfment of SPO
and the density of microglia were not affected by minocycline
treatment (Figures S4E–S4G). These results suggest that micro-
glia selectively engulfed the postsynaptic sites of mossy fiber
synapses during development. Whether presynapses or postsy-
napses are engulfedmay depend on the developmental changes
of synaptic structure in mossy fiber boutons that occur in the
course of synaptic formation (Figure S8A). In P18 MIA offspring,
the volume of CD68 (Figure 3C) and that of engulfed PSD95
puncta (Figure 3D), but not the density of microglia (Figure 3E),
were lower than in control mice, suggesting that the impaired
synaptic engulfment by microglia underlies the increased density
of mossy fiber synapses in MIA offspring. Finally, we examined
whether microglia were involved in the exercise-induced
enhancement of synapse elimination inMIA offspring (Figure 2C).
To test this idea, we gave MIA offspring daily injections of mino-
cycline throughout the exercise period (P30 to P60). The engulf-
ment of the presynaptic protein VGLUT1 by microglia was lower
in the mossy fiber pathway of the MIA offspring than in that of the
control mice (Figure 3F). In contrast, exercise promoted synaptic
engulfment in MIA offspring to a level comparable to that in con-
trol (Figure 3F) without affecting the density of microglia or the
CD68 volume (microglial density: control, 1.40 3 104 ± 0.17 3
104/mm3; MIA, 1.39 3 104 ± 0.15 3 104/mm3; MIA + exercise,
1.59 3 104 ± 0.15 3 104/mm3; MIA + exercise + minocycline,
1.423 104 ± 0.173 104/mm3; CD68 ofmicroglia volume: control,
ission (frequency without DCG-IV�with DCG-IV). n = 12 cells from 8–10mice
ce (1–2 cells/mouse).
e. n = 4 cells from 3 mice (1–2 cells/mouse).
nd SP. Lower right: representative mossy fiber bouton.
boutons/mouse).
1 in the boutons. n = 31 boutons from 3 mice (8–12 boutons/mouse). r = 0.39,
A (C, F, andH), Dunnett’s test (D), Student’s t test (G, I, L, andM), andPearson’s
l, MIA, and MIA + exercise) at each postnatal day. See also Figures S1 and S2.
Figure 3. Adult Exercise Stimulates Microglia to Prune Synapses in MIA Offspring
(A) (i) Representative image of microglia with CD68 and PSD95 immunostaining in the stratum lucidum (SL) of a CX3CR1GFP/+ mouse at P18. (ii) Themicroglia with
signals outside of the image field were removed. (iii) Merged CD68 (iv) and PSD95 (v) signals within the microglia. Arrows indicate PSD95 in microglial lysosomes.
(B) Mossy fiber synapse density at P15 and P20. Minocycline was injected daily from P15 to P19. n = 8 mice (74–93 fields/mouse).
(C) CD68 volume (percentage of microglial volume) at P18. n = 9 mice (4 fields/mouse).
(D) Engulfed PSD95 volume in microglia (percentage of control average) at P18. n = 8 mice (4–6 fields/mouse).
(E) Microglial density in the SL at P18. n = 4 mice (4 fields/mouse).
(F) Engulfed VGLUT1 volume in microglia (percentage of control average) at P60. n = 7–12 mice (6 fields/mouse).
(G) Mossy fiber synapse density at P60. Minocycline was injected daily from P30 to P60. n = 5–10 mice (10–12 fields/mouse).
Mean ± SEM (B–G). *p < 0.05 and **p < 0.01; Tukey’s test after ANOVA (B), Dunnett’s test (F and G), and Student’s t test (C–E). Mice from 3 litters were used for
each condition (control, MIA, MIA + exercise, and MIA + exercise + minocycline). See also Figures S3–S5.
1.62% ± 0.27%; MIA, 2.37% ± 0.19%; MIA + exercise, 2.41% ±
0.60%; MIA + exercise + minocycline, 1.51% ± 0.36%; mean ±
SEM, n = 7–8 mice, p > 0.05, Dunnett’s test). In addition, the
effect of exercise on synaptic engulfment (Figure 3F) was atten-
uated by minocycline. In agreement with these results, minocy-
cline treatment resulted in an increase in synapse density (Fig-
ure 3G). Because minocycline has various biological actions,
we examined whether minocycline directly affects exercise-
induced activity in the granule cells. We found that minocycline
did not affect the granule cell activity (Figure S5), supporting a
more specific action of minocycline on microglia downstream
of neural activity, though other indirect effects of minocycline
cannot be excluded. For example, minocycline given systemi-
cally might affect other cells beyond microglia, including reduc-
tion of infiltrating cells. In the current experimental conditions,
however, we did not detect evidence of contamination of the
Cell Reports 27, 2817–2825, June 4, 2019 2821
Figure 4. Neuronal Activation Enhances Microglial Synapse Engulfment
(A) AAV8-CaMKIIa-eGFP or AAV8-CaMKIIa-hM3Dq-mCherry was injected into the bilateral dentate gyrus at P10.
(B) Mossy fiber pathway stratum lucidum (SL) and CA3 stratum pyramidale (SP) immunostained for GFP and SPO at P31.
(C) Mossy fiber pathway SL immunostained for mCherry and Iba1 at P31.
(D) Images of microglial processes touching or not touching the mossy fiber boutons (arrow).
(E) Rate of the microglial processes touching the mossy fiber boutons. n = 3 mice (89–180 boutons/mouse).
(F) Relationship between the engulfment of VGLUT1 bymicroglia and the volume ofmCherry(+)mossy fiber boutons in the SLwith or without CNO treatment. n = 3
mice (5–6 fields/mouse). Saline: r = 0.071, p = 0.79; CNO: r = 0.74, p = 0.0007.
Mean ± SEM (E). *p < 0.05; Student’s t test (E) and Pearson’s correlation coefficient (F). See also Figures S6 and S7.
infiltrating cells (Figure S3) in both MIA and minocycline-injected
conditions. In addition, the density of Iba1(+) cells was not
changed in MIA and minocycline-injected conditions (control,
1.40 3 104 ± 0.17 3 104/mm3; MIA, 1.39 3 104 ± 0.15 3
104/mm3; MIA + exercise, 1.59 3 104 ± 0.15 3 104/mm3; MIA +
exercise + minocycline, 1.42 3 104 ± 0.17 3 104/mm3; mean ±
SEM, n = 7–8mice, p > 0.05, Dunnett’s test). From these findings,
we speculate that it is likely that minocycline did not affect the
number of infiltrating cells under the conditions and regions of in-
terest used in the current study. Altogether, these results suggest
that exercise activates microglia in adult MIA offspring to engulf
surplus synapses in the mossy fiber pathway.
Neuronal Activation Enhances Microglial SynapseEngulfmentFinally, we examined whether increased activity of granule cells
is sufficient to induce engulfment of the mossy fiber synapses
by microglia, because activity-dependent synapse competition
is a key factor that primes microglia to eat weaker synapses
(Schafer et al., 2012). For this purpose, we used the chemoge-
2822 Cell Reports 27, 2817–2825, June 4, 2019
netic technology designer receptors exclusively activated by
designer drugs (DREADD), a method for remote and transient
manipulation of the activity of cells that express the designer re-
ceptors, namely, mutated human muscarinic receptors (hM3Dq
and hM4Di) that are exclusively activated by the designer drug
clozapine N-oxide (CNO) (Alexander et al., 2009). We first
confirmed that the local injection of the adeno-associated virus
(AAV) into the dentate gyrus (Figure 4A) successfully labeled the
SPO-positive mossy fiber synapses using AAV8-CaMKIIa-eGFP
(Figure 4B). Then, we transfected the granule cells with hM3Dq
in combination with mCherry (27.0% ± 3.7% of granule cells
expressed mCherry, mean ± SEM, n = 6 mice) to induce neural
activity using AAV8-CaMKIIa-hM3Dq-mCherry and confirmed
with c-Fos immunoreactivity that hM3D-expressing granule
cells increased activity in response to CNO injection (Figure S6).
To examine the interaction between the mossy fiber bouton and
the microglia (Figure 4C), we assessed the rate of microglial pro-
cesses touching hM3Dq-expressing mossy fiber boutons (Fig-
ure 4D) and found that the intraperitoneal injection of CNO at
P30 decreased the touching events in 24 h (Figure 4E). We
further found that the microglia engulfed more VGLUT1 puncta
when the total volume of hM3Dq-expressing boutons was
higher in CNO-injected mice (Figure 4F). However, we found
no correlation between the engulfment of VGLUT1 puncta
and the total volume of hM3Dq-expressing boutons in saline-
injected mice (Figure 4F). These results suggest that CNO
injection induced activity-dependent synapse competition be-
tween hM3Dq-expressing and hM3Dq-non-expressing mossy
fiber boutons, resulting in the engulfment of weaker synapses,
i.e., hM3Dq-non-expressing synapses, by microglia. In addition,
we pharmacologically assessed the role of neuronal activity
in the pruning of mossy fiber synapses by using hippocampal
slice cultures (Figure S7) (Kasahara et al., 2016). We confirmed
that neuronal activity was necessary in the developmental elim-
ination of synapses by microglia and sufficient to induce the
elimination of synapses by microglia in slice cultures prepared
from MIA offspring (Figures S7C–S7E). Overall, these results
suggest that induced competition between the granule cells re-
sults in the engulfment of mossy fiber synapses by microglia
(Figure S8B).
DISCUSSION
The present study unveiled the role of microglia in the synapse
surplus and ASD-related behaviors observed in MIA offspring
and its modification by adult voluntary exercise. The modifica-
tion of dentate gyrus-hippocampal CA3 connections may affect
the functional connectivity of the hippocampus with other brain
regions, which is associated with autistic-like behaviors in mice
(Zhan et al., 2014). In addition, the increased density of functional
mossy fiber synapses in adult MIA offspring is consistent with
previous studies that demonstrated increased excitability and
spine density of hippocampal neurons in rodent models of
ASD (Tyzio et al., 2014; Jawaid et al., 2018).
Thoughwe have not assessed themolecular link thatmediates
neuronal activity and microglial activation in the present study,
possible candidates aremolecules involved in the classical com-
plement pathway such as C1q and C3. C1q and C3 have been
suggested to tag relatively weak or inactive synapses to be en-
gulfed bymicroglia in the retino-geniculate connections (Schafer
et al., 2012; Stevens et al., 2007). Thus, it is possible that comple-
ment molecules also contribute to exercise-induced pruning of
mossy fiber synapses, because the phenomenon likely depends
on synaptic competition. The competition between mossy fiber
synapses might be modulated by other molecules, such as
brain-derived neurotrophic factor (BDNF), which is enriched in
mossy fiber boutons (Koyama et al., 2004) and upregulated by
exercise (Voss et al., 2013). Furthermore, BDNF is released
from mossy fiber boutons in response to neuronal activity and
induces the maturation of synapses (Yoshii and Constantine-Pa-
ton, 2010). Therefore, it is possible that exercise-induced granule
cell activation promotes the release of BDNF from mossy fiber
boutons and strengthen some synapses, leading to the synapse
competition. However, changes in hippocampal BDNF levels
have not been reported in mouse models of MIA (Han et al.,
2016, 2017) except for a decrease in aged (22-month-old) MIA
mice (Giovanoli et al., 2015). In addition to these molecules,
newly generated granule cells in adulthood would be a key to
induce synaptic competition. Adult neurogenesis is enhanced
by voluntary exercise (Voss et al., 2013), and adult-born granule
cells exhibit higher excitability than existing mature granule cells
(Danielson et al., 2016). It has been revealed that MIA inhibits
neurogenesis in dentate gyrus during both postnatal days and
adulthood, leading to impaired maturation of newborn granule
cells (Zhang and van Praag, 2015). Thus, enhanced neurogene-
sis by exercise may also contribute to competition between syn-
apses derived from adult-born granule cells and existing mature
granule cells.
Our results revealed that exercise reversed the abnormal be-
haviors and synaptic surplus in adult MIA offspring. We deter-
mined that exercise activated a portion of neurons, resulting in
the engulfment of excess synapses by microglia, likely primed
by synaptic competition. However, whether the CA3 synaptic
surplus is the underlying cause of the behavioral abnormalities
in MIA offspring remains undetermined.
The abnormal behaviors in patients with neurodevelopmental
disorders such as ASDs typically appear by 8 to 10 months
of age and have been recognized to be irreversible after that
period. Reports have shown that autistic-like behaviors may be
ameliorated, but the effect was transient or necessitated genetic
modification (Yizhar et al., 2011; Mei et al., 2016). In contrast, our
findings propose clinically applicable methods to ameliorate
ASD symptoms. Further investigations are needed to better
understand the cellular and molecular mechanisms that are
involved in not only the development but also the reversible as-
pects of ASD symptoms.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Animals
B Organotypic culture of hippocampal slices
d METHOD DETAILS
B Gestational exposure to poly(I:C)
B Running
B Sample preparation and immunohistochemistry
B Stereotaxic surgery for virus infection
B Three-chamber social interaction test
B Grooming test
B Novelty-suppressed feeding test
B Open field test
B Electrophysiology
d QUANTIFICATION AND STATISTICAL ANALYSIS
B Quantification
B Statistical Analysis
d DATA AND SOFTWARE AVAILABILITY
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.
celrep.2019.05.015.
Cell Reports 27, 2817–2825, June 4, 2019 2823
ACKNOWLEDGMENTS
We thank Dr. Crocker, Dr. Kiyama, and Dr. Konishi for providing us with anti-
Siglec-H antibodies and the method for immunohistochemistry with the anti-
body. We thank the members of JCAR (The Japanese Consortium for Autism
Research) for discussion. This research was supported in part by a Grant-in-
Aid for Scientific Research (C) (26460094 to R.K.) and by a Grant-in-Aid for
Scientific Research on Innovation Area ‘‘Glia Assembly’’ (26117504 and
16H01329 to R.K.) from JSPS and by the Brain Science Foundation, Japan
to R.K. and by ERATO (JPMJER1801 to Y.I.) from JST.
AUTHOR CONTRIBUTIONS
M.A. conducted and analyzed the experimental data and wrote the manu-
script. K.S., J.O., and K.M. conducted and analyzed the experiments. K.O.
and Y.M. helped with electrophysiological experiments. R.K. designed and
planned the project and wrote the manuscript. Y.I. discussed the results and
commented on the manuscript.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: April 27, 2018
Revised: March 11, 2019
Accepted: May 1, 2019
Published: June 4, 2019
SUPPORTING CITATIONS
The following reference appears in the Supplemental Information: Wilke et al.
(2013).
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Cell Reports 27, 2817–2825, June 4, 2019 2825
STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
rabbit anti-Synaptoporin - 1:1000 Synaptic Systems Cat# 102 002; RRID:AB_261974
mouse anti-PSD95 – 1:500 Thermo Fisher Scientific Cat# MA1-046; RRID:AB_2092361
guinea pig anti-VGLUT1 – 1:1000 Synaptic Systems Cat# 135304; RRID:AB_887878
mouse anti-VGAT – 1:1000 Synaptic Systems Cat# 131 011; RRID:AB_887872
rabbit anti-Gephyrin – 1: 1000 Synaptic Systems Cat# 147 002; RRID:AB_2619838
rabbit anti-Iba1 – 1:400 Wako Cat# 019-19741; RRID:AB_839504
rat anti-CD68 – 1:200 Bio-Rad Cat# MCA1957GA; RRID:AB_324217
chicken anti-GFP – 1:1000 Abcam Cat# ab13970; RRID:AB_300798
mouse anti-mCherry – 1:1000 Abcam Cat# ab125096; RRID:AB_11133266
guinea pig anti-Homer1 – 1:500 Synaptic Systems Cat# 160 004; RRID:AB_10549720
rabbit anti-DsRed – 1:1000 Takara Bio Cat# 632496; RRID:AB_10013483
rabbit anti-CCR2 – 1:100 Abcam Cat# ab203128
rabbit anti-P2Y12 – 1:500 AnaSpec Cat# AS-55043A; RRID:AB_2298886
mouse anti-IL-1b – 1:100 Cell Signaling Technology Cat# 12242; RRID:AB_2715503
rabbit anti-c-Fos – 1:1000 Santa Cruz Biotechnology Cat# sc-52-G; RRID:AB_2106783
goat anti-c-Fos – 1:100 Santa Cruz Biotechnology Cat# sc-52; RRID:AB_2629503
mouse anti-NeuN – 1:1000 Millipore Cat# MAB377; RRID:AB_2298772
sheep anti-Siglec-H – 1:1000 Dr. Konishi N/A
secondary antibodies: conjugated to AlexaFluor dyes – 1:500 Thermo Fisher Scientific N/A
NeuroTrace 435/455 Blue Fluorescent Nissl Stain – 1:200 Thermo Fisher Scientific N21479
CY3 – 1:1000 Perkin Elmer Cat# NEL704A001KT; RRID:AB_2572409
Bacterial and Virus Strains
AAV8-CaMKIIa-eGFP Addgene 50469
AAV8-CaMKIIa-hM3D(Gq)-mCherry Addgene 50476
Chemicals, Peptides, and Recombinant Proteins
polyinosinic:polycytidylic acid Sigma-Aldrich P9582
minocycline Sigma-Aldrich M9511
clozapine-N-oxide Abcam ab141704
tetrodotoxin Tocris Bioscience 04330-31
picrotoxin Sigma-Aldrich P1675
DCG-IV Tocris Bioscience 0975
Experimental Models: Organisms/Strains
B6.129P2(Cg)-Cx3cr1tm1Litt/J The Jackson Laboratory Stock No. 005582
Thy1-mGFP Dr. Pico Caroni N/A
Software and Algorithms
ImageJ N/A
MATLAB N/A
CONTACT FOR REAGENT AND RESOURCE SHARING
Requests for resources, reagents, or questions about methods should be directed to and will be fulfilled by the Lead Contact, Ryuta
Koyama ([email protected]).
e1 Cell Reports 27, 2817–2825.e1–e5, June 4, 2019
EXPERIMENTAL MODEL AND SUBJECT DETAILS
AnimalsC57BL/6J mice (SLC, Shizuoka, Japan) and mice from the reporter line Thy1-mGFP (Lsi1; a generous gift from Dr. Pico Caroni) and
CX3CR1-GFP (Stock No: 005582, The Jackson Laboratory) were maintained under conditions of controlled temperature and a light
schedule and provided with unlimited food and water. Pregnant mice were used from E12.5 to make the maternal immune activation
(MIA) model (please see Gestational exposure to poly(I:C) in Method Details). All the born pups were used and sacrificed by P67. All
experimental procedures conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and to the
guidelines provided by the University of Tokyo (approval number: P29-15). Unless otherwise noted, males were used.
Organotypic culture of hippocampal slicesHippocampal slice cultures were prepared from P10 mice (males and females) as previously described with minor modifications
(Kasahara et al., 2016). Briefly, the posterior part of the mouse brain was cut into 400-mm-thick transverse slices using a DTK-
1500 vibratome (Dosaka, Kyoto, Japan) in aerated, ice-cold GBSS (consisting of the following (g/l): 7.00 NaCl, 0.212 MgCl2・6H2O, 2.27 NaHCO3, 0.14 MgSO4・7H2O, 0.286 Na2HPO4・12H2O, 0.37 KCl, 0.22 CaCl2・2H2O, 0.33 KH2PO4) containing 25 mM
glucose. The slices were incubated for 30-90 minutes at 4�C with cold incubation medium containing minimum essential medium
(MEM) and Hanks’ balanced salt solution (HBSS) (consisting of the following (g/l): 8.00 NaCl, 0.10 MgCl2・6H2O, 0.35 NaHCO3,
0.10 MgSO4・7H2O, 0.12 Na2HPO4・12H2O, 0.40 KCl, 0.185 CaCl2・2H2O, 0.06 KH2PO4) at a ratio of 2:1, 10 mM Tris and 25 mM
HEPES. The slices were placed on Omnipore membrane filters (JHWP02500; Millipore) in a solution containing 50% MEM, 25%
horse serum (Cell Culture Lab, Cleveland, OH, USA) and 25%HBSS, 10 mM Tris, 25 mMHEPES and 5 mMNaHCO3, supplemented
with 33mMglucose. The slices were then incubated at 37�C in a humidified incubator with 5%CO2 and 95% air. Hippocampal slices
were cultured with medium containing 1 mM tetrodotoxin (TTX; Tocris Bioscience, Bristol, UK) or 50 mMpicrotoxin (PIC; Sigma) from
5 days in vitro (DIV) to 10 DIV.
METHOD DETAILS
Gestational exposure to poly(I:C)Thematernal immune activation (MIA) model wasmade by injecting polyinosinic:polycytidylic acid (poly(I:C)) (Potassium salt; Sigma)
as previously described (Malkova et al., 2012; Naviaux et al., 2013). Pregnant dams received intraperitoneal (i.p.) injection of poly(I:C)
at two doses (3 mg/kg on E12.5 and 1.5 mg/kg on E17.5). The same volume of saline was injected into pregnant dams at the same
time to prepare the control mice. Poly(I:C)-injected offspring were used as MIA offspring and weaned at P21. All the born pups were
alive and normally developed, though some pregnant mice had abortion (the ratio was 11% for Saline group and 33% for MIA group).
No mice were housed alone. Male mice were used for the experiments.
RunningMice in the exercise group were housed in a cage (3–4 mice/cage) with a freely accessible running wheel (130 mm in diameter). The
running wheel was placed in the corner of the cage for 30 days from P30. Minocycline (Sigma, 30 mg/kg) was intraperitoneally
injected once a day beginning on the first day that the running wheel was placed in the cage. The same volume of saline was injected
as a control.
Sample preparation and immunohistochemistryExperimental mice were deeply anesthetized with isoflurane and perfused transcardially with cold phosphate-buffered saline (PBS)
followed by 4% paraformaldehyde (PFA). The brain samples were postfixed with 4% PFA for 2–4 h on ice and subsequently
immersed in 20% and 30% sucrose in PBS for 24 h and 48 h, respectively, at 4�C. Coronal hippocampal sections (40 mm thick)
were preparedwith a cryostat (HM520; Thermo Fisher Scientific,Waltham,MA, USA) at�24�C. For slice cultures, sampleswere fixed
overnight in 4% PFA at 4�C. Floating sections were used for PSD95 staining.
The fixed sampleswere rinsed three timeswith 0.1Mphosphate buffer (PB). Slices were then permeabilized and blocked for 30min
at room temperature in 0.1 M PB with 0.3% Triton X-100 and 10% goat serum. The samples were subsequently incubated with
primary antibodies in 0.1 M PB with 0.3% Triton X-100 and 10% goat serum overnight at 4�C. After the samples were rinsed three
times with 0.1 M PB, they were incubated with secondary antibodies in 0.1 M PBwith 0.3% Triton X-100 and 10% goat serum for 4 h
at room temperature. For slice cultures, the samples were incubated with primary antibodies 2 overnights at 4�C under agitation and
secondary antibodies and NeuroTrace (435/455 blue fluorescent Nissl stain, 1:200; Thermo Scientific, MA, USA) 1 overnight at 4�Cunder agitation. Finally, the samples were rinsed three times with 0.1 M PB and embedded in VECTASHIELD with DAPI (Vector Lab-
oratories, Burlingame, CA, USA) or MOUNTANT (Thermo Scientific). The primary antibodies used in this study were as follows: rabbit
anti-SPO (1:1000; Synaptic Systems, Gottingen, Germany), mouse anti-PSD95 (1:500; Thermo Scientific), guinea pig anti-VGLUT1
(1:1000; Synaptic Systems), mouse anti-VGAT (1:1000; Synaptic Systems), rabbit anti-gephyrin (1:1000; Synaptic Systems), rabbit
anti-Iba1 (1:400;Wako, Osaka, Japan), rat anti-CD68 (1:200; Bio-Rad, CA, USA), chicken anti-GFP (1:1000; Abcam, Cambridge, UK),
mouse anti-mCherry (1:1000; Abcam), guinea pig anti-Homer1 (1:500, Synaptic Systems), rabbit anti-DsRed (1:1000, Takara Bio,
Cell Reports 27, 2817–2825.e1–e5, June 4, 2019 e2
Shiga, Japan), rabbit anti-CCR2 (1:100, Abcam), rabbit anti-P2Y12 (1:500, AnaSpec, CA, USA) andmouse IL-1b (1:100, Cell Signaling
Technology, MA, USA). Secondary antibodies conjugated to Alexa fluor dyes (1:500; Invitrogen, MD, USA) were used.
c-Fos immunostaining was performed on free-floating sections as described below. The samples were incubated for 1 h in 0.2%
Triton X-100 in PBS containing 5% goat serum and with rabbit anti-c-Fos (1:1000; Santa Cruz Biotechnology, CA, USA) and mouse
anti-NeuN (1:1000; Millipore, Bedford, MA, USA) overnight at 4�C. The sections were then incubated with the goat anti-rabbit
biotinylated secondary antibody (1:500; Vector Laboratories) and Alexa fluor dye-conjugated secondary antibodies (1:500; Invitro-
gen) for 4 h at room temperature, with the avidin-biotin complex (1:100; Vector Laboratories) for 1.5 h at room temperature, and
with CY3 (1: 1000; PerkinElmer, MA, USA) for 1 h at room temperature. After three rinses, the samples were embedded. Or the
samples were incubated for 1 h in 0.3% Triton X-100 in PBS containing 5% bovine serum albumin and with goat anti-c-Fos
(1:100; Santa Cruz Biotechnology, CA, USA) and mouse anti-NeuN (1:1000; Millipore, MA, USA) overnight at 4�C. The sections
were then incubated with Alexa fluor dye-conjugated secondary antibodies (1:500; Invitrogen) for 1.5 h at room temperature. After
three rinses, the samples were embedded.
For Siglec-H immunostaining, CX3CR1GFP/+ mice were perfused with cold phosphate-buffered saline (PBS) followed by 2%
paraformaldehyde (PFA). The brain samples were immersed in 30% sucrose in PBS for 24 h at 4�C. Coronal hippocampal
sections (30 mm thick) were prepared with a cryostat and mounted on glass slides. The samples were incubated for 1 h in 0.3%
Triton X-100 in PBS containing 5% bovine serum albumin and with goat anti-GFP, rabbit anti-Iba1 and sheep anti-Siglec-H
(1:1000, provided by Dr. Konishi, assistant professor at Nagoya University) overnight at 4�C. The sections were then incubated
with Alexa fluor dye-conjugated secondary antibodies (1:1000; Invitrogen) for 1 h at room temperature. After three rinses, the samples
were encapsulated.
Stereotaxic surgery for virus infectionMIA offspring were used at P10. Mice were anesthetized using pentobarbital (25 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and placed
in a stereotaxic apparatus. The virus (0.5 mL per side) was injected into the bilateral dentate gyrus (AP: �1.9 mm, LM: ± 0.9 mm,
DV: �1.8 mm) at a rate of 0.25 mL/min using glass pipettes. For the expression of DREADD, we bilaterally injected AAV8-
CaMKIIa-eGFP or AAV8-CaMKIIa-hM3Dq-mCherry (purchased from Addgene) using glass pipettes, which were then left in
place for a few minutes before they were slowly removed. Then, the mice were intraperitoneally injected with the selective ligand
clozapine-N-oxide (CNO; Abcam, 5 mg/ kg) or saline at P30 and sacrificed at P31 for further analysis.
Three-chamber social interaction testA three-chamber test was carried out to investigate the social interaction of mice. The test was performed in a white three-
chamber box (25 3 51 3 25 cm). Age- and gender-matched C57BL/6J mice that had never been exposed to the test mice
were used as stranger mice and individually placed in a chamber. In the other side of the chamber, a cage-mate mouse was
used as a familiar mouse. Both the stranger and familiar mice were placed in wire cups to allow the test mice to freely access
and sniff the mice in the cups. The three-chamber box and cups were cleaned with 70% ethanol and wiped with paper towels
between each trial. In the first 10-min session, a test mouse was placed in the three-chamber box, where two empty cups were
located in both sides of the chamber, and allowed to freely explore to habituate the test mouse. In the second 10-min session, a
stranger mouse and a familiar mouse were placed in the cage on each side. Then, the test mouse was placed in the center
chamber of the three-chamber box and allowed to freely explore the box 10 min. The brightness of the room was 10 lux
throughout the test. The movement of the mice was recorded by a USB webcam and PC-based video capture software. The
recorded video file was further analyzed by ImageJ, and the time spent in each chamber was measured. A preference index
to stranger mice was calculated to assess the social interaction using the following formula: 100 3 time in stranger chamber/
total time in stranger and familiar chamber.
Grooming testA grooming test was performed the day after the three-chamber test to investigate repetitive behaviors. A test mouse was placed in a
normal cage without wood bedding for 20 minutes, in which the first 10 minutes was the habituation session and the last 10 minutes
was the test session. The movement of the test mouse was recorded by a USB webcam and PC-based video capture software. The
recorded video file was further analyzed to manually measure the grooming time during the test session.
Novelty-suppressed feeding testThe novelty-suppressed feeding test was carried out for 5 min in a white polystyrene box (403 403 30 cm) whose floor was covered
with wood bedding. Twenty-four hours before testing, all food was removed from the home cage, but water was freely accessible
during this period. During the test period, a single pellet was placed on a white paper platform located in the center of the test
box at which the light intensity was approximately 1000 lux. The test mouse was placed in a corner of the box, and the latency
from the starting time until the time when the mouse bit the food pellet in the center of the field was measured to examine the level
e3 Cell Reports 27, 2817–2825.e1–e5, June 4, 2019
of anxiety. Immediately after this test, the mice were transferred to their home cage, and the latency to feed in their home cage was
measured to examine their basal appetite.
Open field testThe open field test was conducted using a large, square, white polystyrene box (40 3 40 3 30 cm) with an open top and a floor
covered with clear acrylic sheeting and lasted for 10 min. The arena of the open field included the center zone (27 3 27 cm). The
movement of the test mouse was recorded by a USB webcam and PC-based video capture software, and the recorded video file
was analyzed using ImageJ to measure the total distance traveled.
ElectrophysiologyP30–34 (before exercise) or P60-67 (after exercise) mice were deeply anesthetized with isoflurane, and the brains were quickly
removed and placed in ice-cold, oxygenated (95% O2/5% CO2) artificial cerebrospinal fluid (ACSF) containing 127 mM NaCl,
1.6 mM KCl, 1.24 mM KH2PO4, 1.3 mM MgSO4, 2.4 mM CaCl2, 26 mM NaHCO3, and 10 mM glucose. Transverse slices containing
the CA3 area were cut at a thickness of 400 mm with a vibratome in ice-cold, oxygenated modified ACSF containing 222.1 mM
sucrose, 27 mM NaHCO3, 1.4 mM NaH2PO4, 2.5 mM KCl, 0.5 mM ascorbic acid, 1 mM CaCl2, and 7 mM MgSO4. Slices were
maintained for 30 min at 37�C and then incubated for at least 30 min at room temperature before use.
Slices were transferred to a recording chamber and superfused with oxygenated ACSF containing 0.1 mM picrotoxin (30–33�C,1–3 mL/min). Whole-cell recordings were made from visually identified, pyramidal neurons located in the CA3 region using infrared
differential interference contrast (IR/DIC) techniques. Patch pipettes (3-6MU) were fabricated from borosilicate glass and filled with a
solution containing 127 mM CsMeSO4, 8 mM CsCl, 10 mM HEPES, 1 mM MgCl2, 10 mM phosphocreatine-Na2, 4 mM MgATP,
0.3 mM NaGTP, and 0.2 mM EGTA (pH 7.2–7.3, 280–295 mOsm). mEPSCs were recorded at a holding potential of �70 mV in the
presence of tetrodotoxin (1 mM). mEPSCs were detected using an in-house MATLAB program and were defined as inward currents
with amplitudes > 7 pA. DCG-IV (Tocris Bioscience, 10 mM for slices from P30-34 mice and 1 mM for slices from P60-67 mice) was
applied to block synaptic transmission from mossy fiber synapses. The series resistance was monitored, and if it exceeded 40 MU,
the data were discarded. Data were sampled at 20 kHz and filtered at 2 kHz using an Axopatch 200B, 700B amplifier (Molecular De-
vices), DIGIDATA 1320A, 1440A (Molecular Devices), and pClamp 10.2 (Molecular Devices).
QUANTIFICATION AND STATISTICAL ANALYSIS
QuantificationThe immunostained samples were analyzed with a TCS SP8 (Leica Microsystems, Wetzlar, Germany) or FV1200 (Olympus, Tokyo,
Japan) confocal system under 10 3, 20 3, 60 3 and 100 3 objectives. Z series images were collected with 0.33-mm steps, and 4 Z
sections (1 mm thick) were stacked using ImageJ (NIH) to quantify the synapse density. 11 Z series sections (5 mm thick) for the quan-
tification of synapse engulfment or 21 Z series sections (10 mm thick) for CD68 expression were collected at 0.50 mmsteps. 25 Z series
sections (8 mm thick) for the quantification of post-synaptic volume in mossy fiber bouton were collected at 0.33 mm steps. ImageJ
was used to quantify the synapse density. After the stacked images in the stratum lucidum were cropped, pre- and postsynaptic
signals were detected by determining the threshold of the fluorescent intensities. Then, the number of colocalized signals of
pre- and postsynaptic puncta were counted.
The quantification of synapse engulfment and CD68 expression was carried out as previously described with minor modifications
(Schafer et al., 2012). Microglial, synaptic, and CD68 immunofluorescent signals were detected by determining the threshold of the
fluorescent intensities in ImageJ. Then, colocalized images of microglia and synapses or CD68 were prepared, and the volume of
microglia and the colocalization were measured in the stratum lucidum. To determine the synapse engulfment by microglia, the
following calculation was used: Volume of internalized synaptic puncta (mm3)/ Volume of microglia (mm3). To determine the % of
CD68 expression, the following calculation was used: Volume of internalized CD68 (mm3)/ Volume of microglia (mm3).
For assays of microglia-dependent synaptic pruning in hippocampal slices, microglial, synaptic, and CD68 immunofluorescent
signals were detected by determining the threshold of the fluorescent intensities in ImageJ. Then, colocalized images of microglia,
synapses and CD68 were prepared, and the volume of microglia and the colocalization were measured in the stratum lucidum.
To determine the synaptic engulfment by microglia, the following calculation was used: Volume of internalized synaptic puncta
(mm3)/ Volume of microglia (mm3).
The quantification of post-synaptic volume in mossy fiber bouton of Thy-1 mGFP mice was carried out as follows; GFP-labeled
bouton and postsynaptic immunofluorescent signals were detected by determining the threshold of the fluorescent intensities in
ImageJ. Then, colocalized images of boutons and synapses were prepared, and the area of boutons and the colocalization were
measured in the stratum lucidum. All analyses were carried out in a blinded manner.
Statistical AnalysisThe data are represented as the mean ± standard error of the mean (SEM) or as box and whisker plots showing the distribution and
median (solid line) of the data (box edges indicate 25th and 75th percentiles; whiskers, min and max) and were pooled from at least 3
Cell Reports 27, 2817–2825.e1–e5, June 4, 2019 e4
independent experiments. The data were collected and statistically analyzed independently by 2 researchers in a blinded manner.
Student’s t test or Dunnet’s test, or one-way or two-way analysis of variance (ANOVA) followed by the Tukey test was performed for
statistical analysis unless otherwise described. The Gehan-Breslow test (Figure 1C) was also used.
DATA AND SOFTWARE AVAILABILITY
Reasonable requests for data will be fulfilled by the Lead Contact.
e5 Cell Reports 27, 2817–2825.e1–e5, June 4, 2019