Abstract

   Mutations that inactivate negative translation regulators cause autism
   spectrum disorders (ASD), which predominantly affect males and exhibit
   social interaction and communication deficits and repetitive behaviors.
   However, the cells that cause ASD through elevated protein synthesis
   resulting from these mutations remain unknown. Here we employ
   conditional overexpression of translation initiation factor eIF4E to
   increase protein synthesis in specific brain cells. We show that
   exaggerated translation in microglia, but not neurons or astrocytes,
   leads to autism-like behaviors in male mice. Although microglial eIF4E
   overexpression elevates translation in both sexes, it only increases
   microglial density and size in males, accompanied by microglial shift
   from homeostatic to a functional state with enhanced phagocytic
   capacity but reduced motility and synapse engulfment. Consequently,
   cortical neurons in the mice have higher synapse density, neuroligins,
   and excitation-to-inhibition ratio compared to control mice. We propose
   that functional perturbation of male microglia is an important cause
   for sex-biased ASD.

   Subject terms: Cellular neuroscience, Autism spectrum disorders,
   Molecular neuroscience
     __________________________________________________________________

   The main cell types involved in autism spectrum disorders through
   elevated protein synthesis are not well identified. Here, the authors
   show that overexpression of translation initiation factor eIF4E in
   microglia results in autism-like behaviour in male, but not female,
   mice.

Introduction

   Autism spectrum disorders (ASD) are a group of neurodevelopmental
   disorders with deficits in two core domains: social interaction and
   communication, and repetitive or restrictive behaviors^[54]1,[55]2. ASD
   are frequently associated with comorbidities, such as intellectual
   disability and anxiety^[56]3,[57]4. Prevalence of ASD is 1 in 59
   children under 8 years of age, and males are four times more likely
   than females to be identified with ASD^[58]5. The biological basis for
   this sex bias is not clear. There is a strong genetic basis for
   ASD^[59]6–[60]8. One recognized common biochemical pathway underlying
   ASD is dysregulation of protein synthesis^[61]3.

   Eukaryotic mRNAs contain a modified guanosine (m^7Gppp), termed a cap,
   at their 5′ end. Translation of mRNAs requires binding of a translation
   initiation factor eIF4E to the cap. This factor interacts with the
   scaffolding protein eIF4G, which is essential for formation of the
   translation initiation complex by bridging an mRNA to the
   ribosome^[62]9. Hypophosphorylated forms of eIF4E-binding proteins
   (4E-BPs) repress translation initiation by sequestering eIF4E and
   consequently disrupting formation of the translation initiation
   complex^[63]9,[64]10. Although most mRNAs need basal amounts of eIF4E
   to be translated, eIF4E preferentially increases translation of a
   selective set of mRNAs with extensive secondary structures at their 5′
   untranslated regions^[65]11. The mammalian target of rapamycin complex
   1 (mTORC1) phosphorylates 4E-BPs and releases eIF4E from the 4E-BP
   complex, thus stimulating translation^[66]9,[67]10.

   There are four main negative regulators upstream of mTORC1: PTEN
   (phosphatase and tensin homolog), TSC1 (tuberous sclerosis complex 1),
   TSC2 and neurofibromatosis type 1^[68]3. Inactivating mutations in the
   genes for these proteins cause ASD in a subset of
   patients^[69]12,[70]13. Furthermore, fragile X syndrome is caused by
   silencing of the FMR1 gene, which encodes fragile X mental retardation
   protein (FMRP)^[71]14, and the mTORC1-eIF4E pathway is over-activated
   in fragile X syndrome patients diagnosed with ASD^[72]15. These
   single-gene disorders account for over 3% of all ASD cases^[73]3. These
   discoveries suggest that elevated translation might cause ASD in a
   subset of individuals. Importantly, a causal relationship between
   elevated translation and ASD-like behaviors has been recently
   established in mice. Deletion of the 4E-BP2-coding Eif4ebp2 gene or
   overexpression of eIF4E under the promoter of beta tubulin (βT-Eif4e)
   increases protein synthesis in the mouse brain and leads to ASD-like
   behaviors^[74]16,[75]17.

   Microglia are derived from myeloid progenitors generated in the yolk
   sac and migrate into the brain when neurons and other glial cells only
   begin to be produced during early embryogenesis^[76]18,[77]19. After
   the closure of the blood brain barrier, microglia self-renew without
   the contribution of circulating monocytes in the healthy
   brain^[78]20,[79]21. Until recently, the primary function of microglia
   was thought to migrate to inflammation sites and engulf debris from
   dead or dying cells^[80]22. Recent work indicates that homeostatic
   microglia also play important roles in synaptic development and
   function^[81]23–[82]26.

   As synthesis of synaptic proteins is necessary for enduring synaptic
   plasticity and synaptic dysfunction can lead to ASD, it has been
   proposed that inactivating mutations in negative translation regulators
   cause ASD by enhancing translation of mRNAs in neurons^[83]27,[84]28.
   Because mRNA translation is elevated in all cells of the body in
   Eif4ebp2 knockout and transgenic βT-Eif4e mice^[85]16,[86]17, it
   remains, however, to be determined whether translational dysregulation
   in neurons is sufficient to cause ASD and whether translational
   dysregulation in glial cells contributes to autism manifestations. In
   this study, we elevated mRNA translation by overexpressing eIF4E and
   showed that exaggerated translation in microglia is sufficient to cause
   ASD-like phenotypes in mice via its detrimental impact on
   microglia-neuron interactions.

Results

Intact sociability in mice with elevated neuronal translation

   We generated a conditional eIF4E overexpression allele at the Rosa26
   locus (R26^Eif4e) that expresses Myc-tagged eIF4E in a Cre-dependent
   manner (Supplementary Fig. [87]1a, b). eIF4E-Myc interacted with eIF4G
   as efficiently as untagged eIF4E (Supplementary Fig. [88]1c),
   indicating that the Myc tag does not interfere with the function of
   eIF4E. As it is generally believed that elevated protein synthesis in
   neurons causes ASD-like behaviors^[89]27, we first overexpressed eIF4E
   in neurons by crossing R26^Eif4e mice with Syn1-Cre mice, which
   selectively express Cre in neurons as early as embryonic day
   12.5^[90]29, to generate Syn1-Cre;R26^Eif4e/Eif4e mice (termed NN^4E
   mice thereafter) (Fig. [91]1a). Levels of total eIF4E
   (eIF4E + eIF4E-Myc) in the NN^4E hippocampus were more than twice as
   high as those in control mice (Fig. [92]1b). Interestingly, neuronal
   transgenic eIF4E expression significantly reduced endogenous eIF4E
   levels (Fig. [93]1b and Supplementary Fig. [94]2a). In fact, we found
   that one copy of the R26^Eif4e allele was not sufficient to
   significantly increase levels of total eIF4E in the brain due to this
   negative feedback regulation (1.01 ± 0.10 for Syn1-Cre;R26^Eif4e/+ vs.
   1.00 ± 0.09 for R26^Eif4e/+, p = 0.92, n = 5 mice per genotype). Using
   surface sensing of translation (SUnSET) which measures incorporation of
   puromycin into nascent polypeptides^[95]30, we found that protein
   synthesis rate was doubled in cultured NN^4E hippocampal neurons over
   control neurons (Fig. [96]1c). These results indicate that neuronal
   protein synthesis is increased in NN^4E mice.

Fig. 1. Neuronal eIF4E overexpression elevates anxiety without altering
social interaction.

   [97]Fig. 1
   [98]Open in a new tab

   a Syn1-Cre;R26^Eif4e/Eif4e (NN^4E) mice overexpress eIF4E in neurons,
   while R26^Eif4e/Eif4e mice serve as controls (Ctrl). b Levels of eIF4E
   in hippocampal extracts prepared from control and NN^4E mice. The eIF4E
   immunoblot reveals both endogenous eIF4E (lower band) and overexpressed
   eIF4E-Myc (upper band). Alpha tubulin was used as a loading control.
   n = 3 per genotype. **p = 0.0015 by two-sided t-test. c Protein
   synthesis in cultured hippocampal neurons as revealed by puromycin
   incorporation. n = 3 mice per genotype. **p = 0.0023 and *p = 0.015 by
   two-sided t-test. d Time spent in the central zone during the first
   5 min in open field tests. Male: 15 control mice and 13 NN^4E mice;
   Female: 15 control mice and 13 NN^4E mice. **p = 0.0076, *p = 0.0218 by
   two-sided t-test. e Percentage of marbles buried. n = 15 control mice
   and 13 NN^4E mice. *p = 0.0247 by two-sided t-test. f, g Sociability of
   male (f) and female (g) NN^4E mice as revealed in three-chamber
   sociability tests. Male: 17 control mice and 14 NN^4E mice; Female: 14
   control mice and 13 NN^4E mice. Two-way analysis of variance (ANOVA)
   with Fisher’s LSD post-hoc test: *p < 0.05, **p < 0.01, ***p < 0.001,
   and n.s. not significant. All data are shown as mean ± s.e.m. Source
   data are provided as a Source Data file.

   We assessed if elevated protein synthesis in neurons leads to abnormal
   behaviors in mice. As revealed in rotarod and open-field tests
   (Supplementary Fig. [99]2b, c), NN^4E mice had intact motor function.
   We examined anxiety-like behaviors by performing open field, elevated
   plus maze, and light-dark box tests. Anxious mice tend to avoid open,
   exposed, and brightly illuminated areas. Although NN^4E and control
   mice spent comparable time in the light chamber in light-dark box tests
   (Supplementary Fig. [100]2e), NN^4E mice of both sexes spent
   significantly less time in the center of the open field than control
   mice in the first 5 min of a 30-min open field test (Fig. [101]1d). In
   addition, male NN^4E mice stayed longer in the closed arm of an
   elevated plus maze than control mice (Supplementary Fig. [102]2d).
   These results indicate that elevated neuronal protein synthesis
   increases anxiety. In agreement with the requirement of protein
   synthesis for long-term memory^[103]31, NN^4E mice showed more freezing
   in the contextual chamber 24 h after fear condition training than
   control mice (Supplementary Fig. [104]2g). Conversely, working memory
   was unaffected in NN^4E mice as measured by T-maze alternation
   (Supplementary Fig. [105]2f). We then ran marble burying and 3-chamber
   sociability tests to determine if elevated neuronal protein synthesis
   leads to repetitive behaviors and deficits in social interaction.
   Although female NN^4E mice showed increased repetitive behaviors in
   marble burying tests relative to control mice (Fig. [106]1e), male
   NN^4E mice were unaffected (Supplementary Fig. [107]2h). Unexpectedly,
   NN^4E mice of both sexes stayed longer in the chamber with a holder
   holding a stranger mouse than in the chamber with an inanimate object
   (empty holder) and spent more time in investigating the stranger mouse
   than the empty holder, as did control mice in 3-chamber sociability
   tests (Fig. [108]1f, g). These behavioral results indicate that
   elevated neuronal protein synthesis does not lead to deficits in social
   interaction associated with ASD. This suggests that social interaction
   impairments observed in Eif4ebp2 knockout and transgenic βT-Eif4e
   mice^[109]16,[110]17 are attributable to elevated protein synthesis in
   glial cells.

Elevated microglial translation leads to ASD-like behavior

   We next overexpressed eIF4E in astrocytes (Supplementary
   Fig. [111]3a–c), which is the largest population of glia in the brain,
   using a Cre transgene driven by the astrocyte-specific promoter for
   glial fibrillary acidic protein (GFAP-Cre)^[112]32 which becomes active
   during late embryogenesis and the first postnatal week^[113]33. We
   found that astrocytic eIF4E overexpression did not alter repetitive and
   social behaviors in mice of either sex (Supplementary Fig. [114]3d–f).
   Thus, we shifted our efforts to microglia, which make up ~10% of brain
   cells^[115]34.

   We employed Cx3cr1^CreER mice^[116]25 to overexpress eIF4E in
   microglia. To determine the efficiency for tamoxifen to activate Cre
   recombinase in microglia, we crossed Cx3cr1^CreER/+ mice to
   Rosa26^Ai9/+ mice^[117]35 to generate Cx3cr1^CreER/+;Rosa26^Ai9/+ mice.
   A single tamoxifen injection (180 mg/kg, subcutaneously) was
   administered to Cx3cr1^CreER/+;Rosa26^Ai9/+ mice at P0. Tamoxifen
   activated CreER to excise the loxP-flanked transcription blocker of the
   Rosa26^Ai9 allele in microglia, leading to tdTomato expression
   (Supplementary Fig. [118]4a). There is nearly 100% colocalization
   (n = 3 mice) between tdTomato and the microglial marker, ionized
   calcium binding adapter molecule 1 (Iba1), in the hippocampus and
   cortex (Supplementary Fig. [119]4b), indicating extremely high
   specificity in the expression of the Cx3cr1^CreER gene. Furthermore,
   the recombination activity of the CreER protein is tightly controlled
   by tamoxifen, as we did not detect tdTomato expression in
   Cx3cr1^CreER/+;Rosa26^Ai9/+ mice in the absence of tamoxifen injection
   (Supplementary Fig. [120]4c). This observation indicates that a single
   tamoxifen injection in newborn pups is sufficient to activate Cre
   recombinase selectively in microglia of Cx3cr1^CreER mice.

   Newborn pups from R26^Eif4e/Eif4e × Cx3cr1^CreER/+;R26^Eif4e/Eif4e
   crosses were treated with tamoxifen (180 mg/kg, subcutaneously) to
   generate control (R26^Eif4e/Eif4e) and MG^4E
   (Cx3cr1^CreER/+;R26^Eif4e/Eif4e) mice that should express eIF4E-Myc in
   microglia (Fig. [121]2a). Immunoblotting analysis of brain lysates
   revealed eIF4E-Myc expression in MG^4E mice (Supplementary
   Fig. [122]4d). Immunohistochemistry revealed that eIF4E levels were
   lower in microglia than in neurons in control mice, but the transgenic
   overexpression increased microglial eIF4E levels more than two folds
   (Supplementary Fig. [123]5a). To further assess microglial eIF4E
   overexpression, we employed an immunopanning method^[124]36 to purify
   microglia from control and MG^4E mice at P10 (Supplementary
   Fig. [125]6). Analysis of purified microglia revealed that two copies
   of Cre-excised R26^Eif4e alleles increased levels of total eIF4E by
   21–28% and protein synthesis rate by 28–35% in both male and female
   MG^4E microglia compared to control microglia (Fig. [126]2b).
   Interestingly, eIF4E-Myc expression did not reduce endogenous eIF4E
   levels in microglia (Fig. [127]2b), as it did in neurons (Supplementary
   Fig. [128]2a) and astrocytes (Supplementary Fig. [129]3c). These
   results indicate that transgenic eIF4E-Myc expression elevates
   microglial protein synthesis to a similar extent in both sexes of mice.

Fig. 2. Overexpression of eIF4E in microglia leads to social deficits.

   [130]Fig. 2
   [131]Open in a new tab

   a Genotypes of control (Ctrl) and MG^4E mice. b Microglial eIF4E
   overexpression and protein synthesis in cultured microglia isolated
   from both sexes of control and MG^4E mice. n = 3 per condition.
   Two-sided t-test: puromycin, female **p = 0.0099 and male *p = 0.0217;
   eIF4E, female **p = 0.0088 and male *p = 0.0293. c Time spent in the
   central zone during the first 5 min of open field tests. Male: 15
   control mice and 14 MG^4E mice; Female: 23 control mice and 25 MG^4E
   mice. n.s. not significant by two-sided t-test. d, e Sociability of
   MG^4E mice. Male: 14 control mice and 14 MG^4E mice; Female: 23 control
   mice and 25 MG^4E mice. Two-way ANOVA with Fisher’s LSD post-hoc test
   for chamber time and social investigation: *p < 0.05; **p < 0.01;
   ***p < 0.001; n.s. not significant. Two-sided t-test for social
   preference index: male, **p = 0.0067; female, p = 0.7440. f Social
   habituation test. n = 16 male control and 14 male MG^4E mice. Two-way
   ANOVA with Fisher’s LSD post-hoc test: *p = 0.0159 between control and
   MG^4E mice in trial 1; ^###p < 0.001 between trial1 and trial4 in
   control mice; ^#p = 0.0334 between trial 4 and trial 5 in control mice;
   n.s. not significant for comparisons of trial 1 vs. trial 4 and trial 4
   vs. novel in MG^4E mice. g Habituation index. **p = 0.0013 by two-sided
   t-test. h, i Novel object recognition. n = 12 control mice and 12 MG^4E
   mice. Two-way ANOVA with Fisher’s LSD post-hoc test: *p < 0.05 and n.s.
   not significant (h). Two-sided t-test: *p = 0.0297 (i). j,
   Self-grooming. The 30-min self-grooming test was divided into three
   10-min segments. n = 6 mice per genotype. Two-way ANOVA with Fisher’s
   LSD post-hoc test was used for each 10-min segment, whereas two-sided
   t-test was used for the whole 30-min test period. *p = 0.0276;
   **p = 0.0016; n.s. not significant. Source data are provided as a
   Source Data file.

   There is a difference in the extent of microglial eIF4E overexpression
   between the two aforementioned assays. To understand the discrepancy,
   we established cultures enriched for neurons, astrocytes, or microglia.
   To our surprise, eIF4E levels in cultured microglia were higher than
   those in cultured neurons (Supplementary Fig. [132]5b), although eIF4E
   levels are higher in neurons than in microglia in vivo on the basis of
   immunohistochemistry (Supplementary Fig. [133]5a). This result
   indicates that cell culturing somehow upregulates microglial eIF4E
   expression, which should cause underestimation of transgenic eIF4E
   expression. Therefore, it is likely that microglia in MG^4E mice have
   more than twice as much eIF4E and protein synthesis as those in control
   mice, as do neurons in NN^4E mice and astrocytes in AC^4E mice.

   Microglia are the most abundant CNS macrophages, which also include
   perivascular macrophages, meningeal macrophages and choroid-plexus
   macrophages^[134]37. In addition to CNS macrophages, CX3CR1 is
   expressed in peripheral monocytes and inflammatory
   macrophages^[135]25,[136]37. In contrast to fast turnover of peripheral
   CX3CR1-expressing cells, CNS macrophages are long-living^[137]37. We
   ran behavioral tests when MG^4E mice were at 2–3 months of age when the
   peripheral monocytes present at the time of tamoxifen injection should
   have been turned over, so that peripheral cells express little or no
   elF4E-Myc in tested mice (Supplementary Fig. [138]5c).

   MG^4E mice had normal locomotion in open field tests, indicating normal
   motor function (Supplementary Fig. [139]7a). They did not show
   anxiety-like behaviors in open field tests (Fig. [140]2c), elevated
   plus maze tests, or light-dark box tests (Supplementary Fig. [141]7b,
   c). Interestingly, MG^4E mice displayed sexual dimorphism in 3-chamber
   sociability tests. Male MG^4E mice displayed social interaction
   deficits, as they spent similar amounts of time investigating a
   stranger mouse and an inanimate object (Fig. [142]2d), whereas female
   MG^4E mice had normal social interaction behaviors (Fig. [143]2e). To
   further characterize social behaviors of male MG^4E mice, we performed
   social recognition tests in the home cage-like environment, an assay
   which relies on the mouse’s innate tendency to investigate a novel
   social partner and decrease the investigation of a known social
   stimulus (social habituation)^[144]38. Consistent with the social
   interaction deficit observed in 3-chamber sociability tests, male MG^4E
   mice spent less time investigating a novel stimulus mouse in trial 1
   (Fig. [145]2f). Furthermore, social habituation (trial 4 vs. trial 1)
   and dishabituation (novel stimulation mouse vs. trial 4) were impaired
   in male MG^4E mice (Fig. [146]2f, g), indicating that their ability to
   adapt to novel social stimuli was attenuated, a phenomenon which has
   been linked to the pathognomonic social impairment and behavioral
   rigidity in ASD patients^[147]39. Again, female MG^4E mice performed
   normally in social recognition tests (Supplementary Fig. [148]7f, g).
   These results indicate that elevated protein synthesis in microglia
   leads to social interaction deficits only in male mice.

   We evaluated learning and memory in MG^4E mice by conducting contextual
   fear conditioning and novel object recognition tests. Both sexes of
   control and MG^4E mice showed similar long-term hippocampus-dependent
   contextual fear memory (Supplementary Fig. [149]7d). However, male, but
   not female, MG^4E mice spent less time investigating a novel object
   than control littermates (Fig. [150]2h, i and Supplementary
   Fig. [151]7h), indicating recognition memory deficits in male MG^4E
   mice. Thus, male MG^4E mice exhibit selective deficits in cognitive
   functions.

   To determine whether MG^4E mice display repetitive behaviors, one of
   the core domains of ASD, we performed marble-burying and self-grooming
   tests. Although both sexes of MG^4E mice performed normally in
   marble-burying tests (Supplementary Fig. [152]7e), male, but not
   female, MG^4E mice spent significantly more time grooming than control
   mice (Fig. [153]2j and Supplementary Fig. [154]7i).

   One copy of the Cx3cr1 gene is disrupted in Cx3cr1^CreER/+
   mice^[155]25. To rule out the possibility that the observed ASD-like
   phenotype in MG^4E mice results from Cx3cr1 haploinsufficiency, we
   examined the behaviors of WT and Cx3cr1^CreER/+ mice in three-chamber
   sociability tests (Supplementary Fig. [156]8a–d) and novel object
   recognition tests (Supplementary Fig. [157]8e–j). Both male and female
   Cx3cr1^CreER/+ mice were normal in these behavioral tests.

   Collectively, these results indicate that elevated protein synthesis in
   microglia leads to ASD-like phenotypes in mice, including male bias,
   deficits in social interaction, increased repetitive behaviors, and
   impaired cognitive functions.

Elevated protein synthesis alters microglial morphology

   We next sought to investigate why microglial eIF4E overexpression
   increased protein synthesis in both sexes, but only caused ASD-like
   behaviors in males. We performed Iba1 immunohistochemistry to examine
   the impact of elevated protein synthesis on microglia in the medial
   prefrontal cortex (mPFC), hippocampus and striatum, the three brain
   regions that have been implicated in ASD
   pathophysiology^[158]40–[159]42. We found that microglia in the mPFC
   were more numerous and larger in 2-week-old male MG^4E mice than male
   control littermates (Fig. [160]3a–c). The same phenotype was observed
   in the hippocampus and striatum (Supplementary Fig. [161]9a–f). In
   contrast, elevated microglial protein synthesis did not affect the
   density and size of microglia in the mPFC, hippocampus and striatum of
   2-week-old female MG^4E mice (Fig. [162]3d–f and Supplementary
   Fig. [163]9g–l). Semi-automatic quantitative morphometric 3D
   measurements of microglia confirmed the increase in size and complexity
   of microglia in 2-week-old male MG^4E mice (Fig. [164]3g). By 6 weeks
   of age, microglia in the mPFC, hippocampus and striatum in male MG^4E
   mice remained larger than those of male control mice; however, their
   density became normal or slightly lower compared to control mice
   (Fig. [165]3h–j, n and Supplementary Fig. [166]10a–f). Again, elevated
   protein synthesis had no detectable effect on microglial size and
   density in 6-week-old female mice (Fig. [167]3k–m and Supplementary
   Fig. [168]10g–l). These data indicate that there is a sexual difference
   in microglial response to elevated protein synthesis.

Fig. 3. Iba1 immunohistochemistry reveals altered microglial density and
morphology in male MG^4E mice.

   [169]Fig. 3
   [170]Open in a new tab

   a–c Increased microglial density and size in the mPFC of male MG^4E
   mice. n = 7 control mice and 6 MG^4E mice. Ten microglia from each
   mouse were randomly selected for measurement of cell size (cross
   section area). *p = 0.0342 and ***p < 0.001 by two-sided t-test. Scale
   bar, 100 μm. d–f Comparable microglial density and size in the mPFC
   between female control and MG^4E mice. n = 6 control mice and 5 MG^4E
   mice. Five to ten microglia from each mouse were randomly selected for
   measurement of cell size (cross section area). n.s. not significant by
   two-sided t-test. Scale bar, 100 μm. g Three-dimension reconstruction
   of microglia in the mPFC of 2-week-old male mice. n = 7 mice per group.
   Three to five microglia from each mouse were reconstructed for
   determination of cell size (volume) and number of processes.
   **p = 0.0037 and ***p = 0.0005 by two-sided t-test. Scale bar, 20 μm.
   h–m Microglia density and size in the mPFC of 6-week-old male (h–j) and
   female (k–m) MG^4E mice. Ten microglia in each brain region of each
   mouse were randomly selected for measurement of cell size (cross
   section area). Male, n = 6 mice per genotype; female, n = 5 control
   mice and 6 MG^4E mice. Two-sided t-test: ***p < 0.001 and n.s. not
   significant. Scale bar, 100 µm. n Three-dimension reconstruction of
   microglia in the mPFC of 6-week-old male mice. Four microglia from each
   mouse were reconstructed. Six control mice and five MG^4E mice.
   *p = 0.0385 by two-sided t-test. Scale bar, 20 µm. All data are shown
   as mean ± s.e.m. Source data are provided as a Source Data file.

   Inactivating mutations in the PTEN and FMR1 genes account for a large
   percentage of human syndromic ASD^[171]3. To determine if microglial
   changes observed in MG^4E mice also occur in mice that model these two
   syndromes, we measured microglial density and size in 2-week-old male
   Pten^+/− and Fmr1 knockout (KO) mice. Microglia in the mPFC,
   hippocampus and striatum were significantly larger in Pten^+/− and Fmr1
   KO mice than WT littermates (Fig. [172]4a, b and Supplementary
   Figs. [173]11a, [174]12a). Moreover, microglial density was increased
   in the Pten^+/− mPFC and Fmr1 KO striatum (Fig. [175]4a and
   Supplementary Fig. [176]12a). The lack of elevated microglial density
   in all brain regions of 2-week-old male Pten^+/− and Fmr1 KO mice could
   be due to a smaller degree of protein synthesis increase relative to
   MG^4E mice. We notice that microglial density in tamoxifen-treated male
   R26^Eif4e/Eif4e (control) mice is lower than that in untreated male WT
   mice at 2 weeks of age (compare Figs. [177]3c, [178]4a, b), which is
   possibly due to growth retardation of mice caused by tamoxifen
   injection at P0.

Fig. 4. Altered microglial density and size in Pten^+/− and Fmr1 KO mice.

   [179]Fig. 4
   [180]Open in a new tab

   a Microglial density and size in the mPFC of 2-week-old male and female
   Pten^+/- mice. Male, n = 5 mice per genotype. Female, n = 6 mice per
   genotype. Five to ten microglia from each condition were randomly
   selected for measurement of cell size (cross section area). Two-sided
   t-test: male, cell density *p = 0.0317, cell size **p = 0.001; female,
   cell density p = 0.0725, cell size *p = 0.0232, n.s. not significant.
   Scale bar, 100 µm. b Microglial density and size in the mPFC of
   2-week-old male and female Fmr1 KO mice. Male, n = 7 mice per genotype.
   Female, n = 4 WT mice and 5 KO mice. 5-10 microglia from each condition
   were randomly selected for measurement of cell size (cross section
   area). Two-sided t-test: **p = 0.0046, and n.s. not significant. Scale
   bar, 100 µm. c, d Microglial density and size in the mPFC of 6-week-old
   male Pten^+/− (c) and Fmr1 KO (d) mice. c n = 5 mice per condition; d
   n = 6 WT mice and 5 Fmr1 KO mice. Five to ten microglia from each
   condition were randomly selected for measurement of cell size (cross
   section area). n.s., not significant by two-sided t-test. Scale bar,
   100 μm. All data are shown as mean ± s.e.m. Source data are provided as
   a Source Data file.

   The microglial morphological phenotype observed in 2-week-old male
   Pten^+/− and Fmr1 KO mice also displays sexual dimorphism. While
   microglia in 2-week-old female Pten^+/− mice show some morphological
   alterations, the phenotype is not as prominent as observed in male
   Pten^+/− littermates. Compared to control mice, microglia in 2-week-old
   female Pten^+/− mice were larger in the mPFC and the striatum but not
   in the hippocampus, and their density was not significantly altered in
   all the three brain regions (Fig. [181]4a and Supplementary
   Fig. [182]11b). Furthermore, the density and size of microglia was
   normal in 2-week-old female Fmr1 KO mice (Fig. [183]4b and
   Supplementary Fig. [184]12b). Similar to what was observed in MG^4E
   mice, the morphological phenotype in male Pten^+/− and Fmr1 KO mice is
   attenuated with age, so that the phenotype in these mice disappeared by
   6 weeks of age (Fig. [185]4c, d and Supplementary Figs. [186]13,
   [187]14).

Synaptic alterations in male MG^4E mice

   As microglia play important roles in neuronal development, the observed
   changes in microglial number and morphology could alter synaptic
   development and function in male MG^4E mice, which then leads to
   ASD-like behaviors. To investigate this possibility, we examined
   synaptic structures in layer 2 of the mPFC prelimbic area (PrL) in
   6-week-old male control and MG^4E mice using serial block-face scanning
   electron microscopy (Fig. [188]5a). Three-dimension reconstruction of
   dendrites revealed that male MG^4E mice had higher spine density, with
   contributions from both non-synaptic and synaptic spines compared to
   control mice (Fig. [189]5b). Male MG^4E mice had smaller spines and
   synapses on average (Fig. [190]5c). To examine synaptic maturation, we
   counted multiple-synapse spines (MSS) and multiple-synapse boutons
   (MSB), which are believed to be the structural basis of synaptic
   multiplicity^[191]26. Synaptic multiplicity increases in the
   hippocampus during postnatal development and is considered an indicator
   of synapse maturation^[192]43. We found that male control and MG^4E
   mice had comparable MSS and MSB density in layer 2 of the PrL
   (Supplementary Fig. [193]15a, b), suggesting that reduced synapse size
   in male MG^4E mice is not indicative of immaturity.

Fig. 5. Structural and functional alterations in synapses of MG^4E mice.

   [194]Fig. 5
   [195]Open in a new tab

   a Schematic diagram of mPFC serial block-face scanning electron
   microscopy (SB-SEM). b Spine density in the mPFC of 6-week-old male
   control and MG^4E mice. Representative dendrites reconstructed from
   SB-SEM images show dendritic spines (gray) and presynaptic terminals
   (blue). Graphs show spine density, synaptic spine density and
   percentage of non-synaptic spines. 5 control mice, 40 dendrites and
   1187 spines; 6 MG^4E mice, 30 dendrites and 1120 spines. Two-sided
   t-test (spine density, **p = 0.0017; synaptic spine density,
   *p = 0.0112; non-synaptic spine *p = 0.0253). Scale bar, 2 µm. c Spine
   volume and synaptic size. Representative dendrites reconstructed from
   SB-SEM images show dendritic spines (blue) and postsynaptic density
   (PSD, light yellow). Graphs show PSD size and spine volume. 5 control
   mice, 40 dendrites and 1098 spines; 6 MG^4E mice, 30 dendrites and 999
   spines. Two-sided t test (PSD area, **p = 0.0044; spine volume,
   **p = 0.0037). Scale bar, 2 µm. d Density of asymmetric and symmetric
   synapses in the mPFC of 6-week-old male and female control and MG^4E
   mice. Images show representative asymmetric and symmetric synapses
   (arrows). n = 3 mice and 30 images per mouse for each group.
   **p = 0.0059 by two-sided t-test. Scale bar, 2 µm. e Spine density in
   mPFC layer 5 neurons and hippocampal CA1 neurons of 2-week-old (2 W)
   and 6-week-old (6 W) male control and MG^4E mice. 2 W, 6 mice per
   genotype; 6 W, 6 control mice and 7 MG^4E mice; 2–5 neurons in each
   brain region per mouse. *p = 0.0362, **p = 0.0012 and ***p < 0.001 by
   two-sided t-test. Scale bar, 5 µm. f Levels of neuroligin 1 (NLGN1) and
   neuroligin 2 (NLGN2) in the mPFC and hippocampus. n = 4 control mice
   and 5 MG^4E mice. Two-sided t test (mPFC, *p = 0.0409 for NLGN1 and
   *p = 0.0279 for NLGN2; hippocampus, *p = 0.0207 for NLGN1 and
   *p = 0.0229 for NLGN2). All data are shown as mean ± s.e.m. Source data
   are provided as a Source Data file.

   Because dendritic spines are the postsynaptic sites for the vast
   majority of excitatory synapses^[196]44, higher spine density indicates
   more excitatory synapses. Indeed, transmission electron microscopy
   analysis revealed more asymmetric (excitatory) synapses in the PrL of
   6-week-old male, but not female, MG^4E mice compared to control mice
   (Fig. [197]5d). This analysis also found a normal density of symmetric
   (inhibitory) synapses in the PrL of both male and female MG^4E mice
   (Fig. [198]5d). Thus, exaggerated protein synthesis in microglia
   increases the number of excitatory synapses in the mPFC in male mice.

   Increased spine density has been observed in ASD
   patients^[199]45,[200]46. To determine if spine density is also
   increased in other brain areas of male MG^4E mice and at other ages, we
   employed the Thy1-GFP transgene^[201]47 to label isolated neurons in
   2-week and 6-week-old mice. We found that male, but not female, MG^4E
   mice had higher spine density in pyramidal neurons of mPFC layer 5 and
   hippocampal CA1 area at both ages (Fig. [202]5e and Supplementary
   Fig. [203]16a). These results indicate that elevated microglial protein
   synthesis increases the density of excitatory synapses in multiple
   brain areas.

   We next examined whether levels of synaptic proteins are altered in the
   mPFC and hippocampus of MG^4E mice. We found that adult male and female
   control and MG^4E mice had comparable levels of presynaptic
   synaptophysin and postsynaptic neuroligin 3, neuroligin 4, PSD95 and
   GluA1 in the hippocampus and mPFC (Supplementary Figs. [204]15c,
   [205]15d, [206]16b, [207]16c). Neuroligins are cell-adhesion molecules
   important for synaptogenesis and have been implicated in ASD^[208]48.
   Levels of neuroligins 1–4 are increased in the hippocampus of Eif4ebp2
   knockout and transgenic βT-Eif4e mice, which was interpreted as a
   result of increased mRNA translation in neurons^[209]16. Interestingly,
   levels of neuroligins 1 and 2 in the hippocampus and mPFC were
   significantly higher in male, but not female, MG^4E mice, compared with
   sex-matched control mice (Fig. [210]5f and Supplementary Fig. [211]16b,
   c). This finding suggests that the observed increase in hippocampal
   levels of neuroligins in Eif4ebp2 knockout and transgenic βT-Eif4e mice
   could be a result of microglial alterations rather than increased eIF4E
   activity in neurons. This argument is supported by the observation that
   levels of neuroligins1–4 were comparable between male control mice and
   male NN^4E mice that overexpress eIF4E in neurons (Supplementary
   Fig. [212]17). The increase in levels of neuroligins 1 and 2 is likely
   due to enhanced transcription in neurons, as levels of mRNAs for the
   two proteins show a trend of increase in male MG^4E mice (Supplementary
   Fig. [213]15e).

   To investigate if alterations in synaptic structure and proteins lead
   to abnormal synaptic function in male MG^4E mice, we recorded miniature
   excitatory postsynaptic currents (mEPSCs) and miniature inhibitory
   postsynaptic currents (mIPSCs) in layer 5 pyramidal neurons of the
   mPFC. The amplitude, but not the frequency, of mEPSCs was increased in
   male MG^4E mice relative to control mice (Fig. [214]6a). In contrast,
   the amplitude of mIPSCs were slightly reduced in male MG^4E mice, as
   indicated by a significant but small left shift of its cumulative curve
   (Fig. [215]6b). These results suggest an alteration in the
   excitation/inhibition (E/I) balance, which is believed to contribute to
   ASD^[216]3, in the mPFC. Indeed, we found that the normalized total
   charge transfer in male MG^4E mice relative to control mice was
   significantly larger for mEPSCs than for mIPSCs (Fig. [217]6c). As
   expected on the basis of synapse density and levels of synaptic
   proteins, female control and MG^4E mice had comparable mEPSCs, mIPSCs
   and charge transfer in the mPFC (Supplementary Fig. [218]16d, e).

Fig. 6. Abnormal synaptic function in male MG^4E mice.

   [219]Fig. 6
   [220]Open in a new tab

   a mEPSCs recorded in mPFC layer 5 neurons of male control and MG^4E
   mice at 6–7 weeks of age. n = 20 cells from five control mice and 33
   cells from 8 MG^4E mice. Two-sided t-test for frequency and amplitude:
   **p = 0.0022; n.s. not significant. Two-sided Kolmogorov–Smirnov test
   for cumulative probability of mEPSC amplitude: ***p < 0.001. Scale
   bars, 50 pA (vertical) and 0.5 s (horizontal). b mIPSCs recorded in
   mPFC layer 5 neurons of male control and MG^4E mice at 6–7 weeks of
   age. n = 17 cells from 4 control mice and 25 cells from 6 MG^4E mice.
   Two-sided t-test for frequency (p = 0.6053) and amplitude (p = 0.1097):
   n.s. not significant. Two-sided Kolmogorov–Smirnov test for cumulative
   probability of mIPSC amplitude: ***p < 0.001. Scale bars, 25 pA
   (vertical) and 0.5 s (horizontal). c Relative changes in mEPSC (n = 33
   cells) and mIPSC (n = 25 cells) total charge transfer in MG^4E mice,
   normalized to control mice. p = 0.0453 by two-sided Kolmogorov–Smirnov
   test. All data are shown as mean ± s.e.m. Source data are provided as a
   Source Data file.

   Collectively, our microscopic, biochemical and electrophysiological
   analyses reveal that elevated protein synthesis in microglia leads to
   synaptic alterations that are associated with ASD in male mice only,
   including increased spine density, increased levels of neuroligins and
   E/I imbalance. These synaptic changes likely contribute to deficits in
   social interaction, repetitive behaviors, and cognitive impairments in
   male MG^4E mice.

Microglia are out of homeostatic state in male MG^4E mice

   To understand how microglial eIF4E overexpression alters synaptic
   structure and function, we analyzed hippocampal gene expression in male
   control and MG^4E mice at 2 and 6 weeks of age using RNA-Seq. Weighted
   gene correlation network analysis (WGCNA)^[221]49 revealed 11 modules
   of highly co-expressed genes in control and MG^4E mice (Supplementary
   Fig. [222]18a). Of note, the magenta module displayed a set of
   transcripts that were significantly upregulated in 2-week-old MG^4E
   mice (Fig. [223]7a). Pathway enrichment analysis of genes within the
   magenta module revealed top KEGG pathways related to antimicrobial
   activities, including herpes simplex infection, antigen processing and
   presentation, phagosome, influenza A and autoimmune thyroid disease
   (Fig. [224]7b, c). Some of the disease-associated microglial genes
   found in neurodegenerative conditions^[225]50 were also upregulated in
   2-week-old MG^4E mice (28 out of 138 genes in the magenta module;
   Fig. [226]7c). By 6 weeks of age, the magenta module eigengene was no
   longer significantly upregulated in MG^4E mice, suggesting that this
   set of transcripts were only upregulated during postnatal development.
   Interestingly, the herpes simplex virus infection and autoimmune
   thyroid disease pathways in the magenta module have been reported to be
   risk factors for ASD^[227]51–[228]53. In addition to the upregulated
   genes (Supplementary Fig. [229]18b, c), we identified a group of
   downregulated genes by comparing 2-week-old male MG^4E mice to control
   littermates (Fig. [230]7d and Supplementary Fig. [231]18b, d). Many of
   the downregulated genes (11/40, or 27.5%) are markers for homeostatic
   (M0) microglia^[232]54, including P2ry12, P2ry13, Cx3cr1, Tmem119, and
   Slco2b1 (Fig. [233]7e).

Fig. 7. Gene expression profiles in male control and MG^4E mice.

   [234]Fig. 7
   [235]Open in a new tab

   RNA-seq was performed using RNA samples isolated from hippocampi of
   2-week-old (2 W) and 6-week-old (6 W) male control (Ctrl) and MG^4E
   mice. a (top) Heatmap showing module-trait relationship of magenta
   module. Correlation coefficients between a module eigengene and trait
   (coded from −1 to 1) and corresponding p values (two-sided Student
   asymptotic p value for a given correlation was calculated using
   corPvalueStudent in WGCNA package) are shown in each cell. The color
   indicates the level of correlation. (bottom) Plot showing the
   expression of the module eigengene across sample categories. b Heatmaps
   of genes (138 genes) within magenta module. c Selected KEGG pathways
   and disease-associated microglial genes enriched in magenta module
   (logP values adjusted with Benjamini correction). d Heatmaps of
   downregulated genes (FDR < 0.05) in 2-week-old MG^4E mice. (e Heatmaps
   of downregulated M0-homeostatic microglial genes in 2-week-old MG^4E
   mice. f, g Heatmaps of top 40 M1 (f) and M2 (g) microglial genes in
   2-week-old MG^4E mice (Ctrl and MG^4E, n = 4 mice). h, i RT-PCR
   validation of selected upregulated (h) and down-regulated (i) genes in
   hippocampi of 2-week-old male MG^4E mice. n = 8 per genotype.
   *p < 0.05, **p < 0.01 and ***p < 0.001 by two-sided t-test. j RT–qPCR
   validation of selected genes in hippocampi of 6-week-old male MG^4E
   mice. n = 7 control mice and 8 MG^4E mice. n.s. not significant by
   two-sided t-test. All data are shown as mean ± s.e.m. Source data are
   provided as a Source Data file.

   In addition to M0 state, microglia can achieve two other polarized
   states (M1 and M2) that are characterized by specific molecular
   signatures^[236]54,[237]55. It is proposed that M1 microglia produce
   pro-inflammatory cytokines and M2 microglia are
   anti-inflammatory^[238]55. We found that very few M1 and M2 markers
   were upregulated in 2-week-old male MG^4E mice (Fig. [239]7f, g). This
   indicates that elevated protein translation does not move microglia to
   M1 or M2 polarized states.

   We validated the RNA-Seq results by measuring mRNA levels of a few
   select genes in 2-week and 6-week-old control and MG^4E mice using
   quantitative RT-PCR (Fig. [240]7h–j). The RT-PCR analysis also found
   that the observed gene expression changes were absent in 2-week-old
   female MG^4E mice (Supplementary Fig. [241]18e). Taken together, the
   RNA-Seq and RT-PCR analyses show that elevated protein synthesis shifts
   microglia from the homeostatic state to a functional state with
   properties that have been observed in neurodegenerative conditions.

Male MG^4E microglia with altered phagocytosis and motility

   Microglia are the primary phagocytes in the brain that engulf invading
   pathogens and cellular debris^[242]56. Upregulation of antimicrobial
   pathways including the phagosome pathway (Fig. [243]7c) prompted us to
   hypothesize that the phagocytic capacity of microglia is enhanced in
   2-week-old male MG^4E mice. Indeed, purified microglia from male MG^4E
   mice phagocytized more than twice as many Aβ[(1–42)] aggregates as
   those from male control mice, but microglia from female control and
   MG^4E mice had comparable phagocytosis (Fig. [244]8a).

Fig. 8. Microglial surveillance and synapse engulfment in MG^4E mice.

   [245]Fig. 8
   [246]Open in a new tab

   a Phagocytosis of FAM-Aβ[(1–42)] in cultured control and MG^4E
   microglia. Male, n = 18 for control and n = 28 for MG^4E; female,
   n = 14 for control and n = 13 for MG^4E. **p = 0.0017 and n.s. not
   significant (p = 0.4919) by two-sided t-test. b, c Migration of
   microglia into FAM-Aβ[(1–42)] injection sites in 2-week-old male (b)
   and female (c) control and MG^4E mice. Microglia clustering index is
   defined as (density of Iba1^+ cells in the FAM-Aβ-covered
   area)/(density of Iba1^+ cells in a contralateral site). Male, n = 6
   control mice and 7 MG^4E mice; female, n = 4 per genotype. Two-sided
   t-test: male mice, contralateral microglia density, **p = 0.0013;
   clustering index, **p = 0.0026, n.s. not significant. Scale bars,
   100 μm. d Microglial surveillance in response to ATP treatment in male
   and female MG^4E microglia at P14–P18. Microglial baseline motility was
   recorded for 5 min, then ATP was bath-applied to brain slices and
   recorded for another 10 min. Data was normalized to the mean process
   motility of first 5 min. Male, n = 15 microglia from 3 control mice and
   20 microglia from 3 MG^4E mice; female, n = 18 microglia from 3 control
   mice and 26 microglia from 3 MG^4E mice. Male, two-way ANOVA for
   genotype during ATP treatment, F[(1, 330)] = 9.566, p = 0.002; female,
   two-way ANOVA for genotype during ATP treatment, F[(1, 420)] = 0.045,
   p = 0.831. Scale bar, 10 μm. e Engulfment of Homer1 by microglia. Upper
   panel shows confocal images of Homer1 and Iba1 double
   immunohistochemistry, and lower panel shows 3-D reconstruction of a
   microglial cell and Homer1 immunoreactivity. Arrowheads denote Homer1
   inside the microglia. Male, 18 microglia from 6 control mice and 15
   microglia from 5 MG^4E mice; female, 15 microglia from 5 mice per
   genotype. *p = 0.0378 and n.s., not significant (p = 0.4891) by
   two-sided t-test. Scale bars, 5 μm. All data are shown as mean ± s.e.m.
   Source data are provided as a Source Data file.

   Given that microglia have been shown to prune synapses through
   phagocytosis during postnatal development^[247]23,[248]24, we were
   puzzled by how elevated spine density (Fig. [249]5b, e) and enhanced
   microglial phagocytosis (Fig. [250]8a) co-exist in male MG^4E mice. We
   noticed that the downregulated genes are associated with leukocyte
   cell–cell adhesion, leukocyte migration, and cell–cell adhesion
   (Supplementary Fig. [251]18d). Thus, we examined migration of microglia
   to an injection site of Aβ[(1–42)] aggregates in 2-week-old male
   control and MG^4E mice. We found that the number of microglia migrated
   to the Aβ[(1–42)] injection site was significantly reduced in male, but
   not female, MG^4E mice relative to control mice (Fig. [252]8b, c).
   These results indicate that elevated protein synthesis impairs the
   motility of microglia in male mice.

   Microglial processes display two modes of motility: constant extension
   and retraction to survey the brain (baseline motility) and extension to
   the source of attractants such as ATP released from a damaged site
   (induced motility)^[253]57. Because synapse pruning involves contacts
   of microglial processes with synapses^[254]58, it should be affected by
   the motility of microglial processes. As elevated protein synthesis
   impaired the motility of whole microglia, it could also affect the
   motility of microglial processes. We monitored the baseline motility
   and ATP-induced motility of microglial processes in organotypic
   hippocampal cultures established from P14-P18 control and MG^4E mice
   harboring the CD68-EGFP transgene^[255]59 which is selectively
   expressed in microglia. Irrespective of sex, microglial processes in
   control and MG^4E mice had comparable baseline processes motility
   (Supplementary Fig. [256]18f). However, ATP-induced motility was
   impaired in male, but not female, MG^4E mice (Fig. [257]8d).

   Synapses to be eliminated may release a factor to initiate their
   interactions with microglial processes, and thereby deficits in the
   induced motility in male MG^4E mice would impair engulfment of
   synapses. We assessed synapse engulfment by measuring the
   immunoreactivity of postsynaptic protein Homer1 inside microglia.
   Three-D reconstruction of Iba1 and Homer1 immunoreactivities revealed
   that the volume of Homer1 immunoreactivity inside microglia was
   significantly reduced in male, but not female, MG^4E mice relative to
   control mice (Fig. [258]8e).

   Collectively, these results indicate that induced motility of microglia
   is impaired in male MG^4E mice, which might lead to reduced microglial
   engulfment of synapses and subsequently increased density of excitatory
   synapses, despite of enhanced microglial phagocytosis capacity in these
   mice.

Discussion

   Our results indicate that elevated neuronal protein synthesis does not
   lead to deficits in social interaction, but it produces some
   ASD-related behaviors. NN^4E mice of both sexes displayed elevated
   anxiety, a common ASD comorbidities^[259]4. In addition, as revealed by
   marble burying tests, elevated neuronal protein synthesis increased
   repetitive behavior in female but not male mice. These behavioral
   phenotypes are interesting, given the observation that elevated
   microglial protein synthesis impaired social behaviors without
   elevating anxiety in male mice. These results indicate that different
   ASD-like and related behaviors have distinct cellular basis. They also
   suggest that ASD manifestations could be different in females and
   males.

   How does elevated protein synthesis in microglia lead to ASD-like
   synaptic and behavioral aberrations only in male mice? Our results
   indicate that the key mechanism is a sex-dependent response of
   microglia to elevated protein synthesis. Male microglia alter their
   transcriptome, which leads to changes in their function and morphology,
   but female microglia do not. Our transcriptomic analysis reveals that
   eIF4E overexpression shifts microglia from the homeostatic state to an
   undefined functional state only in male mice. The alterations in male
   microglia should result from elevated translation of some mRNAs
   in-these cells. However, our data do not exclude a remote possibility
   that elevated translation in peripheral immune cells impacts microglia
   differently in males and females through circulating factors, because
   monocytes and peripheral macrophages do overexpress eIF4E in MG^4E mice
   in the first few postnatal weeks.

   Homeostatic microglia are constantly surveying their microenvironment
   with extremely motile processes and their processes rapidly converge at
   the site of injury in the brain^[260]60,[261]61. The motility and
   convergence of microglial processes in response to ATP released from
   damaged cells are dependent on the purinergic receptors such as
   P2Y12^[262]61,[263]62. It is likely that homeostatic microglia employ a
   similar mechanism to reach synapses tagged for elimination. We found
   that eIF4E overexpression downregulates many genes specific to
   homeostatic microglia including genes for purinergic receptors (P2ry12
   and P2ry13)^[264]54 in young male mice. As this gene expression change
   would predict, eIF4E overexpression dramatically diminishes the induced
   motility of microglia. The loss of the induced motility would impair
   the ability of microglia to find synapses tagged for elimination so
   that microglia could not efficiently prune synapses. Indeed, we found
   that microglia in male MG^4E pups contain a lower amount of engulfed
   Homer1 than those in control pups. This could be the main reason for
   observed increases in spine density and synapse density in male MG^4E
   mice. This conclusion does not exclude the possibility that elevated
   microglial protein synthesis increases the rate of spine formation in
   male mice. We found that microglia in male MG^4E mice have more
   processes relative to control mice, which would increase points of
   microglial contacts with neurons. The contact has been found to induce
   synapse formation in the cortex^[265]63. It would be important to
   identify the proteins that lead to alterations in transcription,
   morphology, and function in MG^4E microglia when their synthesis is
   elevated and to understand why elevated synthesis of the proteins only
   affects male microglia in future studies.

   Our RNA-Seq data indicate that microglia in male MG^4E mice upregulate
   expression of several cytokines, including C-X-C motif chemokine 10
   (CXCL10), CXCL16, C-C motif chemokine 12 (CCL12) and interleukin 1
   alpha (Il1α). We speculate that these cytokines could upregulate the
   expression of neuroligins and modify the function of glutamate
   receptors via posttranslational modifications, leading to the observed
   increase in the amplitude of mEPSCs. The increase in the function of
   excitatory synapses, along with increased density of excitatory
   synapses, would lead to E/I imbalance. Deficits in synapse pruning
   would lead to the presence of excess, imprecise and inefficient
   neuronal connections. Both E/I imbalance and synapse pruning impairment
   have been linked to ASD^[266]3 and could be responsible for deficits in
   social interaction and repetitive behaviors in male MG^4E mice.

   It is generally believed that PTEN haploinsufficiency elevates protein
   synthesis by increasing mTORC1 activity^[267]3. However, a recent study
   shows that genetic ablation of mTOR complex 2 (mTORC2), but not mTORC1,
   activity reverses the pathophysiology of Pten^+/− mice^[268]64. It is
   likely that the complete ablation of mTORC1 activity is not the best
   way to reverse mTORC1 hyperactivity in Pten^+/− mice. It is known that
   mTORC2 can fully activate Akt via Ser473 phosphorylation^[269]65,
   thereby increasing the activity of mTORC1, an Akt downstream target.
   Therefore, it could be a good strategy to treat ASD associated with
   elevated protein synthesis by targeting mTORC2 to reduce mTORC1
   activity. There are conflicting reports on the effect of elevated
   protein synthesis on basal synaptic transmission. Enhanced mTORC1
   activity via expression of a constitutively active form of Rheb was
   reported to reduce the frequency of spontaneous EPSCs in pyramidal
   neurons of the anterior cingulate cortex^[270]66. However, increases in
   both the frequency and the amplitude of mEPSCs were found in the
   hippocampal CA1 region of Eif4ebp2 knockout mice, where mRNA
   translation is elevated^[271]16,[272]67. The discrepancy could result
   from differences in the extent of translation enhancement, brain
   region, and recorded synaptic property in these studies. We found that
   elevated protein synthesis in microglia via eIF4E overexpression
   increases the amplitude of mEPSCs in the mPFC without affecting the
   frequency of mEPSCs. It would be interesting to investigate if elevated
   protein synthesis in multiple types of brain cells is required to
   affect both frequency and amplitude of mEPSCs.

   Transcriptomic analyses of post-mortem brains find that genes
   associated with activated microglia are upregulated in the cortex of
   ASD patients^[273]68,[274]69. Furthermore, knockdown of the
   microglia-specific Cx3cr1 gene leads to a transient reduction in
   microglia, deficits in synapse pruning and ASD-like behaviors^[275]26.
   These findings provide some evidence for involvement of microglia in
   ASD; however, ASD-associated causal genetic variants in microglial
   genes have not been identified yet. Protein synthesis is elevated as a
   result of ASD-causing mutations in genes encoding negative translation
   regulators (such as in PTEN, TSC1/2, and FMR1)^[276]12,[277]13,[278]15.
   Our results show that elevating protein synthesis in microglia leads to
   ASD-like synaptic and behavioral aberrations in mice. Therefore, our
   study suggests that ASD-associated mutations in ubiquitously expressed
   genes could lead to ASD through their primary effects on microglia. In
   addition, our findings may provide some insights into the
   pathophysiology of ASD associated with other risk factors.
   Environmental insults, such as microbial infection during pregnancy,
   lead to the emergence of core ASD symptoms in the adult offspring,
   especially in males^[279]70–[280]73. Because microglia are at the
   interface between the brain and environment, our findings suggest that
   environmental insults could increase the risk of ASD by altering
   functional states of microglia in the offspring. We propose that
   augmented responses of microglia to biochemical perturbations induced
   by gene mutations or microbial infection are an important underlying
   pathogenesis for ASD. Elucidation of the sexual difference in
   microglial response to the perturbations in future studies could
   provide innovative strategies for prevention and treatment of ASD.

Methods

Animals

   Syn1-Cre (Stock No: 003966), GFAP-Cre (Stock No: 024098), Thy1-GFP
   (Stock No: 007788), Rosa26^Ai9/+ (Stock No: 007909), Fmr1 knockout
   (Stock No: 003025), CD68-EGFP (Stock No: 026827) and Cx3cr1^CreER/+
   (Stock No: 021160) mouse strains were obtained from the Jackson
   Laboratory. The Pten^+/− strain was from the National Cancer Institute.
   All mice were maintained at 22 °C on a 12-h/12-h light/dark cycle with
   ad libitum access to water and food. Animal procedures were approved by
   the Scripps Florida Institutional Animal Care and Use Committee.

Generation of the R26^Eif4e mouse strain

   To generate a transgenic mouse strain overexpressing eIF4E from the
   Rosa26 locus, we generated a targeting construct by replacing the
   tdTomato coding sequence with the Eif4e coding sequence in the Ai9
   construct^[281]35 (a gift from Hongkui Zeng, Addgene plasmid # 22799).
   To distinguish between endogenous and overexpressed eIF4E, we inserted
   a DNA sequence for the Myc tag
   (gccGAACAAAAACTCATCTCAGAAGAGGATCTGaatagctag) immediately before the
   stop code of the eIF4E coding sequence. The targeting vector was
   linearized and transfected into C57BL/6 embryonic stem cells.
   G418-resistant ES clones were screened with Southern blots. Two
   positive ES clones were injected into blastocysts to obtain chimeric
   mice. The mouse strain was maintained on the C57BL/6 background.

Immunohistochemistry and cell counting

   Mice were deeply anaesthetized with avertin and transcardially perfused
   with phosphate-buffered saline (PBS) and 4% paraformaldehyde (PFA)
   sequentially. Brains were removed, post-fixed in 4% PFA for 6 h, and
   then cryoprotected in 30% sucrose until sectioning. Coronal brain
   sections (40-µm thickness) were obtained using a sliding microtome.
   Brain sections were incubated with rabbit anti-Iba1 (Wako, 019-19741,
   1:500) at 4 °C overnight and then biotinylated goat anti-rabbit
   secondary antibody (1:200, Vector, BA-1000). Sections were developed in
   0.05% DAB (3,3’-diaminobenzidine) and 0.003% hydrogen peroxide in 0.1 M
   Tris–HCl (pH 7.5), mounted onto slides, dehydrated, and coverslipped
   with dibutyl phthalate-xylene mixture. Density of Iba^+ microglia was
   measured under a 20× objective using Stereo Investigator software
   (MicroBrightField Inc.). The analysis was performed blind to genotype.

Tamoxifen injections

   Tamoxifen (Sigma, T5648) was dissolved in corn oil at a concentration
   of 40 mg/ml. P0 mice were subcutaneously injected with tamoxifen
   solution around the lower neck region one time at 180 mg/kg (~7 μl per
   mouse).

Immunocytochemistry

   Cultured microglia were fixed for 20 min with 4% PFA and 4% sucrose at
   room temperature. Cells were washed with PBS and permeabilized with
   0.25% Triton X-100 in PBS. After being washed three times with PBS,
   microglia were incubated with a blocking buffer (PBS containing 5% BSA
   and 0.1% Triton X-100) for one hour at room temperature. Afterwards,
   cells were incubated with the following primary antibodies in the
   blocking buffer at 4 °C overnight: rabbit anti-Iba1 (Wako, 019-19741,
   1:500), mouse anti-CD11b (Invitrogen, 12-0112-82, 1:100), rabbit
   anti-eIF4E (Cell Signaling Technology, #2067, 1:500) and goat anti-Iba1
   (Abcam, ab5076, 1:500). Appropriate DyLight conjugated secondary
   antibodies were used after primary antibodies were washed off with PBS
   three times. Nuclei were counterstained with DAPI (Invitrogen D1306,
   10 mg/ml stock solution, 1:10,000 dilution). Images were acquired using
   a Nikon C2+ confocal microscope.

Three-dimensional reconstruction of microglia

   Coronal sections of the mPFC from 2-week and 6-week-old mice were
   stained with rabbit anti-Iba1 (Wako, 019-19741, 1:500) overnight,
   followed by Alexa Fluor 488-conjugated (711-545-152, Jackson
   ImmunoResearch, 1:500) secondary staining. Images were acquired on a
   Nikon C2+ confocal laser scanning microscope using a 40× oil objective.
   Z-stack images were recorded with a 0.8-μm interval for 27 steps. 3-D
   images of microglia were constructed using Amira software (Thermo
   Fisher Scientific).

Microglial engulfment of Homer1

   Coronal brain sections from 2-week-old male and female mice were
   incubated with rabbit anti-Iba1 (Wako, 019-19741, 1:500) and chicken
   anti-Homer1 (Synaptic Systems, 160006, 1:500) overnight, followed by
   Alexa Fluor 594 (703-585-155, Jackson ImmunoResearch, 1:500) and 649
   (711-605-152, Jackson ImmunoResearch, 1:500) conjugated secondary
   antibodies. Images were acquired on a Nikon C2+ confocal laser scanning
   microscope using a 40× oil objective, laser power and gain were kept
   consistent throughout the experiment. Z-stack images were acquired with
   a 0.8-μm interval for 21 steps. Images were analyzed using Amira
   software (Thermo Fisher Scientific) to create a 3D surface rendering of
   microglia with a threshold to ensure that microglial processes were
   accurately reconstructed. This rendering was used to mask the Homer1
   channel, and the overlapped volume was considered as Homer1 engulfed by
   microglia.

Western blotting

   Hippocampal and prefrontal cortical tissues were dissected on ice using
   a coronal Brain Matrix (Roboz, SA-2175). Dissected brain tissues or
   cultured cells were lysed on ice for 30 min in lysis buffer containing
   10 mM Tris (pH 7.4), 1% Triton X-100, 150 mM NaCl, 10% glycerol, and
   freshly added protease inhibitors (Roche Complete Protease Mini,
   #4693159001) and phosphatase inhibitors (PhosStop pellets, Sigma
   Aldrich, #4906845001). Lysates were centrifuged at 15,000×g for 30 min
   at 4 °C, and supernatants were saved. Protein samples were run on
   SDS-PAGE gels and transferred to PVDF membrane. Membrane was blocked
   with Odyssey Blocking Buffer (Thermo Fisher Scientific). The following
   primary antibodies were used: mouse anti-α-tubulin (Sigma-Aldrich
   T6074, 1:10,000), mouse anti-PSD95 (Thermo Fisher Scientific MA1-045;
   1:1000), rabbit anti-eIF4E (Cell Signaling Technology, #2067, 1:1000),
   mouse anti-β-actin (Sigma-Aldrich A5441, 1:10,000), rabbit anti-GluA1
   (Millipore AB1504, 1:1000), rabbit anti-Myc (Cell Signaling Technology,
   #2278, 1:1000), rabbit anti-synaptophysin (Invitrogen, MA5-14532,
   1:1000), mouse anti-neuroligin 1 (Synaptic Systems, 129111, 1:1000),
   rabbit anti-neuroligin 2 (Synaptic Systems, 129202, 1:1000), mouse
   anti-neuroligin 3 (Synaptic Systems, 129311, 1:1000), mouse
   anti-neuroligin 4 (Synaptic Systems, 129403, 1:1000), mouse anti-GFAP
   (MA5-12023, Thermo Fisher Scientific, 1:1000), rabbit anti-P2Y12
   (702516, Thermo Fisher Scientific, 1:1000), rabbit anti-eIF4G (Cell
   Signaling Technology, #2498, 1:1000), and mouse anti-eIF4E (sc-9976,
   Santa Cruz Biotechnology, 1:100). Appropriate IRDye infrared secondary
   antibodies (LI-COR Biosciences) were used at a dilution of 1:10,000.
   Odyssey Infrared Imaging System (Image Studio Lite Ver 4.0, LI-COR
   Biosciences) was used to detect and quantify signals of target
   proteins.

eIF4E and eIF4E-Myc expressing constructs

   The mouse Eif4e coding sequence alone or extended at its 3′ end with a
   sequence encoding the Myc tag
   (gccGAACAAAAACTCATCTCAGAAGAGGATCTGaatagctag, where the Myc-encoding
   sequence is listed in capital letters) was cloned into pUltra (a gift
   from Malcolm Moore; Addgene plasmid #24129).

Immunoprecipitation

   HEK293 cells were transfected with plasmid constructs (0.6 µg/kb)
   expressing eIF4E or eIF4E-Myc using lipofectamine 3000 (Invitrogen).
   Cells were harvested 48 h post transfection and lysed with a buffer
   containing: 50 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% NP-40,
   2 mM DTT and freshly added protease inhibitors (Roche Complete Protease
   Mini, #4693159001) and phosphatase inhibitors (PhosStop pellets, Sigma
   Aldrich, #4906845001). Cleared cell lysates were incubated with either
   anti-eIF4E antibody (A301-154A, Bethyl Laboratories, 1:100) or IgG on
   ice for 2 h. Protein G-Agarose (11719416001, Roche) was then added to
   each sample, and the mixture was incubated at 4 °C under rotary
   agitation for 4 h. Immunoprecipitates were washed with the buffer five
   times and eluted with the SDS-loading buffer for immunoblotting
   analysis.

Microglial culture

   Purification of primary microglia was performed using an immunopanning
   protocol^[282]36. Briefly, a mouse brain at P10 was digested with 2 ml
   of buffer containing 2.4 mg papain (Worthington, [283]LS003126) and
   1 mg deoxyribonuclease I (Sigma, DN25) at 37 °C for 30 min. The brain
   was then mechanically dissociated by gentle trituration using a 1 ml
   pipette. Cell suspension was transferred to a 10 cm culture dish
   pre-coated with rat anti-mouse CD45 antibody (Invitrogen, Cat.
   17045182) and incubated for 45 min at room temperature. The culture
   dish was then washed with Dulbecco’s PBS 8–10 times. Culture medium
   (DMEM with 10% FBS, 1% GlutaMAX, 1% penicillin/streptomycin) was added
   to the dish, and the dish was returned to an incubator until harvest.

Astrocyte culture

   Astrocytes were isolated from brains of control and AC^4E mice at P3.
   Brains were isolated and mechanically dissociated in HBSS and papain
   (Worthington, [284]LS003126) for 20 min at 37 °C. Afterwards, papain
   was inhibited with Dulbecco’s modified Eagle’s medium (DMEM) containing
   10% FBS. Cell suspensions were subsequently plated into T75 flasks.
   Astrocytes were cultivated till confluency and subsequently shaken for
   18 h (200 rpm). After the medium was removed, astrocytes in flasks were
   trypsinized and plated onto coverslips or plates for further
   experiments.

Primary neuronal culture

   Hippocampal neurons were cultured from P0 newborn mice^[285]74.
   Isolated hippocampi were removed and digested with 20 U/ml of papain in
   Hank’s Balanced Salt Solution (HBSS) at 37 °C for 30 min. Dissociated
   neurons were grown in Neurobasal media A (Invitrogen) supplemented with
   2% B27, 1% GlutaMAX (Gibco) and 1% penicillin-streptomycin at 37 °C and
   5% CO[2] incubator.

Tissue processing for SB-SEM

   Mice were anesthetized and transcardially perfused with 2% PFA and 2.5%
   glutaraldehyde in 0.15 M cacodylate buffer (pH 7.4). The brains were
   removed and stored overnight at 4 °C in fresh perfusion solution. The
   brains were sliced in cold 0.15 M cacodylate buffer into 150-µm coronal
   sections, which were used for dissection of the prelimbic area of the
   mPFC. Tissue blocks were placed in cacodylate buffer containing 2%
   OsO4/1.5% potassium ferrocyanide for 1 h. Samples were placed in 1%
   thiocarbohydrazide (Ted Pella) in ddH[2]O for 20 min and then in 2%
   aqueous OsO4 for 30 min. Tissues were incubated in 1% uranyl acetate at
   4 °C overnight and fresh lead aspartate solution at 60 °C for 30 min to
   enhance membrane contrast. On the next day, samples were dehydrated
   using an ascending series of ethanol (50, 70, 90, and 100%, 5 min
   each), followed by ice-cold dry acetone for 10 min. Tissues were then
   gradually equilibrated with Epon 812 resin (EMS, Hatfield, PA, USA). To
   enhance specimen conductivity^[286]75, samples were embedded in Epon
   812 resin containing 7% (w/v) carbon black powder (a kind gift from Dr.
   Nobuhiko Ohno, Jichi Medical University, Tochigi-ken, Japan), mounted
   on aluminum rivets, and then cured in a dry-oven at 60 °C for 2 days.

Acquisition of SB-SEM datasets and analysis

   A tissue block was trimmed flat using a glass knife. Silver paste was
   applied to the specimen to ground the resin to the aluminum pin. The
   pin was then coated with 10 nm of gold-palladium in a sputter coater to
   further enhance conductivity. Specimens were imaged with a Merlin VP
   field emission scanning electron microscope (Carl Zeiss) equipped with
   3View2 in-chamber ultramicrotome technology and a backscattered
   electron detector (Gatan). Serial images were acquired with a 30-μm
   aperture, high vacuum, acceleration voltage of 2.5 kV, image size of
   5000 by 5000 pixels, dwell time of 3.5 µs, and X–Y resolution of
   10.8 nm at a nominal thickness of 50 nm.

   Eleven stacks (5 stacks for control mice, 6 stacks for MG^4E mice, one
   stack per mouse) of 500 serial images were obtained for layer 2 of the
   prelimbic area of the mPFC. The serial images obtained were processed
   with Image J and Fiji plugins TrakEM2 software
   ([287]http://fiji.sc/wiki/index.php/Fiji). We randomly selected spiny
   dendritic segments that could be followed within the stack and whose
   linear lengths was at least 15 μm. To make sure that our analysis was
   restricted to pyramidal neurons, we avoided all dendritic segments with
   few or no spines. A synapse was defined by the presence of a
   presynaptic bouton with at least three synaptic vesicles within a 50 nm
   distance from the membrane facing a postsynaptic density (PSD). All
   dendritic spines were divided into spines with synapses and spines
   lacking synapses. In total, 70 dendritic branches were segmented in L2.
   Dendritic branches and all of their protrusions were segmented manually
   in the reconstruction software
   ([288]https://synapseweb.clm.utexas.edu/software-0) by three trained
   annotators who were blind to genotype.

Transmission EM analysis

   Both male and female MG^4E and control mice were anesthetized and
   perfused with 2% PFA and 2.5% glutaraldehyde in 0.15 M cacodylate
   buffer (pH 7.4). The brains were sliced into 150-µm coronal sections
   using a vibratome and the prelimbic area of the mPFC was dissected out.
   Tissue blocks were prepared using standard procedures for TEM^[289]76.
   Tissues were postfixed in 2% OsO4 for 1 h, en bloc stained with 1%
   uranyl acetate, dehydrated, and then embedded in Epon 812 resin (EMS).
   Ultrathin (70-nm-thickness) sections were collected on the 200-mesh
   grids and post-stained with uranyl acetate followed by lead citrate.
   For each animal, 30 images were recorded on a Tecnai F20 TEM (FEI)
   (2500× magnification, 120 kV). Counting frame (6 × 6 μm^2) was placed
   on each image and synapses with a PSD as well as clusters of synaptic
   vesicles were identified. Synapses were classified into either
   asymmetric (Gray’s type 1) or symmetric (Gray’s type 2) based on their
   distinct PSD shapes. Synaptic density values were averaged to produce
   animal means. All analyses were performed blind to experimental
   conditions.

Stereotaxic injection of FAM-Aβ

   FAM-Aβ[(1–42)] peptides (Anaspec, AS-23525) were dissolved in
   trifluoroacetic acid (Sigma, T6508), lyophilized in a SpeedVac, and
   resuspended in PBS/DMSO solution at 37 °C overnight to a final
   concentration of 2 μg/μl. FAM-Aβ aggregates (500 nl) were
   stereotaxically injected into hippocampi of control and MG^4E mice at
   P14 using the following coordinates (relative to the bregma): AP
   (anterior posterior), −2.25 mm; ML (medial lateral), ±1.80 mm; DV
   (dorsal ventral), −2.0 mm. Mice were perfused 18–19 h after FAM-Aβ
   injection.

Aβ phagocytosis assay

   FAM-Aβ[(1–42)] aggregates were added to primary microglial culture at a
   concentration of 1.8 μg/ml and incubated for 4 h at 37 °C.
   Extracellular FAM-Aβ[(1–42)] was quenched with 0.2% trypan blue in PBS
   for 1 min. FAM-Aβ[(1–42)] fluorescence signal intensity was measured at
   485 nm excitation and 538 nm emission using a CLARIOstar (BMG LABTECH)
   microplate reader.

Electrophysiology

   Mice at P42–P49 were used for electrophysiological recording. Mice were
   transcardially perfused with oxygen-saturated ice-cold cutting solution
   containing (in mM): 124 choline chloride, 26 NaHCO[3], 2.5 KCl, 3.3
   MgCl[2], 1.2 NaH[2]PO[4], 0.5 CaCl[2] and 1 d-glucose. Brains were
   rapidly removed and placed in ice-cold cutting solution equilibrating
   with 95% O[2] and 5% CO[2]. Medial prefrontal cortical slices (300 μm)
   were obtained using a vibratome (Leica VT 1200S, Germany) and
   transferred to oxygenated artificial cerebrospinal fluid (aCSF)
   composed of (mM): 124 NaCl, 3 KCl, 26 NaHCO[3], 1.25 NaH[2]PO[4], 1
   MgSO[4], 2 CaCl[2], and 10 d-glucose. Brain sections were recovered in
   aCSF at 32 °C for at least 1 h. Afterwards, a slice was gently
   transferred to a recording chamber (RC-27, Warner Instruments, Hamden,
   CT) at room temperature. The chamber was perfused with circulated
   oxygenated aCSF at a flow rate of 2–3 ml/min. Prelimbic layer 5
   pyramidal neurons were visually identified in slices using an
   infrared-differential interference contrast microscope (Scientifica,
   UK). Whole-cell patch-clamp recordings were performed using
   borosilicate glass pipettes (ID: 0.68 mm, OD: 1.2 mm, WPI, Sarasota,
   FL) of 3–5 MΩ pulled with a micropipette puller (P-1000; Sutter
   Instrument, Novato, CA). For mEPSC recording, pipettes were filled with
   internal solution containing (in mM): 115 CsMeSO[3], 20 CsCl, 10 Hepes,
   0.6 EGTA, 4 MgATP, 0.3 Na[3]GTP, 1 QX-314, 2.5 MgCl[2], 10
   Na[2]-Phosphocreatine (pH 7.3 with CsOH, osmolarity 285 mM). For mIPSC
   recording, pipettes were filled with internal solution containing (in
   mM): 100 CsCl, 35 CsMeSO[3], 10 Hepes, 0.5 EGTA, 4 MgATP, 0.3 Na[3]GTP,
   5 QX-314, and 10 Na[2]-Phosphocreatine (pH 7.3 with CsOH, osmolarity
   285 mM). Neurons were held at −70 mV in voltage-clamp mode without
   serious resistance and liquid junction compensation. Neurons with Ra
   <25 MΩ were recorded. mEPSCs were recorded in the presence of 1 μM
   tetrodotoxin and 100 μM picrotoxin. To isolate mIPSCs, we used 1 μM
   tetrodotoxin, 20 μM CNQX and 50 μM APV.

   Experimenters were blind to genotype. Signals were acquired with
   Multiclamp 700B and Digidata 1550A (Molecular Devices, San Jose, CA).
   Data were low-pass filtered at 2.9 kHz and sampled at 10 kHz. mEPSCs
   and fEPSPs were analyzed with Clampfit 10.6 software. Detection
   threshold was set at 5 pA (mEPSC) or 6 pA (mIPSC), and the first 200
   events were sampled per neuron. Total charge transfer was calculated by
   summing the charge transfer of all individual events detected over the
   first 2-min acquisition period for each neuron.

SUnSET

   400-μm-thick hippocampal slices were obtained using a vibratome and
   recovered in aCSF for 2 h at 32 °C. Puromycin (10 μg/ml) was applied to
   slices for 1 h to label newly synthesized proteins. To measure protein
   synthesis in microglia, puromycin (10 μg/ml) was applied to cultured
   microglia for 1 h. Tissues and cultured microglia were then lysed and
   used for Western blot. Mouse anti-puromycin antibody (1:2000,
   Millipore, MABE343) was used to detect puromycin-incorporated proteins.

Behavioral tests

   Male and female mice at 2-month-old of age were tested during the dark
   (active) phase of a 12/12 h reversed light-dark cycle. Mice were moved
   to a holding room in the behavioral testing area at least 1 h ahead of
   behavioral assays. Apparati were cleaned with 1% Micro-90 between each
   trial. Automatic scoring was performed using the Ethovision XT video
   tracking system (Noldus, Netherlands). Mice were tested in batteries
   with at least 3 days between assays. Details of these paradigms are
   listed below.

   For open field test, a mouse was placed in the open field arena
   (43.8 cm × 43.8 cm × 32.8 cm) for 30 min. Total distance moved, center
   zone duration and velocity were recorded automatically.

   For elevated plus maze test, a mouse was placed in the center of the
   maze to start the test. Each trial lasts for 5 min. Total distance
   moved and durations in open and closed arms were recorded
   automatically.

   For light-dark box test, a mouse was placed in the light chamber and
   allowed to explore for 5 min. Latency to enter the dark chamber, time
   in the light chamber and number of crossings between chambers were
   automatically recorded.

   Spontaneous alternation in T-maze was used to assess working memory in
   mice. Briefly, mice received two successive non-rewarded trials in one
   day. On trial one mice were allowed to enter one of the two unfamiliar
   arms. After staying 10 s in the chosen arm, mice were removed from the
   maze and placed back on the start arm for the second trial. A correct
   choice was to explore the arm that was not previously explored.

   Mice were placed on a rotating rod (ENV-577M, Med Associates Inc.) for
   rotarod tests. The speed of rotation was gradually increased from 4 to
   40 rpm over a 5-min period. Mice received three trials per day for two
   days, and each trial was spaced at least 1 h apart. The latency to fall
   was measured.

   For fear conditioning test, a mouse was placed into a Phenotyper
   chamber (29.2 cm × 29 cm × 30.5 cm, Noldus) equipped with an
   electrified floor and speaker. Training consisted of a 150 s baseline
   followed by three 0.75-mA foot shocks. Mice were tested for contextual
   fear memory 24 h after training by returning to their training chambers
   for 5 min. Freezing behavior was automatically recorded by using the
   Ethovision XT video tracking system.

   For marble burying test, mice were placed individually in a cage filled
   with 5 cm of corncob bedding and 20 black marbles arranged on top. The
   test lasted for 30 min, and the number of marbles that were at least
   two-thirds buried at the end of a trial were counted^[290]38.

   For self-grooming test, mice were placed into a new cage with fresh
   bedding but no cardboard or nesting material. Self-grooming behavior
   was video recorded for 30 min and scored manually afterward.
   Experimenters were blind to genotype during scoring.

   A three-chamber arena was used to assess sociability in mice^[291]77. A
   test mouse was placed in the central chamber and allowed to explore the
   empty apparatus for 5 min. Immediately after habituation, a stimulus
   mouse (same-sex, similar age, and unfamiliar mouse) was introduced into
   a wire cage located in one of the two side chambers. An identical empty
   wire cage was placed in the other side chamber. The test mouse was
   allowed to explore the arena for 10 min. Time spent in each chamber and
   time spent in sniffing the stimulus mouse (or empty wire cage) was
   recorded by Ethovision XT video tracking system. In addition to
   analyzing the time spent in each chamber or investigation time, we also
   calculated the social preference index, which represents the difference
   between time spent in investigating the stimulus mouse vs. object,
   divided by total time spent in investigating both targets.

   For social recognition test, a tested mouse was housed alone for 2 h in
   a home cage-like environment^[292]38. A same sex, juvenile (3–4 weeks
   old) stimulus mouse was placed into an acrylic tube (7.25 cm in
   diameter, 12.5 cm tall) with holes at the bottom. Social recognition
   test consisted of 5-min stimulus presentations separated by 10-min
   inter-trial intervals; the first four presentations (H1–H4) were of the
   same stimulus mouse, with the fifth using a novel juvenile
   (non-littermate juvenile). Time spent in sniffing the stimulus mouse
   was manually scored from video. Habituation index is calculated using
   the formula: (H1 investigation time – H4 investigation time)/total H1
   and H4 investigation time.

   Novel object recognition was conducted in a three-chamber arena. A test
   mouse was placed in the middle chamber and allowed to explore the two
   side chambers with two identical objects located in each side chamber
   for 10 min. Immediately after this, one object was replaced with a
   novel object and the test mouse was allowed to explore for another
   10 min. Time spent investigating the object (nose-point tracking) was
   automatically recorded by Ethovision XT video tracking system.
   Discrimination index is defined as (novel object time – familiar object
   time)/total time on two objects.

RNA-Seq analysis

   Total RNA was extracted from dissected hippocampal tissues using a
   Quick-RNA Miniprep Kit (Zymo Research, R1054). RNA was quantified using
   the Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and run on the
   Agilent 2100 Bioanalyzer RNA nano chip (Agilent Technologies, Santa
   Clara, CA) for quality assessment. All RNA samples were of excellent
   quality with RNA Integrity Number (RIN) > 8.0 and were processed for
   mRNA_seq library preparation. The final mRNA libraries were validated
   on the bioanalyzer DNA chips, normalized to 1 nM, pooled equally,
   loaded onto the NextSeq 500 (Illumina) at a final concentration of
   1.8 pM, and sequenced using 2 × 40 bp paired-end chemistry. Library
   preparation and sequencing was performed at the Scripps Florida Genomic
   Core.

   Reads were trimmed off for sequencing adapter and were mapped to the
   mouse genome using the STAR version 2.5.2a aligner. Differential gene
   expression analysis was performed with genes with CPM > 0.5 in at least
   four samples, using Bioconductor packages edgeR^[293]78 with false
   discovery rate (FDR) threshold <0.05
   ([294]http://www.bioconductor.org). Weighted gene co-expression network
   analysis (WGCNA) was performed to find modules of highly correlated
   genes via its R package^[295]49. WGCNA clustering, using the
   “blockwiseModules” function with the network type “signed hybrid”, a
   power parameter of 5 (as established by scale-free topology network
   criteria), and minModSize = 50, minKMEtoStay = 0.3, deepSplit = 2,
   mergeCutHeight = 0.15, dissected the data into 11 modules. The gene set
   enrichment and pathway analysis were performed using online tools
   DAVID^[296]79 ([297]https://david.ncifcrf.gov) and Generic GO Term
   Finder^[298]80.

Quantitative reverse transcription PCR

   Mouse hippocampi were collected at 2-week and 6-week of ages. Total RNA
   was extracted from Trizol homogenates following the manufacturer’s
   protocol. Following DNaseI (New England Biolabs) treatment, cDNA was
   synthesized using M-MuLV reverse transcriptase (New England Biolabs)
   with oligo dT primers. Candidate genes were then quantified by
   real-time PCR using SYBR Green Mix (Roche). The actin gene ACTB was
   used as control. The PCR primers for each gene are listed
   (Supplementary Table [299]1).

Two-photon imaging of microglial motility and data analysis

   We performed two-photon imaging to visualize microglial motility
   according to a previously described protocol^[300]81. Briefly, acute
   brain slices (300 µm thick) were prepared from
   R26^Eif4e/Eif4e;CD68-EGFP (control) or
   Cx3cr1^CreER/+;R26^Eif4e/Eif4e;CD68-EGFP (MG^4E) mice at P14-P18 using
   a vibratome (Leica VT 1200S, Germany) and transferred to oxygenated
   artificial cerebrospinal fluid (aCSF) composed of (mM): 124 NaCl, 3
   KCl, 26 NaHCO[3], 1.25 NaH[2]PO[4], 1 MgSO[4], 2 CaCl[2], and 10
   d-glucose. Brain sections were recovered in aCSF at 32 °C for 1 h.
   Microglia were imaged at a depth of 50–100 µm in the brain slice using
   a Leica TCS SP8 MP multiphoton microscope (25× lens). Stacks of 26
   images with 2-µm intervals were acquired every 60 s for 5 min (base
   line) and for another 10 min after ATP (1 mM) was bath-applied to brain
   slices. Images were analyzed using ProMoIJ, an ImageJ macros that
   perform automatic microglial motility analysis^[301]82. Briefly,
   microglia were cropped and aligned, subsequently individual microglial
   processes were selected for automated analysis of their motility.
   Process motility was analyzed by reconstructing the process 3D skeleton
   and was calculated as the absolute difference of process length between
   two consecutive time frames. Specifically,
   [MATH: <mi mathvariant="normal">Process</mi><mspace
   width="0.25em"></mspace><mi
   mathvariant="normal">motility</mi><mo>=</mo><mfrac><mrow><msqrt><msup><
   mrow><mrow><mo>(</mo><mrow><msub><mrow><mi
   mathvariant="normal">Length</mi></mrow><mrow><mi>f</mi><mo>+</mo><mn>1<
   /mn></mrow></msub><mo>−</mo><msub><mrow><mi
   mathvariant="normal">length</mi></mrow><mrow><mi>f</mi></mrow></msub></
   mrow><mo>)</mo></mrow></mrow><mrow><mn>2</mn></mrow></msup></msqrt></mr
   ow><mrow><mi>t</mi></mrow></mfrac><mo>,</mo> :MATH]

   where f is time frame and t corresponds to the time interval between
   consecutive frames (1 min).

Statistical analysis

   Statistical analyses were performed using SPSS. Shapiro-Wilk (n < 10)
   and D’Agostino and Pearson omnibus (n > 10) normality tests were
   performed to determine if values fit a Gaussian distribution.
   Comparisons between two groups/treatments were performed using
   two-tailed Student’s t-test or Chi-square test as applicable.
   Cumulative curves were compared using Kolmogorov–Smirnov test. For
   multiple comparisons, two-way ANOVA with Fisher’s LSD post hoc test
   were used. The minimal level of significance was set at p < 0.05.
   Statistical parameters are detailed in the legend for each figure.

Reporting summary

   Further information on research design is available in the Nature
   Research Reporting Summary linked to this article.

Supplementary information

   [302]Supplementary Information^ (8.2MB, pdf)
   [303]Peer Review File^ (107KB, pdf)
   [304]Reporting Summary^ (169.4KB, pdf)

Acknowledgements