Neuron

Article

Colony-Stimulating Factor 1 Receptor Signaling Is

Necessary for Microglia Viability, Unmasking
a Microglia Progenitor Cell in the Adult Brain
Monica R.P. Elmore,1,4 Allison R. Najafi,1,4 Maya A. Koike,1 Nabil N. Dagher,1 Elizabeth E. Spangenberg,1 Rachel A. Rice,1
Masashi Kitazawa,3 Bernice Matusow,2 Hoa Nguyen,2 Brian L. West,2 and Kim N. Green1,*
1Department of Neurobiology and Behavior, Institute for Memory Impairments and Neurological Disorders, University of California, Irvine,

Irvine, CA 92697-4545, USA

2Plexxikon Inc., Berkeley, CA 94710, USA
3Department of Molecular and Cell Biology, University of California, Merced, Merced, CA 95343, USA
4Co-first authors

*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.neuron.2014.02.040

SUMMARY The CSF1R is expressed by macrophages, microglia, and

osteoclasts (Patel and Player, 2009), and it has two natural
The colony-stimulating factor 1 receptor (CSF1R) is a ligands: colony-stimulating factor 1 (CSF1) and interleukin-34
key regulator of myeloid lineage cells. Genetic loss of (IL-34) (Lin et al., 2008). CSF1 regulates the proliferation, dif-
the CSF1R blocks the normal population of resident ferentiation, and survival of macrophages (Patel and Player,
microglia in the brain that originates from the yolk 2009), with mice lacking either CSF1 or the CSF1R showing
sac during early development. However, the role of reduced densities of macrophages in several tissues (Li
et al., 2006). Furthermore, CSF1R knockout mice are devoid
CSF1R signaling in microglial homeostasis in the
of microglia (Erblich et al., 2011; Ginhoux et al., 2010) and
adult brain is largely unknown. To this end, we tested
die before adulthood. In the brain, it has been demonstrated
the effects of selective CSF1R inhibitors on microglia that microglia are the only cell type that expresses the
in adult mice. Surprisingly, extensive treatment re- CSF1R under normal conditions (Erblich et al., 2011; Nandi
sults in elimination of
99% of all microglia brain- et al., 2012). In this study, we investigate the effects of
wide, showing that microglia in the adult brain are CSF1R inhibitors on microglial function, and we find that inhi-
physiologically dependent upon CSF1R signaling. bition leads to the elimination of virtually all microglia from the
Mice depleted of microglia show no behavioral or adult CNS, with no ill effects or deficits in behavior or cogni-
cognitive abnormalities, revealing that microglia are tion. Withdrawal of the inhibitor leads to rapid repopulation
not necessary for these tasks. Finally, we discovered with new cells that then differentiate into microglia. Notably,
that the microglia-depleted brain completely repopu- repopulation occurs rapidly from nestin-expressing cells
found throughout the CNS, representing a microglial progeni-
lates with new microglia within 1 week of inhibitor
tor cell.
cessation. Microglial repopulation throughout the
CNS occurs through proliferation of nestin-positive
RESULTS
cells that then differentiate into microglia.
Selective CSF1R Kinase Inhibitors Block Growth of EOC
INTRODUCTION 20 Microglial Cells In Vitro
The EOC 20 microglial cell line has been shown to depend on the
Microglia colonize the CNS during development, originating from addition of spent media from LADMAC cells that produce CSF1.
uncommitted c-Kit+ stem cells found in the yolk sac (Kierdorf We tested several selective CSF1R kinase inhibitors (PLX3397
et al., 2013). These c-Kit+ cells develop into CD45+/c-Kit/ [Artis et al., 2005], PLX647 [Zhang et al., 2013], Ki20227 [Ohno
CX3CR1+ cells that migrate to the CNS and become microglia. et al., 2006], and GW2580 [Conway et al., 2005], and the nonse-
The development of these cells is dependent upon Pu.1 and lective kinase inhibitor dasatinib) on EOC 20 cells grown with
Irf8 (Kierdorf et al., 2013), as well as the colony-stimulating factor either LADMAC spent media or conventional media, to which
1 receptor (CSF1R; Erblich et al., 2011; Ginhoux et al., 2010). After purified CSF1 was added (Figure S1A available online; conven-
migration of these progenitors to the CNS, the blood-brain barrier tional media + CSF1 is shown). In both cases, the inhibitors
(BBB) forms, effectively separating the microglia from the periph- completely arrested cell growth, as determined by a standard
ery. Infiltration of peripheral monocytes or macrophages into the ATP assay (Crouch et al., 1993), with half-maximal inhibitory
CNS does not occur under normal conditions (Ginhoux et al., concentration (IC50) values below 1 mM (Figure S1B) and
2010; Mildner et al., 2007), and thus microglia form an autono- PLX3397 showing the most robust inhibition of all of the com-
mous population. pounds tested.

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CSF1R Inhibitors Dramatically Reduce Microglia marker, we treated 2-month-old CX3CR1-GFP+/ mice with
Numbers in the Adult Brain PLX3397. These mice express GFP in myeloid lineage cells
For the initial in vivo experiments, all drugs were tested at very (e.g., microglia and macrophages). After only 3 days of treat-
high doses, because their ability to penetrate the BBB was ment, GFP+ cells were counted in a 103 field of view from the
unknown. Based on our in vitro experiments, we selected hippocampus, cortex, and thalamus (n = 3 per group), showing
PLX3397 for our in vivo work, because its IC50 values have been >50% reduction in cell numbers (Figures 1R and 1S).
published and shown to potently and selectively inhibit CSF1R
and c-Kit over most other kinases (DeNardo et al., 2011). In addi- Microglial Death with CSF1R Inhibition
tion, the effects of PLX3397 on peripheral myeloid cells have been Given the rapid depletion of microglia from the brain, we
extensively characterized (Abou-Khalil et al., 2013; Chitu et al., reasoned that blocking CSF1R signaling must result in microglial
2012; Coniglio et al., 2012; DeNardo et al., 2011; He et al., cell death, rather than just an inhibition of proliferation. Thus, we
2012; Mok et al., 2014; Prada et al., 2013), where chronic looked for evidence of microglial cell death. We further reasoned
PLX3397 treatment eliminates tumor-associated macrophages, that dying and dead microglia would be most present at 3 and
but has only modest effects on macrophage numbers in other 7 days of PLX3397 treatment, because most microglia are elim-
tissues in wild-type mice (Mok et al., 2014). We also tested inated within the first week. Indeed, we found many examples of
the PLX3397 analog PLX647 (Zhang et al., 2013). PLX3397 or IBA1+ staining that looked like remnants of cells (Figures 1P and
PLX647 was mixed into a standard rodent diet at 1,160 and 1Q, indicated by arrows). Given the enlarged size of surviving mi-
1,000 mg drug per kilogram of chow, respectively, corresponding croglia during CSF1R inhibition, we hypothesize that these cells
to doses of approximately 185 and 160 mg/kg body weight, and may be highly phagocytotic and involved in the clearing of micro-
administered to a lipopolysaccharide (LPS) (0.5 mg/kg) mouse glial corpses from the CNS. However, few remnants were seen at
model of neuroinflammation (Figure S1C). Brains were homoge- 21 days of treatment, suggesting that the CNS has an additional
nized, and western blots were performed using anti-ionized cal- microglia-independent method for eliminating cellular debris. To
cium binding adaptor molecule 1 (IBA1), a marker for microglia. confirm that microglia undergo cell death with CSF1R inhibition,
As expected, LPS-treated mice were found to have elevated we found that many microglia stained for active caspase-3 in the
steady-state levels of IBA1, consistent with increased neuro- same animals, a classic marker of apoptosing cells (Figure S2J–
inflammation (Figures S1D and S1E). Treatment with either S2L). Further evidence of microglial death with CSF1R inhibition,
CSF1R antagonist prevented this LPS-induced IBA1 increase, as opposed to differentiation into alternative cell types, is shown
suggesting that CSF1R signaling is essential for this neuroinflam- in Figure 8.
matory effect. However, quite surprisingly, in the case of PLX3397 We then conducted a 7-day treatment in 2-month-old CX3CR1-
treatment, the IBA1 protein levels decreased to 70% below the GFP+/ mice and performed flow cytometry on processed whole
levels of the PBS-treated controls. Immunostaining for IBA1 in brains. GFP+ cell counts revealed a 90% reduction with 7 days of
the cortex of these animals confirmed these results and further re- PLX3397 treatment (Figure 1T). In addition, more than 20% of
vealed a clear decrease in microglia numbers with inhibitor treat- these GFP+ cells stained positively for propidium iodide (PI), indic-
ments (Figures S1F and S1G), with remaining microglia exhibiting ative of dying microglia (Figure 1U). We also performed stereo-
an enlarged morphology with thickened processes. logical volume measurements of mice treated for 7 days with
Based on these results, PLX3397 produced the most robust PLX3397 and found no significant differences, despite the loss
reductions in brain microglia. Next, we sought to administer of >90% of microglia (Figure 1V).
decreasing concentrations of the compound in chow to deter- To further show that microglia are dependent upon CSF1R
mine a dose regimen for chronic studies. As before, 2-month- signaling for their survival, we treated 2-month-old wild-type
old male mice were treated with vehicle, LPS, or LPS + mice with PLX3397 or vehicle for 7 days (n = 4 per group) and
PLX3397 for 7 days (n = 4 per group). Western blot analysis of counted the number of microglia, as well as mRNA levels for
brain homogenates again showed a robust reduction in microglial markers (Figures 2A–2D). PLX3397 treatment reduced
steady-state levels of IBA1 at all doses, with 290 mg/kg chow microglia numbers in the hippocampus, cortex, and the thal-
PLX3397 still showing maximal effects (Figure S1H and S1I). amus by >90%, as determined by automated counts of IBA1+
Having determined the optimal dosing for all future chronic cell bodies (Figures 2A–2C). mRNA levels of the microglial
studies, we treated 12-month-old wild-type mice with markers AIF1, which encodes IBA1, CSF1R, CX3CR1, FCGR1,
290 mg/kg chow PLX3397 for 0, 1, 3, 7, 14, or 21 days (n = 4– ITGAM, and TREM2 were all significantly reduced (Figure 2D).
5 per group). Immunostaining for IBA1 showed a robust, time- Of the two CSF1R ligands, which are both produced by neurons,
dependent reduction in microglia number, with a 50% reduction CSF1 was unchanged but IL-34 was upregulated (Greter et al.,
in microglia after just 3 days of treatment, and brains were essen- 2012; Nandi et al., 2012).
tially microglia devoid by 21 days in all regions surveyed (Figures
1A–1F and 1J–1N, with quantification in Figure 1O). Morpholog- Effects of Microglial Depletion on Other CNS Cell Types
ical analyses of surviving microglia revealed a larger cell body We explored the effects of treatment on other CNS cell types
(Figure S2E), an increased thickness of processes (Figure S2F) by probing for steady-state levels of astroctytic, oligodendro-
typically associated with a more phagocytotic phenotype (Neu- cytic, and neuronal markers via western blot (Figures S3A
mann et al., 2009), and a reduction in the number of branches and S3B). No changes in markers hexaribonucleotide binding
per microglia (Figure S2H). To determine whether the results protein-3 (NeuN), microtubule-associated protein 2 (MAP2),
could simply be due to downregulation of the IBA1 microglial or oligodendrocyte transcription factor (Olig2) were observed.

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Figure 1. CSF1R Inhibition Eliminates Microglia from the Adult Brain

Twelve-month-old wild-type mice (C57BL/6/129 mix; n = 4–5 per group) were treated with PLX3397 (290 mg/kg chow) for 0, 1, 3, 7, 14, or 21 days.
(A–F) Immunostaining for IBA1 shows robust decreases in microglial numbers, with no detectable microglia present after 21 days of treatment.
(G–I) IBA1 immunostaining shows changes in microglia morphology during treatment, with representative microglia shown from control, 7-, and 21-day treated
mice, imaged from between the blades of the dentate gyrus.
(J–N) Representative IBA1 immunofluorescent staining from the hippocampal region showing 633 z stacks of microglia during treatment. Scale bar represents
20 mm.
(legend continued on next page)

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However, robust increases in the astrocytic markers glial fibril- trial of the Barnes maze (Figures S4H and S4I) or on locomotion,
lary acidic protein (GFAP) and S100 were found. GFAP mRNA as tested with the accelerating rotarod (Figure S4J). Given these
levels were also increased with 7 days of PLX3397 treatment, results in mice depleted of microglia for 21 days, we then set out
as measured via real-time PCR (Figure S3C). To further inves- to determine whether further long-term depletion of microglia
tigate these changes in astrocytic markers, we performed would alter cognition or behavior, because perhaps deficits
immunofluorescent stains for GFAP at all treatment time would take longer than 3 weeks to manifest. To that end, wild-
points. GFAP cell counts showed no differences with treat- type mice were treated with PLX3397 or vehicle for 2 months,
ment, despite the measured increases in mRNA and protein and then cognition and behavior were assessed. A third group
(Figures S3D–S3G). Likewise, S100 cell counts also revealed was administered the cholinergic antagonist scopolamine on
no differences (Figure S3G). No changes in signal intensity testing days to induce cognitive deficits as a positive control
with either GFAP or S100 were measured; likewise, morpho- (n = 10 per group). With long-term microglial elimination, no
logical analyses revealed no changes in astrocyte cell body changes in elevated plus maze were observed (Figures 2E and
volumes, process lengths, or diameters (Figures S3H–S3P). 2F). Likewise, no changes were seen in open field (Figures 2G–
Thus, depletion of microglia results in increased mRNA and 2I) or accelerating rotarod (Figure 2L). Intriguingly, training on
protein for astrocytic markers, but no changes in cell numbers the Barnes maze revealed that microglia-depleted mice were
or morphology. able to learn the task significantly better than microglia-intact an-
imals, as shown by shorter escape latencies on days 2 and 3 of
Elimination of Microglia Does Not Affect the BBB training (Figure 2J), as well as an overall reduction in average
We tested whether microglial elimination could compromise the escape latency across all training days (Figure 2K). Mice treated
integrity of the BBB, using Evans blue. No Evans blue was found with scopolamine were unable to learn the task, as evidenced by
in the brain, and there were no changes in peripheral organs an inability to escape the maze more quickly on subsequent days
in either control mice (n = 4) or microglia-depleted mice (n = 4) (Figure 2J). No differences were found in the probe trial (data not
(Figures S2M and S2N), showing that the BBB remains intact. shown). We then performed contextual fear conditioning, an
additional hippocampal-dependent learning and memory task.
Short- or Long-Term Microglia Depletion Does Not No significant differences were found between microglia-
Affect Cognition or Behavior depleted mice and microglia-intact mice, whereas mice treated
Two-month-old wild-type mice were treated for 21 days with with scopolamine performed significantly worse, and therefore
PLX3397 or vehicle (n = 10 per group). This treatment regimen showed a cognitive deficit as expected (Figure 2M). Thus, mice
was found to deplete >99% of all microglia from the brain, as depleted of microglia for either 21 days or 2 months show no def-
illustrated in Figures S4A and S4B. Mice were first tested on icits in learning, memory, motor function, or behavior; and sur-
the elevated plus maze (Figures S4C and S4D). Microglia- prisingly, mice chronically depleted of microglia showed some
depleted mice spent significantly more time in the closed arms evidence of enhanced learning.
of the maze, a behavior that is typically considered ethologically
relevant in rodents, and could be indicative of increased anxiety. Immune Profiling of the Microglia-Depleted Brain
No differences in the number of arm entries into either the closed To explore how the microglia-depleted brain responds to im-
or open arms were observed. Despite these changes in the mune challenges, we treated 2-month-old wild-type mice for
elevated plus maze, open field analysis revealed no change in 7 days with PLX3397 to deplete their microglia (Figures 3A–
the amount of time spent in the center of the field, showing 3F). We then administered either PBS or LPS (0.25 mg/kg) and
that microglia-depleted mice did not have increased anxiety in sacrificed the animals 6 hr later. mRNA was extracted from
this task (Figure S4G). In addition, no changes in the distance whole brains, converted to cDNA, and then analyzed against a
traveled or velocity were seen (Figures S4E and S4F), indicating panel of 86 immune-related genes (Figures 3G–3I). Overall,
that there were no locomotor differences between groups. depletion of microglia leads to robust reductions in the expres-
Microglial elimination throughout the CNS had no effects on sion of many inflammatory genes, including TNF-a and other cy-
learning and memory, as determined via acquisition and probe tokines. Microglia-expressed genes are also robustly reduced,

(O) Quantification of number of IBA1+ cell bodies from a 103 field of view from the hippocampal regions as a function of time. Statistical analyses were performed
via one-way ANOVA indicating p < 0.0001 for CA1, CA3, and subiculum, comparing all time points, shown as an average for all three regions.
(P and Q) IBA1+ cell debris and nonintact cell parts were observed throughout the brains of mice treated with CSF1R inhibitors. Arrows indicate these microglial
remnants alongside intact microglia.
(R and S) Two-month-old CX3CR1-GFP+/ mice were treated with PLX3397 or vehicle for 3 days (n = 3 per group). Brains were sectioned and the number of GFP+
cell bodies was counted from a 103 field of view of the hippocampus, cortex, and thalamus. Each white dot represents a GFP+ cell body.
(T) Two-month-old CX3CR1-GFP+/ mice were treated with PLX3397 for 7 days or vehicle (n = 3 per group). Brains were then extracted, dissociated into a single-
cell suspension, and then incubated with PI. Flow cytometry was then performed, revealing >90% reduction in the number of GFP+ cells with treatment compared
with vehicle treated.
(U) The fraction of GFP+ cells that were undergoing cell death, as shown by incorporation of PI, was significantly higher with PLX3397 treatment than vehicle,
consistent with microglial cell death with CSF1R inhibition.
(V) Total brain volumes were measured via Cavalieri stereology in 2-month-old wild-type mice treated with PLX3397 or vehicle for 7 days (n = 4 per group). No
significant differences were seen. Asterisk (*) indicates significance (p < 0.05) by unpaired Student’s t test. Error bars indicate SEM. CA, cornus ammonis; DG,
dentate gyrus; O, stratum oriens; R, stratum radiatum; M, molecular layer dentate gyrus; H, hilus.

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Figure 2. CSF1R Inhibition Reduces Microglial Markers and Numbers, but Does Not Affect Brain Volume, Cognition, or Motor Function
(A–D) Two-month-old wild-type mice were treated with PLX3397 for 7 days or vehicle (n = 4 per group). Representative images from brain sections stained with
anti-iBA1 from the hippocampal region of control (A) and PLX3397 (B)-treated mice. (C) Quantification of the number of IBA1+ cell bodies in a 103 field of view
from the hippocampus, cortex, and thalamus shows >90% elimination of microglia with 7 days PLX3397 treatment. (D) Real-time PCR of mRNA extracted from
half brains for microglial markers shows robust reductions in AIF1, CSF1R, CX3CR1, FCGR1, ITGAM, and TREM2.
(E–M) Two-month-old wild-type mice were treated for 2 months with PLX3397 or vehicle to deplete microglia from the CNS (n = 10 per group).
(legend continued on next page)

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including CD4, CD68, CD86, H2-Eb1, which encodes MHC II, numbers (Figure 4H). Thus, the adult CNS has a highly plastic
and PTPRC, which encodes CD45, reinforcing the finding that and dynamic microglial population that can be entirely repopu-
microglia are absent from these treated brains. Responses to lated after microglial elimination, even in the aged brain. mRNA
LPS are dampened for many genes, but chemokine responses profiling of these brains shows loss of microglia markers with
are mixed. In addition, these results demonstrate that microglial CSF1R inhibition, consistent with microglia elimination, followed
elimination is not accompanied by an inflammatory response by by recovery consistent with repopulation (Figure S6A). In addi-
the remaining cells in the CNS, an important feature of the tion, large increases in the chemokines CCL2, CCL3, and
approach to microglial depletion shown here. CCL5 were also seen at the 3-day recovery time point, sug-
gesting strong signaling for repopulation to occur. Counts of re-
Rapid Restoration of CNS Microglia after Drug Removal populating microglia at the 3-day recovery time point showed
Eighteen-month-old wild-type mice were treated with PLX3397 that three mice had robust repopulation, whereas two mice still
for 28 days to eliminate microglia, first to show that microglia lacked microglia (Figure 4H). mRNA levels of AIF1 reflected
are still dependent upon CSF1R signaling in the aged brain, these microglia counts, prompting us to perform correlations
and second, to explore microglial homeostasis in the aged brain. between AIF1 levels and CCL2, CCL3, and CCL5 (Figures
At this point, all mice were switched to vehicle chow and then S6B–S6G). Strong and highly significant correlations were found
sacrificed 0, 3, 7, 14, and 21 days later to assess any microglial between AIF1 levels and CCL2 and CCL5 during the early stages
repopulation (n = 4 per group; Figure 4A). Remarkably, within of repopulation (day 3), and trended toward significance for
3 days IBA1+ cells appear throughout the brain with very different CCL2 at day 7. Because microglial depletion does not alter either
morphologies to resident microglia in control brains (Figures 4B CCL2 or CCL5 (Figure S6A), these increases in chemokines are
and 4D). They are much larger, with only short stubby processes. a consequence of the repopulation process rather than just the
By 7 days of recovery, the total number of microglia exceeds that reappearance of microglia.
of control mice, and their morphologies lie between that of the
cells seen at 3 days and untreated microglia (Figures 4B–4H). Early Microglial Repopulation Events Highlight Robust
By 14 days of recovery, the microglia numbers stabilize to un- Nestin-Expressing Cells
treated levels, and the repopulating microglia resemble normal We next set out to explore the early repopulation events that
ramified microglia. Thus, repopulation of the microglia-depleted occurred between drug withdrawal and the 3-day recovery
brain occurs through rapid increases in cell numbers and differ- time point. We treated 2-month-old CX3CR1-GFP+/ mice with
entiation into microglial morphologies. The cells seen at the 3- PLX3397 for 7 days and then withdrew the drug for 0, 1, 2, or
day recovery time point are unique: they are much larger than 3 days (n = 4 per group; Figure 5A). Seven days of PLX3397 treat-
resident ramified microglia (Figure 4I) and appear throughout ment eliminated
70% of microglia in these CX3CR1-GFP+/
the CNS, rather than in discrete locations (Figure S5). Curiously, mice (Figures 5B–5F). Microglia continued to be eliminated until
we also found that these cells express several markers not seen day 2 of recovery, but by day 3, the microglia population was
in microglia in control brains, nor in surviving microglia while be- quadrupled from that of the previous day (Figure 5H). These
ing treated (Figure 4K). They are very strongly positive for the lec- data highlight a critical period of 48–72 hr for microglial repopu-
tin IB4, as well as CD45. Many of these cells are Ki67+, a marker lation, consistent with the results from the 18-month-old mice
of cell proliferation, and CD34+, a marker of hematopoietic stem (Figure 4). Measurements of PLX3397 in brain tissue revealed
cells (HSCs), whereas
10% of these cells also show c-Kit stain- that the drug was quickly cleared from the brain, with trace
ing, another HSC marker. The majority of cells are also nestin+, a amounts being detected by 1-day recovery (Figure 5I). Thus, mi-
neuroectodermal development marker, a surprising finding given croglial elimination continues in the absence of drug. We also
the myeloid lineage of microglia. However, at day 7, IBA1+ cells performed flow cytometry for GFP+ cells from the liver and
assume a more typical microglia morphology; have repopulated spleen to look at the effects in the periphery (Figure 5G). No sig-
the entire CNS; and are CD45, IB4, CD34, c-Kit, nestin, and Ki67 nificant changes in GFP+ cell numbers were seen in the liver with
negative. By 14 days, cells are morphologically indistinguishable either treatment or repopulation, whereas some reductions were
from resident microglia in control brains and have comparable seen in the spleen by day 2 of recovery, but increased again the

(E and F) Elevated plus maze showed no differences in the frequency of arm entries or time spent in the open or closed arms in microglia-depleted mice versus
controls.
(G–I) Open field analysis showed no differences in the average total distance moved (G), the average velocity (H), or the relative amount of time spent at the edge
of the arena versus the center (I) with microglial elimination. Combined, these two tasks demonstrate that mice depleted of microglia do not show increased
anxiety.
(J and K) Microglia-depleted mice showed reduced escape latencies in the Barnes maze on days 2 and 3 of training compared with controls, as well as a lower
average escape latency for all training sessions. Mice administered scopolamine, a cholinergic antagonist that causes memory deficits, performed more poorly
than both controls and PLX3397-treated mice.
(L) Accelerating rotarod showed no changes in mice depleted of microglia.
(M) As expected, animals treated with scopolamine had reduced memory in a contextual fear conditioning paradigm, but there were no differences in time spent
freezing for controls or microglia-depleted mice.
Asterisk (*) indicates significance (p < 0.05) by unpaired Student’s t test, whereas same capital letter(s) above conditions (K and M) indicates no significant
difference. For comparisons in (J): *p < 0.05, control versus PLX3397; yp < 0.05, control versus control + scopolamine; cp < 0.05, PLX3397 versus control +
scopolamine. For (C)–(M), error bars indicate SEM.

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Figure 3. LPS Challenge in Microglia-Deficient Brains

(A) Schematic of the experimental design: 2-month-old C57BL/6 mice were fed either PLX3397 or control chow for 7 days. On day 7, mice were injected (i.p.) with
either LPS (0.25 mg/kg) or PBS (n = 4 per group). Mice were euthanized 6 hr postinjection.
(B–F) Immunostaining for IBA1 shows robust decreases in microglial numbers for both PLX3397 and PLX3397 + LPS (B–E), which was confirmed by microglial
counts (F).
(G–I) The relative mRNA expression of the microglia-devoid brain in response to LPS for a variety of inflammatory gene markers. Significance is dictated by the
following symbols: *, control versus control + LPS; y, control versus PLX3397; #, control + LPS versus PLX3397 + LPS; f, PLX3397 versus PLX3397 + LPS (for all
comparisons, p < 0.05). Full p values can be found in Table S1. Error bars indicate SEM.

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following day. In accordance with Figure 4, we found that the mi- Aif1, CSF1R, Cx3cr1, and Trem2, providing validation of the
croglia were strongly positive for IB4 at the 2- and 3-day recovery microarray data. We also looked at macrophage-specific genes
time points (Figure S7A); but curiously, repopulating cells were in the microarray data set, including Fn1, Slp1, Saa3, Prg4, Cfp,
negative for CD34 and c-Kit in these younger animals (data not Cd5L, GM11428, Crip1 Pf4, and Alox15 (Gautier et al., 2012), but
shown). In addition, we explored the microglial transcription fac- we found no significant changes (data not shown). In addition,
tor Pu.1, important for myeloid lineage cells to differentiate from the monocyte-specific marker CCR2 was not changed. Thus,
progenitors (Kueh et al., 2013), and we found that staining is these data support the notion that repopulating cells have an
markedly increased in microglia at the 2- and 3-day recovery expression profile of microglia and not of peripheral myeloid
time points (Figure S7B). As in the 18-month-old mice, repopu- cells and that they are derived from proliferation rather than
lating microglia are strongly nestin+ at the 2- and 3-day recovery infiltration.
time points (Figure 5J), with no expression seen in the other
groups. Indeed, western blot of whole-brain homogenates re- Fate Mapping with BrdU Reveals that Repopulating
vealed a dramatic increase (1,000%) in steady-state levels of Microglia Derive from Nonmicroglial Nestin+ Progenitor
nestin at day 3 of recovery (Figure 5K and quantified in Figure 5L). Cells
We also probed for steady-state levels of CSF1R, levels that are Having determined that repopulation occurs from cell prolifera-
reduced at recovery days 0, 1, and 2, but are restored by recov- tion rather than infiltration, we next stained tissue using Ki67 as
ery day 3. In addition, the monocyte marker CCR2 revealed a marker of cell proliferation in our early repopulation time course
no changes, suggesting that repopulation does not occur from in CX3CR1-GFP+/ mice. At the 2-day recovery time point,
peripheral monocytes. the brain contains many microglia-negative but dividing (GFP/
Ki67+) cells throughout, often with two adjacent nuclei, which
Microarray Analysis of Microglia-Depleted and are not seen in control, 0-, or 1-day recovery brains (Figures
Repopulating Brains Reveals Cell Proliferation 6A and 6B with quantification in Figures 6C–6E). By 3 days of re-
To gain insight into the origin(s) and properties of repopulating covery, most Ki67+ cells are now also GFP+, suggesting that
cells, we conducted microarray analysis of mRNA extracted these Ki67+ cells are potential microglia progenitors.
from whole brains of control, recovery day 1, and recovery day Proliferating cells can be labeled with the thymidine analog
3 groups. We selected recovery day 1 as a time point at which bromodeoxyuridine (BrdU), thus allowing us to mark the Ki67+/
microglia are eliminated and the drug is cleared from the system proliferating cells and track their fate, to determine whether
(Figures 5H and 5I). Significant changes in gene expression were they do indeed become microglia. To that end, we tagged prolif-
determined by Cyber T analysis and ranked in order of signifi- erating cells with BrdU (single-injection intraperitoneally [i.p.]) at
cance, with the top 30 gene expression changes shown in Fig- the 2-day recovery time point (Figures 7A–7H). Mice were sacri-
ures S8A–S8C. Reductions in known microglial-associated ficed 5 hr later, and their brains were analyzed to confirm that the
genes were most common in the 1-day recovery group nonmicroglial proliferating cells incorporated BrdU. Indeed, 5 hr
compared with controls, including recently identified micro- postinjection, many nonmicroglial cells had incorporated BrdU
glial-selective genes p2ry13, Siglech, and slc2a5 (Chiu et al., (Figures 7B and 7C, IBA1), whereas only 30% of all BrdU+ cells
2013; Gautier et al., 2012). In addition, reductions in CSF1R, expressed microglial markers (Figures 7B and 7C with quantifi-
ITGAM, and CX3CR1 were observed, consistent with real-time cation in Figures 7G and 7H). BrdU is not incorporated into
PCR data shown in Figure 2D. To build a gene expression profile appreciable numbers of cells in control animals (Figure 7A and
of the repopulating brain and cells, we compared both day 1 re- quantified in Figures 7G and 7H) or in those depleted of microglia
covery (microglia remain depleted) and control brains to day 3 and still on inhibitor (data not shown). Thus, these BrdU-incorpo-
recovery (repopulation has just begun to occur). In both compar- rated nonmicroglial cells correspond to the potential microglia
isons, changes in gene expression associated with cell prolifer- progenitor cells. Having determined that the potential progenitor
ation and cell cycle control were highly prevalent (Figures S8B cells incorporate BrdU, we then repeated the experiment, but
and S8C), including mki67 as well as Ube2c, Ccna2, Prr11, and sacrificed animals 24 hr after BrdU administration, rather than
Top2a. Thus, the expression profile of the repopulating brain 5 hr, to track their fate. After 24 hr, virtually all BrdU-incorporated
supports the notion that repopulation occurs as a result of prolif- cells were microglia (as determined by IBA1; Figures 7E and 7F
eration. In addition, we compared expression of significantly and quantified in Figures 7G and 7H), with very few BrdU+/IBA1
changed myeloid genes in recovery day 1 (microglia depleted) cells observed (Figure 7H). These results demonstrate that the
against recovery day 3 (repopulating) to determine the expres- nonmicroglial proliferating/BrdU-incorporating cells become mi-
sion pattern of the repopulating cells (Figure S8D). Myeloid croglia within 24 hr, and they confirm these cells as microglial
genes were increased, as expected. However, several micro- progenitors.
glial-specific genes have been recently identified that are not ex- Given these findings of potential microglial progenitor cells
pressed in macrophages, and we found that these markers were stimulated to proliferate and then differentiate into microglia,
also significantly increased, including F11r, Gpr165, Gpr84, we reasoned that these progenitors must express some of the
Olfml3, Serpine2, and Siglech (Chiu et al., 2013; Gautier et al., markers that the initial repopulating microglia express, such as
2012). The microglial-specific gene Tmem119 was not detected nestin. Indeed, costaining for Ki67 and nestin in 2-day repopulat-
via microarray. However, real-time PCR of mRNA extracted from ing brains revealed that these newly appeared proliferating cells
3-day repopulating brains revealed increases for both microglial- express nestin, with fine processes radiating out from a cell body
specific genes Tmem119 and Siglech (Figure S9C), as well as (Figures 7K–7N, with magnified images in Figures 7L and 7N),

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CSF1R Regulates Resident Microglia

Figure 4. Rapid Repopulation of the Microglia-Depleted Brain with New Cells that Differentiate into Microglia
(A) To explore microglia homeostasis in the adult brain, 18-month-old wild-type mice were treated with PLX3397 for 28 days to deplete microglia. The inhibitor
was withdrawn, and groups of mice were sacrificed immediately and 3, 7, 14, and 21 days later (n = 4–5 per group).
(B–G) IBA1 immunostaining revealed microglia throughout the untreated (control) brains (B) and elimination of microglia in mice treated with PLX3397 (C). New
IBA1+ cells appeared throughout the CNS at the 3-day recovery time point with very different morphologies to control resident microglia (D). Cell numbers
increased by the 7-day recovery time point, and the morphology of the cells begin to resemble a more ramified state (E). By 14 days of recovery (F) and 21 days of
recovery (G), the IBA1+ cells resemble ramified microglia and have fully repopulated the entire CNS.
(H) Quantification of the number of IBA1+ cells in the hippocampal field.
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lending further credence to the idea that these proliferating CSF1R inhibitor. We treated 2-month-old wild-type mice with
nestin-expressing cells become the repopulated microglia. As 290 mg/kg chow PLX3397 for 14 days to deplete microglia. At
controls, we show that these cells do not express either GFAP this point, this dose was replaced with chow containing 0, 16,
(Figure 7I) or MAP2 (Figure 7J). Finally, we confirmed that the 32, 75, 150, or 290 mg/kg PLX3397 (n = 3 per group; Fig-
BrdU-incorporated nonmicroglial cells found 5 hr after BrdU ure S10A). Three days later, mice were sacrificed, and half brains
administration (Figure 7B) also expressed nestin (Figures 7O taken for PK and mRNA analyses, and repopulation was as-
and 7P). These BrdU-incorporated cells lack the extensive sessed. Plasma and brain PLX3397 levels were measured for
nestin+ processes seen in Figures 7K–7N, suggesting the cells each of the groups (Figure S10B), showing that even in the
are between the stages of DNA synthesis and mitosis/cytoki- 16 mg/kg group, the plasma PLX3397 concentration was greater
nesis, unlike the Ki67+/nestin+ cells that had completed cell divi- than that found in the brain in the 290 mg/kg group (i.e., the con-
sion (Figures 7K–7N). centration needed for microglial elimination). Brain PLX3397
concentrations were very low, with 0.5–0.1 mM measured in
Surviving Microglia Cannot Solely Account for the 75 mg/kg and lower dose groups. Repopulation (as assessed
Repopulation by CSF1R and the microglial-specific markers Siglech and
Although not all microglia are eliminated with CSF1R inhibition, Tmem119) occurred in the 0, 16, 32, and 75 mg/kg groups, but
the numbers and rates of repopulation cannot support repopula- not in the 150 or 290 mg/kg groups, despite peripheral
tion solely from these few surviving cells. Our data from Figure 5 PLX3397 concentrations being much higher than that found in
shows that repopulation begins 48 hr after drug withdrawal and the brains of the 290 mg/kg group (Figure S10B). From these
that there is a substantial increase in repopulating cells by 72 hr. findings, we conclude that repopulation cannot occur from
In fact, the average number of microglia per section quadruples peripheral cells, but must occur from within the CNS.
from approximately 950 cells at 48 hr to roughly 3,700 by 72 hr. Second, although we have already determined that brain-
Proliferation from the surviving 950 cells alone would be unlikely wide CCR2 levels are not altered with repopulation (Figures
to produce 3,700 cells within 24 hr. In this experiment, only 5K and 5L), we further set out to determine whether infiltration

70% of microglia were eliminated; however, we see faster of monocytes could represent a source of repopulating cells.
rates of repopulation with >95% elimination (as shown in Figures To that end, we treated CCR2-RFP+/ mice with PLX3397 for
S9A–S9C). Here, repopulation occurred at a tremendous rate, 7 days, and withdrew drug for 5 days to stimulate repopulation
with
14,000 cells/slice present at 72 hr, despite only
600 (n = 4 per group). Monocytes specifically express red fluores-
cells/slice being present in the depleted brains. If the surviving cent protein (RFP) in these mice (Saederup et al., 2010), which
microglia were to be the only source of repopulation each surviv- we confirmed in the blood of these animals (sample shown
ing cell would have to proliferate every 5–6 hr (Figure S9D), with is obtained from a CCR2-RFP+/ 3 CX3CR1-GFP+/ mouse;
no reductions in cell size and with constant migration away from Figure S10D). However, we were unable to find any RFP-ex-
daughter cells, to account for complete repopulation. The pres- pressing cells within the CNS of control, microglia-depleted,
ence of a progenitor cell in the brain could, however, account for or repopulated animals (Figure S10E), despite robust microglial
the rapid repopulation observed. repopulation, confirming that the repopulating cells do not
derive from monocytes. We were able to find a handful of
Microglia Repopulation Does Not Occur from Peripheral RFP-positive cells in the 12 brains examined, and these were
Cells always found within blood vessels, such as that shown in Fig-
Our data show the presence of a microglial progenitor cell in the ure S10F. To further rule out the possibility of monocytes
adult CNS that can proliferate and then differentiate into the re- contributing to repopulation, we also performed immunostains
populating microglia. However, we also wished to rule out fully for CCR2 in repopulating brains at 0, 1, 2, and 3 days of recovery
the possibility that peripheral cells were able to cross the BBB in the event that monocytes were able to enter the CNS and
and also contribute to repopulation. To that end, we performed then rapidly differentiate into microglia (Figure S10G). Although
two experiments. First, pharmacokinetic (PK) data revealed neurons stained positive for CCR2, as has been reported previ-
that only
5% of plasma PLX3397 enters the CNS, rendering pe- ously (Banisadr et al., 2005; van der Meer et al., 2000), none of
ripheral concentrations
20 times higher. We set out to establish the repopulating cells were CCR2 positive. We also performed
whether repopulation could still occur with concentrations of stains for T cells (anti-CD3) and dendritic cells (anti-CD11c),
PLX3397 that block CSF1 receptors in the periphery, but not in but we found no evidence for either cell type in the repopulating
the CNS, as repopulation is dependent upon withdrawal of the brains (data not shown).

(I) Analysis of cell body size shows that IBA1+ cells at the 3-day recovery time point are much larger than resident microglia. The size of these cells then normalizes
over the following recovery time points.
(J) Representative IBA1+ cells from each of the groups, showing the changes in cell morphology and size that occur during repopulation. Con, control.
(K) Cells at the 3-day recovery time point express a number of unique markers, including CD45, nestin, Ki67, CD34, and c-Kit. They also show high immuno-
reactivity to the IB4 lectin. Notably, IBA1+ cells in control brains and surviving IBA1+ cells in the 0-day recovery group are negative for all these markers. Likewise
cells at the 7-, 14-, and 21-day recovery time points are negative for all these markers, highlighting that the initial repopulating cells have a unique morphology and
phenotype. Same capital letter(s) above conditions (H and I) indicates no significant difference (p > 0.05) via one-way ANOVA with post hoc Newman-Keuls
multiple comparison test. Error bars indicate SEM. Scale bar represents 20 mm.

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Figure 5. Microglial Repopulation Occurs between 48 and 72 hr after CSF1R Inhibitor Withdrawal
(A) To investigate early repopulation events, 2-month-old CX3CR1-GFP+/ mice were treated with PLX3397 for 7 days. The inhibitor was then withdrawn, and
groups of mice were sacrificed 1, 2, and 3 days later (n = 4 per group).
(B–F) Representative sections shown from the hippocampus of each of these groups (B–F), in which microglia express GFP.
(G) Flow cytometry of GFP+ cells from the liver and spleen of these animals. Con, control.
(H) Quantification of whole-brain sections for microglial numbers shows a reduction of
70% with 7 days of PLX3397 treatment. Microglial numbers continue to
decline for 2 days after inhibitor withdrawal but rapidly recover between days 2 and 3, highlighting a crucial time period in repopulation.
(I) Brain levels of PLX3397 show rapid clearance of the drug from the CNS.
(J) GFP+ cells strongly express nestin at the 2- and 3-day recovery time points. The 633 z stacks obtained by confocal microscopy and maximal projections are
shown. Scale bar represents 20 mm. Separate channels and merge are shown for each of the time points in the panels below.
(K) Western blots of whole-brain homogenates show significant increases in nestin and CSF1R with repopulation, but no changes in CCR2.
(L) Quantification of (K) normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. Same capital letter(s) above conditions (G, H, I,
and L) indicates no significance (p > 0.05) via one-way ANOVA with post hoc Newman-Keuls multiple comparison test. Error bars indicate SEM.

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Figure 6. Microglial Repopulation Is Preceded by Proliferation of a Nonmicroglial Cell Type throughout the CNS
(A) Using the same experimental groups as shown in Figure 5, repopulating brains were stained for the cell proliferation marker Ki67. Few Ki67+ cells were seen in
control, 0-, or 1-day recovery groups. A fraction of the GFP+ cells expresses Ki67 at the 2-day recovery time point, and a majority of the GFP+ cells express Ki67 at
the 3-day recovery time point. Of note, Ki67+/GFP cells appeared throughout the CNS at 2 days of recovery, usually with two nuclei adjacent to one another,
suggesting a recent cell division (highlighted by arrows). Far fewer Ki67+/GFP cells were seen at the 3-day recovery time point. The 633 z stacks obtained by
confocal microscopy and maximal projections are shown. Scale bar represents 20 mm.
(B–E) Images of Ki67+ and GFP+ cells from the cortex are shown to illustrate the number of proliferating, but GFP, cells that appear at the 2-day recovery time
point, as quantified in (C) for the hippocampal region, (D) for the piriform cortex, and (E) for the cortex. Error bars indicate SEM.

Fate Mapping Reveals that Microglia Do Not Next, to track the fate of microglia during CSF1R inhibition,
Dedifferentiate into Alternative Cell Types with CSF1R we tagged repopulating microglia with BrdU (as depicted in Fig-
Inhibition and that Repopulated Cells Are Fully ure 8E), via treatment with PLX3397 for 7 days, followed by
Dependent upon CSF1R Signaling 7 days of recovery, during which BrdU was administered daily.
An additional hypothetical explanation for repopulation is that mi- As expected, repopulating microglia incorporated BrdU (Fig-
croglia are not actually dying with CSF1R inhibition, but dediffer- ure 8G and quantified in Figure 8H). Once repopulating microglia
entiating into an alternative cell type that then redifferentiates were tagged with BrdU, we treated mice with PLX3397 for 7 days
back into microglia upon removal of the inhibitor. To address and then explored the CNS for BrdU-labeled cells. This second
this, we have first determined that repopulating cells are also fully treatment eliminated >80% of microglia, including BrdU-incor-
dependent upon CSF1R signaling, as repopulating microglia porated cells (Figure 8H and quantified in Figure 8I), thus con-
strongly express the CSF1R (Figure 8A). We treated 2-month-old firming that microglia are being eliminated from the CNS, rather
wild-type mice with PLX3397 for 21 days to deplete microglia than differentiating into a nonmicroglial cell type.
(‘‘on,’’ n = 4). An additional group was then allowed to repopulate
for 14 days (‘‘on-off,’’ n = 5), and a final group was then treated DISCUSSION
again with PLX3397 for 7 days (‘‘on-off-on,’’ n = 5). As shown
in Figures 8C and 8D, 21 days of PLX3397 treatment elimi- Dependence of Microglia on CSF1R Signaling in the
nated >99% of all microglia from the CNS, and 14 days of recovery Adult Brain
restored numbers of microglia back to resident microglial levels. Studies have highlighted the importance of the CSF1R to the
Crucially, treatment of repopulated microglia with PLX3397 elimi- development of microglia, with mice lacking the CSF1R being
nated >95% of microglia, showing that these cells are also depen- born devoid of microglia (Erblich et al., 2011; Ginhoux et al.,
dent upon CSF1R signaling for their survival. 2010). Unfortunately, these mice have developmental defects

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Figure 7. Fate Mapping Reveals a Nestin-Expressing Microglia Progenitor Cell that Becomes the Repopulating Microglia
(A–F) To determine whether the nonmicroglial Ki67+ proliferating cells were becoming microglia, 2-month-old wild-type mice were treated with PLX3397 or
vehicle to deplete microglia. The inhibitor was withdrawn and BrdU was administered 2 days later to label proliferating cells (n = 3–4 per group). Mice were
sacrificed 5 or 24 hr later (n = 4 per group). Representative images of the cortical region are shown for controls (A) and the 2-day recovery group (B) for IBA1 and
anti-BrdU at 5 hr after BrdU administration. The 633 maximal projection z stacks are shown for the 2-day recovery group + 5 hr BrdU (C), revealing that the
majority of BrdU-incorporated cells are not microglia.
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and usually die by adulthood, by which time some microglia are ditioning (Parkhurst et al., 2013). We do not find these deficits
observed (Erblich et al., 2011). Mice lacking either of the two in our mice, despite lacking microglia for 2 months. These differ-
CSF1R ligands, CSF1 (Wegiel et al., 1998) or IL-34 (Wang ences may be accounted for by the method of microglia elimina-
et al., 2012), also have reduced densities of microglia throughout tion and the acute response of the CNS and surviving microglia
the CNS. Thus, the CSF1R is heavily implicated in the develop- to massive microglial death via diphtheria toxin, whereas the
ment of microglia. However, it is unknown what role the response of the immune system in our paradigm may be more
CSF1R plays in microglia homeostasis and viability in the adult suppressed due to CSF1R inhibition. This study also reported
brain. Our results show that microglia in the adult brain are fully slower repopulation than we see, a finding that may be ac-
dependent upon CSF1R signaling for their survival and that we counted for by the different methods of microglial elimination
can eliminate virtually all microglia from the CNS for extended and the timescales involved that likely activate different signaling
periods through the administration of CSF1R inhibitors. Thus, pathways within the brain. Because administration of CSF1R
CSF1R signaling appears to act as a requisite growth factor inhibitors is potentially translatable to humans for modulation
receptor for microglia, and its blockade drives microglia to their of microglia numbers, a lack of negative effects on cognition is
death. Growth factor withdrawal is known to induce apoptosis in an important observation.
many other cell types, including HSCs (Cornelis et al., 2005) and
macrophages (Chin et al., 1999). Consequently, we can take Rapid Repopulation of the Microglia-Depleted Brain
advantage of this dependency to manipulate microglial levels Having shown that we could eliminate >99% of all microglia from
in the adult brain through administration of CSF1R inhibitors, the adult brain, we asked the question of whether new cells could
allowing studies into microglia function that have not been replace the lost microglia and repopulate the CNS. Microglia
possible before. Moreover, the CSF1R provides an ultimate colonize the CNS during development, before E9 (Ginhoux
drug target for neuroinflammation, in that we can now eliminate et al., 2010). Once the CNS has formed, these microglia are
microglia rather than just suppress aspects of their activity. long lived and have the capacity to divide and self-renew in an
autonomous cell population, but the dynamics and regulation
Role of Microglia in the Healthy Adult Brain of resident microglia numbers are not fully understood. In the pe-
We set out to determine whether microglia play an important role riphery, macrophage populations are thought to be replenished
in cognition and behavior in healthy adult mice. Chronic deple- by circulating monocytes derived from multipotent HSCs found
tion of >99% of all microglia for 3 or 8 weeks in adult mice in the bone marrow, although this view has been challenged
resulted in no deficits in any behavioral cognitive task adminis- (Hashimoto et al., 2013; Sieweke and Allen, 2013). In contrast,
tered, including Barnes maze (a test of spatial learning and the brain is separated from circulation by the BBB, and experi-
memory). In fact, mice depleted of microglia for 8 weeks learned ments have shown that there is little infiltration of peripheral
to escape the Barnes maze significantly faster than control ani- HSCs/monocytes/macrophages into the CNS to help maintain
mals. Finally, no motor deficits were observed in treated mice or replenish microglia under normal, nonirradiated conditions
as determined by accelerating rotarod testing and open field. (Ajami et al., 2011; Greter and Merad, 2013; Mildner et al.,
Therefore, these results show that microglia are not overtly 2007). Thus, we set out to explore whether repopulation could
important in these cognitive tasks, a surprising finding given occur in the adult brain, as well as the consequences of with-
the numerous physical interactions between neurons and micro- drawing CSF1R antagonists once microglia were depleted.
glia, as well as the secreted factors that are released from micro- We initially predicted that the brain would remain absent of mi-
glia. Although our results show that microglia are not overtly croglia for some time, given our current knowledge about the or-
necessary for these behaviors, previous studies have shown igins and proliferative properties of microglia; but remarkably, we
that microglia are crucial during development, with CX3CR1- found that the CNS can fully repopulate with new microglia within
GFP mice showing a transient reduction in microglia during just 7 days. Furthermore, the returning number of microglia is
development (Zhan et al., 2014), leading to long-term deficits identical to that in untreated mice, showing astonishing and pre-
in behavior (Zhan et al., 2014), memory, and long-term potentia- cise regulation of the microglial population within a very short
tion (Rogers et al., 2011). Of note, a study found that short-term period of time. Repopulating microglia derive from proliferation,
depletion of microglia, via genetic expression of diphtheria toxin as shown with Ki67 expression and incorporation of BrdU, rather
receptor and subsequent diphtheria toxin administration for than infiltration of peripheral cells into the CNS. Initially, repopu-

7 days, led to deficits in learning, including contextual fear con- lating microglia show very different morphologies and

(D–F) Representative images of the cortical region are shown for controls (D) and the 2-day recovery group (E) for IBA1 and anti-BrdU at 24 hr after BrdU
administration. The 633 maximal projection z stacks are shown for the 2-day recovery group at 24 hr after BrdU administration (F), revealing that the majority of
BrdU-incorporated cells are now microglia.
(G) Quantification of (A)–(F) shows that only 30% of BrdU-incorporated cells are microglia after 5 hr but that 96% become microglia after 24 hr.
(H) Quantification of the total amount of BrdU+/nonmicroglial cells per field of view shows that most of these cells have differentiated into microglia within 24 hr.
(I–N) CX3CR1-GFP+/ mice were treated for 7 days with PLX3397, and 2 days after inhibitor withdrawal, Ki67+/GFP/nestin+ cells are induced throughout the
CNS (highlighted with white arrowheads in K and M; two different fields of view are shown, with a zoom image of the nestin-expressing cells shown in L and N). In
addition, costains with Ki67 show that the repopulating cells do not express GFAP (I) or MAP2 (J).
(O and P) BrdU-incorporated cells in the 2-day recovery brains (sections obtained from B), also express nestin (microglia only shown in the 633 merge). Same
capital letter above conditions (G and H) indicates no significant difference (p > 0.05) via one-way ANOVA with post hoc Newman-Keuls multiple comparison test.
Error bars indicate SEM. Scale bars represent 20 mm.

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Figure 8. Microglia Are Eliminated with CSF1R Inhibition and Not Dedifferentiated
(A) Repopulating microglia express CSF1R; the 3-day recovery time point is shown.
(B) Schematic of the experimental design: 2-month-old mice were treated for 21 days with PLX3397 to deplete microglia (‘‘on’’). PLX3397 was then removed
from the diet in a second group, and repopulation was allowed for 14 days (‘‘on-off’’). A final group was then treated for a second time with PLX3397 (‘‘on-off-on,’’
n = 4–5 per group) to determine whether repopulated microglia were also eliminated with CSF1R inhibition.
(C) Representative sections from the hippocampal field for IBA1 and NeuN from each of the four groups.
(D) Quantification of IBA1 cells in matching full brain sections shows that repopulating microglia are also fully dependent upon CSF1R signaling.

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expression patterns to resident microglia, such as immunoreac- 2469, or in Dulbecco’s modified Eagle’s medium containing recombinant
tivity for nestin, but they rapidly differentiate into ramified micro- CSF1. Cell numbers were quantified using ATPlite luminescence assay
(Pierce).
glia over a 7- to 14-day period. Crucially, we find that the repo-
pulating brain induces the proliferation of nestin-expressing
Animal Treatments
cells throughout the CNS that appear to become the repopulat-
All rodent experiments were performed in accordance with animal protocols
ing microglia. This finding helps to explain why these initial mi- approved by the Institutional Animal Care and Use Committee at the University
croglia strongly express nestin and how microglia numbers can of California, Irvine (UCI).
be restored in a very short time, given that there are so few sur- LPS Treatment. LPS was administered i.p.
viving microglia. It should be noted that the microglia themselves Evans Blue Dye Administration. To assess BBB integrity, mice were injected
also proliferate, as evidenced by the expression of Ki67 and ob- with Evans blue dye (i.p.) and sacrificed 6 hr later.
BrdU Labeling. BrdU was administered i.p., and mice were sacrificed 5 or
servations of cytokinesis (i.e., Figure 7A, 3-day recovery),
24 hr later.
revealing that repopulation may occur partly from nestin-ex-
pressing proliferating progenitors and partly from the cells that Immunoblotting
were nestin-expressing progenitors and have just become mi- Brain homogenates and immunoblotting were prepared as described previ-
croglia, or from proliferation of surviving cells. ously (Green et al., 2011). Quantitative densitometric analyses were performed
Of note, microglia are of a myeloid lineage rather than the on digitized images of immunoblots with ImageJ (National Institutes of Health).
neuroectodermal lineage that nestin expression would suggest,
leading us to question why the repopulating microglia express Immunostaining
nestin. In explaining this, it is possible to generate microglia Light-level immunohistochemistry was performed using an avidin-biotin
immunoperoxidase technique and was visualized with diaminobenzidine, as
from embryonic stem cells (ESCs) (Beutner et al., 2010); ESCs
described previously (Oddo et al., 2003).
are differentiated to a neuronal, nestin+ lineage and then the
neuronal growth factors are removed, resulting in microglia. Confocal Microscopy
Hence, ESCs need to pass through a nestin+ stage on their Fluorescent immunolabeling followed a standard indirect technique (primary
way to becoming microglia, in line with the cells that we describe antibody followed by fluorescent secondary antibody) as described in Neely
in this study, providing clear evidence that repopulating micro- et al. (2011).
glia strongly express nestin. In addition, several previous studies
have shown subsets of microglia to be able to express nestin un- Flow Cytometry
Using a FACSAria II cell sorter (BD Biosciences), the viable cell population of
der certain conditions, such as in culture (Yokoyama et al., 2004),
interest from an unstained control was gated according to size and granularity
after traumatic brain injury (Sahin Kaya et al., 1999), or after optic
based on forward and side scatter properties. Using these parameters, the
nerve injury (Wohl et al., 2011) where proliferating, BrdU-incor- percentage of GFP+ cells, as well as GFP+/PI+ cells, were quantified for the
porating microglia initially also express nestin. A study has also CX3CR1-GFP+/ mice using FACSDiva software.
highlighted that the CSF1R negatively regulates the expansion
of nestin+ progenitors in the developing brain, an intriguing par- Brain Volume
allel with our own findings in the adult brain and the relationship Brain volumes were obtained via Cavalieri measurements of every sixth sec-
between CSF1R signaling and nestin-expressing progenitors tion per animal.
and microglia (Nandi et al., 2012). Thus, we show that the adult
mRNA Extraction and Real-Time PCR
brain has a remarkable capacity to regulate and renew its micro-
Total mRNA was extracted from frozen half brains, cDNA was synthesized,
glia population, through microglial progenitor cells, and that the
and real-time-PCR was performed with commercially available kits.
CSF1R plays a crucial role in microglial tissue homeostasis.
Behavioral Testing
EXPERIMENTAL PROCEDURES Mouse cognition and behavior were evaluated using the elevated plus
maze, open field, Barnes maze, accelerating rotarod, and contextual fear
Refer to Supplemental Information for additional details. conditioning.

Compounds Microarray
PLX3397 and PLX647 were provided by Plexxikon and formulated in AIN-76A RNA was extracted and purified (as described above) and then processed
standard chow by Research Diets at the doses indicated in the text. PLX3397 at the UCI DNA and Protein Microarray Facility using commercially available
was provided at 290 mg/kg, unless otherwise specified. microarray cards.

EOC 20 Growth Assays Statistics

EOC 20 microglial cells were grown either with the addition of LADMAC Appropriate statistical analyses were carried out to determine significance
cell conditioned media, as a source of CSF1 as described in ATCC CRL- between groups (see Supplemental Information for specific tests used).

(E) Schematic of the experimental design: 2-month-old wild-type mice were treated with PLX3397 for 7 days to deplete microglia. PLX3397 was removed to allow
microglia to repopulate and BrdU was administered daily to tag these new cells. Seven days later, PLX3397 was readministered to BrdU-tagged microglia
containing mice.
(F–H) Representative stainings from the hippocampal region for BrdU and IBA1 show that compared with controls (F), repopulating microglia incorporate BrdU (G)
and that PLX3397 treatment eliminates both IBA1 cells and BrdU-incorporated cells (H).
(I) Quantification of (F)–(H). Same capital letter above conditions (D) indicates no significant differences (p > 0.05) via one-way ANOVA. Error bars indicate SEM.

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CSF1R Regulates Resident Microglia

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M.R.P.E., A.R.N., M.A.K., N.N.D., E.E.S., R.A.R., M.K., B.M., H.N., B.L.W.,
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ACKNOWLEDGMENTS
J. Immunol. Methods 160, 81–88.
Research reported in this publication was supported by the National Institute DeNardo, D.G., Brennan, D.J., Rexhepaj, E., Ruffell, B., Shiao, S.L., Madden,
of Neurological Disorders and Stroke of the National Institutes of Health under S.F., Gallagher, W.M., Wadhwani, N., Keil, S.D., Junaid, S.A., et al. (2011).
award number 1R01NS083801 to K.N.G. and F31NS086409 to R.A.R. Support Leukocyte complexity predicts breast cancer survival and functionally regu-
was further provided through NIH award UL1 TR000153 as well as the White- lates response to chemotherapy. Cancer Discov. 1, 54–67.
hall foundation to K.N.G., the American Federation of Aging Research to Erblich, B., Zhu, L., Etgen, A.M., Dobrenis, K., and Pollard, J.W. (2011).
K.N.G., and the Alzheimer’s Association to K.N.G. M.R.P.E. is supported by Absence of colony stimulation factor-1 receptor results in loss of microglia,
NIH training fellowship AG00538. The content is solely the responsibility of disrupted brain development and olfactory deficits. PLoS ONE 6, e26317.
the authors and does not necessarily represent the official views of the Na-
tional Institutes of Health. We thank Vanessa Scarfone, the Sue and Bill Gross Gautier, E.L., Shay, T., Miller, J., Greter, M., Jakubzick, C., Ivanov, S., Helft, J.,
Stem Cell Research Center Core Facility, and the CIRM Shared Research Lab Chow, A., Elpek, K.G., Gordonov, S., et al.; Immunological Genome
for assistance with flow cytometry. B.M., H.N., and B.L.W. are employees of Consortium (2012). Gene-expression profiles and transcriptional regulatory
Plexxikon. pathways that underlie the identity and diversity of mouse tissue macro-
phages. Nat. Immunol. 13, 1118–1128.
Accepted: February 14, 2014 Ginhoux, F., Greter, M., Leboeuf, M., Nandi, S., See, P., Gokhan, S., Mehler,
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