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Article

Microglial Phagocytosis of

Newborn Cells Is Induced by Endocannabinoids and Sculpts Sex Differences in Juvenile Rat Social Play

Highlights

d Microglia are more phagocytic in the male amygdala during

neonatal development

d Androgen-induced endocannabinoids increase

phagocytosis in males

d Microglia engulf viable newborn astrocytes in a complement-

dependent manner

d Developmental phagocytosis produces a sex difference in

juvenile social play

VanRyzin et al., 2019, Neuron 102, 435–449 April 17, 2019 ª 2019 Elsevier Inc. https://doi.org/10.1016/j.neuron.2019.02.006

Authors

Jonathan W. VanRyzin,

Ashley E. Marquardt,

Kathryn J. Argue, ...,

Sheryl E. Arambula, Matthew N. Hill,

Margaret M. McCarthy

Correspondence mmccarthy@som.umaryland.edu

In Brief

VanRyzin et al. demonstrate that

microglia in the developing amygdala

engulf and kill otherwise viable newborn

astrocytes, establishing sex differences

in social circuits. This process, which

depends on gonadal hormones and

endocannabinoid signaling, promotes

juvenile play by males.

mailto:mmccarthy@som.umaryland.�edu

https://doi.org/10.1016/j.neuron.2019.02.006

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Neuron

Article

Microglial Phagocytosis of Newborn Cells Is Induced by Endocannabinoids and Sculpts Sex Differences in Juvenile Rat Social Play Jonathan W. VanRyzin,1,2 Ashley E. Marquardt,1 Kathryn J. Argue,2 Haley A. Vecchiarelli,3 Sydney E. Ashton,1

Sheryl E. Arambula,2 Matthew N. Hill,3,4 and Margaret M. McCarthy1,2,5,* 1Program in Neuroscience, University of Maryland School of Medicine, Baltimore, MD 21201, USA 2Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD 21201, USA 3Hotchkiss Brain Institute and Mathison Center for Mental Health Research and Education, Cumming School of Medicine,

University of Calgary, Calgary, AB T2N4N1, Canada 4Department of Cell Biology and Anatomy & Psychiatry, University of Calgary, Calgary, AB T2N4N1, Canada 5Lead Contact *Correspondence: mmccarthy@som.umaryland.edu

https://doi.org/10.1016/j.neuron.2019.02.006

SUMMARY

Brain sex differences are established developmen- tally and generate enduring changes in circuitry and behavior. Steroid-mediated masculinization of the rat amygdala during perinatal development pro- duces higher levels of juvenile rough-and-tumble play by males. This sex difference in social play is highly conserved across mammals, yet the mecha- nisms by which it is established are unknown. Here, we report that androgen-induced increases in endocannabinoid tone promote microglia phago- cytosis during a critical period of amygdala develop- ment. Phagocytic microglia engulf more viable newborn cells in males; in females, less phagocy- tosis allows more astrocytes to survive to the juve- nile age. Blocking complement-dependent phago- cytosis in males increases astrocyte survival and prevents masculinization of play. Moreover, increased astrocyte density in the juvenile amygdala reduces neuronal excitation during play. These find- ings highlight novel mechanisms of brain develop- ment whereby endocannabinoids induce microglia phagocytosis to regulate newborn astrocyte number and shape the sexual differentiation of social cir- cuitry and behavior.

INTRODUCTION

Brain sex differences are established by steroid hormone expo-

sure during the perinatal period. The fetal testis produces andro-

gens as early as the second trimester in humans and the latter

third of gestation in rodents, resulting in higher levels of circu-

lating testosterone in males. Testosterone readily gains access

to the brain, where it either acts directly on androgen receptors

or is locally converted into estradiol and acts via estrogen recep-

tors. Activation of steroid hormone receptors initiates the

process of sexual differentiation, whereby region-specific mech-

anisms masculinize the brain and program lasting behavioral

differences between males and females (reviewed in Zuloaga

et al., 2008; McCarthy et al., 2017).

It has long been known that sexual differentiation of the

amygdala mediates a male bias toward greater intensity and

frequency of juvenile rough-and-tumble play (Meaney et al.,

1981; Meaney and McEwen, 1986). We previously discovered

that the developing amygdala of males has fewer newborn cells

than that of females. The sex difference in newborn cell number

and juvenile play are both driven by a parallel and inverse sex

difference in endocannabinoid (eCB) tone, being higher in the

male amygdala (Krebs-Kraft et al., 2010). The eCB system is

active early in brain development and comprises two principal

ligands, 2-arachidonoylglycerol (2-AG) and anandamide (AEA),

which act on the type-1 and type-2 cannabinoid receptors

(CB1R and CB2R; reviewed in Maccarrone et al., 2014). We

previously demonstrated that mimicking the ‘‘male-like’’ eCB

tone in female rat pups via administration of cannabinoid (CB)

receptor agonists masculinizes both newborn cell number and

later juvenile play (Krebs-Kraft et al., 2010; Argue et al., 2017).

However, the mechanism by which eCBs regulate cell number

and impact play circuitry and behavior was unknown. To this

end, we investigated microglia as a putative link between devel-

opmental sex differences in eCB tone and later life social

behavior.

Microglia are the brain’s innate immune cells and are increas-

ingly recognized as important modulators of brain develop-

ment. They both promote and prune synaptic connectivity

(Paolicelli et al., 2011; Schafer et al., 2012; Ji et al., 2013;

Lenz et al., 2013; Squarzoni et al., 2014; Miyamoto et al.,

2016) and regulate progenitor cell populations by providing

trophic support or inducing cell death (Marı́n-Teva et al.,

2004; Sierra et al., 2010; Cunningham et al., 2013; Ueno

et al., 2013; Shigemoto-Mogami et al., 2014). Microglia express

both CB1Rs and CB2Rs (reviewed in Stella, 2009), making

them likely candidates to influence eCB-mediated sexual

differentiation.

Neuron 102, 435–449, April 17, 2019 ª 2019 Elsevier Inc. 435

mailto:mmccarthy@som.umaryland.edu

https://doi.org/10.1016/j.neuron.2019.02.006

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Here, we test the hypothesis that microglia program sex differ-

ences in the developing rat amygdala by phagocytosing

newborn cells. We find that testosterone-induced elevations in

eCB tone drive microglia to engulf viable newborn astrocytes

in a complement-dependent manner. By developmentally elimi-

nating astrocyte precursors, microglia alter neural excitation

selectively in one node of the play circuit. Together, these find-

ings reveal a novel mechanism for establishing developmental

sex differences that involves a convergence of the eCB system

and the brain’s immune system to control cell number and

thereby regulate social behavior.

RESULTS

More Microglia Are Phagocytic in the Developing Male Amygdala We began by characterizing the microglia population in the

developing amygdala over the first postnatal week (Figure 1A),

a time that encompasses the middle and end of the critical

period for sexual differentiation. Using an antibody for ionized

calcium binding adaptor molecule 1 (Iba1) to visualize microglia

via immunohistochemistry, we found more microglia engaged in

phagocytosis (defined by the presence of a phagocytic cup; Fig-

ures 1B and 1C, white arrowheads) in the amygdala of males

from postnatal day 0 (P0) (birth) to P4 than in females (Figure 1D).

Phagocytic microglia made up a substantial portion of the total

microglia population in both males and females (25.9% ± 1.4%

in males and 16.5% ± 1.1% in females on P0; Figures 1E and

1G) and gradually waned as the total microglia number increased

over the first postnatal week (Figure 1F).

To determine whether the observed sex difference in phago-

cytic microglia was a consequence of the cell’s activational

state, we analyzed microglial morphology and CD68 immunore-

activity by confocal microscopy (Figure S1A). We scored micro-

glia along a scale ranging from 0 (highly ramified, low CD68) to 5

(amoeboid, high CD68) on P4, as previously described (Schafer

et al., 2012). The distribution of microglia activation did not differ

between the sexes (Figure S1B), nor were there sex differences

in activation state of phagocytic microglia (Figure S1C). Microglia

at the midpoint of the 0–5 scale were the most frequently phago-

cytic, but some microglia along the entire scale possessed

phagocytic cups.

We next sought to determine whether the sex difference in

phagocytic microglia number could be attributed to intrinsic dif-

ferences in phagocytic activity between males and females. We

analyzed the number of phagocytic cups per microglia in vivo

and found no difference between males and females at P4 (Fig-

ure 1H). Moreover, most phagocytic microglia possessed just

one phagocytic cup (83.6% ± 3.58% in females; 88.5% ±

2.4% in males). Because sex differences can also arise from

the sex chromosomes (McCarthy and Arnold, 2011; e.g., XX

versus XY), we next assessed microglia phagocytic capacity

in vitro to determine whether our findings in vivo could be

attributed to intrinsic differences in genetic composition. We

harvested microglia from sex-specific mixed glia cultures,

incubated them with fluorescent beads, and then analyzed

microglial phagocytosis by flow cytometry (Figure S1D). In the

absence of local cues present in the in vivo environment, micro-

436 Neuron 102, 435–449, April 17, 2019

glia derived from male and female cultures engulfed similar

numbers of fluorescent beads (Figure S1E), and the percentage

of phagocytic microglia did not differ between the sexes

(72.47% ± 3.42% in females; 79.30% ± 2.87% in males; Fig-

ure S1F). Together, these results suggest that microglia in the

developing amygdala are highly phagocytic in vivo and that local

cues in the newborn male amygdala make them more phago-

cytic than in females.

Phagocytic Microglia Engulf Newborn Cells in the Developing Amygdala To identify the targets of microglial engulfment, we measured

the diameter of phagocytic cups in the P4 amygdala using

confocal microscopy (Figure 1I). We found no difference in

average cup diameter between males and females (Figure 1J),

and the large size (8.75 ± 0.31 mm for females; 8.55 ± 0.26 mm

for males) suggested microglia were engulfing objects consis-

tent in size with cell bodies rather than synapses or small cellular

debris. To confirm that microglia were phagocytosing cell

bodies, we analyzed the contents of phagocytic cups at P4 by

co-immunolabeling with Iba1 and DNA binding dyes (Figures

2A–2C). Three-dimensional visualization of phagocytic cups

showed clear engulfment of DAPI (Figure 2A), and as seen in

Figure 2C, the DAPI signal was contained within, not apposed

to, phagocytic cups. The majority of phagocytic cups contained

nuclear material in both males (83.54% ± 2.29%) and females

(77.94% ± 5.42%; Figure 2D). These observations led us to hy-

pothesize that microglia engulf newborn cells. We tested this

possibility by immunolabeling for proliferating cell nuclear anti-

gen (PCNA), a marker for recently divided cells, and again

analyzed the contents of phagocytic cups in males and females

(Figures 2E–2G). Nearly 60% of phagocytic cups co-labeled

with PCNA in both sexes (62.55% ± 4.11% in males;

59.25% ± 4.09% in females; Figure 2H), indicating microglia

predominantly engulf recently born cells in the developing

amygdala. To confirm that the majority of cells being engulfed

were viable as opposed to undergoing apoptosis, we visualized

cleaved caspase-3 (cCasp3) and again analyzed phagocytic

cups (Figures 2I–2K). cCasp3 localized to only 10%–15% of

phagocytic cups (Figure 2L), compared to �60% for PCNA (Fig- ure 2H). Overall, there were �203 fewer cCasp3+ cells than PCNA+ cells in the P4 amygdala (Figures S2A–S2F), indicating

that the differences in newborn cell number cannot be driven

by apoptosis alone.

To further explore the dynamics of microglia interactions with

neighboring cells, we created a ‘‘microglia interactome,’’ in

which we identified individual microglia and measured the num-

ber of DAPI+, PCNA+, or cCasp3+ cells within 10 mm of that mi-

croglia’s processes. We found significantly more DAPI+ and

PCNA+ soma in close proximity to male microglia (Figures 2M,

2N, 2P, and 2Q). In contrast, the few cCasp3+ cells detected

were rarely found near microglia processes (Figures 2O and

2R). Collectively, these data indicate male microglia engage

more cells overall than females and are biased toward newborn

cells as opposed to dying cells. Given the higher number of

phagocytic microglia in males, our data indicate that sex differ-

ences in newborn cell number are largely determined by micro-

glia engulfment in the developing amygdala.

M al

e F

em al

P0 P8

Iba1 Males Females

__ _

_ _

_ __ __ 0 1 2 3 4 5 6

P0 P2 P4 P6 P8

* **

P ha

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gl ia

(x 1

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)

Iba1

_ _ __ __ __ __

P0 P2 P4 P6 P8

** *** **

P ha

go cy

tic m

ic ro

gl ia

(% )

__ __ __

__ _ _

P0 P2 P4 P6 P8

To ta

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lia (x

1 ,0

0 0

)

Iba1__

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100

1 2 3

P er

ce nt

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ha go

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Number of cups

Female Male

P ha

go cy

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up d

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et er

( μm

)

A B C D

H I J

Figure 1. Males Have More Phagocytic Microglia in the Developing Amygdala

(A) Nissl-stained coronal section of the P0 brain. Dashed white line indicates the boundaries of the amygdala used for analysis. Scale bar repre-

sents 500 mm.

(B and C) Maximum intensity projection of a non-phagocytic (B) and phagocytic microglia (C) immunolabeled for Iba1. White arrowheads indicate phagocytic

cups in (C). Scale bars represent 10 mm.

(D–F) Quantification of the number of phagocytic microglia (D; two-way ANOVA sex 3 age interaction F(4, 58) = 4.763; p = 0.00216), the percentage of microglia

that are phagocytic (E; two-way ANOVA sex 3 age interaction F(4, 58) = 12.19; p = 2.96e�07), and the total number of microglia (F; two-way ANOVA main effect of age F(4, 58) = 135.419; p < 2e�16) in the developing amygdala. Holm-Bonferroni post hoc comparison between males and females at each age. p, postnatal day. n = 6 or 7 rats per sex per age.

(G) Representative 203 field of view of Iba1 labeling from the P0 and P8 amygdala in females and males. White arrowheads indicate phagocytic microglia. Scale

bars represent 50 mm.

(H) Quantification of the percentage of phagocytic microglia with 1, 2, or 3 phagocytic cups. n = 4 females (140 cells) and 5 males (236 cells).

(I) Single confocal image through a phagocytic cup. Red bar demonstrates how the diameter of phagocytic cups was measured for (J). Scale bar repre-

sents 2 mm.

(J) Boxplot of the distribution of microglia phagocytic cups diameters. Open circles indicate values for individual phagocytic cups. n = 4 females (96 cups) and 4

males (97 cups).

Red and blue bars represent the mean ± SEM of females and males, respectively. Boxplot represents the median, 25%–75% range, and whiskers show max and

min values. Open circles represent individual data points for each animal or values for each cell (J). *p < 0.05, **p < 0.01, and ***p < 0.001.

See also Figure S5.

requires appropriate initiation and reciprocal response to social

cues (Pellis and McKenna, 1992; Argue and McCarthy, 2015).

Here, we identify the amygdala as an essential node mediating

the sex difference in juvenile social play, which is established

developmentally by the phagocytic action of microglia. Testos-

terone-induced endocannabinoids drive microglia phagocytosis

of newborn astrocytes, which ultimately modulates neuronal ac-

tivity in the MePD and promotes a higher frequency of play.

Androgens Elevate Endocannabinoid Tone in the Amygdala during the Critical Period for Sexual Differentiation Sexual differentiation of the brain begins prenatally, induced

by a testosterone surge produced by the fetal male testis.

, BrdU, and DAPI (left). Representative 203 field of view shows GFAP and BrdU

BrdU, and DAPI colocalization (right). Dashed lines indicate boundaries of the

esent 400 mm (left), 50 mm (middle), and 10 mm (right).

st t(13.904) = 2.8891; p = 0.01196. n = 10 or 11 rats per sex.

, BrdU, and DAPI (left). Representative 203 field of view shows NeuN and BrdU

ght). Dashed lines indicate boundaries of the MePD. White arrowheads indicate

, and 10 mm (right).

rats per sex.

BrdU, and DAPI (left). Representative 203 field of view shows Iba1 and BrdU

ht). Dashed lines indicate boundaries of the MePD. White arrowheads indicate

and 10 mm (right).

ats per sex.

d indicates phagocytic cup. Three-dimensional rendering of a P4 phagocytic

s and grid lines represent 5 mm.

. Grid lines represent 1 mm.

LDH1L1 within the cup. Grid lines represent 2 mm. Voxel size = 0.0124 3

elch’s t test t(5.4415) = �2.7408; p = 0.03728. n = 5 females (120 cups) and

ach animal. MePD, posterodorsal medial amygdala. *p < 0.05 and ***p < 0.001.

Neuron 102, 435–449, April 17, 2019 445

Testosterone circulates and reaches the brain, where it acts

on androgen receptors or is locally converted into estradiol

(McCarthy et al., 2017). Multiple mechanisms are invoked by ste-

roids simultaneously in distinct brain regions in the service of

differentiating a variety of functional endpoints. One such mech-

anism, as identified here, is androgen-dependent upregulation of

eCB tone within the developing amygdala. By birth, this process

is well underway with the male-specific increase in local eCB

content observable at P0 and persisting for the first few days

of life (Krebs-Kraft et al., 2010). However, neither the source of

eCBs nor the mechanism by which androgens lead to their in-

crease (i.e., synthesis or degradation) is clear. Microglia are

capable of producing 2-AG (Carrier et al., 2004; Witting et al.,

2004) and express distinct synthetic and hydrolytic enzymes

(Witting et al., 2004; Viader et al., 2016). Although they would

seem to be likely candidates for mediating differential eCB pro-

duction, our data indicate otherwise, as neonatal microglia

depletion did not alter the developmental eCB tone. This raises

the question: where are neonatal amygdala eCBs coming

from? There are two non-exclusive possibilities. First, mature

neurons and astrocytes could be responsible for the develop-

mental eCB tone, as both are critical to 2-AG metabolism in

the adult brain (Viader et al., 2015, 2016). Second, the newborn

cells themselves may be the source of the eCB tone (Aguado

et al., 2005, 2006). The former hypothesis predicts that earlier

born and mature cells influence the number of cells that subse-

quently develop, possibly as a mechanism for attaining appro-

priate cell numbers and density. Conversely, the latter hypothe-

sis predicts that control of cell number in the developing

amygdala is self-regulated by the pool of stem cells, perhaps

related to their ultimate fate as astrocytes instead of neurons.

Endocannabinoids Provide a Local Cue that Directs Phagocytosis of Viable Newborn Cells in a Complement- Dependent Manner Sex differences in the morphology and activational state of mi-

croglia are evident developmentally in multiple brain regions

(Schwarz et al., 2012; Lenz et al., 2013; Hanamsagar et al.,

2017). We found no differences in microglia number or functional

profile; instead, only the propensity for phagocytosis differed,

being higher in males and promoted by endocannabinoids.

Quantification of CD68+ lysosomal activity has been used to

quantify microglia engulfment (Schafer et al., 2012) and to infer

microglia functional or metabolic state (De Biase et al., 2017). Mi-

croglia along the entire spectrum of functional states were

phagocytic, but the majority were at the midpoint between fully

activated and surveying, reinforcing the notion that microglia

can effectively phagocytose their targets without becoming acti-

vated, as seen in the adult hippocampus (Sierra et al., 2010).

Sex differences can arise both from gonadal hormone expo-

sure and directly from the sex chromosomes. We found that,

when removed from the body and hence any circulating steroids,

microglia did not differ in their intrinsic phagocytic capacity, sug-

gesting the sex difference is the result of the microenvironment

of the developing amygdala, consistent with other findings (De

Biase et al., 2017). We identified eCBs as the microenvironment

cue, with the sex difference in developmental phagocytosis

tightly linked to transient changes in eCB tone. How eCBs induce

446 Neuron 102, 435–449, April 17, 2019

a phagocytic state is unknown. 2-AG can act as a chemoattrac-

tant for microglia, and its role as a ‘‘find me’’ signal has been pro-

posed in the context of neuroinflammation (Walter et al., 2003).

The higher eCB tone may induce more microglial mobility and

motility in males, increasing surveillance and driving microglia to-

ward newborn cells, which are then engulfed due to the high

localization of ‘‘eat me’’ signals. Our findings support this notion,

based on an interactome in which the proximity of microglia to

newborn cells was mapped and measured. On average, more

newborn cells were in close proximity to microglia in males. In

developing neurons, 2-AG is critical for appropriate pathfinding,

directing axonal outgrowth to appropriate targets, and assisting

in the formation of cortical circuits (Mulder et al., 2008; Vitalis

et al., 2008; Wu et al., 2010). Thus, eCB signaling may provide

chemoattractant signals for both neurons and microglia, albeit

for different purposes, in early life. Notably, the effects of eCBs

on microglia in the immature brain equally involve CB1 and

CB2 receptors, with no additive effects, suggesting a conver-

gence on a common signal transduction pathway.

Phagocytosis is usually associated with the engulfment of

dead or dying cells, but phagocytosis of viable cells, termed pri-

mary phagocytosis or ‘‘phagoptosis’’ (reviewed in Brown and

Neher, 2012, 2014), is an evolutionarily conserved mechanism

during brain development that eliminates excess progenitor cells

(Hoeppner et al., 2001; Reddien et al., 2001; Cunningham et al.,

2013). Microglia also actively induce cell death via the produc-

tion of reactive oxygen species in the developing cerebellum

(Marı́n-Teva et al., 2004) and hippocampus, which is dependent

upon CD11b/CR3 (Wakselman et al., 2008). We found tightly

regulated phagoptosis of newborn cells, producing a sex differ-

ence in density of mature astrocytes. Moreover, complement

proteins, recognized by microglial CR3, trigger the phagocytosis

of these otherwise viable newborn cells. We found newborn

cells are more likely to express eat me signals and, when they

do so, at a higher level than non-newborn cells. C3b was sub-

stantially more enriched than C1qA, perhaps reflecting its role

as a ‘‘tagging and/or signal amplification’’ factor in the comple-

ment cascade (Veerhuis et al., 2011). There may also be addi-

tional factors, such as ‘‘don’t eat me’’ signals (Lehrman et al.,

2018), expressed by mature cells that protect them from

phagocytosis.

Juvenile Social Play Is Masculinized by Microglia- Mediated Reduction in Astrocyte Density of the MePD The neural circuitry of play is largely redundant with the canonical

social behavior circuit and intersects with components of the

reward circuit (see Vanderschuren et al., 2016 for review). Both

males and females engage in social play but males more so.

The higher levels of play by males could be a distributed property

of the social and/or reward networks or it could be due to a spe-

cific node that boosts responsiveness to cogent stimuli and pro-

motes more frequent interactions. It has long been known that

the medial amygdala is important to promoting higher levels of

play by males (Meaney et al., 1981, 1983; Meaney and McEwen,

1986), but why that is so and whether it is uniquely so has been

unknown. We have filled that gap, implicating microglial control

of astrocyte density as essential to higher play by males. The

sex difference in the astrocyte population of the MePD could

either impact the formation of this node, perhaps by impacting

synapse formation as in other sexually dimorphic brain regions

(Mong et al., 1999; Amateau and McCarthy, 2002), or MePD

astrocytes may perform ‘‘real-time’’ synaptic gating of social in-

formation during a play bout (Martin-Fernandez et al., 2017). In

support of the latter scenario, male MePD neurons were more

active during play, unless the astrocyte density was increased

by blocking developmental phagocytosis, in which case both

neuronal activity and frequency of play were the same as seen

in females. Independently manipulating microglia phagocytosis,

without changing steroid hormone profile or endocannabinoid

tone, causally connects the density of surviving astrocytes and

the propensity for play. Furthermore, we found no evidence

that developmental phagocytosis programs other critical nodes

of the social and reward circuits, confirming the centrality of

the MePD.

In conclusion, we present a series of novel mechanisms medi-

ating masculinization of the medial amygdala to promote higher

levels of juvenile play. An androgen-induced upregulation of the

endocannabinoid tone promotes microglia phagocytosis of as-

trocytic precursors via a complement-dependent process. This

results in a reduced density of astrocytes in the amygdala of ju-

venile males, which allows for greater neuronal activation that in

turn promotes more intense play. Through this series of cellular

events, a reliable and enduring sex difference is established for

a complex social behavior.

Identifying the biological origins of sex differences provides

new insights into basic mechanisms and sources of individual

variability in brain development. Our data highlight a potential

consequence of cannabis use by pregnant women, as delta-9-

tetrahydrocannibinol (THC) and other cannabinoid molecules

can cross the placenta (Grant et al., 2018) and could thereby in-

fluence brain development in a sex-dependent manner. To date,

prenatal exposure to cannabis has been found to elicit subtle, yet

potentially significant, effects on brain development (Berghuis

et al., 2005; Tortoriello et al., 2014). As Western society enters

a new era with increasing medicalization, decriminalization,

and legalization of marijuana, our results compel us to under-

stand the basic biological mechanisms by which the brain de-

velops and can be perturbed by exogenous neuromodulatory

compounds in a sex-dependent manner.

STAR+METHODS

Detailed methods are provided in the online version of this paper

and include the following:

d KEY RESOURCES TABLE

d CONTACT FOR REAGENT AND RESOURCE SHARING

d EXPERIMENTAL MODEL AND SUBJECT DETAILS

B Animal studies

B Primary cell culture

d METHOD DETAILS

B Animal treatments

B Immunohistochemistry

B Unbiased stereological cell counting

B Image acquisition

B Quantification of microglial activation state

B Quantification of phagocytic cup contents, diameter,

and three-dimensional rendering of microglia

B Quantification of local cell-microglia interactions (‘‘Mi-

croglia Interactome’’)

B Quantification of cell fate

B Juvenile social play testing

B Quantification of zif268 expression following a single

social exposure

B In vitro phagocytosis assay

B Flow cytometry for complement proteins

B Mass spectrometry

B Western blot

d QUANTIFICATION AND STATISTICAL ANALYSIS

SUPPLEMENTAL INFORMATION

Supplemental Information includes six figures and can be found with this

article online at https://doi.org/10.1016/j.neuron.2019.02.006.

A video abstract is available at https://doi.org/10.1016/j.neuron.2019.02.

006#mmc3.

ACKNOWLEDGMENTS

This work was funded by the National Institutes of Health (R01MH52716 and

R01DA039062 to M.M.M.) and an operating grant from the Natural Sciences

and Engineering Council of Canada (NSERC) to M.N.H. Salary support was

provided to M.N.H. in the form of a Tier II Canada Research Chair from the Ca-

nadian Institutes of Health Research (CIHR), and H.A.V. is a Vanier Scholar

(CIHR) and receives studentships from Alberta Innovates and BranchOut

Neurological Foundation. We thank the Confocal Microscopy Core Facility at

the University of Maryland School of Medicine, including J. Mauban, for the

technical assistance. Flow cytometry analyses were performed at the Univer-

sity of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer

Center Flow Cytometry Shared Service, and we thank K. Underwood and X.

Fan for the technical assistance. We would also like to acknowledge the

Southern Alberta Mass Spectrometry Centre, located in and supported by

the Cumming School of Medicine, University of Calgary, for their services in

targeted liquid chromatography tandem mass spectrometry. Finally, we thank

B. Alger, J. Cheer, M.K. Lobo, D. Loane, and R. Ritzel for their helpful com-

ments and critiques of the manuscript.

AUTHOR CONTRIBUTIONS

Conceptualization, J.W.V., K.J.A., and M.M.M.; Investigation, J.W.V., K.J.A.,

A.E.M., H.A.V., S.E. Ashton, and S.E. Arambula; Writing – Original Draft,

J.W.V. and M.M.M.; Writing – Review & Editing, J.W.V., K.J.A., A.E.M., S.E.

Ashton, S.E. Arambula, and M.M.M.; Funding Acquisition, M.N.H. and

M.M.M.; Supervision, M.N.H. and M.M.M.

DECLARATION OF INTERESTS

The authors declare no competing interests.

Received: April 20, 2018

Revised: December 17, 2018

Accepted: February 4, 2019

Published: February 28, 2019

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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies

Goat polyclonal anti-Iba1 Abcam Cat#ab5076; RRID: AB_2224402

Rabbit polyclonal anti-Iba1 Wako Cat#019-19741; RRID: AB_839504

Mouse monoclonal Anti-BrdU BD Biosciences Cat#347580; RRID: AB_400326

Mouse monoclonal anti-CD68 (ED1) Abcam Cat#ab31630; RRID: AB_1141557

Mouse monoclonal anti-CD11b, clone OX-42 Bio-Rad Cat#MCA275GA; RRID: AB_566455

Mouse monoclonal anti-CD11b antibody (OX42) Abcam Cat#ab1211; RRID: AB_442947

Rabbit monoclonal anti-NeuN Abcam Cat#ab177487; RRID: AB_2532109

Rabbit polyclonal Anti-GFAP Abcam Cat#7260; RRID: AB_305808

Mouse monoclonal Anti-PCNA Abcam Cat#ab29; RRID: AB_303394

Rabbit polyclonal anti-ALDH1L1 Abcam Cat#ab87117; RRID: AB_10712968

Rabbit monoclonal anti-Egr1 (zif268) Cell Signaling Technology Cat#4153; RRID: AB_2097038

Rabbit anti-cleaved caspase-3 (Asp175) Cell Signaling Technology Cat#9661; RRID: AB_2341188

Biotinylated goat anti-rabbit Vector Laboratories Cat#BA-1000, RRID: AB_2313606

Biotinylated horse anti-mouse Vector Laboratories Cat#BA-2000; RRID: AB_2313581

Donkey anti-rabbit, Alexa Fluor 488 conjugate Thermo Fisher Scientific Cat#A21206; RRID: AB_2535792

Donkey anti-goat, Alexa Fluor 488 conjugate Thermo Fisher Scientific Cat#A11055; RRID: AB_2534102

Donkey anti-mouse, Alexa Fluor 488 conjugate Thermo Fisher Scientific Cat#A21202; RRID: AB_141607

Donkey anti-rabbit, Alexa Fluor 594 conjugate Thermo Fisher Scientific Cat#A21207; RRID: AB_141637

Donkey anti-goat, Alexa Fluor 594 conjugate Thermo Fisher Scientific Cat#A11058; RRID: AB_2534105

Donkey anti-mouse, Alexa Fluor 594 conjugate Thermo Fisher Scientific Cat#A21203; RRID: AB_2535789

NucRed Dead 647 Invitrogen Cat#R37113

Hoechst 33342 Solution Thermo Fisher Scientific Cat#62249

NeuroTrace 500/525 Green Fluorescent Nissl Stain Thermo Fisher Scientific Cat#N21480

Rat monoclonal anti-F4/80 Santa Cruz Cat#sc-52664; RRID: AB_629466

Rabbit polyclonal Anti-C3b Abcam Cat#ab11887; RRID: AB_298669

Rabbit monoclonal anti-C1qA Abcam Cat#ab189922

Alexa Fluor 647 mouse anti-Ki-67 BD Biosciences Cat#558615; RRID: AB_647130

Alexa Fluor goat anti-rabbit-FITC Thermo Fisher Scientific Cat#11-4839-81; RRID: AB_1210845

Alexa Fluor goat anti-mouse-PE Thermo Fisher Scientific Cat#12-4010-82; RRID: AB_11063706

Rabbit monoclonal anti-CD11b Abcam Cat#ab133357; RRID: AB_2650514

Rabbit polyclonal anti-b actin Abcam Cat#ab8229; RRID: AB_306374

Rabbit polyclonal anti-GAPDH Sigma-Aldrich Cat#G9545; RRID: AB_796208

IRDye 680RD donkey anti-rabbit LI-COR Cat#925-68073; RRID: AB_2716687

IRDye 800CW donkey anti-goat LI-COR Cat#925-68074; RRID: AB_2650427

Chemicals, Peptides, and Recombinant Proteins

Testosterone propionate Sigma-Aldrich Cat#T1875

Flutamide Sigma-Aldrich Cat#F9397

Estradiol benzonate Sigma-Aldrich Cat#E8515

Cannabinoid Receptor agonist ACEA Tocris Cat#1319

Cannabinoid Receptor agonist GP1a Tocris Cat#2764

MAGL inhibitor KML29 Tocris Cat#4872

Cannabinoid Receptor antagonist AM630 Tocris Cat#1120

(Continued on next page)

e1 Neuron 102, 435–449.e1–e6, April 17, 2019

Continued

REAGENT or RESOURCE SOURCE IDENTIFIER

Cannabinoid Receptor antagonists AM281 Tocris Cat#1115

Liposomal coldronate Encapsula Nanoscienes Cat#CLD-8909

FluoSpheres carboxylate-modified

microspheres, 1 mm

Thermo Fisher Scientific Cat#F8816

5-Bromo-20-deoxyuridine (BrdU) Sigma-Aldrich Cat#B5002

Critical Commercial Assays

Neonatal Neuronal Dissociation Kit (P) Miltenyi Bioscience Cat#130-092-628

Experimental Models: Organisms/Strains

Sprague-Dawley rats Charles River Laboratories N/A

Software and Algorithms

StereoInvestigator MBF Bioscience https://www.mbfbioscience.com/stereo-investigator

Imaris Bitplane https://www.bitplane.com/imaris; RRID: SCR_007370

ImageJ NIH https://imagej.net/Welcome; RRID: SCR_003070

Neurolucida MBF Bioscience https://www.mbfbioscience.com/neurolucida; RRID:

SCR_001775

FlowJo X FlowJo https://www.flowjo.com/

ImageStudio LI-COR https://www.licor.com/bio/products/software/

image_studio/

R, version 3.4.4 R Core Team https://cran.r-project.org/

BZ-X Analyzer KEYENCE https://www.keyence.com/ss/products/microscope/

bz-x700/product/index.jsp

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Margaret

McCarthy (mmccarthy@som.umaryland.edu).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Animal studies Adult Sprague-Dawley rats, obtained from Charles River Laboratories, were maintained on a 12:12h reverse light/dark cycle with ad

libitum food and water. Animals were mated in our facility, and pregnant females were allowed to deliver naturally with the day of birth

being designated as postnatal day 0 (P0). On P0, pups were sexed, treated, and culled to no more than 14 pups per dam. Male and

female pups were used in these studies, and treatment groups and sexes were balanced across litters. All animal procedures were

performed in accordance with the Animal Care and Use Committee’s regulations at the University of Maryland School of Medicine.

Primary cell culture To generate sex-specific mixed glia cultures, rat pups were sexed on the day of birth and rapidly decapitated, and their brains were

removed and placed into ice cold HBSS (without divalent ions), stripped of meninges, and minced with a razor blade. Individual brains

were incubated for 15 min in 0.5% trypsin at 37�C, then dissociated by trituration with a pipette. The resulting suspension was pel- leted by centrifugation at 400g for 4 min. Excess supernatant was aspirated and the suspension was resuspended in culture media

(DMEM/F12 supplemented with 0.45% D-glucose, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% heat-inactivated fetal

bovine serum). One P0 rat brain was used to seed a T75 flask, which was incubated at 37�C with 5% CO2. One day after seeding, the culture media was completely replaced to remove unattached cells. Subsequently, 50% of the media was replaced every 4 days,

and cultures were allowed to grow until DIV14. On DIV14, flasks were shaken on a rotating platform at 100 rpm for 1 h to detach micro-

glia from the astrocyte layer. Media was removed from the flask and centrifuged at 400g for 4 min to pellet microglia, and resus-

pended in serum-free culture media (DMEM/F12, 0.45% D-glucose, 100 U/ml penicillin, 100 mg/ml streptomycin).

METHOD DETAILS

Animal treatments The following drugs and doses were used in these studies:

Neuron 102, 435–449.e1–e6, April 17, 2019 e2

mailto:mmccarthy@som.umaryland.edu

https://www.mbfbioscience.com/stereo-investigator

https://www.bitplane.com/imaris

https://imagej.net/Welcome

https://www.mbfbioscience.com/neurolucida

https://www.flowjo.com/

https://www.licor.com/bio/products/software/image_studio/

https://cran.r-project.org/

https://www.keyence.com/ss/products/microscope/bz-x700/product/index.jsp

For studies involving hormonal modulation, drugs were dissolved in sesame oil and delivered subcutaneously. Testosterone pro-

pionate (100 mg; Sigma-Aldrich Cat#T1875), flutamide (100 mg; Sigma-Aldrich Cat#F9397), and estradiol benzoate (10 mg; Sigma-Al-

drich Cat#E8515) were injected in a volume of 0.1 mL per pup per day.

Cannabinoid receptor agonists ACEA (1 mg/kg; Tocris Cat#1319) and GP1a (1 mg/kg; Tocris Cat#2764) and the MAGL inhibitor

KML29 (1 mg/kg; Tocris Cat#4872) were dissolved in ethanol at 5 mg/ml and further diluted in saline and delivered intraperitoneally

in a volume of 0.1 mL per pup per day.

Cannabinoid receptor antagonists AM281 (1 mg/kg; Tocris Cat#1115) and AM630 (1 mg/kg; Tocris Cat#1120) were dissolved in

DMSO at 5 mg/ml and further diluted in saline and delivered intraperitoneally in a volume of 0.1 mL per pup per day.

BrdU (50 mg/kg; Sigma-Aldrich Cat#B5002) was dissolved in saline and delivered intraperitoneally in a volume of 0.1 mL per pup

per day.

Liposomal clodronate (Encapsula Nanosciences Cat#CLD-8909) was delivered by bilateral intracerebroventricular injection (1 mL

per hemisphere) at the following coordinates: �1.0 mm, caudal from bregma; ± 1.0 mm, lateral from midline; �3.0 mm, ventral from surface of the skull.

Anti-CD11b antibody (0.5 mg/ml; OX-42 clone; Bio-Rad Cat#MCA275GA, RRID:AB_566455), anti-F4/80 antibody (0.2 mg/ml;

Santa Cruz Cat#sc-52664, RRID:AB_629466) or phosphate buffered saline vehicle was delivered by intra-amygdalar injection at

the following coordinates: �0.8 mm, caudal from bregma; ± 3.0 mm, lateral from midline; �5.0 mm, ventral from surface of the skull). Intracerebroventricular and intra-amygdalar injections were performed under cryoanesthesia using a 23 gauge Hamilton syringe

attached to a stereotaxic instrument. The time the pups were separated from the dam was kept to a minimum, between 15 min to 1 h

for all procedures.

Immunohistochemistry Rat pups were deeply anesthetized with Fatal Plus (Vortech Pharmaceuticals) and transcardially perfused with phosphate-buffered

saline (PBS; 0.1M, pH 7.4) followed by 4% paraformaldehyde (PFA; 4% in PBS, pH 6.8). Brains were removed and postfixed for 24 h

in 4% PFA at 4�C, then kept in 30% sucrose at 4�C until fully submerged. Coronal sections were cut at a thickness of 45 mm (for developmental studies) or 20 mm (for juvenile studies) on a cryostat (Leica CM2050S) and directly mounted onto silane-coated slides.

For fluorescent imaging, slide-mounted sections were washed in PBS and blocked with either 5% normal goat serum (NGS) or

10% bovine serum albumin (BSA) in PBS + 0.4% Triton X-100 (PBS-T) for 1 h. Slides were incubated in primary antibody solution

(either 5% NGS or 5% BSA in PBS-T) overnight. The following day, slides were incubated in secondary antibody solution (either

5% NGS or 5% BSA in PBS-T) for 2 h and coverslipped with ProLong Diamond Antifade (Thermo Fisher Scientific). For quantification

of DNA material in phagocytic cups, sections were incubated in NucRed Dead 647 (Invitrogen Cat#R37113) or DAPI (Hoechst;

Thermo Fisher Scientific Cat#62249) according to manufacturer’s instructions. Sections used for fluorescent Nissl imaging were

stained with NeuroTrace (Thermo Fisher Scientific Cat#N21480) according to manufacturer’s instructions.

For DAB staining, slide-mounted sections were washed in Tris-buffered saline (TBS; 0.05M, pH 7.6), incubated in 0.3% hydrogen

peroxide in TBS for 30 min at room temperature. Sections were blocked with 5% NGS or 10% BSA in TBS + 0.4% Triton X-100

(TBS-T). Sections were incubated in primary antibody in solution (either 5% NGS in TBS-T or 5% BSA in TBS-T) overnight. The

following day, sections were incubated in biotinylated secondary antibody for 1 h, followed by incubation in ABC reagent (1:500 dilu-

tion; Vectastain Elite ABC Kit, Vector Laboratories) in TBS-T for 1 h, and visualized using DAB chromagen or nickel-enhanced DAB

chromagen (0.05% 3,30-diaminobenzidine, 0.2% nickel (II) sulfate, 0.006% hydrogen peroxide; all from Sigma-Aldrich). The DAB re- action was allowed to proceed until completion, as confirmed under a microscope. Sections were counterstained with either hema-

toxylin or methyl green, dehydrated in an ascending ethanol series, defatted in xylene, and coverslipped with DPX mounting medium.

Antigen retrieval (0.01M sodium citrate, pH 6.0 for 20 min at 99�C) was used prior to the blocking step to enhance immunolabeling for PCNA, ALDH1L1, and zif268. Primary antibodies used included the following: Rabbit anti-Iba1 (1:1000; Wako Cat#019-19741,

RRID:AB_839504), goat anti-Iba1 (1:1000; Abcam Cat#ab5076, RRID:AB_2224402), mouse anti-BrdU (1:500; BD Biosciences

Cat#347580, RRID:AB_400326), mouse anti-CD68 (1:500; Abcam Cat#ab31630, RRID:AB_1141557), mouse anti-CD11b (1:500;

OX-42 clone; Abcam Cat#ab1211, RRID:AB_442947), rabbit anti-NeuN (1:1000; Abcam Cat#ab177487, RRID:AB_2532109), rabbit

anti-GFAP (1:1000; Abcam Cat#7260, RRID:AB_305808), mouse anti-PCNA (1:1000; Abcam Cat#ab29, RRID:AB_303394), rabbit

anti-cleaved caspase-3 (Asp175) (1:500; Cell Signaling Technology Cat#9661, RRID:AB_2341188), rabbit anti-ALDH1L1 (1:1000;

Abcam Cat#ab87117, RRID:AB_10712968), rabbit anti-Egr1 (zif268) (1:1000; Cell Signaling Technology Cat#4153, RRID:

AB_2097038). Secondary antibodies used included the following: biotinylated anti-rabbit (1:500; Vector Laboratories Cat#BA-

1000, RRID:AB_2313606), biotinylated anti-mouse (1:500; Vector Laboratories Cat#BA-2000, RRID:AB_2313581), Alexa Fluor

488- or 594-conjugated antibodies to rabbit (488 Cat#A21206, RRID:AB_2535792; 594 Cat#A21207, RRID:AB_141637), goat (488

Cat#A11055, RRID:AB_2534102; 594 Cat#A11058, RRID:AB_2534105), or mouse (488 Cat#A21202, RRID:AB_141607; 594

Cat#A21203, RRID:AB_2535789) (1:500; Thermo Fisher Scientific, all raised in donkey).

Unbiased stereological cell counting Stereological cell counts were performed using StereoInvestigator (MBF Bioscience) on a computer interfaced with a Nikon Eclipse

E600 microscope and MBF Bioscience CX9000 camera. Every third section (45 mm thick) was used for analysis for a total of

four sections, and the amygdala, prefrontal cortex, nucleus accumbens, and paraventricular nucleus of both hemispheres were

e3 Neuron 102, 435–449.e1–e6, April 17, 2019

quantified. The boundaries of each region were drawn with a 4x objective, referencing a neonatal rat atlas (Ashwell and Paxinos,

2008), and the optical fractionator method was used to quantify microglia and BrdU+ cells at 40x magnification, using a 100 mm x

100 mm counting grid with a 250 mm x 250 mm sampling grid for microglia and a 50 mm x 50 mm counting grid with a 250 mm x

250 mm sampling grid for BrdU+ cells. The optical dissector height was set to 12 mm with a 2 mm guard zone on the top and bottom

for both quantifications. Microglia were counted based on the presence of an observable cell body within the designated counting

region and determined to be phagocytic if the microglia contained an observable phagocytic cup that was distinctly identifiable from

the cell body. BrdU+ cells were counted if the nuclear staining was uniformly dark and was within the designated counting region.

Image acquisition For all experiments, confocal fluorescence images were acquired with the following: Zeiss LSM 710 microscope equipped with 488,

561, and 633 lasers, using a 20x (1.0 NA) water-immersion or 100x (1.46 NA) oil-immersion objective and Zeiss Zen software; Nikon

CSU-W1 or A1 microscope equipped with 405, 488, 561, and 647 lasers, using a 20x (0.75 NA), 60x (1.49 NA) TIRF oil-immersion, or a

100x (1.45 NA) oil-immersion objective and Nikon Elements software. Widefield fluorescence and brightfield images were captured

on a Keyence BZ-X700 microscope using a 4x (0.2 NA), 10x (0.45 NA) or a 20x (0.75 NA) objective and BZ-X Viewer software.

Quantification of microglial activation state 45 mm thick fixed coronal sections were immunolabeled for Iba1 and CD68 and imaged with a Zeiss 710 confocal microscope. Six

fields of view (1 per hemisphere from 3 sections total) were taken with a 20x objective using 2 um z steps through the entire tissue

thickness. Subsequent maximum intensity projections were used to quantify microglia morphology and CD68 expression. The acti-

vation state was categorized on a 0 (lowest activation) to 5 (highest activation) according to the following criteria: morphology was

scored as 0 (5+ processes with at least secondary branches), 1 (1 - 4 processes with at least secondary branches), 2 (1+ processes

with no secondary branches), and 3 (round with no clear processes). CD68 expression was scored as 0 (no clear expression), 1 (punc-

tate expression) or 2 (aggregated expression throughout the cell). For each cell, the morphology and CD68 scores were combined to

produce a final 0-5 score, as described in Schafer et al. (2012).

Quantification of phagocytic cup contents, diameter, and three-dimensional rendering of microglia For three-dimensional rendering, confocal z stacks were taken with a 100x objective to achieve an x-y pixel size between 0.012 -

0.070 mm and a z-step size between 0.050 - 0.200 mm. Images were deconvolved using the automatic deconvolution algorithm in

Nikon Elements, and were reconstructed in three dimensions in Imaris (Bitplane, RRID:SCR_007370). Individual microglial cells

were reconstructed using the Surfaces module to create a volumetric boundary of the cell. The resulting microglial surface was

then used as a mask to process the channels containing the material to be colocalized. After masking, the Surfaces module was again

used to generate a new volume of the engulfed material, and the two surfaces (microglia and engulfed material) were merged to

create the final rendering.

For phagocytic cup analyses, single confocal images were taken with a 20x objective through the middle of the phagocytic cup.

Images were then assessed for colocalization or measured for diameter in ImageJ (RRID:SCR_001775).

Quantification of local cell-microglia interactions (‘‘Microglia Interactome’’) Three-dimensional renderings of microglia were generated using the Surfaces module in Imaris as described above. The Spots mod-

ule was used to mark the location of surrounding cells (DAPI+, PCNA+, cCasp3+), and the DistanceTransform function was used to

calculate the distance of each spot (cell) from the microglia surface. Spots within a 10 mm distance from the microglia surface were

included in analyses. Additionally, the total number of PCNA+ and cCasp3+ cells were quantified using 20x field of view images of the

amygdala using ImageJ (RRID:SCR_001775).

Quantification of cell fate Quantification of the number of BrdU+ cells across the four MeA subregions was performed using Neurolucida software (MBF Biosci-

ence, RRID:SCR_001775) on a computer interfaced with a Nikon Eclipse E600 microscope and MBF Bioscience CX9000 camera.

Contours outlining the boundaries of each subregion were drawn at 4x magnification, and the area of each subregion was recorded.

Sections from three alternate series were used to quantify the total number of BrdU+ cells and the number colocalized with either

GFAP, NeuN or Iba1. Cells in each subregion were counted at 20x magnification across both hemispheres of the MeA. Additionally,

the total number of GFAP+ cells was quantified only in the MePD. The data were normalized to the area of the subregion to account

for any volumetric differences in the subregions between the sexes and averaged across hemispheres to generate a density estimate.

In all cases, BrdU+ cells were counted if the nuclear staining was uniformly dark and present within the boundaries of the designated

subregion. BrdU+ cells were counted as colocalized if a well-defined BrdU+ nucleus was associated with an immunopositive (i.e.,

GFAP+, NeuN+, Iba1+) cell body. For a subset of samples, colocalization criteria was confirmed by confocal microscopy.

Juvenile social play testing Animals were weaned on P21 and housed in same-sex, same-treatment sibling pairs. On P26, animals were tested for 10 min in an

open field (78 3 78 cm, 40 cm high), underlaid with a grid delineating perimeter and center regions. Total locomotion and center time

Neuron 102, 435–449.e1–e6, April 17, 2019 e4

were analyzed to rule out confounds due to differences in activity or anxiety-like behavior on play. Once per day from P27-30, same-

sex, same-treatment non-sibling pairs of animals were placed in an enclosure (49 3 37 cm, 24 cm high) with TEK-Fresh cellulose

bedding (Harland Laboratories). Animals were allowed to acclimate for 2 min, then video recorded for 10 min. Behavior testing

took place during the dark phase of the animal’s light/dark cycle under red light illumination. Videos were scored offline to determine

the number of pounces, pins, and boxing behaviors.

Quantification of zif268 expression following a single social exposure Juvenile animals were given access to a novel sex-, treatment-, and age-matched play partner for 10 min as described above. Imme-

diately following, animals were returned to their original cages. After 1 h, they were euthanized and brains were collected for histo-

logical analysis. Sections were labeled for zif268 and imaged using a Keyence BZ-X700 microscope at 4x magnification. Using the

Hybrid Cell Count module in BZ-X Analyzer software (Keyence), contours were drawn to outline each region of interest, images were

thresholded to account for background signal, and zif268+ cells were quantified. The number of zif268+ cells was normalized to the

area of each contour to account for any volumetric differences.

In vitro phagocytosis assay Microglia harvested from sex-specific mixed glia cultures were plated into 6 well plates at a density of 50,000 cells per well and kept in

serum-free culture media at 37�C with 5% CO2. After 24 h, carboxylated latex beads (FluoSpheres, 1 mm; Thermo Fisher Scientific Cat#F8816) were added to each well in a 1:100 cell:bead ratio and allowed to incubate for 1 h at 37�C with 5% CO2. Immediately following this, plates were placed on ice to halt any further phagocytosis, washed with ice-cold PBS containing 2 mM EDTA, and

vigorously pipetted to detach cells from the plate. Cells were centrifuged at 400g for 4 min, resuspended in FACS buffer (1.0%

BSA, 0.1% sodium azide, in HBSS), and incubated with propidium iodide for flow cytometry analysis. Flow cytometry was performed

on a LSR II (BD Biosciences) with FACSDiva software, and analysis performed using FlowJo X (FlowJo). Live cells were gated based

on propidium iodide staining and bead fluorescence determined using live cells that were not exposed to beads.

Flow cytometry for complement proteins On P4, male and female pups were rapidly decapitated, and the amygdala of both hemispheres was dissected out from the brain on

ice. Tissue was immediately dissociated using the Neonatal Neuronal Dissociation (P) Kit (Miltenyi Bioscience Cat#130-092-628). The

resulting single cell suspension was fixed in 4% paraformaldehyde for 20 min and stored in a 1:1 ratio of glycerol:HBSS at �20�C until staining. Cells were permeabilized with saponin and stained with a combination of the following antibodies: rabbit anti-C3b (1:50;

Abcam Cat#ab11887, RRID:AB_298669), rabbit anti-C1qA (1:50; Abcam Cat#ab189922), and mouse anti-Ki-67-647 (1:10; BD Bio-

sciences Cat#558615, RRID:AB_647130). Unconjugated primary antibodies were labeled using Alexa Fluor goat anti-rabbit-FITC

(Cat#11-4839-81, RRID:AB_1210845) or Alexa Fluor goat anti-mouse-PE (Cat#12-4010-82, RRID:AB_11063706) (1:100; Thermo

Fisher Scientific). Flow cytometry was performed on a LSR II (BD Biosciences) with FACSDiva software, and analysis performed

using FlowJo X. Debris was eliminated based on forward and side scatter gating. Appropriate gates for C3b, C1qA, and Ki-67

were determined using an ‘‘empty channel’’ with the appropriate FMO samples to determine the negative population and

autofluorescence.

Mass spectrometry Lipid extraction and mass spectrometry was performed as previously described (Morena et al., 2015; Qi et al., 2015). On P4, rat pups

were rapidly decapitated and the amygdala dissected out and immediately flash frozen until analysis. Brain tissue was weighed and

placed into borosilicate glass culture tubes containing 2 mL of acetonitrile with 5 pmol of AEA and 5 nmol of 2-AG for extraction. Tis-

sue was homogenized with a glass rod, sonicated for 30 min in ice water, and incubated overnight at �20�C to precipitate proteins. Subsequently, samples were then centrifuged at 1500g to remove particulates, and the supernatants transferred to a new glass tube

and evaporated to dryness under nitrogen gas. The samples were reconstituted in 200 mL of acetonitrile and stored at �80�C until further analysis. Analysis of AEA and 2-AG was performed by liquid chromatography tandem mass spectrometry as previously

described (Qi et al., 2015).

Western blot On P4, rat pups were rapidly decapitated and the amygdala dissected out and immediately flash frozen until analysis. Tissue was

homogenized in RIPA buffer with added phosphatase (Sigma; 1:1000) and protease (Sigma; 1:1000) inhibitors. After homogenization,

samples were centrifuged at 3000 rpm at 4�C for 10 min. The protein supernatant was collected and total protein concentration deter- mined by Bradford assay using an Infinite M1000 Pro (Tecan). 25 mg protein was loaded per sample and run on a 8%–16% tris-glycine

gel (Invitrogen). Protein was transferred to a polyvinyl difluoride membrane (Bio-Rad), blocked for 1 h in Odyssey Blocking Buffer

(LI-COR) diluted 1:1 with TBS and incubated overnight at 4�C with rabbit anti-CD11b (1:1000; Abcam Cat#ab133357, RRID: AB_2650514), goat anti-Iba1 (1:1000; Abcam Cat#ab5076, RRID:AB_2224402), rabbit anti-b actin (1:1000; Abcam Cat#ab8229,

RRID:AB_796208) and rabbit anti-GAPDH (1:20,000; Sigma-Aldrich Cat#G9545, RRID:796208) in diluted Odyssey Blocking Buffer

with 0.1% Tween-20 (BioRad). Subsequently, membranes were rinsed and incubated for 1 h with IRDye 680RD donkey anti-rabbit

(Cat#925-68073, RRID:AB_2716687) and IRDye 800CW donkey anti-goat (Cat#925-68074, RRID:AB_2650427) antibodies

e5 Neuron 102, 435–449.e1–e6, April 17, 2019

(1:20,000; LI-COR) in diluted Odyssey Blocking Buffer with 0.1% Tween-20 and 0.02% sodium dodecyl sulfate (Sigma). Membranes

were imaged in both the 700 and 800 nm channels using an Odyssey CLx scanner (LI-COR) and quantified using ImageStudio soft-

ware (LI-COR). The CD11b signal was normalized to Iba1, GAPDH, and b actin, and the resulting values averaged for each sample.

Each sample was normalized to a standard internal control sample to allow for comparison across membranes.

QUANTIFICATION AND STATISTICAL ANALYSIS

All values are shown as the mean ± SEM, with the exception of boxplots, which depict the median, 25th and 75th percentiles, with

whiskers depicting the minimum and maximum values. Statistical analyses were performed using R (R Core Team, 2018; version

3.4.4). Statistical details of experiments can be found in figure legends and in the text (tests used, exact n, p value). Comparisons

between two experimental groups were performed using a two-tailed Welch’s t test (for independent samples) or paired t test (for

dependent samples). Data including multiple experimental groups were analyzed using one-way or two-way analysis of variance

(ANOVA) when appropriate. In experiments in which females were treated with agents predicted to induce masculinization, Dunnett’s

post hoc comparisons were calculated using female vehicle as a control to test the hypothesis that each treatment masculinized the

end point. In some experiments, post hoc pairwise t tests were calculated for specific comparisons to determine differences in means

between the treated female groups and male and female vehicle groups. Linear correlation was calculated using Pearson’s r. A

p value of < 0.05 was used as the criterion for significance.

Neuron 102, 435–449.e1–e6, April 17, 2019 e6

Neuron, Volume 102

Supplemental Information

Microglial Phagocytosis of Newborn Cells

Is Induced by Endocannabinoids and Sculpts

Sex Differences in Juvenile Rat Social Play

Jonathan W. VanRyzin, Ashley E. Marquardt, Kathryn J. Argue, Haley A. Vecchiarelli, Sydney E. Ashton, Sheryl E. Arambula, Matthew N. Hill, and Margaret M. McCarthy

Figure S1 (Related to Figure 1). Phagocytic microglia are distributed across the activation scale with no sex differences in phagocytic capacity. (A) Maximum intensity projection (MIP) of a phagocytic microglia co-immunolabeled for Iba1, CD68 and DAPI (top). Bottom, single channel MIP of Iba1 (bottom left) and CD68 (bottom right). Scale bar = 10 µm. (B and C) Quantification of microglia activation state on a 0-5 scale based on morphology and CD68 labeling for all microglia (B) and phagocytic microglia (C). n = 4 females (642 cells), 5 males (836 cells) for B; n = 4 females (140 cells), 5 males (236 cells) for C. (D) Gating strategy for the data shown in E and F. Cultured microglia were gated on forward and side scatter to separate cells from debris, then further gated on side scatter to separate single cells. Live cells were selected for based on propidium iodide exclusion and phagocytic cells determined by bead fluorescence. (E) Representative histograms showing the relative distribution of microglia containing 1 or more fluorescent beads from female (red) and male (blue) cultures. Microglia containing zero beads shown in black. Median fluorescence intensity (MFI) of bead+ cells indicated in the upper righthand corner, +/- SEM of MFI. (F) Quantification of the percentage of microglia engulfing 1+ fluorescent beads. n = 6 (females), 7 (males) independent biological replicates. Red/blue bars represent the mean +/- SEM of females/males. Open circles represent individual data points for each animal.

Figure S2 (Related to Figure 2). Newly proliferated cells outnumber dying cells in the P4 amygdala. (A and D) Representative image of a P4 amygdala section immunolabeled for PCNA (A) or cCasp3 (D). Dashed box indicates location of images in B and E. Scale bars = 400 µm. (B and E) Representative 20x field of view from A and D. Scale bars = 100 µm. (C and F) Quantification of the number of PCNA+ (C) or cCasp3+ (F) cells in each 20x field of view. n = 5 rats per sex (6 fields per animal). Bars represent the mean +/- SEM. Open circles represent individual data points for each animal.

Figure S3 (Related to Figure 3). Neither estradiol treatment nor microglia depletion masculinizes eCB content; testosterone and KML29 masculinize newborn cell number in the developing amygdala. (A) Schematic showing the treatment paradigm and timeline for B and C. (B) Quantification of 2-AG levels measured by mass spectrometry in amygdala tissue. ANOVA F(2, 24) = 12.27, p = 0.000214. Dunnett’s post-hoc comparisons to female vehicle. n = 8-11 rats per group. (C) Quantification of AEA levels measured by mass spectrometry in amygdala tissue. n = 8-11 rats per group. (D) Schematic showing the treatment paradigm and timeline for E-G. (E) Quantification of total microglia number following microglia depletion. Welch’s t test t(2.6235) = 18.178, p = 0.0007826. n = 3 rats per group. (F) Quantification of 2-AG levels measured by mass spectrometry in microglia-depleted amygdala tissue. Two-way ANOVA main effect of sex F(1, 40) = 4.586, p = 0.0384. n = 11 rats per group. (G) Quantification of AEA levels measured by mass spectrometry in microglia-depleted amygdala tissue. n = 11 rats per group. (H-J) Gating strategy for the data shown in L and N. Dissociated cells from the P4 amygdala were gated on forward and side scatter to separate cells from debris (H). (I) Representative density plot of a sample labeled for Ki-67 showing both the Ki-67+ and Ki-67- populations. (J) Ki-67+ gate was determined by analyzing fluorescence of an unstained sample in an “empty channel” to identify a negative population and eliminate autofluorescence. (K) Schematic showing the treatment paradigm and timeline for L. (L) Quantification of the percentage of Ki-67+ cells. ANOVA F(2, 19) = 9.318, p = 0.00151. Dunnett’s post-hoc comparisons to female vehicle. n = 6-8 rats per group. (M) Schematic showing the treatment paradigm and timeline for N. (N) Quantification of the percentage of Ki-67+ cells. Welch’s t test t(12.308) = 3.7645, p = 0.002586. n = 8-9 rats per sex. Bars represent the mean +/- SEM. Vertical dashed line indicates separation of male from female groups. Open circles represent individual data points for each animal. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure S4 (Related to Figure 4). Treatment with anti-CD11b, but not anti-F4/80, reduces phagocytosis; CD11b validation and gating strategy by flow cytometry. (A) Schematic showing treatment paradigm and timeline for B. (B) Quantification of the percent of phagocytic microglia between anti-CD11b treated and untreated hemispheres. Paired t test t(3) = 10.131, p = 0.002049. n = 4 males. (C) Schematic showing treatment paradigm and timeline for D and E. (D) Quantification of the number of BrdU+ cells between anti-F4/80 treated and untreated hemispheres. n = 5 males. (E) Quantification of the percent of phagocytic microglia between anti-F4/80 treated and untreated hemispheres. n = 5 males. (F) Western blot analysis of CD11b from P4 amygdala showing expression does not vary between males and females. n = 10 rats per sex. (G) Gating strategy for C1qA flow cytometry analysis. Dissociated cells from the P4 amygdala were gated on forward and side scatter to separate cells from debris. Ki-67+ gates were determined as described in Figure S3H-J; both Ki-67+ and Ki-67- cells were analyzed for C1qA expression. (H) Gating strategy for C3b flow cytometry analysis. Dissociated cells from the P4 amygdala were gated on forward and side scatter to separate cells from debris. Ki-67+ gates were determined as described in Figure S3H-J; both Ki-67+ and Ki-67- cells were analyzed for C3b expression. (I) C1qA+ gate (used in G) was determined by analyzing fluorescence of an unstained sample in an “empty channel” to identify a negative population and eliminate autofluorescence. (J) C3b+ gate (used in H) was determined by analyzing fluorescence of an unstained sample in an “empty channel” to identify a negative population and eliminate autofluorescence. Bars represent the mean +/- SEM. Open circles represent individual data points for each sample. Gray lines connect data points between hemispheres for each individual animal in B, D and E. **p < 0.01.

Figure S5 (Related to Figure 5). No sex difference in density of neonatally-born cells in the MeAD, MeAV and MePV; GFAP+ cell density, but not Iba1+ or NeuN+ density, is higher in female MePD. (A) Nissl stained coronal section of the P26 brain. Dashed white line indicates the boundaries of the MeAD and MeAV used for analysis. (B) Quantification of the density of BrdU+ cells in the MeAD. n = 10-11 rats per sex. (C) Quantification of the density of BrdU+ cells in the MeAV. n = 10-11 rats per sex. (D) Nissl stained coronal section of the P26 brain. Dashed white line indicates the boundaries of the MePD and MePV used for analysis. (E) Quantification of the density of BrdU+ cells in the MePV. n = 10-11 rats per sex. (F) Quantification of the density of GFAP+ cells in the MePD. Welch’s t test t(14.78) = 2.8829, p = 0.01152. n = 10-11 rats per sex. (G) Quantification of the density of Iba1+ cells in the MePD. n = 10-11 rats per sex. (H) Quantification of the density of NeuN+ cells in the MePD. n = 10-11 rats per sex. Bars represent the mean +/- SEM. Open circles represent individual data points for each animal. Abbreviations: anterodorsal medial amygdala (MeAD); anteroventral medial amygdala (MeAV); posterodorsal medial amygdala (MePD); posteroventral medial amygdala (MePV). *p < 0.05.

Figure S6 (Related to Figure 6). Developmental microglia phagocytosis does not program neuronal activation in other nodes of the social behavior circuitry. (A) Quantification of the number of total microglia (left) and phagocytic microglia (right) during development in the PFC. n = 7 rats per sex. (B) Nissl stained coronal section of the P26 brain (top). Dashed box indicates location of the PFC. Representative images of female (bottom left) and male (bottom right) sections immunolabeled for zif268 and DAPI. Dashed lines indicate boundaries of the PFC. Scale bars = 300 µm. (C) Quantification of the density of zif268+ cells in the PFC. n = 16 rats per sex. (D) Quantification of the number of total microglia (left) and phagocytic microglia (right) during development in the NAc. n = 7 rats per sex. (E) Nissl stained coronal section of the P26 brain (top). Dashed box indicates location of the NAc. Representative images of female (bottom left) and male (bottom right) sections immunolabeled for zif268 and DAPI. Dashed lines indicate boundaries of the NAc. Scale bars = 200 µm. (F) Quantification of the density of zif268+ cells in the NAc. Welch’s t test t(25.589) = 2.8081, p = 0.009408. n = 16 rats per sex. (G) Quantification of the number of total microglia (left) and phagocytic microglia (right) during development in the PVN. n = 7 rats per sex. (H) Nissl stained coronal section of the P26 brain (top). Dashed box indicates location of the PVN. Representative images of female (bottom left) and male (bottom right) sections immunolabeled for zif268 and DAPI. Dashed lines indicate boundaries of the PVN. Scale bars = 100 µm. (I) Quantification of the density of zif268+ cells in the PVN. Welch’s t test t(27.247) = 2.4044, p = 0.02326. n = 16 rats per sex. Red/blue bars represent the mean +/- SEM for females/males. Open circles represent individual data points for each animal. Abbreviations: prefrontal cortex (PFC); nucleus accumbens (NAc); paraventricular nucleus (PVN). *p < 0.05 and **p < 0.01.

NEURON14678_proof_v102i2.pdf

Microglial Phagocytosis of Newborn Cells Is Induced by Endocannabinoids and Sculpts Sex Differences in Juvenile Rat Social Play

Introduction
Results

More Microglia Are Phagocytic in the Developing Male Amygdala
Phagocytic Microglia Engulf Newborn Cells in the Developing Amygdala
Testosterone Programs 2-AG Content, Newborn Cell Number, and Phagocytic Profile in the Developing Amygdala to Masculinize J ...
Microglia Engulf Viable Newborn Cells in a Complement-Dependent Manner to Masculinize Juvenile Play
Neonatally Born Cells Largely Differentiate into Astrocytes in the Juvenile Posterodorsal Medial Amygdala
Neonatal Microglia Phagocytosis Programs Juvenile Play Behavior, Astrocyte Density, and Neuronal Activation in the MePD

Discussion

Androgens Elevate Endocannabinoid Tone in the Amygdala during the Critical Period for Sexual Differentiation
Endocannabinoids Provide a Local Cue that Directs Phagocytosis of Viable Newborn Cells in a Complement-Dependent Manner
Juvenile Social Play Is Masculinized by Microglia-Mediated Reduction in Astrocyte Density of the MePD

Supplemental Information
Acknowledgments
Author Contributions
Declaration of Interests
References
STAR★Methods

Key Resources Table
Contact for Reagent and Resource Sharing
Experimental Model and Subject Details

Animal studies
Primary cell culture

Method Details

Animal treatments
Immunohistochemistry
Unbiased stereological cell counting
Image acquisition
Quantification of microglial activation state
Quantification of phagocytic cup contents, diameter, and three-dimensional rendering of microglia
Quantification of local cell-microglia interactions (“Microglia Interactome”)
Quantification of cell fate
Juvenile social play testing
Quantification of zif268 expression following a single social exposure
In vitro phagocytosis assay
Flow cytometry for complement proteins
Mass spectrometry
Western blot

Quantification and Statistical Analysis