Sex Difference in Cell Proliferation in Developing Rat Amygdala Mediated by Endocannabinoids Has Implications for Social Behavior

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Sex difference in cell proliferation in developing rat amygdala mediated by endocannabinoids has implications for social behavior

Desiree L. Krebs-Krafta, Matthew N. Hillb, Cecilia J. Hillardc, and Margaret M. McCarthya,d,1

Departments of aPhysiology and dPsychiatry, University of Maryland School of Medicine, Baltimore, MD 21201; bLaboratory of Neuroendocrinology, The Rockefeller University, New York, NY 10065; and cDepartment of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226

Edited* by Donald W. Pfaff, The Rockefeller University, New York, NY, and approved October 7, 2010 (received for review April 13, 2010) The amygdala is a sexually dimorphic brain region critical for the that endocannabinoid signaling can have dramatic inﬂuences on regulation of social, cognitive, and emotional behaviors, but both neural development. To date, most studies have focused on the the nature and the source of sex differences in the amygdala are CB1 receptor, with minimal investigation of the CB2 receptor largely unknown. We have identiﬁed a unique sex difference in the (14). This focus is in part because CB2 receptor distribution developing rat medial amygdala (MeA) that is regulated by canna- was considered limited to peripheral tissues and immune cells, binoids. Newborn females had higher rates of cell proliferation than although a functional role is emerging for CB2 receptors in neural males. Treatment of neonates with the cannabinoid receptor ago- stem cell proliferation (14, 15). In the current study, we report nist, WIN 55,212–2 (WIN), reduced cell proliferation in females to that a unique female-biased sex difference in cell proliferation in the of males and a wide range of WIN doses had no effect on cell pro- developing MeA that is mediated by the CB2 receptor and liferation in males. The effect of WIN on cell proliferation in the MeA accompanied by a sex difference in endocannabinoid content. was prevented by coinfusions of a CB2 but not CB1 receptor antag- Administration of cannabinoid receptor agonist, WIN 55,212–2 onist. Females had higher amygdala content of the endocannabinoid (WIN) to newborn female rats induced masculinization of juve- degradation enzymes, fatty acid amid hydrolase, and monoacylgly- nile play behavior, supporting the notion that endocannabinoid- cerol lipase than males, and lower amounts of the endocannabinoids

mediated reduction of cell proliferation within the MeA is a criti- NEUROSCIENCE 2-arachidonoylglycerol and N-arachidonylethanolamide (ananda- cal determinant of sexual dimorphism in this behavior. To our mide). Inhibition of the degradation of 2-arachidonoylglycerol in knowledge, this study is unique in reporting a role for endo- females occluded the sex difference in cell proliferation. Analyses cannabinoids in the establishment of a previously unexplored sex of cell fate revealed that females had significantly more newly gen- difference in cell proliferation in the developing brain, with erated glial cells but not more newly generated neurons than males, functional implications. and treatment with WIN significantly decreased glial cell genesis in females but not males. Finally, early exposure to cannabinoids mas- Results culinized juvenile play behavior in females but did not alter this be- Activation of Cannabinoid Receptors Occludes a Sex Difference in Cell fi havior in males. Collectively, our ndings suggest that sex dif- Proliferation in the Developing Amygdala. Injection of the cell cycle ferences in endocannabinoids mediate a sex difference in glial cell marker BrdU from postnatal day (PN)1 to PN4, revealed that fi genesis in the developing MeA that impacts sex-speci c behaviors females had more new cells in the developing amygdala than in adolescence. males on PN4 (Fig. 1). Treatment with the CB1/2 receptor agonist, WIN (1 mg/kg), on PN1 and PN2 occluded the observed sexual differentiation | neurogenesis | development sex difference on PN4, such that females treated with WIN had the same number of new cells as both control and WIN-treated he medial amygdala (MeA) is a sexually dimorphic nucleus crit- males [F(3,12) = 4.64, P < 0.05] (Fig. 1 B and C). WIN treatment Tical for modulating sex differences in juvenile rough-and-tumble had no effect in males. It has been reported previously that adult play (1), and regulation of adult social behaviors, including mating, males have larger MeA volumes than adult females (3, 16), parenting, aggression, and territoriality (2). The overall size of the which we have replicated in 33-d-old animals [F(1,14) = 6.39, rat MeA is larger in adult males than females (3) and is responsive to P < 0.05] (Fig. S1). To ensure that sex differences in cell genesis steroids in adulthood (4). Most of the well-characterized volumetric during the first postnatal week are not the result of bias in- sex differences in the brain are the result of differential cell death troduced by overall volume differences, we also compared the during a perinatal-sensitive period, with more cells dying in one sex MeA volume at PN4 in male and female animals. There was no than the other (5). sex difference or effect of treatment on MeA volume in 4-d-old A large body of evidence has accumulated in the last 10 y sup- animals [F(1,21) = 0.12, P > 0.05) (Fig. S1). To distinguish be- porting the important role of cannabinoid receptors and their en- tween sex differences in cell proliferation versus differential dogenous ligands in regulation of synaptic strength (6). It is clear, in survival of new cells in males versus females, we used a time- particular, that endocannabinoid signaling, via CB1 receptor acti- course sufficiently short to preclude a contribution from cell vation, subserves activity-dependent, retrograde signaling in many death. Females had significantly more BrdU+ cells than males brain regions. Cannabinoid receptors and endogenous cannabi- 8 h after treatment with WIN and 4 h after BrdU, indicating noid ligands are present and active early in brain development (7), and the tissue contents of the primary endocannabinoids N -arachidonylethanolamine (AEA) and 2-arachidonoylglycerol (2- Author contributions: D.L.K.-K. and M.M.M. designed research; D.L.K.-K. and M.N.H. per- AG) vary substantially throughout brain development. The 2-AG formed research; C.J.H. contributed new reagents/analytic tools; D.L.K.-K., M.N.H., and content peaks on the day of birth and dramatically decreases from M.M.M. analyzed data; and D.L.K.-K., C.J.H., and M.M.M. wrote the paper. there, whereas AEA content gradually increases throughout life (8). The authors declare no conflict of interest. Cannabinoid signaling has been implicated in cell proliferation (9), *This Direct Submission article had a prearranged editor. neurogenesis (10), neuronal differentiation (11), and synapto- 1To whom correspondence should be addressed. E-mail: [email protected]. genesis (12). Neural progenitors have functional CB1 and CB2 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. receptors and can synthesize endocannabinoids (13), demonstrating 1073/pnas.1005003107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1005003107 PNAS Early Edition | 1of6 Downloaded by guest on October 6, 2021 Fig. 1. Activating cannabinoid receptors occludes a sex difference in cell proliferation in the developing amygdala. (A). Rat pups were treated once daily from PN1 to PN4 with BrdU and vehicle or WIN (1 mg/kg) on PN0 and PN1, and their brains collected on PN4 for immunohistochemical detection of BrdU. (B). Data are expressed as mean ± SEM [n (total sample size) = 15]. Females have greater BrdU+ cell density than males (*P < 0.05) in the MeA at PN4. Activation of CB1/2 receptors with WIN on PN0 and PN1 obscures the sex difference by reducing the density of new cells in females (#P < 0.05). (C). Representative photomicrographs of BrdU+ cells in the MeA in males and females treated with vehicle or WIN. (D). Newborn males and females were treated with vehicle or WIN at 0 and 4 h in combination with a single injection of BrdU (300 mg/kg) and brains collected at 8 h and processed for detection of BrdU. (E). Data are expressed as mean ± SEM (n = 23). Females have greater BrdU+ cell densities in the MeA than males (*P < 0.05). Treatment with WIN decreased BrdU+ cell density in females (#P < 0.05) but not males (P > 0.05). (F). Data are expressed as mean ± SEM (n = 50). Again, females have greater BrdU+ cell density in the MeA than do males (*P < 0.05). Treatment with WIN (0.5, 1, or 10 mg/kg) decreased the BrdU+ cell density in females (#P < 0.05) but not males.

a sex difference in cell proliferation. This sex difference was Females Have More Endocannabinoid Degradative Enzyme and Lower occluded by WIN [F(1,19) = 4.27, P < 0.05], which reduced the Endocannabinoid Levels than Males in the Developing Amygdala. density of new cells in females to that of males (Fig. 1E). There After determining that both males and females have detectable was no sex difference in the density of pyknotic (i.e., dying) cells CB2 receptor protein in the MeA (Fig. S4), we hypothesized that in the developing amygdala at PN4 [F(1,11) = 0.12, P < 0.05] lower endogenous cannabinoid signaling mediates the enhanced cell genesis in the MeA of females. The endocannabinoids are (Fig. S2). To determine whether males have reduced sensitivity made-on-demand, so their tissue contents are thought to reflect to exogenous cannabinoids, we conducted a dose-response their signaling concentrations (17). In support of our hypothesis, analysis and found that three doses of WIN (0.5, 1.0, or 10 mg/ newborn males had higher 2-AG content in the amygdala than kg) significantly decreased cell proliferation in the developing females on both PN0 and PN4 [PN0: F(1,16) = 23.15, P < 0.05; amygdala of females but had no effect in males [F(3,41) = 3.00, PN4: F(1,14) = 6.66, P < 0.05] (Fig. 3C). Newborn males also P < 0.05] (Fig. 1F). had higher AEA content in the amygdala on the day of birth [F(1,16) = 4.74, P < 0.05] but not on PN4 [F(1,14) = 0.11, P > CB2 Receptor Activation Reduces Cell Proliferation in the Developing 0.05] (Fig. 3C). There were no sex differences in the amygdala Female Amygdala to That of Males. WIN is a nonselective CB1/2 contents of oleoylethanolamide and palmitoylethanolamide, N- receptor agonist; therefore, we determined the receptor mediat- acylethanolamines that do not bind cannabinoid receptors (Fig. ing the effects of WIN by combining treatment with either a se- S5). Endocannabinoid levels are regulated by both biosynthetic lective CB1 or CB2 receptor antagonist to explore which of these and catabolic processes (17). We determined the amounts of the receptors is involved in the regulation of amygdala cell genesis. primary catabolic enzymes for AEA and 2-AG, fatty acid amide There was no effect of either antagonist alone and so the treat- hydrolase (FAAH) and monoacylglycerol lipase (MAGL), re- spectively, using Western blot analysis. Females had significantly ment groups were collapsed (Fig. S3). Coinfusions of WIN and the more FAAH protein than males in the amygdala at PN3 [t(10) = CB2 receptor antagonist AM-630 (3 mg/kg) prevented the WIN- 6.25, P < 0.05] but only a statistical trend at PN11 [t(11) = 4.30, + F induced decrease in BrdU cell density in females [ (7, 50) = P = 0.06] (Fig. 3B). Females also exhibited a statistical trend P < B 2.40, 0.05] (Fig. 2 ). In contrast, coinfusion of WIN and the toward more MAGL protein than males in the amygdala at PN3 CB1 receptor antagonist AM-281 (3 mg/kg) did not prevent the [t(12) = −1.65, P = 0.08] and a difference that reached signifi- WIN-induced decrease in BrdU+ cell density in the MeA of cance at PN11 [t(11) = −11.46, P < 0.05] (Fig. 3B). These females (P > 0.05). findings are consistent with the hypothesis that males have higher

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1005003107 Krebs-Kraft et al. Downloaded by guest on October 6, 2021 Fig. 3. Females have more endocannabinoid degradative enzyme and lower endocannabinoid levels than males in the developing amygdala. (A) The amygdala of males and females were collected and processed for Western blot analysis of FAAH and MAGL protein levels and HPLC quanti- Fig. 2. CB2 receptor activation reduces cell proliferation in the developing fication of AEA and 2-AG at multiple developmental time points. (B) Data amygdala to that of males. (A) Newborn males and females were treated are expressed as mean ± SEM (n = 14 at PN3; n = 12 at PN11). There was with vehicle, WIN (1 mg/kg), the CB1 receptor antagonist AM-281 (3 mg/kg), significantly more FAAH (*P < 0.05) and a tendency toward more MAGL ($P = the CB2 receptor antagonist AM-630 (3 mg/kg), or combinations of WIN and 0.07) in the female amygdala on PN3, and this sex difference persisted until either the CB1 or CB2 receptor antagonist at 0 and 4 h in combination with PN11 for MAGL (*P < 0.05) but only tended to persist for FAAH ($P = 0.06). NEUROSCIENCE a single injection of BrdU and brains were collected at 8 h and processed for (C) Data are expressed as mean ± SEM (n = 18 at PN0; n = 16 at PN4). There detection of BrdU. Data are expressed as mean ± SEM (n = 59). In a separate was significantly more AEA and 2-AG in male amygdala at PN0 (*P < 0.05), experiment, newborn males and females were treated with vehicle, the and the sex difference in 2-AG persisted until PN4 (*P < 0.05). FAAH inhibitor JNJ (20 mg/kg), or the MAGL inhibitor NAM (1 mg/kg) at 0 and 4 h in combination with a single injection of BrdU and brains were collected at 8 h and processed for detection of BrdU. Data are expressed as F(3,31) = 3.53, P < 0.05] (Fig. 4 C and D). The overall density of + mean ± SEM (n = 67). (B). There were more BrdU cells in the female MeA GFAP+ cells was not affected by sex or treatment at either time than male (*P < 0.05) and treatment with WIN decreased BrdU+ cell density # point. However, the number of cells in which BrdU and GFAP in females ( P < 0.05) but not males. Combined treatment with WIN and AM- fi 630, but not AM-281 prevented the sex difference in BrdU+ cell density (*P < were colocalized was signi cantly higher in females and de- 0.05, vs. male-vehicle; %P > 0.05, vs. female-vehicle). (C). There were again creased by WIN treatment in females but not males at both times more BrdU+ cells in the female amygdala than male (*P < 0.05) and in- [PN4: F(3,23) = 3.82, P < 0.05; PN14: F(3,31) = 3.62, P < 0.05] hibition of MAGL decreased the number in females (#P < 0.05) but not males. (Fig. 4 C and D). The number of newly generated neurons was Inhibition of FAAH attenuated BrdU+ cell densities in females ($P > 0.05 vs. assessed by confocal microscopy of double-label immunofluo- control females or males). rescence for BrdU and the neuronal marker, NeuN (Fig. 4E). Although females had more BrdU+ cells in the MeA than males (P < 0.05) and treatment with WIN decreased BrdU+ cells in endocannabinoid-mediated signaling than females and suggest females but not males [F(1,40) = 4.18, P < 0.05] (Fig. 4F), the further that higher expression of the catabolic enzymes in female density of NeuN+ cells and BrdU+/NeuN+ cells were not affected amygdala contributes to this difference. We examined this notion by sex or WIN treatment (Fig. 4F). by treating newborn pups with either a FAAH (JNJ 1661010) or a MAGL [N-arachidonylmaleimide (NAM)] inhibitor before Neonatal Cannabinoid Treatment Masculinizes Play Behavior in Juve- + measurement of the density of BrdU cells. Treatment with the nile Females. As exogenous cannabinoid agonists abolish the sex MAGL inhibitor, but not the FAAH inhibitor, significantly re- difference in cell genesis in the MeA, we examined the effects of duced the density of BrdU+ cells in females but not in male pups neonatal WIN treatment on sex differences in juvenile social [F(5,61) = 3.90, P < 0.05] (Fig. 2C). These data support the hy- play, anxiety, and locomotion in the open-field test. As expected, pothesis that the concentration of 2-AG in the developing amyg- males exhibited more social play behavior than females. How- dala is greater in males than females, in part because of lower ever, treatment with WIN increased the duration of play in MAGL activity, and that this results in reduced cell genesis. females to that of males [F(3,36) = 2.98, P < 0.05] (Fig. 5B). There was no significant effect of sex or WIN treatment in either Cannabinoids Reduce Astrocyte Density in the Developing Female anxiety or locomotor responses in the open-field test (Fig. S6). Amygdala to That Seen in Males. To determine the mature phe- notype of the newly proliferating cells being impacted by canna- Discussion binoids in the developing MeA, we used double-label immu- We report here that more new cells are born in the amygdala of nohistochemistry for BrdU and GFAP, a marker for mature neonatal females than males and that this sex difference is a result astrocytes (Fig. 4B). Consistent with our other findings, newborn of higher rates of cell proliferation in females. Furthermore, the females had more BrdU+ cells in the MeA than males at PN4, sex difference in cell proliferation was caused by enhanced as- and this was still true at 2 wk of age (P < 0.05 compared with trocyte cell genesis in female animals, with no differences in males for both time points). Treatment with WIN significantly neurogenesis between males and females. Our pharmacological decreased the number of BrdU+ cells in females but not males and biochemical data support the hypothesis that greater CB2 at both time points [PN4: F(3,23) = 11.68, P < 0.05; PN14: receptor activation in males, possibly as a result of decreased

Krebs-Kraft et al. PNAS Early Edition | 3of6 Downloaded by guest on October 6, 2021 Fig. 4. Cannabinoids reduce the number of astrocytes in the developing female amygdala to that seen in males. (A) Males and females were treated PN1-4 with BrdU (50 mg/kg) and on PN 0 and PN1 with vehicle or WIN (1 mg/kg) to determine whether WIN treatment would decrease the number of BrdU, GFAP, BrdU/GFAP, NeuN, and BrdU/NeuN immunoreactive cells in the rat MeA at PN4 or PN14. (B) Representative photomicrographs of BrdU+, GFAP+, and BrdU/ GFAP+ cells in the developing MeA. [Scale bars, 250 μm(4×)or25μm(60×).] (C) Data are expressed as mean ± SEM (n = 29). On PN4, females had more BrdU+ and BrdU/GFAP+ cells in the MeA than males (*P < 0.05) and activation of CB1/2 receptors eliminated the sex difference (#P < 0.05, vs. female-vehicle). (D) Data are expressed as mean ± SEM (n = 35). On PN14, females still had more BrdU+ and BrdU/GFAP+ cells than males (*P < 0.05, vs. male-vehicle), and this sex difference was again eliminated by prior treatment with WIN (#P < 0.05, vs. female-vehicle). (E) Representative photomicrographs of BrdU+, NeuN+,and BrdU/NeuN+ cells in the developing MeA. Cells were visualized by fluorescent confocal microscopy on a Grid Confocal at 100×. (Scale bars, 17 μm.) (F) Data are expressed as mean ± SEM (n = 44). Quantification of fluorescently detected BrdU also revealed that females had more BrdU+ cells in the MeA than males (*P < 0.05, vs. male-vehicle) and activation of CB1/2 receptors again eliminated the sex difference (#P < 0.0, vs. female-vehicle). There was no sex difference or effect of neonatal WIN treatment on the density of NeuN+ or BrdU/NeuN+ cells in the MeA.

endocannabinoid catabolism, underlies the sex difference in as- riod could be specific to those developmental ages and underlie trocyte proliferation. In accord with this hypothesis, we have found functional differences associated with development, such as the that treatment of neonatal females with exogenous CB receptor correlation we observed here between cell proliferation and ju- agonists induces a male profile of juvenile play behavior. venile play behavior. The current report of more proliferating cells in the female Our observation that the sex difference in proliferation were than male MeA at PN4 at first appears in contrast to the findings cells that coexpressed an astrocyte—not neuronal—cell marker, in adult rats, where the male MeA is larger than that of females could mediate the known sex difference in synaptic patterning (3, 18), with more and bigger neurons (16, 19), greater dendritic within the mature MeA (18, 22), as has been seen for other brain length, and higher spine density (16, 20). To date, the majority of regions (23). There are conflicting reports on sex differences in sex differences in the size of a particular brain region have been astrocytes in the adult MeA, with some finding higher GFAP attributed to differences in the total number of neurons as a re- content in the MeA of females (24); others report fewer and less sult of differential cell death (5). The findings reported here are complex astrocytes in females (25). Elevated hormones associ- surprising because the bias is toward more cell proliferation in ated with puberty also impact the final differentiation of the females and because very few sex differences in cell proliferation brain (26), including sex differences in adolescent neurogenesis have been reported (21), suggesting that sex differences in pro- in the MeA (27). liferation may not mediate the establishment of sex differences Our data support the hypothesis that differences in endo- in volume of particular brain regions. Moreover, sex differences cannabinoid/CB2 receptor signaling underlie the sex difference in in cell proliferation during the early postnatal and juvenile pe- cell proliferation in the MeA. First, we found that activation of

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1005003107 Krebs-Kraft et al. Downloaded by guest on October 6, 2021 However, many sex differences in the adult brain are the result of the organizational action of neonatal androgens from the testis and brain synthesis of estrogens (33). Neonatal exposure to steroid hormones during a sensitive period permanently alters the morphology and synaptic patterning of the brain and results in sex differences in physiology and behavior in juveniles and adults (33). The play of juvenile males is more frequent and of longer duration than females and neonatal treatment of females with estradiol or testosterone masculinizes these parameters of play (34, 35). Data indicate that the effects of the steroids are mediated by the amygdala (35) and correlate with increased excitatory synapses (22). In the present study, we found that early exposure to cannabinoids masculinizes social play in females, implicating cannabinoid signaling in the establishment of normal Fig. 5. Neonatal cannabinoid treatment masculinizes play behavior in ju- sex differences in the brain. This is in contrast to studies in which venile females. (A) Male and female rats were treated with vehicle or WIN at prenatal cannabinoid exposure is associated with enduring im- PN0 and PN1, weaned on PN21, and housed in same-sex groups of three pairments in cognitive and social functions (36). An important until testing. (B) Data are expressed as mean ± SEM (n = 41). Control males played more frequently than females (*P < 0.05) but WIN treatment in- next step will be determining the effects of gonadal hormones on creased the total occurrence of play behavior in females (#P < 0.05 vs. control the endogenous cannabinoid system and cell genesis to further females) to the level seen in males; WIN treatment had no effect in males. place these findings in the context of organization of sex differences during normal brain development. In summary, we have identified a unique sex difference in cell CB1/2 receptors with WIN had no effect on proliferation in males proliferation that favors females in the developing MeA. Fur- but caused a significant reduction in females. The effect of WIN thermore, these data indicate a pivotal role of endocannabinoids was reversed by coadministration of a CB2, but not a CB1, re- and CB2 receptors in cell proliferation and astrocyte genesis in ceptor antagonist, suggesting that cell proliferation in the female females. Developmental cannabinoid exposure masculinizes as- MeA is suppressed by CB2 receptor activation. These results trocyte numbers in the MeA and juvenile social play behavior.

could be explained in two ways: (i) cell proliferation in males is These findings are unique in implicating cannabinoid regulation NEUROSCIENCE insensitive to CB2 receptor activation, or (ii) CB2 receptor reg- of cell proliferation and cell type as a mechanism for sexual ulation of cell proliferation occurs in both males and females differentiation of the developing brain and behavior. Elucidating but endogenous CB2 signaling is maximally activated in males, the normal function of endogenous signaling molecules that are thereby occluding effects of WIN. In support of the second pos- readily mimicked by drugs of abuse is essential to understanding sibility, Western blot analyses indicate that both males and the consequences of inappropriate exposure, and how those females have CB2 receptor protein. More importantly, male pups consequences can be avoided or best remedied. had significantly higher amounts of the endocannabinoid that activates CB2 receptors, 2-AG, than females, which is consistent Materials and Methods with the hypothesis that the CB2 receptor in males is maximally Subjects. Timed pregnant Sprague-Dawley rats (Harlan) mated in our facility activated. Males also had less of the primary catabolic enzyme for were allowed to deliver normally under standard laboratory conditions. Food and water were available ad libitum. The University of Maryland, Baltimore 2-AG, MAGL, and inhibition of MAGL in females prevented the Institutional Animal Care and Use Committee approved all animal procedures sex difference in cell proliferation in the MeA. These data suggest (SI Materials and Methods). that 2-AG is the endocannabinoid involved and that differences in MAGL activity underlie the sex difference observed. These Drug Preparation and Administration. Male and female rat pups were given findings do not rule out a role for AEA in cell genesis because intraperitoneal injections of BrdU (50 or 300 mg/kg; Sigma) or subcutaneous MAGL inhibition could also affect AEA content (28); however, the injections of vehicle (0.1% DMSO in sterile saline, pH = 7.0), the CB1/2 receptor lack of effect of a FAAH inhibitor argues against a role for AEA. agonist WIN 55–212,2 mesylate (0.5, 1, or 10 mg/kg), the CB1 receptor an- Contrary to expectation, treatment of male pups with the CB2 tagonist AM-281 (3 mg/kg;), the CB2 receptor antagonist AM-630 (3 mg/kg), the FAAH inhibitor 4-(3-phenyl-[1,2,4]thiadiazol-5-yl)-piperazine-1-carbox- receptor antagonist, AM-630, did not increase cell proliferation. ylic acid phenylamide (JNJ 1661010) (20 mg/kg), or the MAGL inhibitor NAM One possibility is that males have delayed maturation of the endo- (1 mg/kg). All drugs, with the exception of BrdU, were purchased from Tocris cannabinoid system and subsequently delayed cell proliferation in Cookson, Inc. comparison with females. This possibility seems unlikely, given males have greater endocannabinoid content, and we detected Immunohistochemistry and Immunofluorescence. Briefly, coronal brain sec- CB2 receptor protein in males by Western blot, although this does tions (45 μm thick) through the rostral-caudal extent of the extended not speak to the functionality of CB2 receptors in males versus amygdala were double-labeled with BrdU and an astrocyte specific marker, fi GFAP, or the mature neuronal maker neuronal nuclear antigen (NeuN). Free- females. More likely is that activation of CB2 receptors is suf - fl cient to serve as a stop signal for cell proliferation, but multiple oating tissue sections were processed for BrdU or BrdU/GFAP immunohistochemistry using protocols our laboratory has previously published using signaling steps in addition to CB2 receptor silencing are required monoclonal anti-BrdU antibody (1:10,000 in PBS-T; Caltag Laboratories) and to enhance cell proliferation. Thus, pharmacological antagonism a polyclonal antibody against GFAP (1:10,000; Sigma) and diaminobenzidine fi of the CB2 receptor is not suf cient to induce cell proliferation (DAB)- and nickel-DAB- H2O2 substrate (21). For detection of new neurons, in the developing amygdala of males, but instead requires en- tissue sections were incubated simultaneously in rat anti-BrdU (1:200; gagement of other as-yet-unidentified parameters, such as the Abcam) and mouse monoclonal anti-NeuN (1:1,000; Chemicon). The BrdU presence of growth factors and activation of signaling cascades primary antibody was used in conjunction with biotinylated anti-rat sec- important for cell genesis. ondary (1:500; Vector) for 2 h and Streptavidin Alexa488 (1:500; Invitrogen). NeuN antibody was used in conjunction with secondary goat anti-mouse In adulthood, females are more susceptible to cannabinoid- fi Alexa Fluor568 (1:500; Invitrogen). See SI Materials and Methods for induced antinociception, locomotion, and memory de cits (29, more information. 30). Thus far, the majority of sex differences in the endocannabinoid system or cannabinoid-induced behavioral effects have Immunohistochemical Analysis. Quantification of DAB stained tissue was been attributed to fluctuations in gonadal steroid levels (31, 32). performed with a Nikon microscope at 60× magnification and Neurolucida

Krebs-Kraft et al. PNAS Early Edition | 5of6 Downloaded by guest on October 6, 2021 + 2 2 software (MicroBrightField, Inc.). The density of BrdU and GFAP Sigma cells 84 pmol of [ H8]anandamide and 186 pmol of [ H8]2-AG. Tissue was ho- was determined in a counting frame (300 × 300 μm) in both hemispheres of mogenized in glass culture tubes and lipids were extracted and stored in the MeA. The density of immunopositive cells was averaged from four sec- methanol until analysis. The contents of the two primary endocannabinoids, tions (45 μm thick) of each brain. AEA and 2-AG, within lipid extracts in methanol from brain tissue were determined using isotope-dilution, liquid chromatography−mass spectrom- Confocal Microscopy. The density of BrdU and NeuN+ cells was determined etry (SI Materials and Methods). using a Volocity Grid Confocal system (SI Materials and Methods). Confocal z-stacks (10–12 μm) were collected sequentially in the two channels at Behavioral Assessment. Play behavior. The social play testing was carried out at + 0.5-μm intervals using a 100× oil objective. The BrdU cells were examined in PN28 to PN38. Briefly, males and females were weaned on P21 and housed in + orthogonal views (0, 90, 180°) to determine whether BrdU cells are colo- same-sex groups of three. Six animals from different sex and treatment calized with NeuN. The density of immunopositive cells in the MeA was conditions were video-recorded in a neutral arena for four 2-min intervals per μ averaged from both hemispheres of four sections (45 m) of each brain day over 5 d. The total number of aggressive play behaviors were scored, × μ using an 80 100- m counting frame. summed, and then averaged per interval by an observer blind to treatment (SI Materials and Methods). Western Blot Analysis. All animals were euthanized by rapid decapitation and Open field. The number of central, intermediate, outer, and total grid amygdala tissue was dissected and processed for Western blot analysis (SI crossings in the open-field arena were recorded for 5 min by an experimenter Materials and Methods). The membranes were incubated in the primary blind to treatment (SI Materials and Methods). antibody, anti-FAAH (1:500; Santa Cruz) or anti-MAGL (1:500; Axxora) and an anti-rabbit HRP-linked secondary antibody (1:3,000; Cell Signaling Technol- Statistical Analyses. The data were expressed as means and SEMs and analyzed ogies). The immunoreactive bands were detected using chemiluminesence using Student’s t tests, one-way ANOVA, or univariate two-factor ANOVA as and the integrative grayscale pixel area-density (IAD) was analyzed with appropriate (SI Materials and Methods). Tukey HSD or Fisher LSD post hoc National Institutes of Health image software. The membranes were then tests were used to detect group differences (See SI Results for detailed incubated in a Ponceau S (Sigma) stain and the density of the largest band on analyses). An α-level of P < 0.05 was required for statistical significance. the membrane was used as a loading control to standardize the total protein values. ACKNOWLEDGMENTS. This research was supported by National Institutes of Health Grants R01 MH052716 (to M.M.M.) and R21 DA022439 (to C.J.H.), and Endocannabinoid Extraction and Analysis. Brain regions were subjected to a Cellular and Integrative Neuroscience Training Grant to the University of a lipid extraction process. Briefly, tissue samples were weighed and placed Maryland T32 NS07375 (to D.L.K.-K.); M.N.H. was supported by a Canadian into borosilicate glass culture tubes containing two ml of acetonitrile with Institutes of Health Research Postdoctoral Fellowship.

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