ARTICLE

Received 25 Apr 2014 | Accepted 16 Jun 2014 | Published 17 Jul 2014 DOI: 10.1038/ncomms5421 Endocannabinoids modulate cortical development by configuring Slit2/Robo1 signalling

Ala´n Alpa´r1,w, Giuseppe Tortoriello1, Daniela Calvigioni1,2, Micah J. Niphakis3, Ivan Milenkovic4, Joanne Bakker2, Gary A. Cameron5,Ja´nos Hanics6, Claudia V. Morris7,Ja´nos Fuzik2, Gabor G. Kovacs4, Benjamin F. Cravatt3, John G. Parnavelas8, William D. Andrews8, Yasmin L. Hurd7, Erik Keimpema1,2,* & Tibor Harkany1,2,*

Local environmental cues are indispensable for axonal growth and guidance during brain circuit formation. Here, we combine genetic and pharmacological tools, as well as systems neuroanatomy in human fetuses and mouse models, to study the role of endocannabinoid and Slit/Robo signalling in axonal growth. We show that excess 2-arachidonoylglycerol, an endocannabinoid affecting directional axonal growth, triggers corpus callosum enlargement due to the errant CB1 cannabinoid receptor-containing corticofugal axon spreading. This phenotype mechanistically relies on the premature differentiation and end-feet proliferation of CB2R-expressing oligodendrocytes. We further show the dependence of both axonal Robo1 positioning and oligodendroglial Slit2 production on cell-type-specific cannabinoid receptor activation. Accordingly, Robo1 and/or Slit2 manipulation limits endocannabinoid modulation of axon guidance. We conclude that endocannabinoids can configure focal Slit2/Robo1 signalling to modulate directional axonal growth, which may provide a basis for understanding impaired brain wiring associated with metabolic deficits and prenatal drug exposure.

1 Division of Molecular Neurobiology, Department of Medical Biochemistry & Biophysics, Karolinska Institutet, Scheeles va¨g 1:A1, SE-17177 Stockholm, Sweden. 2 Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Spitalgasse 4, A-1090 Vienna, Austria. 3 Department of Chemical Physiology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA. 4 Institute of Neurology, Medical University of Vienna, AKH 4J, Wa¨hringer Gu¨rtel 18-20, A-1090 Vienna, Austria. 5 Institute of Medical Sciences, Liquid Chromatography-Mass Spectrometry Section, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK. 6 Department of Anatomy, Histology and Embryology, Semmelweis University, Tu+zolto´ u. 58, H-1094 Budapest, Hungary. 7 Departments of Psychiatry and Neuroscience, Icahn School of Medicine at Mount Sinai, 1470 Madison Avenue, New York, New York 10029, USA. 8 Department of Cell and Developmental Biology, University College London, 21 University Street, London WC1E 6DE, UK. w Present address: Research Group of Experimental Neuroanatomy and Developmental Biology, Hungarian Academy of Sciences, Budapest, Hungary; Department of Anatomy, Histology and Embryology, Semmelweis University, Budapest, Hungary. * T.H. and E.K. share senior authorship. Correspondence and requests for materials should be addressed to T.H. (email: [email protected]).

NATURE COMMUNICATIONS | 5:4421 | DOI: 10.1038/ncomms5421 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5421

xonal pathfinding decisions rely on short- and long-range employed the small molecule JZL184 from embryonic day intercellular cues determining cytoarchitectural arrange- (E)12.5–18.5 to inhibit monoacylglycerol lipase (MGL), the Aments permissive for directional growth in the developing enzyme chiefly responsible for degrading 2-AG in the nervous brain. Guidance cues include a plethora of structurally hetero- system23. The selective inhibition of MGL by JZL184 was geneous proteins, released during defined temporal windows and confirmed with activity-based protein profiling of serine acting within circumscribed spatial domains. Slits, a family of hydrolases in the brains of JZL184 and vehicle-treated dams phylogenetically conserved chemorepellent molecules1, bind and their E18.5 embryos. Consistent with previous reports24, Roundabout (Robo)1/2 receptors2,3 to regulate axonal tract JZL184 displayed a high selectivity for MGL across all detectable formation during forebrain development4–8. Slit/Robo1/2 brain serine hydrolases (Fig. 1a) leading to a significant increase signalling is implicated in midline crossing9–12 and the in 2-AG levels in adult and embryonic cortices (Fig. 1b). While dorsoventral positioning of long-range axons13,14, thus being fatty-acid amide hydrolase (FAAH)25, which catabolizes the indispensable for the basic forebrain wiring diagram and endocannabinoid anandamide (AEA), was partially inhibited by connectivity principles to emerge. Accordingly, Robo1 À / À JZL184 in adult and to a lesser degree, embryonic brain samples, mice present the dysgenesis of major commissural tracts5 and we did not observe a significant increase in AEA levels in fetal or axon guidance errors in the corticofugal system4. Similarly, adult cortices (Fig. 1b). The levels of 2-linoleoyl glycerol (a Slit2 À / À and Slit1 À / À /Slit2 À / À mice show corticothalamic- congener of 2-AG) and oleoylethanolamide (an analogue of and thalamocortical-targeting defects8, as well as optic tract AEA), were both unaffected by JZL184 treatment (Fig. 1b). abnormalities12. In addition, axons coalesce into enlarged Together, these data confirm that JZL184 selectively targets MGL fascicles in Robo1 À / À mice5. Heparin sulphate proteoglycans leading to an accumulation of 2-AG in the brains of gravid adult and transcription factors, including members of the T-box or the mice and their offspring. LIM homeodomain families can influence Slit/Robo1/2 signalling14. Nevertheless, the molecular identity of signalling networks for the upstream and local control of coordinated Slit/ MGL inhibition induces axon fasciculation deficits. We studied Robo1/2 expression, subcellular positioning and signal the morphology of corticofugal axons in developing fetal brains competence during corticogenesis remains relatively poorly after JZL184 exposure, since these tracts contain the highest levels 18 explored. of CB1Rs during embryonic development . JZL184 exposure Signalling lipids have recently emerged as regulators of induced L1 neural cell adhesion molecule (L1-NCAM) þ axons to neuronal development. Endocannabinoids, the natural ligands coalesce into enlarged fascicules (Fig. 2a,b; Supplementary of cannabinoid receptors, modulate both neuronal15 and glial16 Table 1) irrespective of the rostrocaudal axis of the developing differentiation, and are efficacious to facilitate neurite extension brain (Supplementary Fig. 1a–c). The enlarged axon fascicles were 17–19 and growth cone-steering decisions .CB1 cannabinoid found spread in the cortical plate, including intermediate zone þ receptors (CB1Rs) contribute to regulating the fasciculation and territories otherwise devoid of L1-NCAM processes (Fig. 2i,j; 17 pallidal targeting of corticofugal axons . In contrast, CB2 Supplementary Fig. 1a). cannabinoid receptors (CB2Rs) are co-expressed with key The coalescing of axonal fibres upon JZL184 exposure was enzymes of 2-arachidonoylglycerol (2-AG) metabolism in glia20, altered irrespective of their origin or projection targets. In particular, and promote the differentiation of cultured oligodendrocytes21, enlarged fascicles were observed to cross the midline (Fig. 2e,f), the cellular source of myelin sheaths in the nervous system22. populated the suprastriatal domain of the corpus callosum Here, we show that pharmacological enhancement of 2-AG (Fig. 2m,n; Supplementary Fig. 2a) or invaded the striatum bioavailability during corticogenesis in mouse triggers the (Fig. 2o,p). Both corticothalamic and thalamocortical axons were CB2R-dependent differentiation of oligodendrocytes. Premature affected in the striatum, as revealed by TAG-1 (refs 26,27) and oligodendrocyte end-feet proliferation in vivo coincides with C-terminal full length of mouse GPR55 (ref. 17) immuno- CB1R-dependent fasciculation errors of corticofugal projections, histochemistry, respectively (Supplementary Fig. 2a–e,r). These including altered axon bundle formation in the paramedian findings suggest the multimodal interplay of corticofugal and domain of the corpus callosum. We hypothesized that endocan- thalamocortical projections along their reciprocal growth paths nabinoids, by simultaneously engaging cell-type-specific during the mechanism termed axonal ‘handshake’28. cannabinoid receptors, might orchestrate chemorepulsion of Next, we examined mice carrying green fluorescent protein cortical axons via a mechanism operating in trans, with (GFP)-tagged cytosolic tau in neurons29 in the presence or absence gfp/gfp 29 þ oligodendrocytes producing surplus chemorepellents. By imple- of CB1Rs (tau ::CB1R) to show that radial glia (RC2 )fibres menting a multi-parametric approach, encompassing neuroanat- and post-mitotic Brn1 þ pyramidal cells16 arranged perpendicular omy in human and mouse fetuses, messenger RNA (mRNA) to corticofugal axons and adopted alternative paths (Fig. 2i,j). We À / À profiling, biochemistry, in vitro analysis and genetic models, we then administered JZL184 to CB1R fetuses to test the reliance identify oligodendrocyte-derived Slit2 acting at Robo1 receptors of pharmacologically induced axon guidance errors on CB1Rs, in corticofugal axons downstream from the respective activation selectively localized to the corticofugal system by mid-gestation18. 18,30 of CB2Rs and CB1Rs upon pharmacological manipulation of In accord with previous reports ,CB1R deletion alone induced À / À 2-AG degradation as the molecular underpinning of endocanna- axon fascicle enlargement (Fig. 2c,g,k,q). In CB1R animals, binoid-induced corticofugal axon guidance errors. We find JZL184 did not affect the diameter of first-order fascicles cannabinoid receptor activation to facilitate Slit2 accumulation (18.78±1.66 mm (JZL184) versus 16.04±1.28 mm (vehicle) À / À in oligodendrocyte end feet, and Robo1 accumulation in axonal (mean±s.e.m.; n ¼ 3 mice per group); Fig. 2d,h,l,q). CB1R growth cones. Overall, these data suggest that endocannabinoid mice treated with JZL184 presented significantly increased axon signalling can regulate Slit/Robo interactions during the establish- spreading in the intermediate zone/corpus callosum ment of forebrain axonal tracts. (203.81±8.44 mm (JZL184) versus 157.27±6.28 mm (vehicle), n ¼ 3micepergroup,Po0.05 (Student’s t-test); Fig. 2q). This was associated with altered distribution of radial glia processes and Results Brn1 þ pyramidal cell radially migrating towards the cortical JZL184 increases 2-AG levels in fetal and adult cortices.To plate—to avoid axon-packed microdomains—in taugfp/gfp:: À / À enhance endogenous 2-AG signalling in gravid mice, we CB1R reporter mice (Fig. 2l).

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Maternal Maternal cortex cortex Embryonic cortex (E18.5) 300 25 600 180 ** 250 150 250 20 500 200 400 120 150 15 150 300 90 100 10 100 200 60 5 75 50 100 30 AEA content (pmol/g tissue) 2-AG content (nmol/g tissue) 2-AG OEA content (pmol/g tissue) 0 0 0 0 FAAH Vehicle Vehicle Vehicle Vehicle JZL184 JZL184 JZL184 50 JZL184 Embryonic cortex 50 10 200 100

37 40 * 150 75 MAGL ABHD6 30 5 100 50 20

Embryonic cortex 50 25 25 10

(kDa) 2-LG content (pmol/g tissue) 2-LG content (pmol/g tissue) OEA content (pmol/g tissue) 2-AG content (nmol/g tissue) 2-AG 0 AEA content (pmol/g tissue) 0 0 0

JZL184 –+–+–+–+ Vehicle Vehicle Vehicle Vehicle JZL184 JZL184 JZL184 JZL184 Figure 1 | JZL184 increases 2-AG levels in maternal and embryonic brains. (a) Ex vivo activity-based protein profiling of brain-derived serine hydrolases in vehicle and JZL184 (40 mg kg À 1)-treated adult and fetal cortices. (b) Select 2-monoacyl glycerol and N-acyl amide levels in maternal and fetal brains correlatively measured after JZL184 treatment. Data were expressed as means±s.e.m., n ¼ 2 per group (mothers), n ¼ 6 per group (fetuses); **Po0.01, *Po0.05 (Student’s t-test).

The dynamic control of axonal growth and fasciculation regulated kinase 1/2 (Erk1/2) phosphorylation as a read-out36 involves the elimination of functionally redundant or mistargeted upon acute agonist challenge (WIN55,212-2, 100 nM) of cultured processes over time. Mature axons become embedded in cortical neurons pre-treated with JZL184 for 4 days to address extracellular matrix, a three-dimensional scaffold of molecularly whether JZL184 induced CB1R desensitization. JZL184 alone did heterogeneous proteins31, to maintain their positions in axon not induce constitutive Erk1/2 phosphorylation, and AM251, a fascicles. Here, we find that neurocan, a chondroitin sulphate CB1R antagonist, did not modify JZL184 effects. Notably, JZL184 proteoglycan whose expression is essential for cell adhesion and allowed WIN55,212-2-induced rapid Erk1/2 phosphorylation slows neurite extension32, is excluded from enlarged axonal (Fig. 2t,u) with comparable temporal dynamics to that in fascicles upon JZL184 treatment (Fig. 2m–p), suggesting altered control (Supplementary Fig. 4c), which is compatible with neurite motility and pathfinding. The JZL184-induced earlier data showing that only prolonged (48 days) agonist 36 remodelling of the corticofugal system also impacted the stimulation induces CB1R desensitization in vitro . These data coincident patterning of thalamocortical afferents suggest that supra-physiological 2-AG signals upon JZL184 (Supplementary Fig. 2a–e), robustly affecting their ‘handshake’28. application during corticogenesis alter axon fasciculation via Nevertheless, the JZL184-induced axonal phenotype was neuronal CB1Rs, while a non-CB1R-mediated mechanism might absent in constitutive MGL À / À mice (Supplementary Fig. 3). account for the increased axonal spread in the prospective corpus Chronic 2-AG signalling in MGL À / À mice is characterized by callosum. CB1R downregulation and desensitization, suggesting compensa- 23 tion for increased CB1R signalling events . Inhibition of MGL by Oligodendrocyte end feet proliferate among misrouted axons. JZL84 in our experimental model (E12.5-E18.5) would likely Excessive spreading of corticofugal axons committed to the À / À induce excessive CB1R signalling, resulting in physiological prospective corpus callosum in JZL184-treated CB1R mice À / À alterations otherwise compensated for in MGL mice. Thus, led us to hypothesize that a CB1R-independent mechanism can we conclude that excess 2-AG availability brought about by the alter the pathfinding of corticofugal axons. Therefore, we tested pharmacological disruption of their degradation during defined whether premature differentiation of oligodendrocytes, which periods of corticogenesis is sufficient to remodel the corticofugal postnatally produce myelin to ensheath callosal axons22 and co- 20 projection system. express CB1Rs and CB2Rs , could participate in axonal misrouting. JZL184 facilitated the accumulation of 0 0 0 Unchanged CB1R signal transduction upon JZL184 exposure. oligodendrocytes that expressed 2 ,3 -cyclic-nucleotide 3 - Repeated JZL184 administration can induce cannabinoid receptor phosphodiesterase (CNPase)20, and formed micro-islands desensitization and functional antagonism23. Therefore, we first interspersed with corticofugal axons in both wild-type and À / À sampled mRNA (Supplementary Table 2) and protein expression CB1R mice (Fig. 3a–d). Oligodendrocyte differentiation of molecular constituents of the 2-AG signalling cassette (Fig. 2s; was reflected by the increased density (Fig. 3e) and size Supplementary Fig. 4). Neither mRNA nor protein levels for sn-1- (Fig. 3f) of their CNPase þ end feet targeted towards nearby diacylglycerol lipases a and b (DAGL), synthesizing 2-AG33, axons. The JZL184-induced morphogenesis of oligodendrocytes 34,35 À / À MGL and ABHD6, involved in 2-AG degradation , and CB1Rs was CB1R independent since it also occurred in CB1R mice changed significantly in JZL184-exposed fetal cortices (density: 37. 7±3.1 (JZL184) versus 14.7±2.7 (vehicle) end feet/ (Supplementary Fig. 4a,b). We then used extracellular signal- 1,000 mm2, Po0.05; Fig. 3e). By western blotting, we

NATURE COMMUNICATIONS | 5:4421 | DOI: 10.1038/ncomms5421 | www.nature.com/naturecommunications 3 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5421

–/– –/– WT/vehicle WT/JZL184 CB1R /vehicle CB1R /JZL184 300 25 * * i j k l * 20 m) d d d d m) 200 μ μ 15 10 /veh. /JZL184 100 * /vehicle /JZL184 spread ( fascicle ( –/– –/– –/– –/– R R 5 R R Corpus callosum 1 1 1 1 WT/vehicle WT/JZL184 CB CB WT/ve. WT/JZL184 CB CB L1-NCAM 0 Diameter of individual 0 ) CB R–/–/vehicle CB R–/–/JZL184 1.2 2 600 20

FC WT/vehicleFC WT/JZL184 1 1 m * m) * μ FC μ

) 15 2 0.8 400 10

cc cc cc cc 0.4 Area of 200 Diameter of size (mm 5 GW GW GW Striatal surface WT/vehicle WT/JZL184 WT/veh. WT/JZL184 WT/vehicle WT/JZL184 striatal fascicle ( L1-NCAM 0 0 striatal fascicle ( 0

2 2.5 JZL184+WIN55

-actin) 1.5 2 β d 1 c.+WIN55 d 1.5 JZL184 d 0.5 +AM251 Normalized fold

α β 1 d GFP/GFP Protein expression c.+AM251

Tau / expression (pErk/Erk) (normalized to 0

RC2/Brn1 DAGL DAGL MGL ABHD6 05’10’ 30’

30 min WT/vehicle WT/JZL184 pErk1/2 cc/IZ cc/IZ

Erk1/2

cpu cpu o p JZL184 – – – +++ WIN55 – + ––+ – Neurocan/ AM251 ––+ ––+ L1-NCAM

Figure 2 | JZL184 alters callosal axon fasciculation and pathfinding. (a–d) Corticofugal axonal phenotypes and (e–h) their midline crossing in vehicle- or JZL184-treated mouse embryos at E18.5. (i–l) A taugfp/gfp reporter mouse29 was used for the genetic tagging of corticofugal axons. Axon fasciculation À / À errors present in CB1R mice (k), were further increased by JZL184 (surface area occupied by corticofugal axons (l). (m–p) Neurocan was excluded À / À from enlarged axon fascicles in JZL184-exposed brains. (q) Corpus callosum spread in both wild-type and CB1R mice. (r) Striatal surface size, and the cross-sectional surface area and diameter of axon fascicles in JZL184- or vehicle-treated mice. (s)DAGLa,DAGLb, MGL and ABHD6 protein levels in JZL184-treated (red circles) versus control (open circles) cortices on E18.5. (t,u) Erk1/2 phosphorylation upon acute WIN55,212-2 and/or AM251 stimulation in drug naive and JZL184 pre-treated (4 days) neurons. Data were expressed as means±s.e.m., n ¼ 3/group (q,r). In vitro experiments were performed in duplicates with nZ2 samples per experiment run in parallel. *Po0.05 (Student’s t-test). cc, corpus callosum; cpu, caudate putamen; FC, fiber crossing; GW, glial wedge; IZ, intermediate zone. Scale bar, 300 mm(a–h,m,n), 10 mm(i–l,o,p). quantitatively confirmed that JZL184 significantly elevated induced oligodendroglia positioning and differentiation in CNPase and myelin basic protein levels in the fetal cerebrum conjunction with 2-AG’s cell-type-specific action via CB1Rs and (Fig. 3g,h). These results outline a multicomponent mechanism CB2Rs could orchestrate in trans Slit/Robo signalling. Therefore, with 2-AG simultaneously activating CB1Rs in axons, and CB2Rs we first examined whether CB1Rs and Robo1 receptors coexist in oligodendrocytes in JZL184-exposed fetuses. Nevertheless, a in long-range axons. By using serial sections from human causal relationship between altered axonal navigation and glial fetuses at gestational weeks 20–23 (Supplementary Table 3), we proliferation remains unknown. Here, we reasoned that find the expression patterns of CB1Rs and Robo1 receptors endocannabinoid-induced glial differentiation might instruct the to overlap in forebrain axonal tracts, particularly axons coursing production of a chemorepellent for the focal remodelling of the in the internal capsule (Fig. 4a–d). Multiple immunofluorescence corticofugal tract (Fig. 3i). labelling substantiated that CB1Rs and Robo1 receptors indeed co-distributed along the same axons in humans (Fig. 4e–g). Likewise, corticofugal axons in the E18.5 fetal mouse brain þ CB1Rs coexist with Robo1/2 receptors in corticofugal axons. contained both receptors (Fig. 4h–m). In addition, CB1R Neurons and glia interact to form chemical conduits for axonal cortical axons in the corpus callosum harboured Robo2 growth during brain development14. Slit/Robo interplay is receptors (Fig. 4n–p). fundamental for the development of major axonal tracts, including corticofugal projections4,5, with midline glia being a primary source of chemorepellent Slit1/2 (ref. 37). However, the Robo1 accumulates in JZL184-exposed neuronal growth cones. upstream regulation of Slit/Robo signalling in axon guidance is Axonal growth is a dynamic process, with growth cones con- not entirely understood. We hypothesized that endocannabinoid- tinuously sampling the local microenvironment and responding

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WT/vehicle WT/JZL184 separately from Robo1 (Fig. 5f; Supplementary Fig. 5a). Next, we purified growth cone particles (GCPs)19 from fetal mouse cortices treated en-block with JZL184 (100 nM) up to 80 min to highlight the in vivo relevance of our findings. Robo1 receptor accumulated in JZL184-exposed GCPs (Fig. 5g), lending further support to the concept that superfluous 2-AG signalling can CNPase/L1-NCAM redistribute Robo1 receptors in a CB1R-dependent fashion. Furthermore, we suggest that axonal transport contributes to –/– –/– CB1R vehicle CB1R JZL184 Robo1 accumulation in growth cones, since we detected a JZL184-induced increase in Robo1 receptor immunofluorescence in the distal (presumed motile)30 part of the neurite adjacent to the growth cone (Fig. 5h). JZL184-induced alterations in Robo1, but not Robo2 protein expression, were rescued by OMDM188, an inhibitor of DAGLs producing 2-AG40, implicating 2-AG dependence of Robo1 signalling (Supplementary Fig. 4d,e). Therefore, we focused on the endocannabinoid dependence of Slit2/Robo1 interactions. Vehicle JZL184 ** To confirm the reliance of our proposed mechanism on MGL

+ * *

) 2

+ 4 2 4 ** activity, we transfected primary cortical neurons with MGL short m μ -actin) interfering RNA (siRNA) and assessed Robo1 receptor intensity β 1.5 m) 3 μ in the growth cone. Transient MGL knockdown recapitulated the 2 2 1 accumulation of Robo1 immunoreactivity within the growth cone (Fig. 5i,j; Supplementary Fig. 5d,e), which was CB1R dependent profiles ( JZL184 JZL184

–/– –/– 1 –/– –/– Protein expression 0.5 given its sensitivity to AM251, a CB1R inverse agonist (Fig. 5k). In R R R R Number of CNPase profiles per 100 1 1 1 1 (normalized to addition, the lack of Robo1 receptor responsiveness in the growth Diameter of CNPapase WT/JZL184 CB CB WT/JZL184 CB CB 0 WT 0 WT CNPase MBP À / À 0 cones of primary cortical cultures from CB1R mice reinforced the critical involvement of CB1Rs in this regulatory Control JZL184 process (Fig. 5i–n). CB1R activation in developing neurons triggers molecularly diverse second-messenger cascades41. Here, CNPase 48 kDa we found that JZL184-induced Robo1 receptor accumulation in MBP 18 kDa β growth cones was occluded by the administration of both U0126 -actin 46 kDa and SP600125, MEK1/2 and c-Jun N-terminal kinases (JNK) inhibitors, respectively (Fig. 5o,p). If a CB R-mediated increase of Robo1 signalling is of

Vehicle 1 JZL184 pathophysiological significance, then Robo/Slit mRNA levels þ Figure 3 | JZL184 induces oligodendrocyte differentiation. CNPase might be expected to coordinately change in the cortices of oligodendroyctes in the prospective corpus callosum were infrequently human fetal subjects exposed in utero to cannabis, relative to À / À detected in wild-type (a) and CB1R (c) embryos (E18.5; open controls. By using the NanoString technology (Supplementary arrowheads). (b,d) JZL184-induced oligodendrocyte differentiation, which Table 4), we found that Robo1 receptor mRNA expression tended À / À progressed in CB1R mice (solid arrowheads pinpoint end feet). to be significantly higher in cannabis-exposed subjects (e,f) Quantitative data on oligodendrocyte morphology. (g,h) CNPase and (F1,26 ¼ 4.156; P ¼ 0.056; Supplementary Fig. 1d). This change myelin basic protein (MBP) levels in the cortices of JZL184-treated mouse was driven by a significant impact of developmental growth embryos. (i) Schema of JZL184-induced remodelling of axonal pathfinding measures. Similarly for Slit1 mRNA, there was a significant upon premature oligodendrocyte differentiation. Data were expressed as confound of development resulting in only a trend effect means±s.e.m., n ¼ 5/6 (JZL184/vehicle), *Po0.05 (Student’s t-test). (P ¼ 0.093) for the contribution of cannabis exposure. Interest- Scale bar, 4 mm(a). ingly, there was a significant correlation (r ¼ 0.576; P ¼ 0.002) between the expression of Robo1 and Slit1 mRNAs in the human fetal cortex, suggesting a functional interaction irrespective of to chemotactic cues by initiating turning decisions38. Robo1/2 cannabis predisposition. localization to growth cones underpins Slit/Robo signalling during axonal pathfinding13,39. On the basis of our pharmacological data (Supplementary Fig. 4d), we hypothesized JZL184 promotes Slit2 production in oligodendrocytes. Genetic that modifications to axonal growth trajectories might be deletion of Slit1 and/or Slit2 induces midline crossing errors8,12. mediated by the CB1R-dependent repartitioning of Robo1 Therefore, we hypothesized that oligodendrocytes could produce receptors in axonal growth cones. Therefore, we confirmed the excess Slits in a CB2R-dependent fashion to impact corticofugal co-localization of CB1Rs and Robo1 receptors in vitro (Fig. 5a), axon fasciculation. Owing to methodological constraints to and validated Robo1/2 mRNA and protein expression by PCR histochemically detect Slit1, we adopted a candidate approach (Fig. 5b) and western blotting (Fig. 5c). Upon JZL184 treatment and focused on Slit2 throughout our proof-of-concept of cultured cortical neurons, Robo1 receptors accumulated in experiments. We first prepared oligodendrocyte cultures, which growth cones, specified as the fluorescence ratio between the only sporadically contained astroglia (Fig. 6a) to demonstrate that growth cone and adjoining neurite shaft (Fig. 5d). This response CNPase þ oligodendrocytes are a likely source of Slit2 in vitro. 30 was O-2050 sensitive , confirming CB1R involvement. In We extended these data by comparing glial fibrillary acidic contrast, Robo2 receptor distribution remained unchanged protein (GFAP) þ astroglia and oligodendroglia cultures (Fig. 6b), (Fig. 5e). We also found a decreased co-localization coefficient and showing that oligodendroglia selectively contain detectable for Robo1 and CB1R, reinforcing increased Robo1 receptor Slit2 mRNA transcripts and Slit2-like protein (Fig. 6c; import if CB1R levels remained stationary and partition Supplementary Figs 5b and 6). Slit2-like immunoreactivity was

NATURE COMMUNICATIONS | 5:4421 | DOI: 10.1038/ncomms5421 | www.nature.com/naturecommunications 5 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5421

CB1R Robo1 Human fetus

cc cc

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ne cpu cpu k ne ne hc ne CB R Robo1 CB R/Robo1 hc 1 1 E18.5 WT

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Robo2 CB1R Robo2/CB1R

Figure 4 | CB1Rs co-localize with Robo1 receptors in human and mouse corticofugal axons. (a,b) The regional distribution of CB1R and Robo1 receptor immunoreactivities in human fetal serial sections (closed arrowheads). (c,d)CB1R and Robo1 receptor immunoreactivities in corticofugal fibres in the internal capsule at high magnification. (e–g) Co-localization of CB1R and Robo1 receptors in human fetal tissues (internal capsule) by high-resolution laser- scanning microscopy (open arrowheads). Robo1 (h–m), as well as Robo2 (n–p), were co-expressed with CB1Rs in mouse callosal axons (arrowheads). cc, corpus callosum; cfa, corticofugal axons; cp, cortical plate; cpu, caudate putamen; hc, hippocampus; IZ, intermediate zone; ne, neuroepithelium. Scale bar, 3 mm (a), 200 mm(h,n), 40 mm(c), 7 mm(k).

þ À seen only in oligodendrocytes (but not astroglia or neurons; In JZL184-treated brains, CNPase /CB1R oligodendroglial Fig. 6a) in vitro and mainly localized to their soma (Fig. 6d) and end feet juxtaposed Robo1/CB1R double-labelled corticofugal membranes of their processes (Fig. 6e,f). We confirmed our Slit2 axons (Fig. 6q), and accommodated enlarged fascicles by forming detection in oligodendrocytes by demonstrating significantly proportionately larger glial scaffolds (that is, no CNPase labelling diminished Slit2-like content in oligodendroglia from Slit2 À / À within individual fascicles; Fig. 6q). These oligodendroglia end mice (Supplementary Fig. 6a–e). In addition, we validated our feet harboured Slit2-like immunoreactivity, which frequently staining protocols by co-labelling for an antibody revealing a juxtaposed nearby axons (Fig. 6r). These data together with phylogenetically conserved sequence from the Drosophila Slit Robo1/CB1R (Fig. 5a) and Robo2/CB1R (Fig. 5a) co-localization ortholog (Fig. 6c). These data identify oligodendrocytes as a and Robo1/Robo2 mRNA (Fig. 5b) and protein (Fig. 5c) candidate source of Slit2. expression in cultured cortical neurons suggest that endocanna- Excess 2-AG facilitates oligodendrocyte differentiation in binoids are poised to modulate Slit/Robo signalling, and support a culture21 and in vivo (Fig. 3). Here, we show that JZL184 model in which ectopic Slit2 presentation by cortical oligoden- þ increased Slit2 expression in cultured oligodendrocytes (Fig. 6g–i), drocytes participates in inducing growth errors of CB1R axons and increased Slit2-like immunoreactivity in their end feet in vivo (Fig. 6s). (Fig. 6j). JZL184’s effect on Slit2-like labelling in oligodendroglia 21 end feet was reversed by AM630, a CB2R antagonist (Fig. 6k–n), suggesting that Slit2 production and subcellular translocation JZL184 fails to affect axonal growth in Robo1 À / À mice.If might be regulated by agonist (for example, 2-AG) signalling at focal Robo1/Slit2 interactions are indeed modulated by 2-AG À / À CB2Rs. To verify the effects of JZL184 on Slit2 production, we signalling, then JZL184 administration to Robo1 (ref. 42) transfected cultured oligodendrocytes with MGL siRNA, which mice will reveal incomplete cortical phenotypes due to the neuron recapitulated the increase of Slit2-like immunoreactivity in versus oligodendroglia-driven arch of the proposed signalling axis oligodendrocyte end feet (Fig. 6o,p). (Fig. 7a). Robo1 À / À mice per se present an axon fasciculation

6 NATURE COMMUNICATIONS | 5:4421 | DOI: 10.1038/ncomms5421 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5421 ARTICLE

1.4 ∼ 220 kDa 1.2 Robo1 1.0 Robo2 Robo1 50 kDa TUJ1 0.8 RT+ RT– RT+ RT– ∼220 kDa (ratio) 0.6 200 bp Robo2 0.4 100 bp Vehicle 50 kDa Robo1 i.r. intensity 0.2 GC TUJ1 JZL184 start 0 0 10 20 30 40 50 60 70 80 90100 CB RCBR 1 1 locus on axon (% of length) Robo1 Robo2 250 1.5 * * 1.5 Control siRNA 200

1 1 150

100

0.5 0.5 (gray scales) Intensity ratio

Intensity ratio 50 Robo1 i.r. intensity growth cone per shaft growth cone per shaft 0 Vehicle +O-2050 +O-2050 0 0 Vehicle 0 2 46810 12 14 16 18 20 Robo1 Robo2 JZL184 JZL184 μm from growth cone tip

GCPs 250 * MGL siRNA 1 200 ** vs control

0.8 150 R 1 JZL184 (25’) JZL184 (80’) Vehicle Robo1 100 0.6 (gray scales) 50 Robo1 i.r. intensity Robo1/CB Colocalization 0.4 TUJ1 0 JZL184 02468101214 16 18 20 CB R/Robo1 CB R/Robo2 0.2 μ 1 1 Vehicle 1.0 0.9 1.7 m from growth cone tip

l Control JZL184 Control 250 80 * * MGL siRNA + AM251 200 60 150 CB1R/Robo1 40 100 JZL184 (gray scales)

growth cone 50 20 Robo1 i.r. intensity Robo1 intensity –/– –/– 0 CB1 Control Robo1 0 CB1 0 2 4 6 8 10 12 14 16 18 20 TUJ1 μm from growth cone tip JZL184 CB R–/– CB R–/– JZL184 JZL184+U0126 1 1 2.0 * * 1.5 * *

1.6 1.0 1.2

0.8 JZL184+SP600125 0.5 (GC per axon) Robo1 i.r. ratio (GC per axon) Robo1 i.r. ratio –/– 0.4 –/– Control Control CB1 + U0126 CB1 0 0 +SP600125 JZL184 JZL184

Figure 5 | CB1R stimulation regulates subcellular Robo1 positioning. (a) Robo1 and Robo2 immunoreactivity in the somata and neurites (including growth þ cone; arrowheads) of CB1R cortical neurons in vitro.(b,c) Cultured cortical neurons expressed both Robo1/Robo2 mRNA and protein. (d) Robo1 and Robo2 (e), fluorescence intensity in growth cones treated with JZL184 or O-2050, a CB1R antagonist. (f) Co-localization coefficient of Robo1 and CB1Rin growth cones upon JZL184 exposure. (g) JZL184 induced Robo1 accumulation in growth cones ex vivo in isolated growth cone particles (GCPs). (h) Robo1 immunoreactivity adjacent to the growth cone in JZL184-treated neurons. (i–k) Robo1 immunoreactivity in growth cones upon siRNA-mediated MGL silencing in cultured primary neurons. (l–n) Effect of the genetic ablation of CB1Rs in cortical neurons on Robo1 receptor expression in growth cones after JZL184 or vehicle administration. (o,p) The effect of Erk1/2 (U0126) and JNK1 (SP600125) inhibition on Robo1 distribution. Data were expressed as means±s.e.m., from triplicate experiments (sample size: 8–25 neurons per group in morphometric analysis), *Po0.05 (Student’s t-test). Scale bar, 10 mm (a), 5 mm(l,o).

5 phenotype . If Robo1 receptors are downstream from CB1Rs, affected by JZL184 (Fig. 7c,d,f). In contrast, JZL184 provoked then JZL184 likely would fail to affect the pre-existing CNPase þ oligodendroglial end-feet differentiation in Robo1 À / À axonal phenotype in Robo1 À / À mice. Indeed, the spread of mice. Yet oligodendroglial end feet were interspersed with callosal axons5 (Fig. 7b) remained unaffected in JZL184-treated individual corticofugal axons, confirming the loss of a Robo1 À / À mice. As such, neither the diameter of individual and chemorepulsive cue (Fig. 7e,g). These data dissociate between enlarged axon fascicles nor their interbundle distance were the loss of CB1R-driven and Robo1 receptor-dependent axon

NATURE COMMUNICATIONS | 5:4421 | DOI: 10.1038/ncomms5421 | www.nature.com/naturecommunications 7 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5421

Oligodendrocyte s Vehicle Astro Olig Astro Olig 200 kDa 50 kDa Slit2 GFAP Slit2 * 48 kDa 200 kDa JZL184 s 42 kDa CNPase Slit 42 kDa CNPase/ 42 kDa β Slit2/GFAP β-actin -actin Slit2 Slit2 Slit2

* 1.2 Vehicle JZL184 JZL184 + ** AM630 1.5 1

Slit2 0.8 1 β-actin 0.6

intensity of end 0.4 0.5 intensity in end feet Slit2

feet/intensity of soma feet/intensity 0.2 Vehicle JZL184 (normalized to shaft) Slit2 0 JZL184 Vehicle JZL184 JZL184+AM630 0 vehicle Slit2

Scrmbl siRNA MGL siRNA a 0.9 * +JZL184 y 0.8 z x 0.7 d a 0.6 cfa 0.5 Olig 0.4 0.3 ibs Cfa intensity in end feet 0.2 d

(normalized to shaft) CB R/ 0.1 1 GFP/Slit2/ Slit2 CNPase/ Slit2/L1/ CB1R Robo1 0 siRNA Scrambled MGL siRNA Hoechst Robo1 CNPase/ CB2R(?) Slit2

Figure 6 | JZL184 alters Slit2 expression and localization in oligodendrocytes. (a) Oligodendrocytes (solid arrowheads), but not astrocytes, (open arrowheads) contained Slit2. (b) GFAP and CNPase were used to validate the purity of isolated astrocytes and oligodendrocytes, respectively. (c) Oligodendrocytes, but not astrocytes, contained mature Slit2. An additional antibody raised against a phylogenetically conserved epitope of Drosophila Slit confirmed this finding (Slit). (d–f) Slit2-like immunoreactivity localized to oligodendrocyte somas (s) and processes. Membranous staining pattern is shown in both e and f (arrowheads). Asterisks (*) indicate location of the nucleus. (g,h) Oligodendrocyte differentiation and increased Slit2 immunoreactivity, particularly in end feet (arrowheads), upon JZL184 exposure. (i) Slit2 protein content in oligodendrocytes after JZL184 treatment. (j) JZL184-induced Slit2 accumulation in oligodendrocyte end feet as measured by quantitative morphometry. (k–n) Effect of AM630, a CB2R antagonist, on JZL184-induced Slit2 accumulation in oligodendrocyte end feet (open and closed arrowheads). (o,p) The effect of siRNA-mediated MGL inhibition on Slit2 expression in end feet (arrowheads). (q) CNPase þ oligodendroglial end feet in the interbundle space (ibs) and in apposition to enlarged fascicles of Robo1 þ corticofugal axons (cfa) in JZL184-treated mouse embryos. (r) Three-dimensional reconstruction of Slit2 in oligodendrocyte end feet (arrowheads) adjacent to corticofugal axons. (s) Schematic of JZL184-induced remodelling of axonal pathfinding upon premature oligodendrocyte differentiation. Data were expressed as means±s.e.m., n ¼ 8–32 cells per group for each condition in duplicate experiments; *Po0.05 (Student’s t-test). Scale bar, 10 mm(a,h,o,q), 2 mm(m).

guidance deficits and CB2R-mediated focal chemorepulsion and We used siRNA knockdown of Robo1 in neurons co-cultured oligodendroglial differentiation in vivo. with oligodendrocytes in the presence of JZL184 for 4 days to test whether JZL184-mediated neurite repulsion relied on Robo1 receptors. Co-transfection with a GFP plasmid and post hoc Slit2/Robo1 signals mediate JZL184-induced chemorepulsion. labelling for Robo1 receptors (Supplementary Fig. 5c) confirmed We devised a genetically amenable co-culture system to show that successful neuronal Robo1 silencing. Robo1 knockdown over- Slit2 þ oligodendrocytes affect directional growth decisions of came JZL184-induced repulsion and adopted differentiated þ þ Robo1 /CB1R neurites in vitro. Neurites regularly contacted growth cone morphologies (Fig. 8c). Consequently, neurites of oligodendrocytes under control conditions (Fig. 8a). JZL184 Robo1 siRNA-transfected neurons approached oligodendrocytes, administration in vitro enhanced Slit2 expression and mobilized leaving the growth cone-oligodendrocyte distance significantly Slit2 to submembranous positions in oligodendrocytes (Fig. 8b), reduced (4.97±0.69 mm (Robo1 siRNA) versus 18.21±2.06 mm with coincident repulsive responses of nearby neurites as (scrambled siRNA), Po0.05; Fig. 8d). Further, JZL184 adminis- shown by their increased distance to apposing glial surfaces tration increased the number of branching points on approaching (15.65±1.01 mm (JZL184) versus 9.74±1.11 mm (vehicle), neurites in the close vicinity of oligodendocytes (Fig. 8e), Po0.05; Fig. 8d). suggesting that an increased number of axon collaterals might,

8 NATURE COMMUNICATIONS | 5:4421 | DOI: 10.1038/ncomms5421 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5421 ARTICLE

Neuron Oligodendrocyte 150 JZL184 JZL184 2-AG 2-AG

o 100

CB1R Robo1 CB2R i

50 + JZL184 JNK, ERK1/2 (% of wild type) –/– Slit2 –/–

Fasciculation Corpus callosum spread Robo1 0 Robo1 CNPase L1-NCAM

Robo1–/– JZL184 Robo1–/– JZL184 14 10 7 * * 12 6 * 8 10 5 8 6 4 m) m) μ μ ( ( 6

3 JZL184 4 vehicle JZL184 JZL184 –/– JZL184 –/– –/– R Bundle spread 4 2 –/– –/– R 1 1

Interbundle distance 2 CNPase CNPase endfeet ratio WT JZL184 WT vehicle CB between/within bundles Robo1 2 CB 1 L1-NCAM L1-NCAM Robo1 Robo1 WT JZL184 WT JZL184 n.d. n.d. 0 0 0

Figure 7 | JZL184-induced oligodendroglial repositioning in Robo1 À / À mice. (a) Schematic overview of 2-AG signalling converging on axonal growth and fasciculation through Slit/Robo signalling. (b) Callosal axon fasciculation in Robo1 À / À mice treated with JZL184. Note that untreated Robo1 À / À mice presented a fasciculation phenotype5.(c,d) Diameter of individual bundles and their interbundle distance upon JZL184 treatment in Robo1 À / À embryos. (e–g) Oligodendroglia end-feet infiltrated axonal bundles in JZL184-treated Robo1 À / À mice. This was undetected (ND) in wild-type or À / À CB1R embryos. Data were expressed as means±s.e.m., from n ¼ 3–5 embryos per genotype; *Po0.05 versus respective wild-type (WT) controls (Student’s t-test). Scale bar, 10 mm(f,g).

Vehicle JZL184 * * Control CB1R 20 gc m) 2-AG μ Forward growth Slit2 Robo1 CB R 10 2

+ Robo1 RNAi 2-AG GC-glia distance ( + scrambled RNAi + scrambled Olig TUJ1/Slit2/Hoechst 0

JZL184+neuron-specific gc1 Olig 1.8 * JZL184 Robo1-siRNA/GFP CB1R 1.6 gc 1.4 2-AG 1.2 Robo1 Slit2 1.0 gc1 DIC/GFP/TUJ1 Repulsive 0.8 guidance gc2 gc2 0.6 0.4 Olig CB2R

Number of branching points Number of branching 0.2

DIC/GFP/TUJ1 JZL184 2-AG GFP/Robo1/Hoechst 0 Olig

Figure 8 | JZL184 induces growth cone repulsion via Slit2/Robo1 signalling. (a) Neurites (arrowheads) contacting oligodendrocytes when co-cultured. (b) Neurites (open arrowheads) excluded from the vicinity of oligodendrocytes upon JZL184 application. Note the intensely Slit2 þ peripheral domain of the oligodendrocyte (Olig) shown. (c) A neurite (arrowheads) of a Robo1 siRNA/GFP co-transfected neuron (gc1 in green), but not of a non-transfected neuron (gc2 in red), overcame chemorepulsion. Insets (gc1,gc2) depict high-resolution images of the growth cones shown in (c). (d) Growth cone distances to the most proximal surface of the nearest oligodendrocyte. (e) JZL184 induced branching of neuronal processes in the vicinity of oligodendrocytes in vitro.(f,g) Schema of Slit2/Robo1 signalling downstream from cannabinoid receptors. Data were expressed as means±s.e.m., triplicate experiments with n ¼ 3 samples run in parallel; n ¼ 6–9 observations per group. *Po0.05 (Student’s t-test). Scale bar, 8 mm(a1,b), 3 mm (insets).

NATURE COMMUNICATIONS | 5:4421 | DOI: 10.1038/ncomms5421 | www.nature.com/naturecommunications 9 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5421 at least in part, contribute to changes in callosal microanatomy. exist. These include the elongating corticofugal axons themselves, Cumulatively, these data identify endocannabinoids as candidates which contain DAGLa (and b)17,18. If so, Slit2/Robo1 signalling to upstream regulate Slit2/Robo1 interactions during directional might represent a cell-autonomous amplification mechanism axonal growth (Fig. 8f,g). whereby DAGL-derived 2-AG could activate CB1Rs within growth cones30, facilitating Robo1 trafficking. Recent data suggest that 2-AG can diffuse extracellularly and evoke Discussion physiological responses in a radius of 20–100 mm (refs 48,49). Endocannabinoids affect neuronal differentiation, including Thus, DAGL activity in axonal growth cones appears sufficient to 17,19,43 directional axonal growth during corticogenesis . generate focal 2-AG microgradients to activate CB2Rs on Endocannabinoid signalling appears particularly efficacious to neighbouring oligodendrocytes to stimulate Slit2 production modulate axonal growth since CB1Rs are selectively enriched in and release (Fig. 8f,g). At the level of signal transduction, navigating axons, and are co-targeted with DAGLs to advancing CB1Rs and Robo1/2 can coordinately activate downstream growth cones30,43. Establishment of major axonal tracts critically effectors within axonal growth cones, which can act as relies on chemorepulsive Slit gradients4,5,7. In fact, midline reinforcers of the individual receptors’ signal outputs. Members crossing of commissural axons particularly depends on focal Slit/ of the Rho family of GTPases, which can be activated by either Robo signalling between neurons and glia44. The architecture of receptor in various tissues19,50,51 and whose activity is À / À 18 corticofugal axons is altered in CB1R mice , a phenotype indispensable for cytoskeletal instability allowing growth cone reminiscent to those of Robo1 À / À /Robo2 À / À mutants4. Here, motility52, can be central to orchestrating endocannabinoid- we resolve this similarity by dissecting a molecular interplay induced modifications to cytoskeletal remodelling during between endocannabinoids and the Slit/Robo signalling axis. Our repulsive growth cone turning. An advantage of this model, data suggest that the lack of CB1R-induced Robo1 accumulation combining elements of autocrine endocannabinoid and paracrine in growth cones might disrupt, at least in part, axonal navigation Slit2/Robo1 signalling, is that it ascertains the ‘on-demand’ and contribute to the altered topography of corticofugal axons upstream coupling of endocannabinoids to Slit2/Robo1 signalling, À / À in CB1R mice. However, by showing that increased 2-AG outlining a temporally defined, spatially restricted and highly levels upon MGL inhibition coincidently induce CB2R- dynamic mechanism to modulate growth cone-steering decisions. mediated oligodendrocyte differentiation and CB1R-dependent We also show that CB1R–Robo1 receptor co-localization is axonal misrouting in the corpus callosum, we characterize preserved during mammalian evolution, as evidenced by our an intercellular signalling network in which a single histochemical data from midgestational human fetuses. More- endocannabinoid ligand engages molecularly distinct over, Robo1 and Slit mRNA expression in human fetal brains are cannabinoid receptors in neurons and glia to upstream control developmentally regulated and appear tightly coupled. Maternal chemorepellent production (Slit2 in glia) and signalling (Robo1 in cannabis smoking significantly increased the amount of Robo1 neurons). Whether changes in axonal topography in the corpus mRNA in the human fetal cerebrum. We propose that the callosum of JZL184-treated mice reflect an increased number of molecular re-configuration of Slit/Robo signalling networks by axon collaterals or instead are due to the spatial reorganization of phytocannabinoids via the CB1R can be a candidate molecular the same number of axons (because of, for example, changes to mechanism to explain altered synaptic neurotransmission, and extracellular matrix composition) as in controls remains consequently, behavioural and cognitive deficits in children unknown. Our in vitro data on the JZL184-induced increase of exposed in utero to cannabis53,54. Considering the continued neurite branching points supports the former scenario, and increase of cannabis smoking during adolescence41, the erroneous suggests that an increased numbers of axon collaterals recruitment of spatially segregated CB1Rs and CB2Rs to regulate compacting in callosal fascicles might significantly increase Slit2/Robo1 interactions during the postnatal refinement of network instability due to erroneous synaptic wiring. Our neuronal circuits might also introduce unwanted modifications results in cultured neurons are also compatible with those from to the cortical wiring diagram. Besides cannabis, fetal infections Robo1 À / À mutant embryos, where glial repositioning around are associated with the induction of endocannabinoid signalling enlarged axon fascicles could argue for the disruption of a critical in placental macrophages, contributing excess endocannabinoids glial–neuronal signal interface. to the fetal circulation55. This cascade can change the temporal 23 We employed JZL184, a selective MGL inhibitor , to increase and subcellular recruitment of CB1Rs and CB2Rs, imposing endocannabinoid levels in the fetal brain. The selectivity of this pathological challenges on the signalling mechanism we compound was verified using activity-based protein profiling after uncovered. Overall, we suggest that a hierarchical interplay in vivo drug application, as well as in in vitro experiments using between endocannabinoid and Slit/Robo signalling networks is a genetically or RNA-silenced primary cultures. Since JZL184 primary regulatory node in the developing corticofugal system. application does not desensitize CB1R unless used for extended periods36, our data suggest that ectopic, cell type- and cannabinoid receptor subtype-dependent signalling events took Methods gfp/gfp À / À place in JZL184-exposed fetuses, sufficient to affect axonal growth Animals and tissue preparation. Tau ::CB1R mice were generated by gfp/ þ À / þ first crossing tau and CB1R mice (both on C57BL/6 background) to and guidance. This included an increased transport of Robo1 gfp/ þ À / þ select tau ::CB1R colony founders. Next, we crossed heterozygous off- along the axon culminating in peak expression levels and gfp/gfp À / þ spring to generate tau ::CB1R parents for in vivo analysis. Given that tau externalization in the growth cone, likely modulated via is dispensable for neuronal development, taugfp/gfp mice did not carry any devel- 29 mitogen-activated protein kinase and JNK signalling, as well as opmental abnormality . We used mice heterozygous for CB1Rs as parents throughout the experiments, allowing direct comparison of littermate taugfp/ an increased Slit2 expression on an enlarged oligodendroglial gfp À / À gfp/gfp þ / þ þ / þ À / À ::CB1R and tau ::CB1R offspring. MGL and MGL end-feet arbour. Considering that CB1Rs are broadly distributed littermates of both sexes from heterozygous pregnancies23 were analysed on E16 45,46 À / þ gfp/ in the developing nervous system , including descending and postnatal day (P)20 (Supplementary Fig. 1). Pregnant CB1R , tau 45,47 gfp À / þ À / þ 5 þ / À 56 projections to the spinal cord , our results might have ::CB1R , Robo1 , Slit2 and wild-type C57BL/6 mice were profound implications for the overall organization of axonal injected with JZL184 (40 mg kg À 1 body weight, intraperitoneally daily)24 or vehicle intraperitoneally during E12.5–18.5. Male mouse embryos (n46/pregnancy, n42 connectivity in the nervous system. pregnancies/analysis) were collected at E18.5. Whole brains were collected for Although we did not seek to identify the cellular origin of histochemistry, protein biochemistry/western blotting, mass spectrometry, JZL184- signal-competent 2-AG, a number of alternative sources may binding assay or quantitative RNA profiling. Experiments on live animals

10 NATURE COMMUNICATIONS | 5:4421 | DOI: 10.1038/ncomms5421 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5421 ARTICLE conformed to the 86/609/EEC directive and were approved by the regional Mixtures of primary antibodies were applied for 48 h at 4 °C (anti-Robo1 diluted authority (Stockholm Norra Djuretiska Na¨mnd; N512/12). 1:500 and anti-CB1R diluted 1:1,000). A mix of secondary antibodies conjugated with Alexa Fluor 488 (Invitrogen/Molecular Probes; 1:1,000) and DyLight 650 (Jackson; 1:400) was applied for 2 h at room temperature. The antibodies were Human fetal tissue preparation and histochemistry. Three fetal brains with tested for optimal dilution, and secondary antibodies were tested for cross- normal development (between 20–23 gestational weeks) were selected from the reactivity and non-specific staining. Sections were imaged and analysed using a Brain Bank of the Institute of Neurology, Medical University of Vienna, Austria. Zeiss LSM780 confocal microscope. Fetal brain tissue was obtained from spontaneous or medically induced abortions. Only cases without genetic disorders, head injury or neurological diseases were included. These cases showed no chromosome aberrations or post-mortem auto- Western blotting. Tissue samples were lysed in modified radio- lysis. Neuropathological examination excluded major central nervous system immunoprecipitation assay buffer containing 1 mM NaF, 1 mM Na3VO4, 0.1% malformations, severe hypoxic/ischaemic encephalopathy, intraventricular hae- N-octyl-b-D-glucopyranoside and a mixture of protease inhibitors (Complete, morrhages, severe hydrocephalus and meningitis or ventriculitis. Demographic Roche). Cell debris and nuclei were pelleted by centrifugation (800g, 10 min at 4 °C). Protein concentrations were determined by Bradford’s colorimetric method60. data are summarized in Supplementary Table 3. Tissues were obtained and used À 1 compliant with the Declaration of Helsinki and following institutional guidelines. Samples were diluted to a final protein concentration of 2 mg ml , denatured in The study was performed in the course of an approved study by the Ethical 5  Laemmli buffer and analysed by SDS–polyacrylamide gel electrophoresis on 6 Committee of the Medical University of Vienna (No.104/2009). or 8% resolving gels. After transferring onto Immobilon-FL polyvinylidene difluoride membranes (Millipore), membrane-bound protein samples were blocked in 3% bovine serum albumin and 0.5% Tween-20 diluted in Tris-buffered saline for Immunohistochemistry, imaging and quantitative morphometry. Embryonic 1.5 h, and subsequently exposed to primary antibodies (Supplementary Table 1; brains were immersion fixed in 4% paraformaldehyde in phosphate buffer (0.1 M, Supplementary Fig. 7) overnight at 4 °C. Appropriate combinations of horseradish pH 7.4, PB) overnight. Tissue samples were cryoprotected in 30% sucrose in peroxidase-conjugated secondary antibodies (Jackson; from goat, rabbit, guinea pig physiological saline for at least 48 h before cryostat serial sectioning (16 mm or mouse hosts; 1:10,000 for 2 h) were used for enhanced chemiluminescence signal thickness), and thaw-mounted on SuperFrost þ glass slides. Multiple immuno- detection. Image acquisition and analysis were performed on a Bio-Rad XRS þ fluorescence histochemistry was carried out according to published protocols19,57. imaging platform. TUJ1 served as loading control, which showed corresponding Sections were washed in PB and pre-treated with 0.3% Triton X-100 (in PB) for 1 h quantities of b-actin and gapdh (Supplementary Fig. 4f). at 22–24 °C to enhance the penetration of primary antibodies. Non-specific immunoreactivity was suppressed by incubating our specimens in a cocktail of 10% PCR. To discriminate the sex of mouse fetuses, we used primers to amplify Y and X normal donkey serum (Jackson), 5% bovine serum albumin (Sigma) and 0.3% chromosome-specific genomic DNA sequences. The Sry gene (forward: 50- Triton X-100 (Sigma) in PB for 1 h at 22–24 °C. Sections were exposed (16–72 h at GTTCAGCCCTACAGCCACAT-30, reverse: 50-CAGCTGCTTGCTGATCTCTG- 4 °C) to select combinations of primary antibodies (Supplementary Table 1) diluted 30, amplicon size: 197 bp (Y chromosome)) and DXNds3 microsatellite (forward: in PB to which 0.1% normal donkey serum and 0.3% Triton X-100 had been added. 50-GAGTGCCTCATCTATACTTACAG-30, reverse: 50-TCTAGTTCATTGTT- After extensive rinsing in PB, immunoreactivities were revealed by carbocyanine GATTAGTTGC-30, amplicon size: 244 bp (X chromosome)) were amplified. For (Cy)2, 3 or 5-tagged secondary antibodies raised in donkey (1:300 (Jackson), 2 h at quantitative PCR, E18 embryonic mouse brains were microdissected on ice to 22–24 °C). Hoechst 33342 (Sigma), a nuclear dye, was applied to reveal tissue isolate pallial regions, and snap frozen in liquid N until processing. RNA was architecture. Sections were coverslipped with Dako Fluorescent mounting medium 2 extracted using the RNeasy mini kit (Qiagen) with a DNase I step performed to (Dako). eliminate traces of genomic DNA30 and reverse transcribed using a high-capacity Sections were inspected and images acquired on a 710LSM confocal laser- cDNA reverse transcription kit (Applied Biosystems). Reactions were performed scanning microscope (Zeiss). Image surveys were generated using the tile scan after an initial denaturation at 95 °C for 10 min followed by 40 cycles of 95 °C for function at  10 primary magnification (Plan-Neofluar  10/0.30). High- 15 s denaturation, annealing and extension at calculated temperatures (60 s) and a resolution images were acquired with optical zoom ranging from  1.5–3.0 at  63 dissociation stage (from 60 to 95 °C with 0.5-°C steps for 10 s each; MyiQ primary magnification (Plan-Apochromat  63/1.40, and 0.5–0.7 mm optical slice Apparatus, Bio-Rad), with primer pairs amplifying short fragments for each gene thickness) to limit signal detection. Emission spectra for each dye were limited as (Supplementary Table 2). Samples without template or reverse transcriptase served follows: Cy2 (505–530 nm), Cy3 (560–610 nm) and Cy5 (650–720 nm). The as negative controls. Expression levels were normalized to the housekeeping gene coexistence of immunosignals was verified by capturing serial orthogonal z-image (HKG) encoding glyceraldehyde-3-phosphate dehydrogenase (gapdh) obtained for stacks at  63 primary magnification (up to  3 optical zoom) and accepted if every sample in parallel assays15. these were present without physical signal separation in r1.0-mm optical slices, and overlapped in all three (x, y and z) dimensions within individual cellular domains. Human fetal NanoString analysis. Midgestational fetal brain subjects (18–22 Morphometric analysis of fetal mouse brains was aided by the ZEN2010 weeks of gestation), collected after saline-induced elective abortions under Insti- software and included: (i) the transverse diameter (mm) of L1-NCAM þ tutional Review Board approval at SUNY Downstate Medical Center, Brooklyn, À / À corticofugal axons (‘corpus callosum spread’) in fetal wild-type and CB1R and New York, were fixed in 1% paraformaldehyde in 0.05 M PB for 24 h, frozen in dry MGL À / À brains after JZL184 or vehicle administration, (ii) the density and (iii) ice-cooled isopentane, cryosectioned in the coronal plane (20 mm) and stored at proximity (mm) of first-order fascicles of 43 mm in diameter, (iv) the density (n), À 30 °C (ref. 61). Tissue isolations from the cortex were carried out with micro- (v) diameter (mm), (vi) embedding of CNPase þ oligodendrocyte end-feet RNAqueous lysis solution (Life Technologies) and gene expression analysed using interspersing callosal axons and (vi) calibrated fluorescence intensities using an the nCounter System from NanoString Technologies62 at the Gene Expression arbitrary grey-scaled unit scale in images acquired with identical laser illumination Shared Resource Facility at Icahn School of Medicine. Briefly, the cell lysates were and capture settings. Robo1 receptor immunoreactivity along growth cones and hybridized to a NanoString code-set containing barcode-labelled probes for axons of primary cortical neurons was measured in ImageJ 1.45 s (NIH). Validation ROBO1, SLIT1 and three HKGs: TBP, Hsp90ab1 and ActB (Supplementary of the Slit2 antibody was performed on 600-nm-thin serial optical slices of Table 4), followed by scanning of the immobilized mRNA transcripts in the embryonic brain sections obtained from Slit2 À / À and littermate wild-type mice. nCounter Digital Analyzer. The raw data files were normalized to the TBP HKG via The number of Slit2 þ profiles was counted and their density calculated on NanoString nCounter software, and statistical analysis was performed using the CNPase þ oligodendrocyte end feet or perinuclear loci. Multi-panel images were JMP statistical program (SAS Institute, Cary, NC). A general linear stepwise assembled in CorelDraw X5. regression was used to calculate statistical significance and identify covariates. Variables were clustered as (i) fetal age, (ii) fetal body weight and foot length (‘developmental measures’) and (iii) cannabis exposure. A univariate model was Human fetal immunohistochemistry and analysis . Three-micrometer tissue first performed with each cluster of variables and any variable that showed Po0.10 sections of formalin-fixed, paraffin-embedded tissue blocks containing the corpus was included in the stepwise regression, in addition to cannabis exposure callosum and the hippocampal formation were mounted on pre-coated glass slides 61 58,59 addition . Variables with Po0.05 were included in the final regression model (Star Frost) . Shortly, after deparaffinization and rehydration, the sections were as covariates of cannabis exposure. pre-treated in low pH EnVision FLEX antigen retrieval solution at 98 °C for 20 min (PTLink; Dako) and subsequently incubated with rabbit anti-Robo1 (ab7279; Abcam; 1:500) or guinea pig anti-CB1R (Dr M. Watanabe 1:1,000) antibodies. Liquid chromatography tandem mass spectrometry. 2-AG, anandamide (AEA), The DAKO EnVision detection kit, peroxidase/DAB and rabbit/mouse (K5007, 2-linoleoyl glycerol and oleoylethanolamide concentrations were determined using ready-to-use, Dako) was used to visualize antibody binding. Sections were a solid-phase extraction liquid chromatography tandem mass spectrometry counterstained with haematoxylin, dehydrated in ascending concentrations of method30,63. The final JZL184 (40 mg kg) injection was given to pregnant females ethanol, cleared with xylene and covered with Consil-Mount (Shandon; Thermo on E18.5 2 h before dissection of maternal and fetal brains. Embryonic hemisected Scientific). Representative images containing the area of interest were exported brains (n ¼ 6 pups from two separate litters) were homogenized in ice-cold 2 from scanned sections (NanoZoomer 2.0, Hamamatsu). acetonitrile containing [ H4]anandamide at 6 pmol as internal standard, made up For immunofluorescent co-localizations, 5-mm-thick sections were pre-treated to 70% water and centrifuged at 13,000 r.p.m. for 5 min. The supernatant was with 1% sodium borohydride, rinsed extensively in 25 mM Tris-Buffer (pH 7.5) applied to Strata-X polymeric reverse-phase cartridges (Phenomenex), methanol- and blocked in 20% normal horse serum (Vector Laboratories) for 30 min. eluted analytes were dried under N2 and the residue was re-suspended in the

NATURE COMMUNICATIONS | 5:4421 | DOI: 10.1038/ncomms5421 | www.nature.com/naturecommunications 11 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5421 mobile phase. A Surveyor LC system coupled to a TSQ Quantum mass oligodendrocyte cultures, Slit2 immunoreactivity was measured in end feet and spectrometer (Fisher Thermo Scientific) was used for analysis. The analytes were along processes (n430 cells per group on two coverslips from duplicate eluted from an ACE 5-m C8 column (150 Â 2.1 mm; Hichrom Ltd.) under isocratic experiments). Immunoreactivity was normalized to vehicle-treated end feet. conditions with a mobile phase consisting of 15% water, 85% methanol and 0.5% Distances between neurites and oligodendrocytes were measured in neuron–glia formic acid at a flow rate of 200 mlminÀ 1 at 30 °C. Mass spectrometry analysis was co-cultures (n420/ group on two coverslips from duplicates). performed using electrospray ionization in positive ion mode. Quantification was undertaken using single-reaction monitoring of the parent ion–product ion 2 GCP isolation Cortical hemispheres from E18.5 mouse embryos (n ¼ 5 embryos transitions for 2-AG and [ H4]AEA of mass-to-charge ratio (m/z)ofm/z 379 to . m/z 287 and m/z 352.2 to m/z 66.2, respectively. Two peaks were present in the per treatment) were vibratome sectioned (200–300 mm thickness), pooled (n ¼ 5 chromatogram for 2-AG because a 1(3)-isomer exists in equilibrium with 2-AG. embryos per treatment) and exposed to JZL184 (100 nM in DMEM/F12/B27 as The areas of both peaks were combined to calculate [2-AG] by weighted linear above) for 25 or 80 min at 37 °C in vitro. Control samples were incubated with vehicle for 80 min. GCPs were isolated from vehicle- or JZL184-treated samples regression analysis against suitable calibration standards using Xcalibur 2.0.6 64 software (Fisher Thermo Scientific). Results were expressed as nanomoles/ according to published protocols . Robo1 protein levels of GCPs were determined picomoles per gram of tissue. by western blotting (Supplementary Table 1). b-III-tubulin served as internal loading control30.

Ex vivo activity-based protein profiling. Frozen, hemisected brains from E18.5 mouse embryos (B35 mg; other hemisphere from mass spectrometry analysis) or a Statistics. Data were analysed using Statistical Package for the Social Sciences section of the cortex from the corresponding mother (B90 mg) were washed with (version 21.0, SPSS Inc.). Pair-wise group comparisons in histochemical and cold Dulbecco’s phosphate-buffered saline (DPBS) (2 Â 1.0 ml) and homogenized western blotting experiments were evaluated using the Student’s t-test (on using a Branson Sonifier 250 probe sonicator (10 pulses, 50% duty cycle, output independent samples). Data were normalized to controls as indicated. Data were setting ¼ 3.0) in cold DPBS (10 mlmgÀ 1 tissue). Homogenates derived from adult expressed as means±s.e.m. A P-value of 0.05 was considered statistically mouse brain samples were centrifuged (3,000g, 3 min) at 4 °C and the pellet was significant. discarded. The total protein concentration for each tissue homogenate was then determined using the DC Protein Assay (Bio-Rad), and each proteome was diluted to 1.0 mg ml À 1 in DPBS. Proteomes (50 ml) were subsequently treated with FP- References Rhodamine (1.0 ml, 50 mM in DMSO), incubated for 30 min at room temperature 1. Brose, K. et al. Slit proteins bind Robo receptors and have an evolutionarily and quenched with 4 Â SDS loading buffer (17 ml). Following separation of pro- conserved role in repulsive axon guidance. Cell 96, 795–806 (1999). teins by SDS–polyacrylamide gel electrophoresis (10% acrylamide gel) and fluor- 2. Kolodkin, A. L. & Tessier-Lavigne, M. Mechanisms and molecules of neuronal escent gel imaging (Hitachi FMBio II flatbed scanner), serine hydrolase activities wiring: a primer. Cold Spring Harb. Perspect. Biol. 3, a001727 (2011). were determined by measuring the fluorescent intensity of target gel bands using 3. Hohenester, E. Structural insight into Slit-Robo signalling. Biochem. Soc. Trans. ImageJ 1.45s (ref. 24). 36, 251–256 (2008). 4. Lopez-Bendito, G. et al. Robo1 and Robo2 cooperate to control the guidance of Primary cultures, pharmacology and transfection. The pallium of embryonic major axonal tracts in the mammalian forebrain. J. Neurosci. 27, 3395–3407 mice was dissected at E16.5. For neuronal cultures, cortical cells from C57BL/6 (2007). À / À 5. Andrews, W. et al. Robo1 regulates the development of major axon tracts and and CB1R mice were enzymatically dissociated and plated at a density of 25–50 000 cells per well (morphometry) or 106 cells per ell (biochemistry) in interneuron migration in the forebrain. Development 133, 2243–2252 (2006). 30 poly-D-lysine-coated 24-well or 6-well plates, respectively . Primary neuron 6. Kadison, S. R., Murakami, F., Matise, M. P. & Kaprielian, Z. The role of floor cultures were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F12 plate contact in the elaboration of contralateral commissural projections within (1:1) containing B27 supplement (2%), L-glutamine (2 mM), penicillin the embryonic mouse spinal cord. Dev. Biol. 296, 499–513 (2006). (100 U ml À 1) and streptomycin (100 mgmlÀ 1; all from Invitrogen) and exposed to 7. Long, H. et al. Conserved roles for Slit and Robo proteins in midline drugs applied alone or in combination (JZL 184: 100 nM, Cayman Chemical; commissural axon guidance. Neuron 42, 213–223 (2004). O-2050: 200 nM, Tocris Bioscience; AM630: 200 nM, Tocris Bioscience; AM251: 8. Bagri, A. et al. Slit proteins prevent midline crossing and determine the 200 nM, Tocris Bioscience; UO126: 10 mM, Tocris Bioscience; SP600125: 5 mM, dorsoventral position of major axonal pathways in the mammalian forebrain. Tocris Bioscience). Primary cortical neurons from C57BL6/N fetuses were Neuron 33, 233–248 (2002). transfected on 1 day in vitro by Lipofectamine 2000 (5 ml; Invitrogen) with a pool of 9. Kidd, T., Bland, K. S. & Goodman, C. S. Slit is the midline repellent for the robo MGL-specific siRNAs (sc-72278; 50 pmol per well) or non-targeting siRNA receptor in Drosophila. Cell 96, 785–794 (1999). (sc-36869; 50 pm per well; Santa Cruz Biotechnology) for 30 min. Neurons were left 10. Simpson, J. H., Bland, K. S., Fetter, R. D. & Goodman, C. S. Short-range and to recover for 3 days before fixation in 4% paraformaldehyde. long-range guidance by Slit and its Robo receptors: a combinatorial code of For glial cultures, neocortices of P2 mouse pups were dissected. Cells were Robo receptors controls lateral position. Cell 103, 1019–1032 (2000). dissociated similarly, transferred to T75 flasks (3 Â 107 cells per flask) and 11. Rajagopalan, S., Nicolas, E., Vivancos, V., Berger, J. & Dickson, B. J. Crossing maintained in DMEM containing 10% fetal bovine serum, L-glutamine (2 mM), the midline: roles and regulation of Robo receptors. Neuron 28, 767–777 À 1 À 1 penicillin (100 U ml ) and streptomycin (100 mgml ). After 7–9 days, T75 (2000). flasks were placed on a shaker at 190 r.p.m. at 37 °C for 1 h. The microglial fraction 12. Plump, A. S. et al. Slit1 and Slit2 cooperate to prevent premature midline was discarded and cells were allowed to equilibrate for 2–6 h in fresh medium. crossing of retinal axons in the mouse visual system. Neuron 33, 219–232 Subsequently, flasks were placed onto an orbital shaker at 210 r.p.m., 37 °C for (2002). 16–18 h to separate oligodendrocytes. The supernatant (oligodendroglial fraction) 13. Nguyen Ba-Charvet, K. T. et al. Slit2-Mediated chemorepulsion and collapse of was collected, centrifuged, re-suspended and seeded at a density of 25,000 cells per 6 developing forebrain axons. Neuron 22, 463–473 (1999). well (morphometry) and 10 cells per well (biochemistry) in poly-D-lysine-coated 14. Ypsilanti, A. R., Zagar, Y. & Chedotal, A. Moving away from the midline: new 24-well and 6-well plates, respectively. Cultures were then treated with JZL184 developments for Slit and Robo. Development 137, 1939–1952 (2010). (100 nM) every other day for 72 h or with MGL-specific siRNA/non-targeting 15. Berghuis, P. et al. Endocannabinoids regulate interneuron migration and siRNA as described above. Cell pellets from the same fraction were taken and morphogenesis by transactivating the TrkB receptor. Proc. Natl Acad. Sci. USA processed for western blotting and PCR analysis simultaneously. Astrocytes were 102, 19115–19120 (2005). collected in fresh medium and processed for protein and mRNA analyses. 16. Aguado, T. et al. The endocannabinoid system promotes astroglial For neuron–glia co-cultures, oligodendrocytes were plated at a density of 25,000 26, cells per well in DMEM containing 10% fetal bovine serum. After 1 day, half of the differentiation by acting on neural progenitor cells. J. Neurosci. 1551–1561 medium was removed and replaced by DMEM/F12 (1:1) culture medium (2006). supplemented with B27 (2%) and neurons were seeded at a density of 25,000 cells 17. Wu, C. S. et al. Requirement of cannabinoid CB(1) receptors in cortical per well. In another set of experiments, Lipofectamine 2000 (5 ml; Invitrogen) was pyramidal neurons for appropriate development of corticothalamic and used to transfect co-cultures with either non-targeting (scrambled) siRNA (sc- thalamocortical projections. Eur. J. Neurosci. 32, 693–706 (2010). 36869) or a pool of Robo1-specific mouse siRNAs (sc-42253; both at 100 pmol per 18. Mulder, J. et al. Endocannabinoid signaling controls pyramidal cell well; Santa Cruz Biotechnology) for 4 h. Co-cultures were treated with JZL184 specification and long-range axon patterning. Proc. Natl Acad. Sci. USA 105, (100 nM) every other day for 72 h. 8760–8765 (2008). Cells on coverslips were immersion fixed on ice with 4% paraformaldehyde in 19. Berghuis, P. et al. Hardwiring the brain: endocannabinoids shape neuronal 0.1 M PB and processed as above for immunocytochemistry. Morphological connectivity. Science 316, 1212–1216 (2007). parameters were determined by analysing calibrated images in the ZEN2010 20. Molina-Holgado, E. et al. Cannabinoids promote oligodendrocyte progenitor imaging software module (Zeiss; n430 cells per group)18,30. In primary neuronal survival: involvement of cannabinoid receptors and phosphatidylinositol-3 cultures, the intensity of Robo1 and Robo2 imunoreactivities along the neurite kinase/Akt signaling. J. Neurosci. 22, 9742–9753 (2002). (n420 per marker and experimental group on two coverslips of two independent 21. Gomez, O. et al. The constitutive production of the endocannabinoid experiments each) was measured by obtaining a plot profile starting at the growth 2-arachidonoylglycerol participates in oligodendrocyte differentiation. Glia 58, cone and continuing along the total axon back to the soma. In primary 1913–1927 (2010).

12 NATURE COMMUNICATIONS | 5:4421 | DOI: 10.1038/ncomms5421 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5421 ARTICLE

22. De Robertis, E., Gerschenfeld, H. M. & Wald, F. Cellular mechanism of 49. Wilson, R. I. & Nicoll, R. A. Endogenous cannabinoids mediate retrograde myelination in the central nervous system. J Biophys. Biochem. Cytol. 4, signalling at hippocampal synapses. Nature 410, 588–592 (2001). 651–656 (1958). 50. Yang, L. & Bashaw, G. J. Son of sevenless directly links the Robo receptor to rac 23. Schlosburg, J. E. et al. Chronic monoacylglycerol lipase blockade causes activation to control axon repulsion at the midline. Neuron 52, 595–607 (2006). functional antagonism of the endocannabinoid system. Nat. Neurosci. 13, 51. Wong, K. et al. Signal transduction in neuronal migration: roles of GTPase 1113–1119 (2010). activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway. Cell 24. Long, J. Z. et al. Selective blockade of 2-arachidonoylglycerol hydrolysis 107, 209–221 (2001). produces cannabinoid behavioral effects. Nat. Chem. Biol. 5, 37–44 (2009). 52. Yuan, X. B. et al. Signalling and crosstalk of Rho GTPases in mediating axon 25. Cravatt, B. F. et al. Molecular characterization of an enzyme that degrades guidance. Nat. Cell Biol. 5, 38–45 (2003). neuromodulatory fatty-acid amides. Nature 384, 83–87 (1996). 53. Huizink, A. C. & Mulder, E. J. Maternal smoking, drinking or cannabis use 26. Wolfer, D. P., Henehan-Beatty, A., Stoeckli, E. T., Sonderegger, P. & Lipp, H. P. during pregnancy and neurobehavioral and cognitive functioning in human Distribution of TAG-1/axonin-1 in fibre tracts and migratory streams of the offspring. Neurosci. Biobehav. Rev. 30, 24–41 (2006). developing mouse nervous system. J. Comp. Neurol. 345, 1–32 (1994). 54. Huizink, A. C. Prenatal cannabis exposure and infant outcomes: overview of 27. Denaxa, M., Chan, C. H., Schachner, M., Parnavelas, J. G. & Karagogeos, D. studies. Prog. Neuropsychopharmacol. Biol. Psychiatry 52, 45–52 (2013). The adhesion molecule TAG-1 mediates the migration of cortical interneurons 55. Bambang, K. N. et al. Immunity and early pregnancy events: are from the ganglionic eminence along the corticofugal fiber system. Development endocannabinoids the missing link? J. Reprod. Immunol. 96, 8–18 (2012). 128, 4635–4644 (2001). 56. Yeh, M. L. et al. Robo1 modulates proliferation and neuogenesis in the 28. Molnar, Z. & Blakemore, C. How do thalamic axons find their way to the developing neocortex. J. Neurosci. 34, 5717–5731 (2014). cortex? Trends Neurosci. 18, 389–397 (1995). 57. Lendvai, D. et al. Neurochemical mapping of the human hippocampus reveals 29. Tucker, K. L., Meyer, M. & Barde, Y. A. Neurotrophins are required for nerve perisynaptic matrix around functional synapses in Alzheimer’s disease. Acta growth during development. Nat. Neurosci. 4, 29–37 (2001). Neuropathol. 125, 215–229 (2013). 30. Keimpema, E. et al. Differential subcellular recruitment of monoacylglycerol 58. Kanaumi, T., Milenkovic, I., dle-Biassette, H., Aronica, E. & Kovacs, G. G. lipase generates spatial specificity of 2-arachidonoyl glycerol signaling during Non-neuronal cell responses differ between normal and Down syndrome axonal pathfinding. J. Neurosci. 30, 13992–14007 (2010). developing brains. Int. J. Dev. Neurosci. 31, 796–803 (2013). 31. Luxardi, G. et al. Glypicans are differentially expressed during patterning 59. Iyer, A. et al. Developmental patterns of DR6 in normal human hippocampus and neurogenesis of early mouse brain. Biochem. Biophys. Res. Commun. 352, and in Down syndrome. J. Neurodev. Disord. 5, 10 (2013). 55–60 (2007). 60. Bradford, M. M. A rapid and sensitive method for the quantitation of 32. Friedlander, D. R. et al. The neuronal chondroitin sulfate proteoglycan microgram quantities of protein utilizing the principle of protein-dye binding. neurocan binds to the neural cell adhesion molecules Ng-CAM/L1/NILE and Anal. Biochem. 72, 248–254 (1976). N-CAM, and inhibits neuronal adhesion and neurite outgrowth. J. Cell Biol. 61. Hurd, Y. L. et al. Marijuana impairs growth in mid-gestation fetuses. 125, 669–680 (1994). Neurotoxicol. Teratol. 27, 221–229 (2005). 33. Bisogno, T. et al. Cloning of the first sn1-DAG lipases points to the spatial and 62. Geiss, G. K. et al. Direct multiplexed measurement of gene expression with temporal regulation of endocannabinoid signaling in the brain. J. Cell Biol. 163, color-coded probe pairs. Nat. Biotechnol. 26, 317–325 (2008). 463–468 (2003). 63. Giuffrida, A., Rodriguez de, F. F. & Piomelli, D. Quantification of bioactive 34. Blankman, J. L., Simon, G. M. & Cravatt, B. F. A comprehensive profile of brain acylethanolamides in rat plasma by electrospray mass spectrometry. Anal. enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol. Chem. Biochem. 280, 87–93 (2000). Biol. 14, 1347–1356 (2007). 64. Lockerbie, R. O., Miller, V. E. & Pfenninger, K. H. Regulated plasmalemmal 35. Marrs, W. R. et al. The serine hydrolase ABHD6 controls the accumulation and expansion in nerve growth cones. J. Cell Biol. 112, 1215–1227 (1991). efficacy of 2-AG at cannabinoid receptors. Nat. Neurosci. 13, 951–957 (2010). 36. Straiker, A., Min, K. T. & Mackie, K. Fmr1 deletion enhances and ultimately desensitizes CB1 signaling in autaptic hippocampal neurons. Neurobiol. Dis. 56, Acknowledgements 1–5 (2013). This work was supported by the Swedish Medical Research Council (T.H.), Swedish 37. Rothberg, J. M., Jacobs, J. R., Goodman, C. S. & rtavanis-Tsakonas, S. slit: an Brain Research Foundation (‘Hja¨rnfonden’; T.H.), Novo Nordisk Foundation (T.H.), extracellular protein necessary for development of midline glia and Petrus & Augusta Hedlunds’ Foundation (T.H.), EU-FP7 project DEVELAGE (N278486, commissural axon pathways contains both EGF and LRR domains. Genes Dev. G.G.K.), National Brain Research Program of the Hungarian Academy of Sciences (A.A.), 4, 2169–2187 (1990). National Institutes of Health (DA023214, T.H. and Y.L.H.; DA030359, Y.L.H; F31- 38. Carmeliet, P. & Tessier-Lavigne, M. Common mechanisms of nerve and blood DA031559, C.V.M.) and the Wellcome Trust (#089775, J.G.P. and W.D.A.). The content vessel wiring. Nature 436, 193–200 (2005). of this report is solely the responsibility of the authors and does not necessarily represent 39. Hivert, B., Liu, Z., Chuang, C. Y., Doherty, P. & Sundaresan, V. Robo1 and the official views of the US National Institutes of Health. We thank M. Watanabe, K. Robo2 are homophilic binding molecules that promote axonal growth. Mol. Mackie and F. Murakami for antibodies, Y.A. Barde for taugfp/ þ mice, K. Mackie for Cell Neurosci. 21, 534–545 (2002). MGL À / À tissues, F. Molina-Holgado for the oligodendrocyte isolation protocol, O.K. 40. Ortar, G. et al. Tetrahydrolipstatin analogues as modulators of Penz for technical assistance, M. Tessier-Lavigne, L. Erskine, R.A. Ross and members of endocannabinoid 2-arachidonoylglycerol metabolism. J Med. Chem. 51, the Harkany laboratory for discussions and feedback and the National Institute of Mental 6970–6979 (2008). À / À Health for the transfer of CB1R colony founders. Laser-scanning microscopy was 41. Keimpema, E., Mackie, K. & Harkany, T. Molecular model of cannabis performed at the Click Imaging Core Facility of the Karolinska Institutet. sensitivity in developing neuronal circuits. Trends Pharmacol. Sci. 32, 551–561 (2011). 42. Andrews, W. et al. The role of Slit-Robo signaling in the generation, migration and morphological differentiation of cortical interneurons. Dev. Biol. 313, Author contributions 648–658 (2008). A.A. and T.H. conceived the general framework of this study. A.A., E.K., B.F.C., G.G.K., 43. Argaw, A. et al. Concerted action of CB1 cannabinoid receptor and deleted in J.G.P., Y.L.H. and T.H. designed experiments. A.A., G.T., M.J.N., D.C., I.M., J.B., G.A.C., colorectal cancer in axon guidance. J. Neurosci. 31, 1489–1499 (2011). W.D.A., J.H., C.V.M., J.F. and E.K. performed experiments. A.A., G.T., M.J.N., G.A.C. 44. Kinrade, E. F. & Hidalgo, A. Lateral neuron–glia interactions steer the response and E.K. analysed data. A.A., E.K. and T.H. wrote the manuscript. All Authors com- of axons to the Robo code. Neuron Glia Biol. 1, 101–112 (2004). mented on and approved the submission of this manuscript. 45. Buckley, N. E., Hansson, S., Harta, G. & Mezey, E. Expression of the CB1 and CB2 receptor messenger RNAs during embryonic development in the rat. Neuroscience 82, 1131–1149 (1998). Additional information 46. Berrendero, F., Sepe, N., Ramos, J. A., Di Marzo, V. & Fernandez-Ruiz, J. J. Supplementary Information accompanies this paper at http://www.nature.com/ Analysis of cannabinoid receptor binding and mRNA expression and naturecommunications endogenous cannabinoid contents in the developing rat brain during late gestation and early postnatal period. Synapse 33, 181–191 (1999). Competing financial interests: The authors declare no competing financial interests. 47. Diaz-Alonso, J. et al. The CB1 cannabinoid receptor drives corticospinal motor Reprints and permission information is available online at http://npg.nature.com/ neuron differentiation through the Ctip2/Satb2 transcriptional regulation axis. reprintsandpermissions/ J. Neurosci. 32, 16651–16665 (2012). 48. Keimpema, E. et al. Diacylglycerol lipase alpha manipulation reveals How to cite this article: Alpa´r, A. et al. Endocannabinoids modulate cortical develop- developmental roles for intercellular endocannabinoid signaling. Sci. Rep. ment by configuring Slit2/Robo1 signalling. Nat. Commun. 5:4421 doi: 10.1038/ 3, 2093 (2013). ncomms5421 (2014).

NATURE COMMUNICATIONS | 5:4421 | DOI: 10.1038/ncomms5421 | www.nature.com/naturecommunications 13 & 2014 Macmillan Publishers Limited. All rights reserved.