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Received 25 Apr 2014 | Accepted 16 Jun 2014 | Published 17 Jul 2014

DOI: 10.1038/ncomms5421

Endocannabinoids modulate cortical development by conguring Slit2/Robo1 signalling

Aln Alpr1,w, Giuseppe Tortoriello1, Daniela Calvigioni1,2, Micah J. Niphakis3, Ivan Milenkovic4, Joanne Bakker2, Gary A. Cameron5, Jnos Hanics6, Claudia V. Morris7, Jnos 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-specic cannabinoid receptor activation. Accordingly, Robo1 and/or Slit2 manipulation limits endocannabinoid modulation of axon guidance. We conclude that endocannabinoids can congure focal Slit2/Robo1 signalling to modulate directional axonal growth, which may provide a basis for understanding impaired brain wiring associated with metabolic decits and prenatal drug exposure.

1 Division of Molecular Neurobiology, Department of Medical Biochemistry & Biophysics, Karolinska Institutet, Scheeles vag 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, Wahringer Grtel 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+zolt 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: mailto:[email protected]

Web End [email protected] ).

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Axonal pathnding decisions rely on short- and long-range intercellular cues determining cytoarchitectural arrangements permissive for directional growth in the developing

brain. Guidance cues include a plethora of structurally heterogeneous proteins, released during dened temporal windows and acting within circumscribed spatial domains. Slits, a family of phylogenetically conserved chemorepellent molecules1, bind Roundabout (Robo)1/2 receptors2,3 to regulate axonal tract formation during forebrain development48. Slit/Robo1/2 signalling is implicated in midline crossing912 and the dorsoventral positioning of long-range axons13,14, thus being indispensable for the basic forebrain wiring diagram and connectivity principles to emerge. Accordingly, Robo1 /

mice present the dysgenesis of major commissural tracts5 and axon guidance errors in the corticofugal system4. Similarly, Slit2 / and Slit1 / /Slit2 / mice show corticothalamicand thalamocortical-targeting defects8, as well as optic tract abnormalities12. In addition, axons coalesce into enlarged fascicles in Robo1 / mice5. Heparin sulphate proteoglycans and transcription factors, including members of the T-box or the

LIM homeodomain families can inuence Slit/Robo1/2 signalling14. Nevertheless, the molecular identity of signalling networks for the upstream and local control of coordinated Slit/ Robo1/2 expression, subcellular positioning and signal competence during corticogenesis remains relatively poorly explored.

Signalling lipids have recently emerged as regulators of neuronal development. Endocannabinoids, the natural ligands of cannabinoid receptors, modulate both neuronal15 and glial16 differentiation, and are efcacious to facilitate neurite extension and growth cone-steering decisions1719. CB1 cannabinoid receptors (CB1Rs) contribute to regulating the fasciculation and pallidal targeting of corticofugal axons17. In contrast, CB2 cannabinoid receptors (CB2Rs) are co-expressed with key enzymes of 2-arachidonoylglycerol (2-AG) metabolism in glia20, and promote the differentiation of cultured oligodendrocytes21, the cellular source of myelin sheaths in the nervous system22.

Here, we show that pharmacological enhancement of 2-AG bioavailability during corticogenesis in mouse triggers the CB2R-dependent differentiation of oligodendrocytes. Premature oligodendrocyte end-feet proliferation in vivo coincides with CB1R-dependent fasciculation errors of corticofugal projections, including altered axon bundle formation in the paramedian domain of the corpus callosum. We hypothesized that endocannabinoids, by simultaneously engaging cell-type-specic cannabinoid receptors, might orchestrate chemorepulsion of cortical axons via a mechanism operating in trans, with oligodendrocytes producing surplus chemorepellents. By implementing a multi-parametric approach, encompassing neuroanatomy in human and mouse fetuses, messenger RNA (mRNA) proling, biochemistry, in vitro analysis and genetic models, we identify oligodendrocyte-derived Slit2 acting at Robo1 receptors in corticofugal axons downstream from the respective activation of CB2Rs and CB1Rs upon pharmacological manipulation of 2-AG degradation as the molecular underpinning of endocannabinoid-induced corticofugal axon guidance errors. We nd cannabinoid receptor activation to facilitate Slit2 accumulation in oligodendrocyte end feet, and Robo1 accumulation in axonal growth cones. Overall, these data suggest that endocannabinoid signalling can regulate Slit/Robo interactions during the establishment of forebrain axonal tracts.

ResultsJZL184 increases 2-AG levels in fetal and adult cortices. To enhance endogenous 2-AG signalling in gravid mice, we

employed the small molecule JZL184 from embryonic day(E)12.518.5 to inhibit monoacylglycerol lipase (MGL), the enzyme chiey responsible for degrading 2-AG in the nervous system23. The selective inhibition of MGL by JZL184 was conrmed with activity-based protein proling of serine hydrolases in the brains of JZL184 and vehicle-treated dams and their E18.5 embryos. Consistent with previous reports24, JZL184 displayed a high selectivity for MGL across all detectable brain serine hydrolases (Fig. 1a) leading to a signicant increase in 2-AG levels in adult and embryonic cortices (Fig. 1b). While fatty-acid amide hydrolase (FAAH)25, which catabolizes the endocannabinoid anandamide (AEA), was partially inhibited by JZL184 in adult and to a lesser degree, embryonic brain samples, we did not observe a signicant increase in AEA levels in fetal or adult cortices (Fig. 1b). The levels of 2-linoleoyl glycerol (a congener of 2-AG) and oleoylethanolamide (an analogue of AEA), were both unaffected by JZL184 treatment (Fig. 1b). Together, these data conrm that JZL184 selectively targets MGL leading to an accumulation of 2-AG in the brains of gravid adult mice and their offspring.

MGL inhibition induces axon fasciculation decits. We studied the morphology of corticofugal axons in developing fetal brains after JZL184 exposure, since these tracts contain the highest levels of CB1Rs during embryonic development18. JZL184 exposure induced L1 neural cell adhesion molecule (L1-NCAM) axons to coalesce into enlarged fascicules (Fig. 2a,b; Supplementary

Table 1) irrespective of the rostrocaudal axis of the developing brain (Supplementary Fig. 1ac). The enlarged axon fascicles were found spread in the cortical plate, including intermediate zone territories otherwise devoid of L1-NCAM processes (Fig. 2i,j;

Supplementary Fig. 1a).

The coalescing of axonal bres upon JZL184 exposure was altered irrespective of their origin or projection targets. In particular, enlarged fascicles were observed to cross the midline (Fig. 2e,f), populated the suprastriatal domain of the corpus callosum (Fig. 2m,n; Supplementary Fig. 2a) or invaded the striatum (Fig. 2o,p). Both corticothalamic and thalamocortical axons were affected in the striatum, as revealed by TAG-1 (refs 26,27) and C-terminal full length of mouse GPR55 (ref. 17) immunohistochemistry, respectively (Supplementary Fig. 2ae,r). These ndings suggest the multimodal interplay of corticofugal and thalamocortical projections along their reciprocal growth paths during the mechanism termed axonal handshake28.

Next, we examined mice carrying green uorescent protein (GFP)-tagged cytosolic tau in neurons29 in the presence or absence of CB1Rs (taugfp/gfp::CB1R)29 to show that radial glia (RC2 ) bres and post-mitotic Brn1 pyramidal cells16 arranged perpendicular to corticofugal axons and adopted alternative paths (Fig. 2i,j). We then administered JZL184 to CB1R / fetuses to test the reliance of pharmacologically induced axon guidance errors on CB1Rs, selectively localized to the corticofugal system by mid-gestation18.

In accord with previous reports18,30, CB1R deletion alone induced axon fascicle enlargement (Fig. 2c,g,k,q). In CB1R / animals,

JZL184 did not affect the diameter of rst-order fascicles(18.781.66 mm (JZL184) versus 16.041.28 mm (vehicle) (means.e.m.; n 3 mice per group); Fig. 2d,h,l,q). CB1R /

mice treated with JZL184 presented signicantly increased axon spreading in the intermediate zone/corpus callosum (203.818.44 mm (JZL184) versus 157.276.28 mm (vehicle), n 3 mice per group, Po0.05 (Students t-test); Fig. 2q). This

was associated with altered distribution of radial glia processes and Brn1 pyramidal cell radially migrating towards the cortical plateto avoid axon-packed microdomainsin taugfp/gfp::

CB1R / reporter mice (Fig. 2l).

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Figure 1 | JZL184 increases 2-AG levels in maternal and embryonic brains. (a) Ex vivo activity-based protein proling of brain-derived serine hydrolases in vehicle and JZL184 (40 mg kg 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 meanss.e.m., n 2 per group (mothers), n 6 per group (fetuses); **Po0.01,

*Po0.05 (Students t-test).

The dynamic control of axonal growth and fasciculation involves the elimination of functionally redundant or mistargeted processes over time. Mature axons become embedded in extracellular matrix, a three-dimensional scaffold of molecularly heterogeneous proteins31, to maintain their positions in axon fascicles. Here, we nd that neurocan, a chondroitin sulphate proteoglycan whose expression is essential for cell adhesion and slows neurite extension32, is excluded from enlarged axonal fascicles upon JZL184 treatment (Fig. 2mp), suggesting altered neurite motility and pathnding. The JZL184-induced remodelling of the corticofugal system also impacted the coincident patterning of thalamocortical afferents (Supplementary Fig. 2ae), robustly affecting their handshake28.

Nevertheless, the JZL184-induced axonal phenotype was absent in constitutive MGL / mice (Supplementary Fig. 3).

Chronic 2-AG signalling in MGL / mice is characterized by CB1R downregulation and desensitization, suggesting compensation for increased CB1R signalling events23. Inhibition of MGL by JZL84 in our experimental model (E12.5-E18.5) would likely induce excessive CB1R signalling, resulting in physiological alterations otherwise compensated for in MGL / mice. Thus, we conclude that excess 2-AG availability brought about by the pharmacological disruption of their degradation during dened periods of corticogenesis is sufcient to remodel the corticofugal projection system.

Unchanged CB1R signal transduction upon JZL184 exposure. Repeated JZL184 administration can induce cannabinoid receptor desensitization and functional antagonism23. Therefore, we rst sampled mRNA (Supplementary Table 2) and protein expression of molecular constituents of the 2-AG signalling cassette (Fig. 2s; Supplementary Fig. 4). Neither mRNA nor protein levels for sn-1-diacylglycerol lipases a and b (DAGL), synthesizing 2-AG33,

MGL and ABHD6, involved in 2-AG degradation34,35, and CB1Rs changed signicantly in JZL184-exposed fetal cortices (Supplementary Fig. 4a,b). We then used extracellular signal-

regulated kinase 1/2 (Erk1/2) phosphorylation as a read-out36 upon acute agonist challenge (WIN55,212-2, 100 nM) of cultured cortical neurons pre-treated with JZL184 for 4 days to address whether JZL184 induced CB1R desensitization. JZL184 alone did not induce constitutive Erk1/2 phosphorylation, and AM251, a CB1R antagonist, did not modify JZL184 effects. Notably, JZL184 allowed WIN55,212-2-induced rapid Erk1/2 phosphorylation (Fig. 2t,u) with comparable temporal dynamics to that in control (Supplementary Fig. 4c), which is compatible with earlier data showing that only prolonged (48 days) agonist stimulation induces CB1R desensitization in vitro36. These data suggest that supra-physiological 2-AG signals upon JZL184 application during corticogenesis alter axon fasciculation via neuronal CB1Rs, while a non-CB1R-mediated mechanism might account for the increased axonal spread in the prospective corpus callosum.

Oligodendrocyte end feet proliferate among misrouted axons. Excessive spreading of corticofugal axons committed to the prospective corpus callosum in JZL184-treated CB1R / mice led us to hypothesize that a CB1R-independent mechanism can alter the pathnding of corticofugal axons. Therefore, we tested whether premature differentiation of oligodendrocytes, which postnatally produce myelin to ensheath callosal axons22 and co-express CB1Rs and CB2Rs20, could participate in axonal misrouting. JZL184 facilitated the accumulation of oligodendrocytes that expressed 20,30-cyclic-nucleotide 30-phosphodiesterase (CNPase)20, and formed micro-islands interspersed with corticofugal axons in both wild-type and CB1R / mice (Fig. 3ad). Oligodendrocyte differentiation was reected by the increased density (Fig. 3e) and size (Fig. 3f) of their CNPase end feet targeted towards nearby axons. The JZL184-induced morphogenesis of oligodendrocytes was CB1R independent since it also occurred in CB1R / mice (density: 37. 73.1 (JZL184) versus 14.72.7 (vehicle) end feet/

1,000 mm2, Po0.05; Fig. 3e). By western blotting, we

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Figure 2 | JZL184 alters callosal axon fasciculation and pathnding. (ad) Corticofugal axonal phenotypes and (eh) their midline crossing in vehicle- or JZL184-treated mouse embryos at E18.5. (il) A taugfp/gfp reporter mouse29 was used for the genetic tagging of corticofugal axons. Axon fasciculation errors present in CB1R / mice (k), were further increased by JZL184 (surface area occupied by corticofugal axons (l). (mp) Neurocan was excluded 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 meanss.e.m., n 3/group (q,r). In vitro experiments were

performed in duplicates with nZ2 samples per experiment run in parallel. *Po0.05 (Students t-test). cc, corpus callosum; cpu, caudate putamen; FC, ber crossing; GW, glial wedge; IZ, intermediate zone. Scale bar, 300 mm (ah,m,n), 10 mm (il,o,p).

quantitatively conrmed that JZL184 signicantly elevated CNPase and myelin basic protein levels in the fetal cerebrum (Fig. 3g,h). These results outline a multicomponent mechanism with 2-AG simultaneously activating CB1Rs in axons, and CB2Rs in oligodendrocytes in JZL184-exposed fetuses. Nevertheless, a causal relationship between altered axonal navigation and glial proliferation remains unknown. Here, we reasoned that endocannabinoid-induced glial differentiation might instruct the production of a chemorepellent for the focal remodelling of the corticofugal tract (Fig. 3i).

CB1Rs coexist with Robo1/2 receptors in corticofugal axons. Neurons and glia interact to form chemical conduits for axonal growth during brain development14. Slit/Robo interplay is 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 upstream regulation of Slit/Robo signalling in axon guidance is not entirely understood. We hypothesized that endocannabinoid-

induced oligodendroglia positioning and differentiation in conjunction with 2-AGs cell-type-specic action via CB1Rs and

CB2Rs could orchestrate in trans Slit/Robo signalling. Therefore, we rst examined whether CB1Rs and Robo1 receptors coexist in long-range axons. By using serial sections from human fetuses at gestational weeks 2023 (Supplementary Table 3), we nd the expression patterns of CB1Rs and Robo1 receptors to overlap in forebrain axonal tracts, particularly axons coursing in the internal capsule (Fig. 4ad). Multiple immunouorescence labelling substantiated that CB1Rs and Robo1 receptors indeed co-distributed along the same axons in humans (Fig. 4eg). Likewise, corticofugal axons in the E18.5 fetal mouse brain contained both receptors (Fig. 4hm). In addition, CB1R cortical axons in the corpus callosum harboured Robo2 receptors (Fig. 4np).

Robo1 accumulates in JZL184-exposed neuronal growth cones. Axonal growth is a dynamic process, with growth cones continuously sampling the local microenvironment and responding

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Figure 3 | JZL184 induces oligodendrocyte differentiation. CNPase oligodendroyctes in the prospective corpus callosum were infrequently detected in wild-type (a) and CB1R / (c) embryos (E18.5; open arrowheads). (b,d) JZL184-induced oligodendrocyte differentiation, which progressed in CB1R / mice (solid arrowheads pinpoint end feet).

(e,f) Quantitative data on oligodendrocyte morphology. (g,h) CNPase and myelin basic protein (MBP) levels in the cortices of JZL184-treated mouse embryos. (i) Schema of JZL184-induced remodelling of axonal pathnding upon premature oligodendrocyte differentiation. Data were expressed as meanss.e.m., n 5/6 (JZL184/vehicle), *Po0.05 (Students t-test).

Scale bar, 4 mm (a).

to chemotactic cues by initiating turning decisions38. Robo1/2 localization to growth cones underpins Slit/Robo signalling during axonal pathnding13,39. On the basis of our pharmacological data (Supplementary Fig. 4d), we hypothesized that modications to axonal growth trajectories might be mediated by the CB1R-dependent repartitioning of Robo1 receptors in axonal growth cones. Therefore, we conrmed the co-localization of CB1Rs and Robo1 receptors in vitro (Fig. 5a), and validated Robo1/2 mRNA and protein expression by PCR (Fig. 5b) and western blotting (Fig. 5c). Upon JZL184 treatment of cultured cortical neurons, Robo1 receptors accumulated in growth cones, specied as the uorescence ratio between the growth cone and adjoining neurite shaft (Fig. 5d). This response was O-2050 sensitive30, conrming CB1R involvement. In contrast, Robo2 receptor distribution remained unchanged (Fig. 5e). We also found a decreased co-localization coefcient for Robo1 and CB1R, reinforcing increased Robo1 receptor import if CB1R levels remained stationary and partition

separately from Robo1 (Fig. 5f; Supplementary Fig. 5a). Next, we puried 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 ndings. Robo1 receptor accumulated in JZL184-exposed GCPs (Fig. 5g), lending further support to the concept that superuous 2-AG signalling can redistribute Robo1 receptors in a CB1R-dependent fashion.

Furthermore, we suggest that axonal transport contributes to Robo1 accumulation in growth cones, since we detected a JZL184-induced increase in Robo1 receptor immunouorescence 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.

To conrm the reliance of our proposed mechanism on MGL activity, we transfected primary cortical neurons with MGL short interfering RNA (siRNA) and assessed Robo1 receptor intensity in the growth cone. Transient MGL knockdown recapitulated the accumulation of Robo1 immunoreactivity within the growth cone (Fig. 5i,j; Supplementary Fig. 5d,e), which was CB1R dependent given its sensitivity to AM251, a CB1R inverse agonist (Fig. 5k). In addition, the lack of Robo1 receptor responsiveness in the growth cones of primary cortical cultures from CB1R / mice reinforced the critical involvement of CB1Rs in this regulatory process (Fig. 5in). CB1R activation in developing neurons triggers molecularly diverse second-messenger cascades41. Here, we found that JZL184-induced Robo1 receptor accumulation in growth cones was occluded by the administration of both U0126 and SP600125, MEK1/2 and c-Jun N-terminal kinases (JNK) inhibitors, respectively (Fig. 5o,p).

If a CB1R-mediated increase of Robo1 signalling is of pathophysiological signicance, then Robo/Slit mRNA levels might be expected to coordinately change in the cortices of human fetal subjects exposed in utero to cannabis, relative to controls. By using the NanoString technology (Supplementary Table 4), we found that Robo1 receptor mRNA expression tended to be signicantly higher in cannabis-exposed subjects (F1,26 4.156; P 0.056; Supplementary Fig. 1d). This change

was driven by a signicant impact of developmental growth measures. Similarly for Slit1 mRNA, there was a signicant confound of development resulting in only a trend effect (P 0.093) for the contribution of cannabis exposure. Interest

ingly, there was a signicant correlation (r 0.576; P 0.002)

between the expression of Robo1 and Slit1 mRNAs in the human fetal cortex, suggesting a functional interaction irrespective of cannabis predisposition.

JZL184 promotes Slit2 production in oligodendrocytes. Genetic deletion of Slit1 and/or Slit2 induces midline crossing errors8,12. Therefore, we hypothesized that oligodendrocytes could produce excess Slits in a CB2R-dependent fashion to impact corticofugal axon fasciculation. Owing to methodological constraints to histochemically detect Slit1, we adopted a candidate approach and focused on Slit2 throughout our proof-of-concept experiments. We rst prepared oligodendrocyte cultures, which only sporadically contained astroglia (Fig. 6a) to demonstrate that CNPase oligodendrocytes are a likely source of Slit2 in vitro.

We extended these data by comparing glial brillary acidic protein (GFAP) astroglia and oligodendroglia cultures (Fig. 6b), and showing that oligodendroglia selectively contain detectable

Slit2 mRNA transcripts and Slit2-like protein (Fig. 6c; Supplementary Figs 5b and 6). Slit2-like immunoreactivity was

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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 bres in the internal capsule at high magnication. (eg) Co-localization of CB1R and Robo1 receptors in human fetal tissues (internal capsule) by high-resolution laser-scanning microscopy (open arrowheads). Robo1 (hm), as well as Robo2 (np), 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).

seen only in oligodendrocytes (but not astroglia or neurons; Fig. 6a) in vitro and mainly localized to their soma (Fig. 6d) and membranes of their processes (Fig. 6e,f). We conrmed our Slit2 detection in oligodendrocytes by demonstrating signicantly diminished Slit2-like content in oligodendroglia from Slit2 /

mice (Supplementary Fig. 6ae). In addition, we validated our staining protocols by co-labelling for an antibody revealing a phylogenetically conserved sequence from the Drosophila Slit ortholog (Fig. 6c). These data identify oligodendrocytes as a candidate source of Slit2.

Excess 2-AG facilitates oligodendrocyte differentiation in culture21 and in vivo (Fig. 3). Here, we show that JZL184 increased Slit2 expression in cultured oligodendrocytes (Fig. 6gi), and increased Slit2-like immunoreactivity in their end feet (Fig. 6j). JZL184s effect on Slit2-like labelling in oligodendroglia end feet was reversed by AM630, a CB2R antagonist21 (Fig. 6kn), suggesting that Slit2 production and subcellular translocation might be regulated by agonist (for example, 2-AG) signalling at CB2Rs. To verify the effects of JZL184 on Slit2 production, we transfected cultured oligodendrocytes with MGL siRNA, which recapitulated the increase of Slit2-like immunoreactivity in oligodendrocyte end feet (Fig. 6o,p).

In JZL184-treated brains, CNPase /CB1R oligodendroglial end feet juxtaposed Robo1/CB1R double-labelled corticofugal axons (Fig. 6q), and accommodated enlarged fascicles by forming proportionately larger glial scaffolds (that is, no CNPase labelling within individual fascicles; Fig. 6q). These oligodendroglia end feet harboured Slit2-like immunoreactivity, which frequently juxtaposed nearby axons (Fig. 6r). These data together with Robo1/CB1R (Fig. 5a) and Robo2/CB1R (Fig. 5a) co-localization and Robo1/Robo2 mRNA (Fig. 5b) and protein (Fig. 5c) expression in cultured cortical neurons suggest that endocannabinoids are poised to modulate Slit/Robo signalling, and support a model in which ectopic Slit2 presentation by cortical oligodendrocytes participates in inducing growth errors of CB1R axons in vivo (Fig. 6s).

JZL184 fails to affect axonal growth in Robo1 / mice. If focal Robo1/Slit2 interactions are indeed modulated by 2-AG signalling, then JZL184 administration to Robo1 / (ref. 42)

mice will reveal incomplete cortical phenotypes due to the neuron versus oligodendroglia-driven arch of the proposed signalling axis (Fig. 7a). Robo1 / mice per se present an axon fasciculation

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Figure 5 | CB1R stimulation regulates subcellular Robo1 positioning. (a) Robo1 and Robo2 immunoreactivity in the somata and neurites (including growth 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), uorescence intensity in growth cones treated with JZL184 or O-2050, a CB1R antagonist. (f) Co-localization coefcient of Robo1 and CB1R in 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. (ik) Robo1 immunoreactivity in growth cones upon siRNA-mediated MGL silencing in cultured primary neurons. (ln) 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 meanss.e.m., from triplicate experiments (sample size: 825 neurons per group in morphometric analysis), *Po0.05 (Students t-test). Scale bar, 10 mm (a), 5 mm (l,o).

phenotype5. If Robo1 receptors are downstream from CB1Rs, then JZL184 likely would fail to affect the pre-existing axonal phenotype in Robo1 / mice. Indeed, the spread of callosal axons5 (Fig. 7b) remained unaffected in JZL184-treated

Robo1 / mice. As such, neither the diameter of individual and enlarged axon fascicles nor their interbundle distance were

affected by JZL184 (Fig. 7c,d,f). In contrast, JZL184 provoked CNPase oligodendroglial end-feet differentiation in Robo1 /

mice. Yet oligodendroglial end feet were interspersed with individual corticofugal axons, conrming the loss of a chemorepulsive cue (Fig. 7e,g). These data dissociate between the loss of CB1R-driven and Robo1 receptor-dependent axon

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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 conrmed this nding (Slit). (df) 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. (kn) 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 oligodendroglial end feet in the interbundle space (ibs) and in apposition to enlarged fascicles of Robo1 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 pathnding upon premature oligodendrocyte differentiation. Data were expressed as meanss.e.m., n 832 cells per group for each condition in duplicate experiments; *Po0.05 (Students t-test). Scale bar, 10 mm (a,h,o,q), 2 mm (m).

guidance decits and CB2R-mediated focal chemorepulsion and oligodendroglial differentiation in vivo.

Slit2/Robo1 signals mediate JZL184-induced chemorepulsion. We devised a genetically amenable co-culture system to show that Slit2 oligodendrocytes affect directional growth decisions of

Robo1/CB1R neurites in vitro. Neurites regularly contacted oligodendrocytes under control conditions (Fig. 8a). JZL184 administration in vitro enhanced Slit2 expression and mobilized Slit2 to submembranous positions in oligodendrocytes (Fig. 8b), with coincident repulsive responses of nearby neurites as shown by their increased distance to apposing glial surfaces(15.651.01 mm (JZL184) versus 9.741.11 mm (vehicle), Po0.05; Fig. 8d).

We used siRNA knockdown of Robo1 in neurons co-cultured 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 labelling for Robo1 receptors (Supplementary Fig. 5c) conrmed successful neuronal Robo1 silencing. Robo1 knockdown overcame JZL184-induced repulsion and adopted differentiated growth cone morphologies (Fig. 8c). Consequently, neurites of Robo1 siRNA-transfected neurons approached oligodendrocytes, leaving the growth cone-oligodendrocyte distance signicantly reduced (4.970.69 mm (Robo1 siRNA) versus 18.212.06 mm (scrambled siRNA), Po0.05; Fig. 8d). Further, JZL184 administration increased the number of branching points on approaching neurites in the close vicinity of oligodendocytes (Fig. 8e), suggesting that an increased number of axon collaterals might,

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Figure 7 | JZL184-induced oligodendroglial repositioning in Robo1 / mice. (a) Schematic overview of 2-AG signalling converging on axonal growth and fasciculation through Slit/Robo signalling. (b) Callosal axon fasciculation in Robo1 / mice treated with JZL184. Note that untreated Robo1 /

mice presented a fasciculation phenotype5. (c,d) Diameter of individual bundles and their interbundle distance upon JZL184 treatment in Robo1 / embryos. (eg) Oligodendroglia end-feet inltrated axonal bundles in JZL184-treated Robo1 / mice. This was undetected (ND) in wild-type or

CB1R / embryos. Data were expressed as meanss.e.m., from n 35 embryos per genotype; *Po0.05 versus respective wild-type (WT) controls

(Students t-test). Scale bar, 10 mm (f,g).

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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 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 meanss.e.m., triplicate experiments with n 3 samples run in parallel; n 69 observations per group. *Po0.05 (Students t-test). Scale bar, 8 mm (a1,b), 3 mm (insets).

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at least in part, contribute to changes in callosal microanatomy. Cumulatively, these data identify endocannabinoids as candidates to upstream regulate Slit2/Robo1 interactions during directional axonal growth (Fig. 8f,g).

DiscussionEndocannabinoids affect neuronal differentiation, including directional axonal growth during corticogenesis17,19,43. Endocannabinoid signalling appears particularly efcacious to modulate axonal growth since CB1Rs are selectively enriched in navigating axons, and are co-targeted with DAGLs to advancing growth cones30,43. Establishment of major axonal tracts critically relies on chemorepulsive Slit gradients4,5,7. In fact, midline crossing of commissural axons particularly depends on focal Slit/ Robo signalling between neurons and glia44. The architecture of corticofugal axons is altered in CB1R / mice18, a phenotype reminiscent to those of Robo1 / /Robo2 / mutants4. Here, we resolve this similarity by dissecting a molecular interplay between endocannabinoids and the Slit/Robo signalling axis. Our data suggest that the lack of CB1R-induced Robo1 accumulation in growth cones might disrupt, at least in part, axonal navigation and contribute to the altered topography of corticofugal axons in CB1R / mice. However, by showing that increased 2-AG levels upon MGL inhibition coincidently induce CB2R-

mediated oligodendrocyte differentiation and CB1R-dependent axonal misrouting in the corpus callosum, we characterize an intercellular signalling network in which a single endocannabinoid ligand engages molecularly distinct cannabinoid receptors in neurons and glia to upstream control chemorepellent production (Slit2 in glia) and signalling (Robo1 in neurons). Whether changes in axonal topography in the corpus callosum of JZL184-treated mice reect an increased number of axon collaterals or instead are due to the spatial reorganization of the same number of axons (because of, for example, changes to extracellular matrix composition) as in controls remains unknown. Our in vitro data on the JZL184-induced increase of neurite branching points supports the former scenario, and suggests that an increased numbers of axon collaterals compacting in callosal fascicles might signicantly increase network instability due to erroneous synaptic wiring. Our results in cultured neurons are also compatible with those from Robo1 / mutant embryos, where glial repositioning around enlarged axon fascicles could argue for the disruption of a critical glialneuronal signal interface.

We employed JZL184, a selective MGL inhibitor23, to increase endocannabinoid levels in the fetal brain. The selectivity of this compound was veried using activity-based protein proling after in vivo drug application, as well as in in vitro experiments using genetically or RNA-silenced primary cultures. Since JZL184 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 place in JZL184-exposed fetuses, sufcient to affect axonal growth and guidance. This included an increased transport of Robo1 along the axon culminating in peak expression levels and externalization in the growth cone, likely modulated via mitogen-activated protein kinase and JNK signalling, as well as an increased Slit2 expression on an enlarged oligodendroglial end-feet arbour. Considering that CB1Rs are broadly distributed in the developing nervous system45,46, including descending projections to the spinal cord45,47, our results might have profound implications for the overall organization of axonal connectivity in the nervous system.

Although we did not seek to identify the cellular origin of signal-competent 2-AG, a number of alternative sources may

exist. These include the elongating corticofugal axons themselves, which contain DAGLa (and b)17,18. If so, Slit2/Robo1 signalling might represent a cell-autonomous amplication mechanism whereby DAGL-derived 2-AG could activate CB1Rs within growth cones30, facilitating Robo1 trafcking. Recent data suggest that 2-AG can diffuse extracellularly and evoke physiological responses in a radius of 20100 mm (refs 48,49).

Thus, DAGL activity in axonal growth cones appears sufcient to generate focal 2-AG microgradients to activate CB2Rs on neighbouring oligodendrocytes to stimulate Slit2 production and release (Fig. 8f,g). At the level of signal transduction, CB1Rs and Robo1/2 can coordinately activate downstream effectors within axonal growth cones, which can act as reinforcers of the individual receptors signal outputs. Members of the Rho family of GTPases, which can be activated by either receptor in various tissues19,50,51 and whose activity is indispensable for cytoskeletal instability allowing growth cone motility52, can be central to orchestrating endocannabinoid-induced modications to cytoskeletal remodelling during repulsive growth cone turning. An advantage of this model, combining elements of autocrine endocannabinoid and paracrine Slit2/Robo1 signalling, is that it ascertains the on-demand upstream coupling of endocannabinoids to Slit2/Robo1 signalling, outlining a temporally dened, spatially restricted and highly dynamic mechanism to modulate growth cone-steering decisions.

We also show that CB1RRobo1 receptor co-localization is preserved during mammalian evolution, as evidenced by our histochemical data from midgestational human fetuses. Moreover, Robo1 and Slit mRNA expression in human fetal brains are developmentally regulated and appear tightly coupled. Maternal cannabis smoking signicantly increased the amount of Robo1 mRNA in the human fetal cerebrum. We propose that the molecular re-conguration of Slit/Robo signalling networks by phytocannabinoids via the CB1R can be a candidate molecular mechanism to explain altered synaptic neurotransmission, and consequently, behavioural and cognitive decits in children exposed in utero to cannabis53,54. Considering the continued increase of cannabis smoking during adolescence41, the erroneous recruitment of spatially segregated CB1Rs and CB2Rs to regulate

Slit2/Robo1 interactions during the postnatal renement of neuronal circuits might also introduce unwanted modications to the cortical wiring diagram. Besides cannabis, fetal infections are associated with the induction of endocannabinoid signalling in placental macrophages, contributing excess endocannabinoids to the fetal circulation55. This cascade can change the temporal and subcellular recruitment of CB1Rs and CB2Rs, imposing pathological challenges on the signalling mechanism we uncovered. Overall, we suggest that a hierarchical interplay between endocannabinoid and Slit/Robo signalling networks is a primary regulatory node in the developing corticofugal system.

Methods

Animals and tissue preparation. Taugfp/gfp::CB1R / mice were generated by rst crossing taugfp/ and CB1R / mice (both on C57BL/6 background) to select taugfp/ ::CB1R / colony founders. Next, we crossed heterozygous offspring to generate taugfp/gfp::CB1R / parents for in vivo analysis. Given that tau is dispensable for neuronal development, taugfp/gfp mice did not carry any developmental abnormality29. We used mice heterozygous for CB1Rs as parents throughout the experiments, allowing direct comparison of littermate taugfp/

gfp::CB1R / and taugfp/gfp::CB1R / offspring. MGL / and MGL /

littermates of both sexes from heterozygous pregnancies23 were analysed on E16 and postnatal day (P)20 (Supplementary Fig. 1). Pregnant CB1R / , taugfp/

gfp::CB1R / , Robo1 / 5, Slit2 / 56 and wild-type C57BL/6 mice were injected with JZL184 (40 mg kg 1 body weight, intraperitoneally daily)24 or vehicle intraperitoneally during E12.518.5. Male mouse embryos (n46/pregnancy, n42 pregnancies/analysis) were collected at E18.5. Whole brains were collected for histochemistry, protein biochemistry/western blotting, mass spectrometry, JZL184-binding assay or quantitative RNA proling. Experiments on live animals

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conformed to the 86/609/EEC directive and were approved by the regional authority (Stockholm Norra Djuretiska Namnd; N512/12).

Human fetal tissue preparation and histochemistry. Three fetal brains with normal development (between 2023 gestational weeks) were selected from the Brain Bank of the Institute of Neurology, Medical University of Vienna, Austria. 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-lysis. Neuropathological examination excluded major central nervous system malformations, severe hypoxic/ischaemic encephalopathy, intraventricular haemorrhages, severe hydrocephalus and meningitis or ventriculitis. Demographic data are summarized in Supplementary Table 3. Tissues were obtained and used compliant with the Declaration of Helsinki and following institutional guidelines. The study was performed in the course of an approved study by the Ethical Committee of the Medical University of Vienna (No.104/2009).

Immunohistochemistry, imaging and quantitative morphometry. Embryonic brains were immersion xed in 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4, PB) overnight. Tissue samples were cryoprotected in 30% sucrose in physiological saline for at least 48 h before cryostat serial sectioning (16 mm thickness), and thaw-mounted on SuperFrost glass slides. Multiple immunouorescence histochemistry was carried out according to published protocols19,57.

Sections were washed in PB and pre-treated with 0.3% Triton X-100 (in PB) for 1 h at 2224 C to enhance the penetration of primary antibodies. Non-specic immunoreactivity was suppressed by incubating our specimens in a cocktail of 10% normal donkey serum (Jackson), 5% bovine serum albumin (Sigma) and 0.3% Triton X-100 (Sigma) in PB for 1 h at 2224 C. Sections were exposed (1672 h at 4 C) to select combinations of primary antibodies (Supplementary Table 1) diluted in PB to which 0.1% normal donkey serum and 0.3% Triton X-100 had been added. After extensive rinsing in PB, immunoreactivities were revealed by carbocyanine (Cy)2, 3 or 5-tagged secondary antibodies raised in donkey (1:300 (Jackson), 2 h at 2224 C). Hoechst 33342 (Sigma), a nuclear dye, was applied to reveal tissue architecture. Sections were coverslipped with Dako Fluorescent mounting medium (Dako).

Sections were inspected and images acquired on a 710LSM confocal laser-scanning microscope (Zeiss). Image surveys were generated using the tile scan function at 10 primary magnication (Plan-Neouar 10/0.30). High-

resolution images were acquired with optical zoom ranging from 1.53.0 at 63

primary magnication (Plan-Apochromat 63/1.40, and 0.50.7 mm optical slice

thickness) to limit signal detection. Emission spectra for each dye were limited as follows: Cy2 (505530 nm), Cy3 (560610 nm) and Cy5 (650720 nm). The coexistence of immunosignals was veried by capturing serial orthogonal z-image stacks at 63 primary magnication (up to 3 optical zoom) and accepted if

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.

Morphometric analysis of fetal mouse brains was aided by the ZEN2010 software and included: (i) the transverse diameter (mm) of L1-NCAM corticofugal axons (corpus callosum spread) in fetal wild-type and CB1R / and

MGL / brains after JZL184 or vehicle administration, (ii) the density and (iii) proximity (mm) of rst-order fascicles of 43 mm in diameter, (iv) the density (n),(v) diameter (mm), (vi) embedding of CNPase oligodendrocyte end-feet interspersing callosal axons and (vi) calibrated uorescence intensities using an arbitrary grey-scaled unit scale in images acquired with identical laser illumination and capture settings. Robo1 receptor immunoreactivity along growth cones and axons of primary cortical neurons was measured in ImageJ 1.45 s (NIH). Validation of the Slit2 antibody was performed on 600-nm-thin serial optical slices of embryonic brain sections obtained from Slit2 / and littermate wild-type mice.

The number of Slit2 proles was counted and their density calculated on CNPase oligodendrocyte end feet or perinuclear loci. Multi-panel images were assembled in CorelDraw X5.

Human fetal immunohistochemistry and analysis. Three-micrometer tissue sections of formalin-xed, parafn-embedded tissue blocks containing the corpus callosum and the hippocampal formation were mounted on pre-coated glass slides (Star Frost)58,59. Shortly, after deparafnization and rehydration, the sections were 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.

The DAKO EnVision detection kit, peroxidase/DAB and rabbit/mouse (K5007, ready-to-use, Dako) was used to visualize antibody binding. Sections were counterstained with haematoxylin, dehydrated in ascending concentrations of ethanol, cleared with xylene and covered with Consil-Mount (Shandon; Thermo Scientic). Representative images containing the area of interest were exported from scanned sections (NanoZoomer 2.0, Hamamatsu).

For immunouorescent co-localizations, 5-mm-thick sections were pre-treated with 1% sodium borohydride, rinsed extensively in 25 mM Tris-Buffer (pH 7.5) and blocked in 20% normal horse serum (Vector Laboratories) for 30 min.

Mixtures of primary antibodies were applied for 48 h at 4 C (anti-Robo1 diluted 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 tested for optimal dilution, and secondary antibodies were tested for cross-reactivity and non-specic staining. Sections were imaged and analysed using a Zeiss LSM780 confocal microscope.

Western blotting. Tissue samples were lysed in modied radio-immunoprecipitation assay buffer containing 1 mM NaF, 1 mM Na3VO4, 0.1%

N-octyl-b-D-glucopyranoside and a mixture of protease inhibitors (Complete, Roche). Cell debris and nuclei were pelleted by centrifugation (800g, 10 min at 4 C). Protein concentrations were determined by Bradfords colorimetric method60. Samples were diluted to a nal protein concentration of 2 mg ml 1, denatured in5 Laemmli buffer and analysed by SDSpolyacrylamide gel electrophoresis on 6

or 8% resolving gels. After transferring onto Immobilon-FL polyvinylidene diuoride membranes (Millipore), membrane-bound protein samples were blocked in 3% bovine serum albumin and 0.5% Tween-20 diluted in Tris-buffered saline for1.5 h, and subsequently exposed to primary antibodies (Supplementary Table 1; Supplementary Fig. 7) overnight at 4 C. Appropriate combinations of horseradish peroxidase-conjugated secondary antibodies (Jackson; from goat, rabbit, guinea pig or mouse hosts; 1:10,000 for 2 h) were used for enhanced chemiluminescence signal detection. Image acquisition and analysis were performed on a Bio-Rad XRS imaging platform. TUJ1 served as loading control, which showed corresponding quantities of b-actin and gapdh (Supplementary Fig. 4f).

PCR. To discriminate the sex of mouse fetuses, we used primers to amplify Y and X chromosome-specic genomic DNA sequences. The Sry gene (forward: 50-

GTTCAGCCCTACAGCCACAT-30, reverse: 50-CAGCTGCTTGCTGATCTCTG-30, amplicon size: 197 bp (Y chromosome)) and DXNds3 microsatellite (forward: 50-GAGTGCCTCATCTATACTTACAG-30, reverse: 50-TCTAGTTCATTGTTGATTAGTTGC-30, amplicon size: 244 bp (X chromosome)) were amplied. For quantitative PCR, E18 embryonic mouse brains were microdissected on ice to isolate pallial regions, and snap frozen in liquid N2 until processing. RNA was extracted using the RNeasy mini kit (Qiagen) with a DNase I step performed to eliminate traces of genomic DNA30 and reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Reactions were performed after an initial denaturation at 95 C for 10 min followed by 40 cycles of 95 C for 15 s denaturation, annealing and extension at calculated temperatures (60 s) and a dissociation stage (from 60 to 95 C with 0.5-C steps for 10 s each; MyiQ Apparatus, Bio-Rad), with primer pairs amplifying short fragments for each gene (Supplementary Table 2). Samples without template or reverse transcriptase served as negative controls. Expression levels were normalized to the housekeeping gene (HKG) encoding glyceraldehyde-3-phosphate dehydrogenase (gapdh) obtained for every sample in parallel assays15.

Human fetal NanoString analysis. Midgestational fetal brain subjects (1822 weeks of gestation), collected after saline-induced elective abortions under Institutional Review Board approval at SUNY Downstate Medical Center, Brooklyn, New York, were xed in 1% paraformaldehyde in 0.05 M PB for 24 h, frozen in dry ice-cooled isopentane, cryosectioned in the coronal plane (20 mm) and stored at

30 C (ref. 61). Tissue isolations from the cortex were carried out with micro-RNAqueous lysis solution (Life Technologies) and gene expression analysed using the nCounter System from NanoString Technologies62 at the Gene Expression Shared Resource Facility at Icahn School of Medicine. Briey, the cell lysates were hybridized to a NanoString code-set containing barcode-labelled probes for ROBO1, SLIT1 and three HKGs: TBP, Hsp90ab1 and ActB (Supplementary Table 4), followed by scanning of the immobilized mRNA transcripts in the nCounter Digital Analyzer. The raw data les were normalized to the TBP HKG via NanoString nCounter software, and statistical analysis was performed using the JMP statistical program (SAS Institute, Cary, NC). A general linear stepwise regression was used to calculate statistical signicance 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 rst performed with each cluster of variables and any variable that showed Po0.10 was included in the stepwise regression, in addition to cannabis exposure addition61. Variables with Po0.05 were included in the nal regression modelas covariates of cannabis exposure.

Liquid chromatography tandem mass spectrometry. 2-AG, anandamide (AEA), 2-linoleoyl glycerol and oleoylethanolamide concentrations were determined using a solid-phase extraction liquid chromatography tandem mass spectrometry method30,63. The nal JZL184 (40 mg kg) injection was given to pregnant females on E18.5 2 h before dissection of maternal and fetal brains. Embryonic hemisected brains (n 6 pups from two separate litters) were homogenized in ice-cold

acetonitrile containing [2H4]anandamide at 6 pmol as internal standard, made up to 70% water and centrifuged at 13,000 r.p.m. for 5 min. The supernatant was applied to Strata-X polymeric reverse-phase cartridges (Phenomenex), methanoleluted analytes were dried under N2 and the residue was re-suspended in the

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mobile phase. A Surveyor LC system coupled to a TSQ Quantum mass spectrometer (Fisher Thermo Scientic) was used for analysis. The analytes were eluted from an ACE 5-m C8 column (150 2.1 mm; Hichrom Ltd.) under isocratic

conditions with a mobile phase consisting of 15% water, 85% methanol and 0.5% formic acid at a ow rate of 200 ml min 1 at 30 C. Mass spectrometry analysis was performed using electrospray ionization in positive ion mode. Quantication was undertaken using single-reaction monitoring of the parent ionproduct ion transitions for 2-AG and [2H4]AEA of mass-to-charge ratio (m/z) of m/z 379 to m/z 287 and m/z 352.2 to m/z 66.2, respectively. Two peaks were present in the chromatogram for 2-AG because a 1(3)-isomer exists in equilibrium with 2-AG. The areas of both peaks were combined to calculate [2-AG] by weighted linear regression analysis against suitable calibration standards using Xcalibur 2.0.6 software (Fisher Thermo Scientic). Results were expressed as nanomoles/ picomoles per gram of tissue.

Ex vivo activity-based protein proling. Frozen, hemisected brains from E18.5 mouse embryos (B35 mg; other hemisphere from mass spectrometry analysis) or a section of the cortex from the corresponding mother (B90 mg) were washed with cold Dulbeccos phosphate-buffered saline (DPBS) (2 1.0 ml) and homogenized

using a Branson Sonier 250 probe sonicator (10 pulses, 50% duty cycle, output setting 3.0) in cold DPBS (10 ml mg 1 tissue). Homogenates derived from adult

mouse brain samples were centrifuged (3,000g, 3 min) at 4 C and the pellet was 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 1 in DPBS. Proteomes (50 ml) were subsequently treated with FP

Rhodamine (1.0 ml, 50 mM in DMSO), incubated for 30 min at room temperature and quenched with 4 SDS loading buffer (17 ml). Following separation of pro

teins by SDSpolyacrylamide gel electrophoresis (10% acrylamide gel) and uorescent gel imaging (Hitachi FMBio II atbed scanner), serine hydrolase activities were determined by measuring the uorescent intensity of target gel bands using ImageJ 1.45s (ref. 24).

Primary cultures, pharmacology and transfection. The pallium of embryonic mice was dissected at E16.5. For neuronal cultures, cortical cells from C57BL/6 and CB1R / mice were enzymatically dissociated and plated at a density of 2550 000 cells per well (morphometry) or 106 cells per ell (biochemistry) in poly-D-lysine-coated 24-well or 6-well plates, respectively30. Primary neuron cultures were maintained in Dulbeccos modied Eagles medium (DMEM)/F12 (1:1) containing B27 supplement (2%), L-glutamine (2 mM), penicillin(100 U ml 1) and streptomycin (100 mg ml 1; all from Invitrogen) and exposed to drugs applied alone or in combination (JZL 184: 100 nM, Cayman Chemical;

O-2050: 200 nM, Tocris Bioscience; AM630: 200 nM, Tocris Bioscience; AM251: 200 nM, Tocris Bioscience; UO126: 10 mM, Tocris Bioscience; SP600125: 5 mM,

Tocris Bioscience). Primary cortical neurons from C57BL6/N fetuses were transfected on 1 day in vitro by Lipofectamine 2000 (5 ml; Invitrogen) with a pool of

MGL-specic siRNAs (sc-72278; 50 pmol per well) or non-targeting siRNA (sc-36869; 50 pm per well; Santa Cruz Biotechnology) for 30 min. Neurons were left to recover for 3 days before xation in 4% paraformaldehyde.

For glial cultures, neocortices of P2 mouse pups were dissected. Cells were dissociated similarly, transferred to T75 asks (3 107 cells per ask) and

maintained in DMEM containing 10% fetal bovine serum, L-glutamine (2 mM), penicillin (100 U ml 1) and streptomycin (100 mg ml 1). After 79 days, T75 asks were placed on a shaker at 190 r.p.m. at 37 C for 1 h. The microglial fraction was discarded and cells were allowed to equilibrate for 26 h in fresh medium. Subsequently, asks were placed onto an orbital shaker at 210 r.p.m., 37 C for 1618 h to separate oligodendrocytes. The supernatant (oligodendroglial fraction) was collected, centrifuged, re-suspended and seeded at a density of 25,000 cells per well (morphometry) and 106 cells per well (biochemistry) in poly-D-lysine-coated 24-well and 6-well plates, respectively. Cultures were then treated with JZL184 (100 nM) every other day for 72 h or with MGL-specic siRNA/non-targeting siRNA as described above. Cell pellets from the same fraction were taken and processed for western blotting and PCR analysis simultaneously. Astrocytes were collected in fresh medium and processed for protein and mRNA analyses.

For neuronglia co-cultures, oligodendrocytes were plated at a density of 25,000 cells per well in DMEM containing 10% fetal bovine serum. After 1 day, half of the medium was removed and replaced by DMEM/F12 (1:1) culture medium supplemented with B27 (2%) and neurons were seeded at a density of 25,000 cells per well. In another set of experiments, Lipofectamine 2000 (5 ml; Invitrogen) was used to transfect co-cultures with either non-targeting (scrambled) siRNA (sc-36869) or a pool of Robo1-specic mouse siRNAs (sc-42253; both at 100 pmol per well; Santa Cruz Biotechnology) for 4 h. Co-cultures were treated with JZL184 (100 nM) every other day for 72 h.

Cells on coverslips were immersion xed on ice with 4% paraformaldehyde in0.1 M PB and processed as above for immunocytochemistry. Morphological parameters were determined by analysing calibrated images in the ZEN2010 imaging software module (Zeiss; n430 cells per group)18,30. In primary neuronal cultures, the intensity of Robo1 and Robo2 imunoreactivities along the neurite (n420 per marker and experimental group on two coverslips of two independent experiments each) was measured by obtaining a plot prole starting at the growth cone and continuing along the total axon back to the soma. In primary

oligodendrocyte cultures, Slit2 immunoreactivity was measured in end feet and along processes (n430 cells per group on two coverslips from duplicate experiments). Immunoreactivity was normalized to vehicle-treated end feet. Distances between neurites and oligodendrocytes were measured in neuronglia co-cultures (n420/ group on two coverslips from duplicates).

GCP isolation. Cortical hemispheres from E18.5 mouse embryos (n 5 embryos

per treatment) were vibratome sectioned (200300 mm thickness), pooled (n 5

embryos per treatment) and exposed to JZL184 (100 nM in DMEM/F12/B27 as 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 according to published protocols64. Robo1 protein levels of GCPs were determined by western blotting (Supplementary Table 1). b-III-tubulin served as internal loading control30.

Statistics. Data were analysed using Statistical Package for the Social Sciences (version 21.0, SPSS Inc.). Pair-wise group comparisons in histochemical and western blotting experiments were evaluated using the Students t-test (on independent samples). Data were normalized to controls as indicated. Data were expressed as meanss.e.m. A P-value of 0.05 was considered statistically signicant.

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Acknowledgements

This work was supported by the Swedish Medical Research Council (T.H.), Swedish Brain Research Foundation (Hjarnfonden; T.H.), Novo Nordisk Foundation (T.H.), Petrus & Augusta Hedlunds Foundation (T.H.), EU-FP7 project DEVELAGE (N278486, G.G.K.), National Brain Research Program of the Hungarian Academy of Sciences (A.A.), National Institutes of Health (DA023214, T.H. and Y.L.H.; DA030359, Y.L.H; F31-DA031559, C.V.M.) and the Wellcome Trust (#089775, J.G.P. and W.D.A.). The content of this report is solely the responsibility of the authors and does not necessarily represent the ofcial views of the US National Institutes of Health. We thank M. Watanabe, K. Mackie and F. Murakami for antibodies, Y.A. Barde for taugfp/ mice, K. Mackie for

MGL / tissues, F. Molina-Holgado for the oligodendrocyte isolation protocol, O.K. Penz for technical assistance, M. Tessier-Lavigne, L. Erskine, R.A. Ross and members of the Harkany laboratory for discussions and feedback and the National Institute of Mental Health for the transfer of CB1R / colony founders. Laser-scanning microscopy was performed at the Click Imaging Core Facility of the Karolinska Institutet.

Author contributions

A.A. and T.H. conceived the general framework of this study. A.A., E.K., B.F.C., G.G.K., 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., 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. and E.K. analysed data. A.A., E.K. and T.H. wrote the manuscript. All Authors commented on and approved the submission of this manuscript.

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How to cite this article: Alpr, A. et al. Endocannabinoids modulate cortical development by conguring Slit2/Robo1 signalling. Nat. Commun. 5:4421 doi: 10.1038/ ncomms5421 (2014).

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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 CB₂ R-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.

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Title

Endocannabinoids modulate cortical development by configuring Slit2/Robo1 signalling

Author

Alpár, Alán; Tortoriello, Giuseppe; Calvigioni, Daniela; Niphakis, Micah J; Milenkovic, Ivan; Bakker, Joanne; Cameron, Gary A; Hanics, János; Morris, Claudia V; Fuzik, János; Kovacs, Gabor G; Cravatt, Benjamin F; Parnavelas, John G; Andrews, William D; Hurd, Yasmin L; Keimpema, Erik; Harkany, Tibor

Pages

4421

Publication year

2014

Publication date

Jul 2014

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Nature Publishing Group

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20411723

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Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms5421

ProQuest document ID

1545511672

Endocannabinoids modulate cortical development by configuring Slit2/Robo1 signalling

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