We set ourselves the goal to establish mouse models amenable to study the molecular underpinnings of life‐long circuit modifications in offspring upon maternal THC exposure. We took note of the fact that cannabis can introduce epigenetic modifications (Dinieri et al , 2011 ), attenuating synaptic neurotransmission. If THC impedes the structural organization of neuronal networks then synapse re‐positioning (i.e. the erroneous recruitment of synaptic afferents to somatodendritic domains on postsynaptic neurons) is likely an early developmental event (Keimpema et al , 2011 ), which might stabilize over time. Precluding the attainment of cell‐type‐specific neuronal structure would inevitably rely on cannabinoid receptor‐mediated signalling events (Berghuis et al , 2007 ) with profound modifications to the cytoskeleton, particularly microtubule integrity. We have mapped Superior Cervical Ganglion 10/stathmin‐2 (SCG10) and identified its loss as THC's molecular target in the mouse and human fetal nervous systems, and used SCG10 to acutely reset cytoskeletal dynamics upon THC‐induced CB 1 R activation.

THC promiscuously binds to members of the cannabinoid receptor family (Howlett, 2002 ), G protein‐coupled receptors (GPCRs), whose cell‐type‐specific segregation furnishes intercellular interactions using endocannabinoids as physiological ligands (Kano et al , 2009 ; Pertwee et al , 2010 ; Di Marzo, 2011 ). Yet THC's mode of action on neuronal fate decisions, including the identity of the cannabinoid receptor mediating THC's effect in the developing nervous system, remains unknown (Keimpema et al , 2011 ). Considering that neurogenesis precedes the production of astrocytes and oligodendrocytes (Schmechel & Rakic, 1979 ; Kessaris et al , 2008 ) [the latter becoming molecularly specified in the forebrain only during late gestation (Hardy & Friedrich, 1996 )], the type 1 cannabinoid receptor (CB 1 R) expressed by developing cortical neurons (Berghuis et al , 2007 ; Keimpema et al , 2011 ), particularly during the axonal growth process (Berghuis et al , 2007 ), emerges as a likely candidate. sn ‐1‐Diacylglycerol lipases α and β (Bisogno et al , 2003 ) in growth cones generate 2‐arachidonoylglycerol (2‐AG) to focally activate CB 1 Rs (Keimpema et al , 2010 ), allowing protrusive endocannabinoid signalling upon activating Rho‐family GTPases (Berghuis et al , 2007 ), a prerequisite of axonal motility (Yuan et al , 2003 ). Therefore, THC, a partial CB 1 R agonist as predicted by postnatal pharmacology (Pertwee et al , 2010 ), could disable the spatial specificity of endocannabinoid cues in the developing brain by indiscriminately activating CB 1 Rs, even at unfavorable subcellular positions (Keimpema et al , 2011 ). In doing so, THC might provoke spatially and temporally segregated signalling events to disrupt neuronal fate decisions and specification, particularly synaptic wiring, thus recapitulating corticofugal axon fasciculation and targeting defects in CB 1 R −/− mice (Berghuis et al , 2007 ; Diaz‐Alonso et al , 2012 ). However, the molecular configuration of signalling cascades hijacked by THC to imprint permanent wiring errors in the fetal cerebrum remains elusive.

The prevalence of recreational cannabis use continues to increase with adolescents and young adults being primary affected (Substance Abuse & Mental Health Service Administration, 2010 ). Cannabis exposure of infants in utero or cannabis use in teenagers [peaking at 15–17 years of age (Substance Abuse & Mental Health Service Administration, 2010 )] can coincide with critical periods of brain development when neuronal connectivity is prenatally established (Kostovic & Jovanov‐Milosevic, 2006 ) or postnatally refined to increase modularity and integrative capacity (Dennis et al , 2013 ). Accordingly, prospective longitudinal assessments (Goldschmidt et al , 2004 ; Willford et al , 2010 ; Day et al , 2011 ) suggests that cannabis use during pregnancy can increase the risk for ill‐behaviors (Goldschmidt et al , 2004 ; Day et al , 2011 ), cognitive deficit (Huizink & Mulder, 2006 ), drug seeking (Day et al , 2006 ), attention deficit (Leech et al , 1999 ), and anxiety and depression (Leech et al , 2006 ) among affected neonatal or adolescent offspring. Large‐scale population analysis associates growth retardation with maternal cannabis use (El Marroun et al , 2009 ), particularly since there is an efficient cross‐placental transfer of Δ 9 ‐tetrahydrocannabinol (THC) (Grotenhermen, 2003 ), the major psychoactive constituent of Cannabis spp. Nevertheless, a gap of knowledge exists regarding the neuronal basis of cannabis‐induced developmental deficits in the nervous system.

CB 1 Rs are predominantly presynaptic in the adult brain (Kano et al , 2009 ). Likewise, cell‐surface CB 1 Rs are only found on the axons of developing neurons (McDonald et al , 2007 ). The domain‐specific cell‐surface targeting of CB 1 Rs allowed us to assay whether the molecular pathway we uncovered is specific to elongating axons. To this end, we first determined THC‐induced changes to SCG10 and acetylated tubulin contents in secondary, short neurites lacking CB 1 Rs, putative primordial dendrites ( Supplementary Fig S6C ). THC induced tubulin acetylation in both the growth cones and neurite stems ( P < 0.05 versus vehicle‐treated control; Supplementary Fig S6C 1 and C 2 ). In contrast, SCG10 levels remained unchanged ( Supplementary Fig S6C 3 and C 4 ). Next, we co‐localized acetylated tubulin and post‐synaptic density protein 95 (PSD95), a marker of excitatory post‐synapses (Tomasoni et al , 2013 ), to dissect THC effects at established synapses. THC did not induce tubulin acetylation in either PSD95 + post‐synapses or apposing pre‐synaptic terminals ( Supplementary Fig S6D–D 4 ). In sum, these experiments establish the axonal specificity of CB 1 R‐dependent SCG10 degradation, and specifically implicate this mechanism in the modulation of axonal growth and guidance.

THC treatment for 24 h reduced SCG10 and to a lesser extent CB 1 R immunoreactivity (Berghuis et al , 2007 ) in growth cones ( Supplementary Fig S6B–B 2 ). If the loss of SCG10 in growth cones relates to diminished axonal growth then SCG10 degradation can be expected to progress in situ in the growth cone. We sought to address this hypothesis by isolating growth cone particles (GCPs) from late‐gestational mouse cortices (Berghuis et al , 2007 ) and exposing intact GCPs to THC (10 min) in vitro . The selective loss of higher‐molecular‐weight SCG10 isoforms demonstrated that local SCG10 degradation persists in growth cones (Fig 7 C). Similarly, acute THC treatment of cortical neurons diminished SCG10 in the central growth cone domain (Fig 7 D and D 1 ), which coincided with acetylated tubulin, a marker of stabilized and aged microtubules (Maruta et al , 1986 ), and F‐actin accumulation (Fig 7 D 2 ). Overt cytoskeletal stability increased the appearance of growth cone with splayed out microtubules (Fig 7 D 3 ), and increased density of exploratory filopodia [11.9 ± 1.2 μm (THC) versus 7.3 ± 1.1 μm (control), P < 0.01; Fig 7 D 4 ], leaving the growth cone surface area unaffected (Fig 7 D 5 ). Notably, THC also increased the formation of F‐actin‐rich filopodia in the distal motile axon segment (Fig 7 E and E 1 ) suggesting that THC can enhance the formation of ectopic axon collaterals. These data demonstrate that THC modulation of CB 1 R activity in motile growth cones can inhibit forward motility and steering decisions.

If the CB 1 R‐JNK‐SCG10 pathway alone is sufficient to account for the THC‐induced cytoarchitectural modifications of cortical neurons then overexpression of a functionally inactive, pseudophosphorylated SCG10 mutant (Tararuk et al , 2006 ; Westerlund et al , 2011 ) (i.e. aspartate substitution of Ser62 and Ser73 (SCG10‐DD)) would phenocopy THC effects. Consistent with this hypothesis, SCG10‐DD overexpression appeared to outcompete endogenous SCG10 in neurites (Fig 7 A), reduced neurite outgrowth and occluded THC‐induced modifications to axonal morphology (84.0 ± 8.7 μm [THC+EGFP‐C1] versus 92.6 ± 9.6 μm [THC+SCG10‐DD], P > 0.5; Fig 7 A and A 1 ). PC12 cells transfected with SCG10‐DD for 24‐72 h were used to show that SCG10‐DD is overexpressed at the expense of endogenous (wild‐type) SCG10. By Western blotting PC12 cell lysates, we separated GFP‐tagged SCG10‐DD from unlabeled endogenous SCG10 and found the progressive reduction of the latter SCG10 form as a factor of time (Fig 7 A 2 ). In addition, siRNA‐mediated knockdown of SCG10 in cultured cortical neurons reduced neurite outgrowth (Fig 7 B–B 2 ) to an extent similar to that seen upon SCG10‐DD overexpression. Overall, these data highlight the central role of SCG10 degradation in axonal growth defects imposed by prenatal THC exposure.

The above in vitro data suggest a central role for JNK1 in regulating CB1R activity‐dependent SCG10 degradation. However, these data are limited in addressing THC‐induced SCG10 degradation in the corticofugal projection system. Therefore, we prepared organotypic slices from E14.5 mouse forebrains to preserve cellular and axonal arrangements along the corticothalamic projection (Fig 6 A) to test acute THC (10 μM, 30 min) on axonal SCG10 availability. We show that both SCG10 fluorescence intensity [56.17 ± 4.97% of vehicle‐treated controls; n = 25 (THC) versus n = 29 (vehicle); P < 0.001] and spatial extent [“area coverage”: 75.62 ± 6.09% of vehicle‐treated controls; n = 25 (THC) versus n = 29 (vehicle); P < 0.01] were significantly reduced upon THC application (Fig 6 B,B 1 and B 3 ). SP600125 significantly attenuated the acute loss of SCG10 in corticofugal axons [fluorescence intensity: 81.75 ± 5.23% of control (THC+SP600125); area coverage: 97.76 ± 3.95% of control (THC+SP600125)]. L1‐NCAM, which remained unaffected by either THC or SP600125 treatment (Fig 6 B 2 and B 4 ), was used to reliably discern corticofugal axons and to normalize SCG10 fluorescence intensity.

SCG10 undergoes fast anterograde axonal transport (Shin et al , 2012 ) and accumulates in the central domain of advancing growth cones (Grenningloh et al , 2004 ), where microtubules are highly dynamic. Therefore, we quantitatively sampled SCG10 distribution in THC‐exposed neurites and growth cones. Consistent with previous findings (Grenningloh et al , 2004 ), SCG10 exhibits quasi‐random distribution along the entire neurite under control conditions (Fig 5 E). Both acute (10 μM, 10 min, Fig 5 E and E 1 ) and prolonged (2 μM, 24 h, Supplementary Fig S6A and A 1 ) THC stimulation induced SCG10 loss from neurite shafts, with residual SCG10 localized to neurite branch forks particularly after 24 h THC treatment ( Supplementary Fig S6A and A 1 ). The above data on the molecular regulation of SCG10 availability and function is compatible with reduced neurite outgrowth of THC‐treated cortical neurons [113.7 ± 9.5 μm (THC) versus 141.0 ± 10.2 μm (control), P < 0.05; Fig 5 F and F 1 ], and demonstrates the THC‐induced reconfiguration of a key maintenance pathway for microtubule instability (Shin et al , 2012 ).

Our data suggest that SCG10 undergoes JNK‐dependent degradation. JNK can directly phosphorylate SCG10 (Tararuk et al , 2006 ) and target it for degradation (Shin et al , 2012 ). Alternatively, JNK effects may be more indirect. We addressed this question by exploiting the decreased gel mobility (increased molecular weight) of phosphorylated SCG10 (Shin et al , 2012 ). We argued that JNK inhibition should lead to the accumulation of non‐phosphorylated SCG10. Conversely, proteasome inhibition should preserve phosphorylated SCG10. Indeed, SP600125 promoted the preferential accumulation of lower molecular weight, predominantly non‐phosphorylated SCG10 (Shin et al , 2012 ) (Fig 5 C). Moreover, by selective isolation of phosphoproteins we show that higher‐molecular‐weight (polyphosphorylated) SCG10 outweighs the relative abundance of mono‐ or non‐phosphorylated SCG10 species in lactacystin‐exposed neurons in a time‐dependent fashion (Fig 5 D). CB 1 R phosphorylation, marking GPCR internalization (Daigle et al , 2008 ), was only detected > 1 h, suggesting that SCG10 degradation is an early event downstream from CB 1 R activation (Fig 5 D). The different time‐course and extent of CB 1 R phosphorylation relative to elements of its signal transduction machinery served as negative control in phosphoprotein isolation. Cumulatively, phosphoprotein profiling demonstrates that phosphorylated SCG10 accumulates in THC‐treated neurons upon proteasomal inhibition.

The maintenance of microtubule instability in elongating axons is a prerequisite of growth advance (Grenningloh et al , 2004 ). The THC‐induced loss of SCG10 in vivo is mechanistically appealing since it could limit the rate of microtubule reorganization, introduce errors to axonal morphology and slow neurite outgrowth. Therefore, we hypothesized that THC‐induced CB 1 R activation can recruit JNK (Rueda et al , 2000 ) [or extracellular signal‐regulated kinase 1/2 (Erk1/2), (Derkinderen et al , 2003 ; Berghuis et al , 2007 )] to phosphorylate and direct SCG10 to proteasomal degradation (Fig 5 A). Consistent with these expectations, THC induced rapid (10 min) JNK and Erk1/2 phosphorylation [JNK: 1.4 ± 0.1 fold (THC), Erk1/2: 1.3 ± 0.1 (THC) fold relative to vehicle] in cultured cortical neurons (Fig 5 B). JNK/Erk1/2 phosphorylation coincided with reduced SCG10 levels upon THC (10 μM) exposure (Fig 5 C and C 1 ). The THC‐induced decrease of SCG10 (10 min: 73.8 ± 4.4%, 30 min: 77.2 ± 3.8% of control) was transient (Fig 5 C 1 ), likely reflecting the time course of THC‐induced CB 1 R desensitization (Hsieh et al , 1999 ). In acute in vitro experiments, WIN55,212‐2 (CB 1 R agonist) reproduced (84.6 ± 7.6%), while AM 251 (CB 1 R antagonist) blocked (103.5 ± 11.2%) the THC‐induced loss of SCG10, confirming CB 1 R involvement. SP600125 (5 μM, JNK inhibitor), but not U0126 (10 μM, Erk1/2 inhibitor (Tararuk et al , 2006 ); Supplementary Fig S5A ), prevented the rapid loss of SCG10 in cultured neurons exposed to THC [SP600125: 105.1 ± 13.1% of control, P < 0.05 versus THC (10 min); U0126: 89.5 ± 2.5% of control, P > 0.1 versus THC (10 min)]. Notably, lactacystin, which irreversibly inactivates the 26S proteasome (Keimpema et al , 2010 ), rescued SCG10, and stabilized both its non‐phosphorylated and phosphorylated isoforms (Fig 5 C and C 1 ). These data are consistent with the molecular framework involving JNK recruitment to agonist‐activated CB 1 Rs to promote SCG10 degradation. The accumulation of acetylated tubulin, a marker of long‐lived, stable microtubules (Maruta et al , 1986 ), in THC‐induced neurons (Fig 5 C 2 ; Supplementary Fig S5B and B 1 ) supports that SCG10 degradation is a candidate mechanism for THC to impair axonal growth.

The dynamic instability of microtubules (i.e. the innate ability of microtubules to abruptly switch between states of growth and shortening) (Grenningloh et al , 2004 ; Manna et al , 2007 ) and the integrity of the microtubule network (Shin et al , 2012 ) in developing neurons are paramount for the growing axon to maintain forward growth and execute steering decisions. SCG10 is a neuron‐specific member of the stathmin family, and triggers microtubule disassembly by binding tubulin dimers in a ternary complex, and by promoting minus end disassembly (Manna et al , 2007 ). SCG10 is increasingly recognized as an axonal substrate of c‐Jun N‐terminal kinase (JNK) (Westerlund et al , 2011 ; Shin et al , 2012 ). Phosphorylation by JNK1, the active brain‐specific JNK isoform (Bjorkblom et al , 2005 ; Westerlund et al , 2011 ), at Ser62 and Ser73 negatively regulates SCG10 activity (Grenningloh et al , 2004 ; Tararuk et al , 2006 ), and promotes its proteasomal degradation in mechanically‐injured axons (Shin et al , 2012 ).

Experimental models in mice might carry evolutionary bias, curtailing translational significance by being of limited relevance to human nervous system development. Therefore, we used in situ hybridization to first show the growth‐associated increase of SCG10 mRNA expression, irrespective of maternal cannabis use, in the fetal human hippocampus ( F = 8.579, P = 0.008; Supplementary Fig S4A and B ). Next, we assessed the distribution and density of SCG10 mRNA in the primordial hippocampus and parahippocampal gyrus of electively aborted second trimester human fetuses exposed prenatally to cannabis [ n = 12; confirmed by routine meconium toxicology (Hurd et al , 2005 )] versus age‐matched controls ( n = 12). We find highest SCG10 mRNA expression in the CA1‐CA3 hippocampal subfields with moderate mRNA hybridization signal detected in the dentate gyrus in normal fetuses (Fig 4 A). In contrast, SCG10 mRNA expression was significantly reduced in cannabis‐exposed subjects (4.32 ± 0.31 [cannabis] versus 5.33 ± 0.28 ln(dpm/mg tissue) [control], P < 0.001; Fig 4 A 1 ). Fetal growth retardation is a critical consequence of maternal cannabis smoking (Hurd et al , 2005 ). Therefore, we used multivariate analysis to control for covariates (fetal foot length and body weight). Both fetal foot length ( F = 17.725, P < 0.001; Fig 4 B) and fetal body weight ( F = 11.566, P = 0.002; Supplementary Fig S4B ) exhibited significant correlation with hippocampal SCG10 mRNA expression. Nevertheless, even with the consideration of these covariates in the statistical model there remained a significant difference in SCG10 mRNA expression levels between cannabis‐exposed and control subjects ( F = 14.669, P = 0.012). Subsequently, we asked whether SCG10 protein content is also reduced in cannabis‐exposed fetuses. After successfully extracting proteins from tissue samples of the above subject cohort, we found significantly reduced SCG10 protein levels in the fetal hippocampus upon maternal cannabis smoking [0.27 ± 0.06 (cannabis, n = 11) versus 0.50 ± 0.05 (control, n = 14), integrated and normalized density (arbitrary units), P < 0.01; Fig 4 C). Inclusion of fetal foot length in our multivariate model did not change group significance ( F 1,23 = 8.33, P < 0.01; Fig 4 C 1 ). Next, non‐parametric correlation analysis was carried out to reveal a close positive relationship between SCG10 mRNA and protein levels (Spearman's ρ = 0.52, P < 0.02), lending further support to the cannabis‐induced developmental deregulation of SCG10 expression in the fetal human cerebrum. Cumulatively, our findings in experimental models and human fetal brains identify SCG10 as a bona fide target of THC. Moreover, our data are compatible with the hypothesis that SCG10 expression in the developing human cerebrum coincides with the formation of intra‐ and extracortical axonal trajectories during weeks 20–29 (Kostovic & Judas, 2010 ), and is mandatory to maintain dynamic microtubule instability required for axonal growth (Grenningloh et al , 2004 ).

SCG10 is expected to be broadly expressed during corticogenesis, coincident with the onset of THC administration, if its loss is to underpin THC‐induced modifications of axonal extension. Indeed, we detected SCG10 mRNA by E14 with its expression level gradually increasing until birth (Fig 3 B 2 ). SCG10 mapped to long‐range forebrain projections ( Supplementary Fig S3B–D ), was enriched in growth cone‐like structures (Fig 3 C), and co‐distributed with CB 1 Rs in both the intermediate zone of the cerebral cortex (Fig 3 D–D 2 ) and the primordial hippocampus ( Supplementary Fig S2C and C 1 ). CB 1 Rs are expressed during the radial migration and morphogenesis of pyramidal cells in the cerebral cortex (Mulder et al , 2008 ). Here, we validated SCG10 localization by showing its enrichment in cells with morphologies reminiscent of pyramidal cells in the cortical plate (Westerlund et al , 2011 ) (Fig 3 D and D 1 ). SCG10 is localized in the cytosol (Grenningloh et al , 2004 ). By using high‐resolution laser scanning microscopy we show that approximately 40% of SCG10 + and CB 1 R + puncta are closely associated, with approximately 3% directly overlapping in corticofugal axons (Fig 3 D 2 and D 3 ). This finding establishes that SCG10 is proximal to CB 1 Rs in corticofugal axons, and could be a downstream target of this GPCR in particular neurite domains. These data, together with retained cerebral SCG10 mRNA and protein expression postnatally in the brain of THC‐exposed fetuses ( Supplementary Fig S1G and G 1 ), highlight SCG10 as a developmentally regulated candidate protein whose loss can confer THC effects on neuronal morphology.

Brain‐specific SCG10 was found particularly reduced in THC‐exposed brains (Fig 3 A 2 ), with a simultaneous reduction of its protein (Fig 3 B) and mRNA expression (Fig 3 B 1 ). SCG10 is an appealing target since its microtubule destabilizing activity (Morii et al , 2006 ; Manna et al , 2007 ) chiefly contributes to the maintenance of cytoskeletal instability required for axonal growth (Stein et al , 1988 ; Grenningloh et al , 2004 ; Tararuk et al , 2006 ), and synaptic plasticity at Schaffer collaterals (Peng et al , 2004 ). SCG10 is regulated by Rho‐family GTPases (e.g. Rho6/Rnd1) (Li et al , 2009 ), which were previously implicated in CB 1 R‐induced growth cone collapse (Berghuis et al , 2007 ).

We sought to identify the molecular effector(s) whose altered expression in THC‐exposed fetuses can impair axonal growth and guidance. We used isobaric tagging for relative and absolute quantification (iTRAQ) to carry out quantitative proteomics (Shirran & Botting, 2010 ) on cortices from male fetuses at E18.5 (Fig 3 A), analysing the resultant peptides by both LC‐MALDI/MS/MS and nLC‐ESI/MS/MS mass spectrometry to profile THC‐sensitive proteins. We identified 35 functionally heterogeneous proteins (out of 837 identified hits), as suggested by their classification according to protein function ontology (Fig 3 A 1 ; Supplementary Fig S3A ), whose levels changed significantly upon THC treatment.

CB 1 R −/− fetuses present enlarged corticofugal axon fascicles indistinguishable from AM 251‐treated fetuses (Mulder et al , 2008 ) (Fig 2 B–B 2 ). Therefore, we hypothesized that CB 1 R −/− fetuses must develop a compound phenotype if THC acts via cannabinoid receptors other than CB 1 R. We tested this by quantitative morphometry of CB 1 R −/− and wild‐type littermate brains (from heterozygous crosses). THC failed to modify the corticofugal axon phenotype of CB 1 R −/− fetuses [fascicle diameter: 13.73 ± 0.11 (THC) versus 14.17 ± 0.62 μm (vehicle), P = 0.60; Fig 2 B–B 2 ]. Our data imply that THC can impair the establishment of the corticofugal tract by disrupting endocannabinoid signaling at CB 1 Rs. This concept is supported by results of mRNA and protein analysis for CB 1 Rs, DAGLα (Bisogno et al , 2003 ) and monoacylglycerol lipase (MGL) (Dinh et al , 2002 ), metabolizing 2‐AG, in THC‐exposed fetuses, demonstrating coincidently reduced receptor and ligand availability upon THC administration (Fig 2 C and C 1 ). Nevertheless, CB 1 Rs in THC‐exposed brains did not desensitize, as suggested by unchanged Erk1/2 phosphorylation levels upon acute WIN55,212‐2 (500 nM) challenge (Fig 2 D) relative to control.

In developing neurons, THC might act as a “functional antagonist” since it can displace the binding of high‐efficacy endocannabinoids, dampening their signaling efficacy (Paronis et al , 2012 ). If so, THC could modify the intrinsic program of neuronal fate specification via a receptor‐mediated mechanism. To determine the molecular identity of THC‐activated cannabinoid receptor(s) and the downstream signal transduction machinery impairing the cortical wiring map, we administered THC (3 mg/kg; i.p.), WIN55,212‐2 (5 mg/kg; CB 1 R agonist) and AM 251 (5 mg/kg; CB 1 R antagonist) in gravid mice from E5.5‐17.5. Since genetic disruption or pharmacological manipulation of CB 1 Rs introduces corticofugal axon fasciculation errors [i.e. enlargement with reduced myelination (Mulder et al , 2008 )], we sampled the diameter of corticofugal axons in drug‐exposed fetuses at E18.5. THC significantly increased the diameter of first‐order fascicles relative to vehicle controls ( P < 0.001, n > 300 fascicles from n = 4–7 fetuses/group; Fig 2 A and A 1 ). AM 251, but not WIN55,212‐2, promoted the formation of enlarged fascicles in vivo ( Supplementary Fig S2A and A 1 ), which was recapitulated by in vitro AM 251 exposure of cortical neurons producing endocannabinoids (Keimpema et al , 2010 ) ( Supplementary Fig S2B and B 1 ). The AM 251‐induced axonal redistribution suggests CB 1 R involvement.

Maternal THC exposure can induce epigenetic modifications, such as repressive histone methylation (Dinieri et al , 2011 ). Here, we demonstrate by quantitative PCR and Western blotting that maternal THC administration did not alter the expression of vesicular glutamate, GABA and acetylcholine transporters, the SNARE component vesicle‐associated membrane protein‐2 (Fig 1 F, Supplementary Fig S1B and C ) or CB 1 R mRNA ( Supplementary Fig S1D ) in the hippocampus and neocortex of adult THC‐exposed offspring. In sum, retained presynaptic protein expression levels and CB 1 R mRNA expression favor the conceptual framework of developmentally regulated circuit reorganization upon maternal THC exposure.

We hypothesized that if THC impairs endocannabinoid‐mediated mechanisms of cortical development then this would manifest as the altered distribution of CB 1 R + afferents and synapses (Berghuis et al , 2007 ; Keimpema et al , 2010 , 2011 ; Diaz‐Alonso et al , 2012 ), which adopt strict layer (L) specificity physiologically (Bodor et al , 2005 ). Therefore, we began to administer THC at a dose of 3 mg/kg (i.p., daily), which did not change maternal behavior or physical measures (Mato et al , 2004 ) as would be predicted for high‐dose THC intoxication, from embryonic day (E)5.5–17.5 (Berghuis et al , 2005 ), and allowed offspring to mature under conventional husbandry conditions (without postnatal re‐introduction of the drug) until postnatal day (P) 10 or 120. THC did not affect maternal bodyweight, the male:female sex ratio of the offspring [50.9% males (THC) versus 50.7% males (vehicle)] or the size of litters [5.9 ± 0.4 (THC) versus 6.3 ± 0.5 (vehicle)]. Male fetuses and offspring were analyzed because of their preferential sensitivity to cannabis (Hurd et al , 2005 ). By P10, THC‐exposed offspring showed a reduced area of superficial LI/II otherwise receiving peak density of CB 1 R + inputs (Bodor et al, 2005 ) (84.8 ± 2.6% of control, P < 0.01, Supplementary Fig S1A and A 1 ). CB 1 Rs are expressed by both perisomatically‐targeting cholecystokinin‐containing interneurons (Bodor et al , 2005 ) and pyramidal cells (Kano et al , 2009 ) in the postnatal cerebral cortex. At P120, we first showed the disruption of perisomatic baskets (Fig S1A and A 1 ), confinements of inhibitory synapses around pyramidal cell somata (Fig 1 B and B 1 ), recapitulating earlier findings in interneuron‐specific CB 1 R knock‐outs (Berghuis et al , 2007 ). Second, and reminiscent to the cortical reorganization of CB 1 R + inputs, we found a significant increase in the density of CB 1 R + boutons in the stratum radiatum of the hippocampal CA1 subfield in mice prenatally exposed to THC [7782 ± 409 (THC) versus 6860 ± 150 (vehicle) boutons/mm 2 , P < 0.05, Fig 1 C–C 2 ], suggesting synapse mistargeting in THC‐exposed brains (Keimpema et al , 2011 ). Since many of these boutons are terminal specializations of Schaffer collaterals, a major glutamatergic pathway originating from CA3 pyramidal cells and whose activity is modulated by endocannabinoids (Takahashi & Castillo, 2006 ), we tested whether the sign of synaptic plasticity upon Schaffer collateral stimulation is altered in THC‐exposed offspring. Upon using 900 pulses at 1 Hz to induce long‐term depression (LTD) (Dudek & Bear, 1993 ), orthodromic stimulation‐evoked LTD significantly diminished in THC‐exposed offspring relative to controls [ n = 7 (vehicle) versus n = 6 (THC); Fig 1 D–D 2 ], as measured in the CA1 stratum radiatum ( Supplementary Fig S1E ). Likewise, the long‐term depression of neuronal population activity, measured in the CA1 stratum pyramidale and expressed as the population spike amplitude, was occluded upon low‐frequency Schaffer collateral stimulation [ n = 6 (vehicle) versus n = 6 (THC); Supplementary Fig S1E 1 –E 3 ]. Moreover, we found increased paired‐pulse facilitation at Schaffer collateral inputs (Fig 1 E, Supplementary Fig S1F ), suggesting increased and deregulated presynaptic activity, compatible with the hypothesis of long‐lasting modifications to CB 1 R signaling in THC‐exposed brains. These data suggest that administration of THC during pregnancy can induce long‐term structural and functional modifications of the cortical circuitry.

Discussion

Our results, for the first time, define a specific molecular target for THC in the developing central nervous system, whose modifications can directly and permanently impair the wiring diagram of neuronal networks during corticogenesis. These finding have direct and increasing human relevance since selective cultivation of Cannabis subspecies recently significantly altered their phytocannabinoid contents, with particularly pronounced increases in THC content (Pitts et al, 1992; Pijlman et al, 2005). We first mount compelling experimental support to the hypothesis that, when available for prolonged periods in vivo, THC disrupts endocannabinoid signaling at their cognate CB 1 R. Our mRNA and protein profiling of molecular components controlling 2‐AG metabolism suggest that THC not only can act as a “functional antagonist” (i.e. displacement of endocannabinoid binding to the CB 1 R), but can disrupt 2‐AG signaling by reducing both CB 1 R and DAGLα expression during cortical development. We attribute the discrepancy between MGL mRNA and protein expression levels to MGL being prone to posttranslational modifications facilitating its proteasomal degradation (Keimpema et al, 2013). Next, we identify SCG10 as a novel signaling node of morphogenic CB 1 R signaling since (i) SCG10 is ideally poised to link cell‐surface CB 1 R activation and pleiotropic downstream mitogen‐activated protein kinase (particularly JNK) activation (Rueda et al, 2000; Berghuis et al, 2007; Keimpema et al, 2011) to cytoskeletal instability (Keimpema et al, 2011), and (ii) the rapid degradation of SCG10 can allow ectopic growth with ensuing modifications to axodendritic morphology in vitro recapitulating corticofugal growth defects in vivo. This mechanism appears to be restricted to the THC‐induced reorganization of axonal morphology, which is compatible with the sole localization of CB 1 Rs to growth cones and presynapses in the developing (Keimpema et al, 2010) and adult nervous systems (Kano et al, 2009), respectively. Nevertheless, we recognize that CB 1 R activation might engage multiple co‐existent signaling pathways (e.g. RhoGTPases (Berghuis et al, 2007; Nithipatikom et al, 2012)) to couple cytoskeletal reorganization to the orchestration of axonal morphology.

THC exposure altered the levels of 35 proteins in the fetal cerebrum. Previously, a total of 49 differentially‐regulated genes were identified by cDNA microarrays in nervous tissues from adult rats treated with THC for 24 h, 7 or 21 days (Kittler et al, 2000), including molecular constituents of endocannabinoid and lipid biosynthesis, and signal transduction machineries. Conspicuously, neural cell adhesion molecule and myelin basic protein, implicated in axonal growth (Yuan et al, 2003; Keimpema et al, 2011) and myelination by oligodendrocytes were also altered. Here, our protein profiling by mass spectrometry combined with cell biology provides a new perspective on developmentally‐regulated candidates, and suggests that the reorganization of synaptic structure and plasticity is an inherent feature of THC action (Keimpema et al, 2011). This is further supported by the observation that SCG10 expression prevails during adulthood and aging in neuronal clusters with long‐lasting synaptic plasticity (Peng et al, 2004). In using iTRAQ for quantitative candidate discovery we reasoned that mRNA may not be translated into biologically active protein. Moreover, we conceptually approached the 17% decrease in cortical SCG10 amount as an indication that a subset of cortical neurons selectively, rather than all of them indiscriminately, down‐regulated SCG10 protein expression. Indeed, SCG10 is primarily expressed in a contingent of neurons in the fetal cortical plate that adopted pyramidal cell‐like positioning and axodendritic morphology. Linking SCG10 deregulation to pyramidal cell development is not unexpected since genetic deletion of CB 1 Rs in pyramidal cells destined to superficial cortical layers leads to axon fasciculation and targeting errors (Mulder et al, 2008). Moreover, since CB 1 Rs are not expressed in cortical proliferative zones (Goncalves et al, 2008; Mulder et al, 2008), CB 1 R‐mediated adverse THC effects will likely impact cortical wiring rather than neuronal diversification in the fetal cerebrum. Nevertheless, we find similarities in the molecular identity and functions of THC's differentially regulated targets in developmental and adult settings (Kittler et al, 2000), such as proteins implicated in secondary metabolism, protein folding and signal transduction (Supplementary Fig S3A). Our data in conjunction with large‐scale analysis from adult brain (Kittler et al, 2000) reinforce that THC's mode of action involves the disrupted development and/or postnatal maintenance of synapses critical for highly ordered executive and cognitive functions.

Microtubules in developing neurons mediate growth cone steering and forward movement (Grenningloh et al, 2004; Morii et al, 2006; Tararuk et al, 2006; Manna et al, 2007). The dynamic instability of microtubules, and the integrity of the microtubule network determines the rate of axonal transport (Grenningloh et al, 2004; Shin et al, 2012). Within growth cones, microtubule plus end switching between growth and shortening states by the process of dynamic instability underpins receptor‐mediated reorganization (Manna et al, 2007). The stathmin family of proteins share a C‐terminal stathmin domain to bind α/β‐tubulin dimers to form ternary T2S complexes (Manna et al, 2007). While stathmin is ubiquitously expressed, SCG10, SCLIP, RB3, RB3′ and RB3″ are neuron‐specific proteins (Grenningloh et al, 2004; Tararuk et al, 2006). These predominantly cytosolic proteins can be differentially modulated to meet the cells' developmental potential and morphogenic demands. Notably, their complementary organ system distribution could constitute a first‐order effector system diversifying endocannabinoid and cannabis effects downstream from CB 1 Rs. In fact, the signaling cascade we describe is, to the best of our knowledge, the first signaling axis directly linking a GPCR to SCG10 as molecular effector.

The considerably different subcellular distribution of stathmin and SCG10 (Grenningloh et al, 2004) could be permissive for the domain‐specific modulation of CB 1 R‐mediated signals on the neuronal cytoskeleton. Such a molecular platform is particularly relevant to brain development since advancing growth cones are a primary site for CB 1 R localization (Berghuis et al, 2007; Keimpema et al, 2010, 2011) and signaling (Berghuis et al, 2007) to promote forward axonal movement. SCG10, unlike stathmin, contains an N‐terminal membrane‐associating domain (Manna et al, 2007), which mediates its fast anterograde transport [at a velocity of ∼1 μm/s (Shin et al, 2012)] to growing tips of axons and dendrites. SCG10 accumulates in the central domain of growth cones where microtubules are highly dynamic (Grenningloh et al, 2004). Here, we define an integrated signaling axis: (i) triggered by THC, (ii) transduced by CB 1 R, (iii) via JNKs, and (iv) executed by SCG10. Biochemical indices of SCG10 degradation are supported by the loss of SCG10 immunosignal from THC‐stimulated neurites and growth cones in vivo and in vitro, and stabilization of acetylated tubulin in the central growth cone domain. Since the frequency of posttranslational modification of tubulin directly correlates with its “age” (i.e. stability) (Maruta et al, 1986), and increased plus end activity in microtubules underpins ectopic filopodiagenesis (Grenningloh et al, 2004), our data identify CB 1 R activity as a driving force of SCG10 availability. Accordingly, depletion of SCG10 from the active growth cone domain increased the proportion of looped/spread growth cone morphologies, suggestive of their altered rate of motility, and reduced neurite outgrowth upon lessening of microtubule dynamics. Retention of residual SCG10 at branch forks can confer minimal essential cytoskeletal dynamics required to maintain cellular integrity and axonal arborization.

SCG10 exists in multiple phosphoisoforms in the neonatal rat brain (Tararuk et al, 2006; Westerlund et al, 2011). SCG10 activity is strongly regulated by phosphorylation of its four serine residues involving protein kinase A (Ser50/97), mitogen‐activated protein kinases (Ser62/73) or CDK5/p25 (Ser73) (Grenningloh et al, 2004). JNKs phosphorylate SCG10 at residues Ser62/73 (Westerlund et al, 2011), likely generating mono‐ and polyphosphorylated SCG10 (Shin et al, 2012) to faciliate its degradation. In general, phosphorylation inhibits the microtubule destabilizing activity of SCG10 suggesting that this protein may link extracellular signals to the rearrangement of the neuronal cytoskeleton. Site‐directed mutagenesis in which serines are replaced by phosphorylation‐mimicking aspartate (D) residues (Tararuk et al, 2006; Westerlund et al, 2011) showed that the relative importance of each site to SCG10 function might vary, but that its overall activity decreases with increased phosphorylation (Grenningloh et al, 2004). SCG10‐S62D/S73D is a functionally inactive, pseudophosphorylated mutant, mimicking JNK‐phosphorylated SCG10 (Westerlund et al, 2011), whose dominant‐negative action can consequently stabilize microtubules. Here, we find that SCG10‐DD overexpression limits neurite outgrowth in competition with endogenous (wild‐type) SCG10, and occludes THC effects. Our findings suggest that SCG10 is a key substrate whose phosphorylation downstream from agonist‐activated CB 1 Rs reduces an essential level of cytoskeletal instability required to maintain the rate of neurite outgrowth. Overall, SCG10 loss is not the trigger for developmental axonal growth errors, but rather is a permissive factor that allows growth‐associated protein stability pathways to initiate ectopic growth.

JNKs are essential regulators of morphogenesis during early development and, besides regulating transcription, are functional in the cytoplasm where SCG10 localizes (Tararuk et al, 2006). JNK is constitutively phosphorylated in the axon, with the kinetics of its spatiotemporal dephosphorylation considered rate limiting. Recently, MAP kinase phosphatase 1 was shown to mediate JNK dephosphorylation (Jeanneteau et al, 2010), allowing prolonged stathmin dephosphorylation, stabilization, and excess microtubule stability. Since cannabinoid receptors, particularly CB 2 Rs (Romero‐Sandoval et al, 2009), can activate MAP kinase phosphatase 1 in a cell type‐specific fashion, the balance of JNK phosphorylation/dephosphorylation will ultimately determine SCG10 availability and actions. JNK1 is the predominant physiologically active form of JNK in the brain (Tararuk et al, 2006) with JNK1−/− mice displaying severely disrupted commissure formation due to the loss of axonal microtubule integrity (Fig 6D and D 1 ) (Chang et al, 2003; Westerlund et al, 2011). SCG10 is the preferred neuron‐specific JNK1 substrate at low concentrations (Tararuk et al, 2006) because, unlike stathmin, its N‐terminal extension allows for efficient JNK1 binding. Notably, SCG10 phosphorylation at Ser62/Ser73 is significantly depleted in JNK1−/− cortex (Tararuk et al, 2006), and disrupts cell cycle exit and radial migration of cortical neurons (Westerlund et al, 2011). Considering that CB 1 Rs affect long‐distance neuronal migration, the present and previous (Tararuk et al, 2006; Berghuis et al, 2007; Keimpema et al, 2011; Westerlund et al, 2011) data cumulatively infer a signaling framework, which is sufficient to account for migration and connectivity deficits in CB 1 R−/− mice or upon exposure to cannabinomimetics or phytocannabinoids.

Recreational cannabis use is particularly prevalent in the young adult age group, including women of child‐bearing age (Substance Abuse & Mental Health Service Administration, 2010). Although recent population statistics revealed significant fluctuations due to geoeconomic variables, > 10% of pregnancies in US and Europe are associated with maternal cannabis exposure (Substance Abuse & Mental Health Service Administration, 2010). Cannabis spp. reportedly contain > 400 bioactive components, with THC being its primary psychoactive constituent. During the past decades, selective agriculture of Cannabis spp. resulted in increased THC content at the expense of cannabidiol (Pitts et al, 1992; Pijlman et al, 2005). In the context of the present study, this is of particular concern since we predict that higher THC concentrations will be, upon efficient cross‐placental transfer (Grotenhermen, 2003), increasingly detrimental for fetal development and postnatal health. Therefore, irrespective of the legal status of cannabis, caution must be exercised to hinder fetal cannabis exposure due to its unequivical impact on the establishment of synaptic connectivity in neuronal networks underpinning memory encoding, cognition and executive skills. Moreover, abnormal synaptic organization, even if remaining latent for long periods, might be prone to “circuit failure” if provoked. A “double hit” scenario of cortical failure when a labile network advances into a runaway cascade upon a secondary insult therefore might account for the increased incidence of schizophrenia, depression and addiction in offspring prenatally exposed to cannabis (Substance Abuse & Mental Health Service Administration, 2010; Keimpema et al, 2011).