Programming of Neural Cells by (Endo)Cannabinoids: from Physiological Rules to Emerging Therapies

REVIEWS

THE ENDOCANNABINOID SYSTEM Programming of neural cells by (endo) cannabinoids: from physiological rules to emerging therapies

Mauro Maccarrone1,2, Manuel Guzmán3, Ken Mackie4, Patrick Doherty5 and Tibor Harkany6,7 Abstract | Among the many signalling lipids, endocannabinoids are increasingly recognized for their important roles in neuronal and glial development. Recent experimental evidence suggests that, during neuronal differentiation, endocannabinoid signalling undergoes a fundamental switch from the prenatal determination of cell fate to the homeostatic regulation of synaptic neurotransmission and bioenergetics in the mature nervous system. These studies also offer novel insights into neuropsychiatric disease mechanisms and contribute to the public debate about the benefits and the risks of cannabis use during pregnancy and in adolescence.

Morphogenetic signals The capacity of the brain for information processing endocannabinoid signalling seems to be a multimodal Gradients of molecules that relies on the number and the molecular and functional communication cassette that is involved in pattern- determine the position of diversity of neurons, their topographically precise con- ing neuronal connections, the synaptic efficacy of specialized cellular subtypes nectivity and their metabolic and signalling interac- which, once they are mature, is often also modulated and instruct their 7 communication and functional tions with glial cells. The developing nervous system by endocannabinoids (FIG. 1a). The widespread nature role during histogenesis. encompasses a range of interacting signalling path- of endocannabinoid modulation of both excitatory and ways, which are individually controlled along unique inhibitory synaptic neurotransmission in the postnatal Sphingolipids temporal and spatial scales. During brain development, brain9 and spinal cord13 (FIG. 1b) suggests that develop- Molecules that contain the organic aliphatic amino alcohol neural progenitor cell proliferation and asymmetric mental rules might place these small signalling lipids into sphingosine or a structurally division, and the positioning and molecular diversifi- a crucial arch of the molecular machinery controlling similar molecule as a cation of neuronal and glial progenies, are modulated synaptic neurotransmission. backbone. by both cell-autonomous and cell–cell interactions of The family of endocannabinoids and their structural morphogenetic signals that are crucial to building com- analogues14 potentially includes hundreds of bioactive Endocannabinoids Endogenous compounds that plex tissues. Among these structurally and functionally molecules. This Review focuses on 2-arachidonoylglyc- bind to cannabinoid 1 diverse signalling domains, bioactive signalling lipids erol (2-AG), which is the most abundant mammalian receptors (CB1Rs) and/or (for example, phospholipids, sphingolipids, glycolipids endocannabinoid that affects synaptic neurotransmis- CB2Rs with high affinity, and and prostanoids) are widely recognized as crucial for sion15,16, and to a lesser extent on anandamide (AEA)17, are able to evoke a 9 neuronal and glial differentiation, as well as for synaptic which is a mixed endovanilloid and endocannabinoid Δ -tetrahydrocannabinol-like 1–3 behavioural tetrad. plasticity . ligand. 2-AG, AEA and related lipids can stimulate Recent work has highlighted similar but unexpect- cannabinoid 1 receptors (CB1Rs) and CB2Rs and the edly diverse roles in the developing nervous system nuclear fatty acid receptors peroxisome proliferator- for the endocannabinoid family of small signalling activated receptor-α (PPARα) and PPARγ (REF. 18). lipids, N-acyl-amines and 2-acyl-glycerols, which 2-AG generally has higher efficacy at CB1R and CB2R, typically contain an arachidonoyl moiety4,5. Indeed, whereas AEA is a low-efficacy agonist that can function Correspondence to evidence suggests that there is a continuum of action as a partial agonist (or even antagonist) in tissues with M.M. and T.H. by endocannabinoids, overarching the early stages of low receptor reserve or at receptors that are inefficiently e-mails: m.maccarrone@ embryo development and implantation6, nervous sys- coupled to downstream effectors. AEA and 2-AG can unicampus.it; Tibor. 7 8 [email protected]; Tibor. tem development , bioenergetics and intercellular stimulate PPARs at high concentrations, although 9,10 [email protected] communication in the adult, including adult neu- related molecules such as N-oleoylethanolamine do so doi:10.1038/nrn3846 rogenesis11,12. More specifically for the nervous system, more potently. Moreover, AEA can engage transient

786 | DECEMBER 2014 | VOLUME 15 www.nature.com/reviews/neuro

sn-1-diacylglycerol lipase-α receptor potential cation channel subfamily V mem- gliomas. Finally, we highlight differential sensitivities of (DAGLα). Two isoforms of ber 1 (TRPV1) and G protein-coupled receptor 55 precisely controlled physiological processes versus dis- DAGL, DAGLα and DAGLβ, (GPR55)18. ease pathomechanisms to help to put the ongoing public which probably evolved Although gaps persist in our knowledge of endocan- debate about the use of Δ9-THC during pregnancy and in evolutionarily through gene duplication, are chiefly nabinoid action during formation of the subcortical adolescents into a more realistic context. responsible for 2-AG forebrain, midbrain and spinal cord, the studies high- synthesis. lighted in this section define multiple steps of cortical Molecular logic of endocannabinoid action and cerebellar development modulated by endocan- Much of what we know about the molecular organization Tripartite synapse nabinoids, which have been elucidated through the use of endocannabinoid signalling during neuronal develop- A concept of synaptic 19–24 FIG. 1a anatomy that includes the of an impressive range of genetic and cellular tools ment (schematically depicted in ) comes from the 25,26 presynaptic terminal, and evolutionary deductions , as well as experimen- comparative analysis of successive developmental stages postsynaptic specialization tal models in invertebrates27, non-mammalian verte- in rodents11,22,32,33,46,48 (FIG. 2a–d). Arrangements at mature and astroglial end-feet that brates28,29, rodents30–34 and human foetal tissues35–38. synapses are such that 2-AG synthesis is postsynap- isolate the synaptic cleft as a 15,16,49 sn-1-diacylglycerol lipase- discrete entity. The evidence for endocannabinoid involvement in tic with α (DAGLα) enriched fundamental developmental processes comes from the in the ‘perisynaptic annulus’ (that is, 50–100 nm from combination of sophisticated mouse genetics21,39, neu- the postsynaptic density50), which allows the fast cou- rophysiology40 and the study of gene polymorphisms pling of postsynaptic metabotropic receptor activation for CB1R, CB2R, α/β-hydrolase domain-containing to Ca2+ and phospholipase Cβ-dependent 2-AG synthe- 12 (ABHD12) and fatty acid amide hydrolase (FAAH) sis51 (FIG. 1b). Diffusing in a retrograde manner across the in humans with diseases that are thought to have a synapse, endocannabinoids activate presynaptic CB1Rs,

developmental origin, such as schizophrenia, bipolar typically signalling via Gi proteins to inhibit synaptic neu- disorder, drug addiction, metabolic disorders and rotransmission (FIG. 1b). 2-AG is then mainly inactivated neurodegeneration41–45. These studies support causality by presynaptic monoacylglycerol lipase (MAGL)52, with between dysregulated endocannabinoid signalling and a possible contribution from ABHD6 and ABHD12 (REFS neuropsychiatric illnesses. Thus, the interdisciplinary 45,53), which are partitioned to postsynaptic sites (FIG. 1b). nature of this Review provides a translational frame- The situation with AEA is more complex. AEA can be work that bridges (at least) two key areas of neuro produced postsynaptically to inhibit synaptic transmis- science: neurophysiology; and psychiatric and addiction sion in a retrograde manner as in the case of 2-AG54. research. Alternatively, N-arachidonoylethanolamine-selective Endocannabinoid signalling at CB1R and CB2R in phospholipase D (NAPE-PLD) may produce AEA neural progenitor cells and postmitotic neurons11,24,46 presynaptically, which then functions at postsynaptic is recognized as the primary molecular substrate for TRPV1 (REFS 55,56) and is inactivated by postsynaptic phytocannabinoids47, most prominently for psycho FAAH57. Notably, and in accordance with the tripartite active Δ9-tetrahydrocannabinol (Δ9-THC). In this synapse hypothesis, perisynaptic astrocytes often con- context, and after discussing the multifarious roles of tain MAGL (FIG. 1b), thus forming a barrier that may endocannabinoid signalling in the prenatal and peri- limit 2-AG spread beyond its intended site of action natal brain, we address endocannabinoid contributions (20–100 µm in a temperature-sensitive manner)58,59. to regulating adult neurogenesis, as cannabis use might At least four premises should be considered to appre- affect the continued production of new neurons in ado- ciate the unique modes of endocannabinoid signal- lescents. We examine the molecular mechanisms and ling during brain development, and these are discussed the health benefits of exploiting the endocannabinoid in this section. First, in differentiating neural tissues, system as a druggable target in pathologies that arise endocannabinoids can adopt a primarily autocrine (cell- from the loss of cell proliferation control, particularly autonomous) mechanism of action19,48,60, in contrast to retrograde signalling by endocannabinoids at adult synapses, which is a paracrine mechanism9. If receptor location Author addresses remains constant, this gives positional differences in the localization of enzymes to influence 2-AG bioavailability. 1 4 School of Medicine and Center of Department of Psychological and Brain An example is DAGLα, which switches from an axonal Integrated Research, Sciences, Indiana University, 702 N to a postsynaptic localization48,51 following differentia- Campus Bio-Medico University of Rome, Walnut Grove Avenue, Bloomington, tion of the presynapse and commencement of synaptic Via Alvaro del Portillo 21, Indiana 47405–2204, USA. 51 I-00128 Rome, Italy. 5Wolfson Centre for Age-Related neurotransmission . For cell-autonomous endocannabi- 2European Center for Brain Research/ Diseases, King’s College, London SE1 1UL, noid signals to drive neural progenitor cell proliferation, Santa Lucia Foundation, United Kingdom. including pathological situations during tumorigenesis Via del Fosso di Fiorano 65, 6Division of Molecular Neurobiology, (see below), the intracellular transport and partition- I-00143 Rome, Italy. Department of Medical Biochemistry and ing of DAGLα are proximal to the localization of CB1Rs 3Department of Biochemistry and Biophysics, Scheeles väg 1:A1, Karolinska and CB2Rs46, which are poised for receptor activation by Molecular Biology I and Centro de Institutet, SE-17177 Stockholm, Sweden. lateral ligand diffusion in the plasmalemma (FIGS 1,3a). 7 Investigación Biomédica en Red sobre Department of Molecular Neurosciences, DAGLα, and to a lesser extent NAPE-PLD, are found Enfermedades Neurodegenerativas, Center for Brain Research, Medical in lipid rafts61 — membrane specializations that allow Complutense University, University of Vienna, Spitalgasse 4, CB1R accumulation and focus signal transduction by E-28040 Madrid, Spain. A-1090 Vienna, Austria. the recruitment of effector kinases62. Following neuronal

NATURE REVIEWS | NEUROSCIENCE VOLUME 15 | DECEMBER 2014 | 787

a Developing synapse Growth cone Radial glia polarization and during directional axonal growth, 2-AG 48 FAAH is protected from ligand degradation by a mechanism NAPE-PLD that increases MAGL protein turnover63 in motile neurite Na+ and Ca2+ CB2R domains, particularly in the growth cone (FIG. 3a). High TRPV1 levels of axonal MAGL function to prevent the premature, ectopic activation of CB1Rs (and perhaps CB2Rs64) dur- TRKs GPR55 CB1R ing axonal transport. Thus, endocannabinoid ‘hot spots’ DAGL α Lipid raft AEA MAGL and DAGLβ exist for focal signalling events. Second, neural progenitor cells, in contrast to differentiated neurons, commonly co- 2–AG FAAH FAAH express CB1Rs and CB2Rs. Pharmacological and histo- ? chemical evidence show predominant CB2R expression in 11 NAPE-PLD ? the subventricular zone of the cerebral cortex (FIG. 2a). On the basis of simultaneous expression of CB2R and DAGLα MAGL FAAH MAGL and DAGLβ in neural progenitor cells, it is probable that autocrine endocannabinoid signalling supports asymmet- Progenitor cell Prospective postsynapse ric cell division, cell cycle exit and long-range migration b Mature synapse Presynapse Microglia of the ensuing progenies11,46,60. Following commitment to 11 Adiposome ? ABHD12 a neuronal fate, a CB2R-to-CB1R switch occurs : CB1R levels become upregulated at the expense of CB2R levels + MAGL ER NAPE-PLD Na and (FIG. 2b,d). Although direct experimental evidence for cau- Ca2+ AIT sality is lacking, experimental data from various vertebrate ABHD12 28,29,65,66 Adiposome ? species suggest that early CB1R expression might EMT NAPE-PLD promote neuronal polarization and the commencement of long-range cell migration. The downregulation of DAGLα MAGL ABHD6 and DAGLβ expression that occurs following neuronal ABHD6 Na+ and Ca2+ MAGL specification can be viewed as a pivotal step to increase FAAH FAAH the reliance of postmitotic neurons on extracellular 2-AG produced by pyramidal cells in the cortical plate51, func- ABHD6 tioning as positional cues (FIG. 2a,c).Third, as neurogenesis precedes gliogenesis67, the spatial spread of endocannabi- Adiposome ABHD12 ? noids in the foetal brain is less restricted than in the adult Astroglia Postsynapse brain. Thus, physiologically relevant ligand concentrations could affect, in bulk, cohorts of migrating neurons Figure 1 | Molecular architecture of the endocannabinoid system during Nature Reviews | Neuroscience or axons during pathfinding63 (FIG. 2a, c). This suggests that synaptogenesis and at mature synapses. Neuronal and glial components of there are wider roles for endocannabinoids in cell–cell developing and mature synapses are shown. The molecular architecture shown here is for a ‘stereotypical’ synapse that uses endocannabinoid signalling. There are differences interactions than was previously thought. Nevertheless, it in neurotransmitter system-specific and developmentally regulated enzyme and/or remains to be established whether these signalling lipids receptor expression and function at different types of synapse and at different stages of preferentially modulate contact guidance or indeed are development. For example, monoacylglycerol lipase (MAGL) is excluded from motile even extracellularly released. Finally, enzymatic inactiva- growth cones until synaptogenesis commences48,68. a | At the developing synapse, tion is a rate-limiting step for endocannabinoid signalling anandamide (AEA) and 2-arachidonoylglycerol (2-AG) initiate downstream signalling by in the developing nervous system48,68. Considerable levels binding to their target receptors: cannabinoid 1 receptor (CB1R), CB2R, of FAAH and MAGL have been shown in cultured neu- G protein-coupled receptor 55 (GPR55) and transient receptor potential cation channel ral progenitor cells19,20 and radial glia48,69,70, which generate subfamily V member 1 (TRPV1). Their availability is determined by biosynthesis enzymes postmitotic cortical neurons71,72 and function as cellular (N-acylphosphatidylethanolamine-specific phospholipase D, NAPE-PLD, sn-1-diacylg- scaffolds for radial migration of neurons73. As radial glia lycerol lipase-α (DAGLα) and DAGLβ195) and degrading enzymes (fatty acid amide hydrolase (FAAH) and MAGL). It is not yet known whether alternative hydrolysis enzymes produce a checkerboard-like patterned map, converting (for example, α/β-hydrolase domain-containing 6 (ABHD6) and ABHD12) also play a part neural progenitor cell distribution along the ventricu- at this stage in development. Unlike other endocannabinoid-binding receptors, CB1Rs lar wall into a three-dimensional neuronal protomap in are preferentially recruited to, and signal within, cholesterol-enriched membrane the cortical plate72, this cell type is ideally positioned to microdomains termed lipid rafts62. b | At the mature synapse, the availability of AEA and limit endocannabinoid diffusion (FIG. 2a). Furthermore, 2-AG is controlled by ABHD6 and ABHD12 hydrolases, in addition to FAAH and MAGL, thalamocortical axons accumulate MAGL33,48, which and also by transmembrane (endocannabinoid transmembrane transporter (EMT)) and produces corridors for CB1R-containing corticotha- 196 intracellular (AEA intracellular transporter (AIT)) transport mechanisms (for example, lamic axons during the corticothalamic–thalamocortical 199 fatty acid-binding proteins , heat shock protein 70 (REF. 197) and FAAH-like AEA handshake.This MAGL localization can both limit axonal transporter198) and storage organelles (adiposomes or lipid droplets)199. There is spread in the prospective internal capsule (FIG. 3b), as well compelling evidence that key receptor and enzyme components of the endocannabinoid system have distinct subcellular distribution, both intracellularly and as delineate migratory routes for CB1R-expressing cor- 65,74 extracellularly on presynaptic and postsynaptic neurons, microglia and astrocytes. tical interneurons (FIG. 2a,c). Overall, the molecular CB2Rs are mainly expressed following brain injury200. Question marks indicate as yet architecture of endocannabinoid signalling networks unknown roles and compartmentalization of ABHD12. AIT, AEA intracellular transporter; can modulate cell proliferation, as well as the migration, EMT, putative endocannabinoid transmembrane transporter; ER, endoplasmic reticulum; polarization and synaptogenesis of postmitotic neurons TRKs, tyrosine receptor kinases. in the cerebral cortex.

788 | DECEMBER 2014 | VOLUME 15 www.nature.com/reviews/neuro

+ REF. 18 Subventricular zone Cannabinoid receptor signalling in foetal brain activation of some K channels (reviewed in ). A progenitor cell-rich part of Although the role of CB1R, CB2R, TRPV1 and GPR55 In stem cells, glial cells and neurons, CB1Rs also acti- the cerebral cortex that lines has been examined in prenatal and/or postnatal neu- vate the phosphatidylinositol 3-kinase (PI3K)–AKT the dorsolateral surface of the rodevelopment, including cell survival and tumori- (also known as PKB)–mammalian target of rapamycin lateral ventricle and retains 75–81 neurogenic capacity genic transformation , a role for endocannabinoid (mTOR) pathway along with several mitogen-activated throughout life. interactions with PPARs remains to be established. protein kinase (MAPK) pathways (for example, extra- Given the predominance of CB1R mRNA and protein cellular signal-regulated kinase (ERK), p38, and JUN Corticothalamic– expression in the foetal nervous system of rodents46,82 N-terminal kinase (JNK)38,84). Although these signal- thalamocortical handshake and humans83, we focus on signal transduction, heter- ling events have recently been reviewed in detail69,85, Where corticothalamic and thalamocortical axons with omerization and upstream regulation of CB1R levels important concepts are that PI3K and AKT recruitment opposite growth trajectories and activity. contributes to neuronal polarization by promoting neu- cross one another. 86 CB1Rs primarily couple to Gi/o proteins and com- rite outgrowth , mTOR signalling regulates a proneural monly signal by inhibition of adenylyl cyclase and of transcriptional cascade of PAX6 and TBR2 (also known certain voltage-dependent Ca2+ channels, as well as by as EOMES) for expansion of the cortical progenitor a b c SMS MZ MZ

CP SMS CP Pyramidal cell HC • DAGL-rich IZ • CB1R dominates IV CP DMS GE IZ

Postmitotic neuron SPT • DAGL downregulated • CB1R dominates E14.5 110µm E14.5 30µm IZ GFP DAGLα d CB1R mRNA

DMS CP Progenitor cell GABA interneuron NE SVZ • DAGL-rich CB1R dominates • CB2R dominates

PU VZ

Radial glial cell Apoptotic Levels of 2-AG Gestational • DAGL (?) • FAAH cell death week 20 200µm • MAGL (?) Low High Figure 2 | Molecular architecture of endocannabinoid signalling that leave the ganglionic eminence (ge) and migrate towards the cerebral 65 during corticogenesis, including neurogenesis and neuronal cortex . c | In mid-gestational mouse brain,Nature DAGL Reviewsα is expressed | Neuroscience at high migration. a | During mid or late gestation in rodents, 2-arachidonoyl levels in pyramidal cells and targeted to their axons. GFP-labelled glycerol (2-AG)-rich cortical microdomains are thought to repulse GABAergic interneurons203 can also be seen migrating in the superficial postmitotic neurons that express cannabinoid 1 receptors (CB1Rs), migratory stream (sms) and deep migratory stream (dms). Yellow colour including radially migrating pyramidal cells and tangentially migrating shows the tight spatial arrangement of DAGL-expressing neuronal GABA interneurons in the cerebral cortex. sn-1-diacylglycerol lipase structures around GFP-labelled GABAergic interneurons, although the (DAGL) expression in the foetal ventricular proliferative zone and in the GFP-positive cells lacks appreciable DAGL expression. d | CB1R mRNA cortical plate can produce physiologically relevant extracellular 2-AG expression concentrates in the CP and proliferative germinal layers concentrations (pink shading). This molecular arrangement, which (ne)36,46 in the human foetal brain (second trimester). Figure part b is produces ‘corridors’ sparse in 2-AG (white areas), could explain some modified, with permission, from Endocannabinoid signaling controls features of the spatially segregated radial migration of pyramidal cells and pyramidal cell specification and long-range axon patterning. Proc. Natl. tangential migration of interneurons. Radial glia function as scaffolds for Acad. Sci. USA 105, 8760–8765 © (2008) Proc. Natl Acad. Sci. USA. Figure migrating neurons and can synthesize and subsequently degrade part c is courtesy of T. H. and E. Keimpema, Karolinska Institutet, Sweden. endocannabinoids, thus promoting endocannabinoid-mediated radial Figure part d is courtesy of Y. L. Hurd, Icahn School of Medicine at Mount detachment of neurons for final positioning. b | At embryonic day 14.5 Sinai, New York, USA. FAAH, fatty acid amide hydrolase; IZ, intermediate (E14.5), cannabinoid 1 receptor (CB1R) mRNA is predominantly expressed zone; MAGL, monoacylglycerol lipase; MZ; marginal zone; LV, lateral by neurons in the cortical plate (CP) and hippocampal primordium (hc). ventricle; SMS, superficial migratory streams; spt, septum; SVZ, Arrows denote CB1R mRNA expression in what are probably interneurons subventricular zone; VZ, ventricular zone.

NATURE REVIEWS | NEUROSCIENCE VOLUME 15 | DECEMBER 2014 | 789

a Neurite outgrowth c Synapse formation

2-AG Intercellular Cell-autonomous FGF NGF GLU GABA CB1R FGFR TRKA DAGLα

CB1R Dendritic 2-AG spine Lipid raft P MAGL P P P PIP2 DAG DAGLα P P α γ P BRCA1 β P GTP PLCγ Gi P PI3K AKT JNK1 RHO

PAX6 CREB Stathmin2 Immature Mature

Cytoskeletal Somato- instability DAGLα dentritic domain 'domain’ DAGLα- b Neurite outgrowth rich Direction of corticofugal E16.5 axon outgrowth ctx 1 Corticofugal axon 2-AG cfa Iv CB1R MAGL transport hc MAGL-rich DAGL 3 f 2 th Stabilized axon Thalamocortical Presynapse tca Growth axon DAGLα-rich MAGL- Breakdown cone rich of 2-AG 2 MAGL MAGL CB1R 400µm Direction of thalamocortical β3 tubulin axon growth

Figure 3 | Design logic of endocannabinoid signalling during neurite thalamocortical axons are CB1R negativeNature but MAGL Reviews positive | Neuroscience (stained outgrowth and synaptogenesis. a | Signal transduction mechanisms green)48. The right-hand panel shows that corticofugal axons express implicated in the cannabinoid 1 receptor (CB1R)-mediated control of DAGLs51 and can use paracrine 2-AG signalling for fasciculation (step 1). cortical neuron specification and morphological differentiation are In turn, autocrine 2-AG signalling in corticofugal axons might be shown. Activation of tyrosine kinase receptors (particularly the fibroblast sufficient to promote their elongation (step 2). This molecular layout is growth factor receptor (FGFR) and the high-affinity nerve growth factor compatible with MAGL-positive thalamocortical axons limiting the (NGF) receptor TRKA) and their activity-dependent phosphorylation are spatial spread of 2-AG (step 3; the dashed line indicates 2-AG thought to induce 2-arachidonoylglycerol (2-AG) production via inactivation), thus controlling the distribution of corticofugal fascicles sequential activation of phospholipase Cγ (PLCγ), which produces and confining their growth trajectories to a subpallial corridor. diacylglycerol (DAG). DAG is then converted to 2-AG by sn-1-diacylglyc- Accordingly, pharmacological inhibition of MAGL activity during erol lipase-α (DAGLα). The dashed arrow in the plasma membrane corticogenesis disrupts the formation of the corticofugal projection indicates lateral 2-AG diffusion that can activate CB1Rs in an autocrine system201. c | The subcellular switch of DAGL and MAGL during neuronal manner. Signalling via G proteins recruited to CB1Rs following agonist polarization and synaptogenesis is shown. In immature neurons, DAGLα binding regulates neuronal morphology by, for example, the is localized to the primary neurite (quiescent axon) and the growth cone, phosphorylation of JUN N-terminal kinases (JNKs; for example, JNK1)38, and is probably involved in autocrine signalling. However, once synapses which triggers the rapid degradation of stathmin 2 to alter cytoskeletal are formed, DAGLα expression is excluded from more proximal parts of stability. Alternatively, CB1R activation can modulate the activity of the axon and is redistributed to the somatodendritic axis of neurons. By RHO-family GTPases, particularly RHOA, to induce growth cone repulsion contrast, MAGL becomes enriched at the presynapse, where it probably and collapse21,31. Neurotrophins and 2-AG signalling can coincidently functions as a ‘stop’ signal to limit 2-AG-mediated neurite elongation48. activate phosphatidylinositol 3-kinase (PI3K)–AKT signalling. This, in turn, The precisely timed molecular reconfiguration of 2-AG signalling influences the activity of the transcriptional regulators PAX6 and cyclic supports a continuum of endocannabinoid actions during neuronal AMP response element-binding protein (CREB) and their control of neural differentiation, leading up to the retrograde control of synaptic progenitor cell proliferation and fate decisions (reviewed in REF. 86). neurotransmission (inset). DAGLα is selectively enriched in the Cytoplasmic breast cancer-associated protein 1 (BRCA1; which is perisynaptic annulus of dendritic spines (pink shading) apposing activated by PI3K signalling) is one of the candidate E3 ubiquitin ligases glutamatergic afferents49,202. cfa, corticofugal axon; ctx, cerebral cortex; f, 68,86 controlling monoacylglycerol lipase (MAGL) degradation . b | The fimbria; hc, hippocampus; lv, lateral ventricle; PIP2, phosphatidylinositol left-hand panel shows the spatial segregation of molecular determinants 4,5-bisphosphate; tca, thalamocortical axon; th, thalamus. Figure Part b, of 2-AG signalling during the corticothalamic-thalamocortical axonal left-hand panel, reproduced with permission, and right-hand panel, ‘handshake’. Corticofugal axons are CB1R positive (stained red), whereas adapted with permission, Society for Neuroscience.

790 | DECEMBER 2014 | VOLUME 15 www.nature.com/reviews/neuro

cell pool in mice32,46,87, and JNK1 links CB1R activity to also to adjust responsiveness to, for example, neurotro- cytoskeletal instability to determine the morphological phin and netrin signalling through Ca2+-dependent cyclic phenotypes of neurons38 (FIG. 3a). AMP production. Moreover, and in contrast to the usual Additional modes of CB1R (and probably CB2R) pro-survival actions of AEA through CB1R, TRPV1 acti- signalling add to the complexity of endocannabinoid vation by AEA and related lipids can lead to cell death signalling and cell stage-specific influence. These addi- via the ATF3-dependent ER stress pathway75. As such, tional modes of signalling include sequestration of G the existence of a signalling axis between AEA-activated proteins88, shifting the balance of signalling between TRPV1 controlling 2-AG biosynthesis and biological GPCRs that co-exist at the same synapse, or heteromeri- activity at CB1Rs in the foetal brain, such as in adult zation with other GPCRs89, inducing G protein switching striatum99, remains to be determined. Therefore, future and differential pharmacology90. From a developmental research might be increasingly directed towards under- standpoint, CB1R interplay with neurotrophin signal- standing the differential involvement of multireceptor ling seems to be of particular importance. First, CB1Rs mechanisms in the wiring of neuronal networks by deter- can transactivate certain receptor tyrosine kinases91, mining how the balance of receptor activities pertains particularly TRKB in the absence of brain-derived neu- to unwanted changes in neurogenesis, gliogenesis and rotrophic factor (BDNF)92 by a mechanism involving neuronal differentiation. SRC kinase activation, and can signal via β-arrestins93. Second, BDNF can sensitize CB1Rs and induce signal Endocannabinoids and neural development transduction (by ERK and AKT phosphorylation) at oth- The bioavailability of 2-AG and AEA strikingly diverges erwise sub-threshold levels of exogenously applied ago- as the embryo develops100,101. AEA predominates in the nists94. Third, 2-AG signalling at CB1Rs can function as blastocyst and early embryonic stages, and is required for an essential effector of neurotrophin signalling68,95. This embryo implantation and the maintenance of pregnancy6. concept (FIG. 3a) was introduced when fibroblast growth By contrast, 2-AG levels gradually increase as tissue dif- factor receptor (FGFR) activation by neural cell adhe- ferentiation progresses, including in the nervous system, sion molecules was shown to couple to neurite outgrowth in which DAGLα and DAGLβ expression in developing in a CB1R-dependent manner95. Mechanistically, FGFR axonal tracts during the mid- and late-gestational periods activation induces phospholipase Cγ (PLCγ) activation, exceeds that in the later stages of development48,51 (FIG. 2a). which generates arachidonoyl-containing diacylglycerol A series of elegant experiments from the laboratory of (DAG) for conversion to 2-AG by DAGLα and DAGLβ Ismael Galve-Roperh combined mouse genetics and and exerts cell-autonomous actions on CB1Rs in motile in vitro models to show that endocannabinoids control the growth cones. A similar mechanism was more recently size of the neural progenitor cell pool in the developing described for TRKA (also known as high-affinity nerve forebrain and are particularly important for adopting neu- growth factor receptor) signalling68 via the PI3K path- ronal versus astroglial cell fate in the hippocampus and way, leading to increased expression of DAGL, MAGL cerebral cortex19,20,87,102. These studies revealed that neural and CB1R. Notably, increased 2-AG content and focal progenitor cells express CB2Rs, along with CB1Rs, and signalling in growth cones was maintained by the coordi- that CB2R activation by non-psychoactive cannabinoids nated, nerve growth factor (NGF)-dependent expression and endocannabinoids and downstream PI3K–AKT— of E3 ubiquitin ligases, including breast cancer-associated mTOR complex 1 (mTORC1) signalling modulates the protein 1 (BRCA1)68,86, to mediate MAGL degradation expansion of the neural progenitor cell pool in vitro and (FIG. 3a). As such, both DAGL and CB1R inhibition in vivo102,103. Although there are considerable gaps in our abolish neurotrophin-induced neurite outgrowth68,95,96 current knowledge about the integration of endocannabi- in cerebellar and subcortical neurons, which supports noid signals in neurogenic niches at the receptor level and the hypothesis that neurotrophin signalling uses endo- the specific contributions of 2-AG, AEA and other endocannabinoids to regulate neurite extension. Similarly, cannabinoids, the consensus view is that neurogenic fate netrin signalling at its receptor, deleted in colorectal decisions coincide with a CB2R-to-CB1R switch in post- cancer (DCC), is modulated by CB1Rs and CB2Rs31,64, mitotic neurons11. This could explain the predominance of which affects netrin-induced growth cone steering deci- CB1R expression (FIG. 2b) and signalling during neuronal sions and directional axonal growth in the developing differentiation22,28,33,60,92 in all vertebrates studied28,29,46, visual system. The pathophysiological implications of including humans36 (FIG. 2d). these interactions are considerable, including disrupted The event of receptor switching parallels the down- neuronal migration92, faulty growth cone turning deci- regulation of DAGL expression in postmitotic neurons60, sions21,31,64 and incomplete hemispheric segregation of which allows the cessation of proliferation-promoting visual pathways31. autocrine 2-AG signalling (FIG. 2a). Rather, reliance of TRPV1 channels97 may be located on the plasma the differentiating progeny on paracrine (target-derived) membrane or on intracellular membranous compart- positional endocannabinoid signals increases. Such a Cytoskeletal instability ments such as the endoplasmic reticulum (ER)98. In both mechanism is probably relevant for radially migrating The dynamic reorganization of cases, TRPV1 activation by AEA will increase levels of pyramidal cells to migrate from the cortical progenitor the cytoskeleton by cytoplasmic Ca2+, which thus stimulates diverse Ca2+- cell niche towards the prospective cortical plate along a differentially altering the elongation and shortening of dependent signalling pathways. Activation of plasma vertically decreasing 2-AG gradient. This also implies that the ‘+’ and ‘-’ ends of membrane TRPV1 will also tend to depolarize the cell, CB1Rs transduce repulsive signals to maintain the direc- mictotubules. which may be important not only for differentiation but tionality of neuronal migration. A similar mechanism

NATURE REVIEWS | NEUROSCIENCE VOLUME 15 | DECEMBER 2014 | 791

Table 1 | Key models used to investigate endocannabinoid signalling* corticofugal axons. This is supported by the axonal localization of CB1Rs, which suggests that autocrine and par- Model or Phenotype Refs 48 approach acrine modes of action can promote neurite elongation , fasciculation and directionality29,31 (FIG. 3b). Recent experi- −/− CNR1 (globally) Decreased neurogenesis 46 mental — particularly in vitro — evidence suggests that Migratory neuron misrouting 21 CB1Rs, CB2Rs64, TRPV1 and GPR55 couple to neurite Axonal growth and guidance errors 46 growth by modulating lateralized Ca2+ signalling105 in CNR1−/− (in Migratory neuron misrouting 21 axonal growth cones, which represent a direct link to interneurons) cytoskeletal reorganization following exogenous ligand Altered synapse distribution 21 exposure in various cell systems80,81,106–110. In this section, CNR1−/− (in Premature cell cycle exit 87 we focus on CB1R-mediated axonal growth, the physi- pyramidal cells) Decreased neural progenitor cell proliferation 46 ological importance of which is supported by genetic and pharmacological analyses (FIG.4; TABLE 1). Initially, Corticofugal axon fasciculation errors 32 CB1Rs, even though they are signalling competent111, Deficient fine motor functions (postnatally) 32 were thought to be expressed at ‘atypical locations’ in FAAH−/− Augmented neurogenesis 46 foetal brains (that is, in the white matter instead of at 100,111 Increased neural progenitor cell proliferation 20 synaptic sites) . In subsequent studies, the selective exposure of CB1Rs on the axonal surface was shown23,112 Increased radial migration in cortex 20 and their activation was shown to induce repulsive growth DAGLA−/− Altered synapse distribution 63 cone turning and eventual collapse, at least in vitro21,31. MAGL−/− No developmental phenotype 201 The molecular mechanism of CB1R-mediated cytoskel- GPR55−/− No developmental phenotype 33 etal instability in growth cones is thought to involve RHO-family GTPases21, RAS and PI3K–AKT–β-catenin Impaired movement coordination (postnatally) 138 signalling95,113 (FIG. 3a). In single-cell systems21,31, CB1R- Δ9-THC Interneuron misplacement 92 mediated repulsive growth cone turning involves neur- Axon fasciculation error 38 ite extension, whereas growth cone collapse is followed Impaired synaptic plasticity (postnatally) 38 by neurite retraction, growth cone reinstatement and renewed extension and motility along alternative paths. Altered drug-seeking behaviour (postnatally) 142 The highly dynamic steering behaviours of advancing SR141716A Axon fasciculation error 46 growth cones in the presence of repulsive cues might be AM251 Axon fasciculation error 38 translated to the complex three-dimensional structure of the developing cerebrum, in which precisely configured URB597 No developmental phenotype; impaired 137 learning and memory (postnatally) 2-AG sources are organized in corridor-like patterns such that repeated cycles of repulsion-alternative pathfinding WIN55,212-2 Disrupted neuronal migration 132 responses, which result in net neurite elongation, propel Impaired synaptic neurotransmission and 133, 134 CB1R+ axons along their growth trajectory. These consid- learning deficits (postnatally) erations, together with CB1R expression being restricted JZL184 Axonal growth and guidance defects (prenatally) 63, 201 to developing neurons destined to the cerebral cortex46,65, CNR1, cannabinoid receptor 1 gene; DAGLA, sn-1-diacylglycerol lipase-α gene; FAAH, fatty suggest that endocannabinoid signalling is particularly acid amide hydrolase gene; GPR55, G protein-coupled receptor 55 gene; MAGL, important for cortical development and that the targeting monoacylglycerol lipase gene. *Existing studies20,21,32,33,38,46,68,87,92,132–134,138,142,201 focused on forebrain development, particularly corticogenesis. Prenatal treatments with cannabinoid of long-range axons leaving the cerebral cortex (termed receptor ligands (SR141716, AM251 (antagonists) and WIN55,2122 (agonist)), FAAH (URB597) corticofugal axons) might switch from endocannabinoid- or MAGL (JZL184) inhibitors are listed. dependent to non-endocannabinoid-mediated forms of axon guidance after passing the pallio–subpallial bound- might operate in some, if not all, GABA interneurons65 ary. The fact that thalamocortical axons express MAGL migrating tangentially in the superficial and deep migra- (FIG 3b) raises the possibility that, during the process of the tory streams from subcortical ganglionic eminences thalamocortical–corticothalamic handshake when axons towards endocannabinoid-rich cortical domains48 along opposite paths use in trans signalling to maintain (FIG. 2a,c). We propose that GABA interneurons use 2-AG directional motility in the internal capsule, MAGL- signals to avoid the DAGLα- and DAGLβ-rich cortical mediated 2-AG inactivation can change axonal growth plate (note that pyramidal cells populate the cortical rate, modulate directionality and determine the size of plate days earlier than interneurons104) and migrate in axon fascicles by adjusting the number of axons that endocannabinoid-sparse cortical corridors instead. This navigate together. Although data using JZL184, which is hypothesis also takes into account that focal endocan- a MAGL inhibitor, in cell culture systems suggests a role nabinoid gradients are probably present (see below) to for MAGL in growth rate control48, in vivo support for control the direction of cell migration92, and integrates this hypothesis is not yet available. Moreover, and even Fasciculation radial glia as a cell contingent that terminates long-range though the extracellular matrix proteins glypican (a hep- The process by which migration by endocannabinoid inactivation46. aran sulphate proteoglycan) and neurocan (a chondroitin developing axons follow a primary pioneer axon, thus In addition to its probable contribution to shaping the sulphate proteoglycan) have been proposed to be down- 38 maintaining strict directionality path of migrating GABA interneurons, DAGL expres- stream targets of CB1Rs during axonal growth , mech- towards a distal target. sion in pyramidal cells can also subserve the growth of anistic insights in endocannabinoid-mediated axon

792 | DECEMBER 2014 | VOLUME 15 www.nature.com/reviews/neuro

fasciculation remain elusive. Nevertheless, a key obser- differentiation in both autocrine and paracrine con- vation is that, once synaptic wiring occurs, DAGL51 texts. Although autocrine signalling might function and MAGL48 become redistributed in neurons (FIG. 3c), in glial progenitor cells to expand available cell pools in with DAGL exclusively present in the somatodendritic gliogenic niches of the brain, paracrine signalling, with domain of neurons and MAGL assuming a presynaptic DAGL-containing axons serving as the cellular sources localization. Although we do not yet know the molecu- of 2-AG, can promote the recruitment of astroglia and lar filters that exclude these enzymes from particular oligodendrocytes to specific neuronal foci for metabolic subcellular compartments, this molecular reorganiza- purposes or to myelinate long-range axons to increase tion is clearly crucial for the retrograde control of syn- their conductance velocity. aptic neurotransmission to become operational in the juvenile brain114–116. Cannabis exposure during pregnancy Developmental neurobiology commonly probes the rel- Endocannabinoid contributions to gliogenesis evance of any signalling system to brain development Although most studies have focused on the role of endo- by a combination of genetic (knockout) models and cannabinoids in neuronal differentiation, essential data pharmacological tools. The >400 bioactive components suggest that these signalling lipids are also important in in Cannabis spp.47 make the use of pharmacological regulating astrogliogenesis, oligodendrogliogenesis and approaches particularly important in relation to endo- differentiation19,117–121. Indeed, CB1R activation promotes cannabinoid signalling. In this section, we summarize astroglial differentiation of mouse neural progenitor cells data from knockout models, from antagonism of CB1Rs in culture19,122,123 and in the postnatal and adult mouse and from modulating endocannabinoid metabolism. hippocampus in vivo124. Furthermore, CB1R stimulation enhances the survival of rat astrocytes in culture and in Genetic models. The lack of lethality or an otherwise the adult rat hippocampus in vivo, probably by a focal robust nervous system phenotype in constitutive CNR1- signalling mechanism (that is, cultured astroglia express knockout mice131 prompted initial scepticism about endo- CB1R, CB2R, MAGL and FAAH, which raises the possi- cannabinoids having a mandatory neurodevelopmental bility of both autocrine and paracrine signalling19,20) and role. The first wave of conditional knockout models21,32,46 reliance on the PI3K–AKT pathway (FIG. 3a). and detailed morphological analyses21,32,68 have begun to The enzymes responsible for 2-AG synthesis and change this view (FIG. 4; TABLE 1) and confirmed that can- degradation were found in oligodendrocytes118,119, which nabinoid receptors and metabolic enzymes have a par- produce high levels of 2-AG. As oligodendrocytes also ticularly robust capacity for compensation and adaptation. express CB1R and CB2R and respond to MAGL inhibi- These findings also suggest that pharmacological chal- tion (by JZL184) by rapid morphological differentiation, lenges might similarly provoke unwanted developmental particularly the formation of highly ramified processes118, complications. it is probable that autocrine 2-AG signalling dominates in driving oligodendroglial maturation. Moreover, agonist CB1R modulation. Accordingly, a bolus injection of activation of both CB1Rs and CB2Rs enhances the sur- SR414716, which is a CB1R antagonist, into the lat- vival of oligodendrocyte progenitor cells in culture. These eral ventricle of mouse fetuses (at embryonic day 14)46 effects are induced by the CB1R- and CB2R-mediated recapitulated the arrest of neuroblast migration24 from activation of the ERK119 and PI3K–AKT signalling the subventricular zone, the ipsilateral cortical delami- pathways in vitro118,125. These data were corroborated nation and the growth defects of corticofugal axons by studies showing that CB1R and CB2R activation also that are seen in mice lacking CB1R in pyramidal cells46 promote oligodendrogenesis in the subventricular zone (FIG. 4). Moreover, perinatal injections of AM251, which of the postnatal rodent brain in vivo117, particularly fol- is another CB1R antagonist, altered the wiring diagram lowing hypoxic–ischaemic damage in neonatal126 and of the barrel cortex by mistargeted axons, thus modify- adult animals, and following viral encephalitis-induced ing the whisker map and compromising somatosensory disease127. Although the above observations seem com- information processing30. Similarly, injection of pregnant pelling, some investigators have been unable to reproduce dams with WIN55,2122, which is a potent and effica- these findings, at least when attempting to differentiate cious agonist at both CB1R and CB2R, dysregulated mouse neural progenitor cells to generate astroglia or oli- TBR1 and TBR2 expression132. This treatment also aug- 128,129 Neuroblast godendroglia in vitro . Nonetheless, the recent find- mented both radial and tangential neuronal migration A postmitotic cell in an ing that haemopressin, which is a putative endogenous in the cerebral cortex with a particular increase in the undifferentiated form migrating CB1R inverse agonist, might increase oligodendrogial number of Cajal–Retzius cells in the cortical marginal towards its final position, where differentiation of neural progenitor cells isolated from zone132, which could cause the altered glutamate neu- it becomes a neuron. 130 133 the subventricular zone of neonatal mice suggests that rotransmission and impaired postnatal learning and 134,135 Functional antagonist spatially restricted, cell stage-specific endocannabinoid memory encoding . A drug that is signals could differentially affect the proliferative capac- pharmacologically classified as ity of neural progenitor cells and could typify the cellular Manipulating endocannabinoid metabolism. Chronic an agonist; it induces a identity of ensuing progenies. MAGL inhibition by JZL184, which produces multifold phenotype similar to that provoked by an antagonist, Neurogenesis precedes gliogenesis during brain increases in embryonic 2-AG levels, modifies the devel- 67 often by downregulation of development . Therefore, we propose that endocan- opment of the cortical circuitry by acting as a functional signalling. nabinoid signalling can be relevant to gliogenesis and antagonist, with long-lasting consequences for CB1R

NATURE REVIEWS | NEUROSCIENCE VOLUME 15 | DECEMBER 2014 | 793

a Wild-type b CB1Rf/f,Nex-Cre/+ outcome of longitudinal human studies in children who P2 present compulsive and depressive behaviours and deficits in recall and visual memory when they have been prenatally exposed to cannabis139,140.

Transgenerational cannabis effects. Research of the cfa cfa effects of cannabis on brain development has so far only 9 34,38,141,142 cpu cpu focused on the analysis of the effect of Δ -THC , which is the major psychoactive component. Pioneering studies from the Hurd laboratory have shown increased 25µm 25µm 25µm 25µm heroin seeking in offspring prenatally exposed to 142,143 CB1R Golga 5 L1-NCAM THC , transgenerational imprinting via epigenetic mechanisms through coincidently enhanced transcrip- Figure 4 | Defective development of the corticofugal system following genetic tional repression and reduced transcriptional activa- Nature Reviews | Neuroscience manipulation of CB1Rs. Conditional deletion204 of cannabinoid 1 receptors (CB1Rs) tion34, as well as misplacement of CB1R+ hippocampal from mouse cortical pyramidal cells results in errant corticofugal axon fasciculation interneurons in neonatal offspring92. The neuronal compared with wild-type controls (shown by arrows). Note that enlarged axon fascicles basis for THC-induced drug-seeking behaviours in (encircled) were also seen in extracortical areas, such as the striatum (cpu), where local the offspring might be due to the altered neuropep- CB1R expression was unaffected. L1NCAM, L1 neural cell adhesion molecule. Image is tide (dynorphin and enkephalin) expression in mes- reproduced, with permission, from Endocannabinoid signaling controls pyramidal cell 144 specification and long-range axon patterning. Proc. Natl. Acad. Sci. USA 105, 8760-8765 ocorticolimbic reward circuits and the erroneous 38 © (2008) Proc. Natl Acad. Sci. US A. synaptic wiring of glutamatergic cortical neurons . Modifications to glutamatergic axons were shown to originate during neuronal development and to be due (and perhaps CB2R) availability and signal compe- to the CB1R-dependent degradation of brain-specific tence68,136. Although in vivo confirmation is lacking, on stathmin 2 (also known as SCG10), which determines the basis of the dependence of neuronal differentiation microtubule elongation by sequestering tubulin dimers on precisely timed 2-AG signalling and of the axonal in a ternary T2S complex145. The loss of stathmin 2 thus mistargeting phenotype in DAGLA−/− brains in vivo63, we compromises cytoskeletal instability in a molecular predict that pharmacological inhibition of DAGLs will mechanism involving the phosphorylation and activa- be detrimental to nervous system development. tion of JNKs, particularly JNK1, which phosphorylate Endocannabinoid signalling is a wide-ranging neural stathmin 2 targeting it for proteasomal degradation146 cell communication system with many molecular nodes (FIG. 3a). As reduced cytoskeletal instability leads to that allow alternative metabolic checkpoints to tightly erroneous neuronal morphologies, particularly neurite control endocannabinoid concentrations (‘homeo- outgrowth, the Δ9-THC-induced loss of SCG10 com- static balance’) and to facilitate broad interactions with promised corticofugal development. Δ9-THC-induced coexistent signalling cassettes (see interactions with wiring deficits seemed to be permanent, as adult off- neurotrophins and netrins above). Thus, it can be pos- spring showed synaptic rearrangements, particularly tulated that, instead of provoking a gross neurodevel- CB1R localization, and reduced long-term plasticity in opmental phenotype (‘direct hit’), pharmacological or the hippocampus38. genetic manipulation of select molecular constituents in Pharmacological manipulation of the endocannabi- endocannabinoid signalling networks will induce sub- noid system is also detrimental in neonates and dur- tle changes in many neurons and synapses, which only ing adolescence. In a provocative series of experiments, manifest as a disease following exposure to a secondary Esther Fride and her colleagues suggested that a single stressor (‘double hit’). This concept is compatible with injection of SR141716 in newborn mice compromises recent studies showing that prenatal or perinatal admin- suckling behaviours so severely that it leads to the istration (from embryonic day 10 to postnatal day 7) of death of the offspring147. Similarly, prolonged Δ9-THC URB597, which is a FAAH inhibitor137, or following administration in neonates induced learning impair- GPR55 deletion138, does not markedly affect neurogen- ments that lasted into adulthood148. For the adolescent esis or axonal development (TABLE 1). However, both period (that is, postnatal days 28–45 in mice), Δ9-THC strategies induced long-lasting behavioural deficits, par- administration impairs social behaviour and cogni- ticularly depression-like symptoms and memory impair- tion, with concomitant alterations in neurochemical ment, which last into adulthood of the affected offspring. indices of both glutamatergic and GABAergic neuro- The lack of a clear anatomical phenotype in these models transmission149,150 (for example, vesicular transporters might be explained by moderate GPR55 mRNA expres- and postsynaptic and SNAP receptor (SNARE) com- sion relative to CNR1 mRNA levels in the brain138 (which plex components). Although most of these data only would reduce the effect of these molecular targets) and/ allow circumstantial conclusions to be made on the or the 1,000-fold lower levels of AEA than 2-AG in the basis of cross-correlations, it is tempting to speculate Psychoactive component developing brain48,101, (which would probably trigger that pharmacological manipulation of endocannabinoid The molecular component of a plant extract that can provoke functional substitutions by other cannabinoid-sensing signalling during crucial periods of synaptogenesis and/ acute changes in perception of receptors and 2-AG). Nevertheless, the behavioural defi- or postnatal pruning might precipitate or predispose an behaviour. cits that have been observed are in agreement with the individual to neuropsychiatric disease-like phenotypes.

794 | DECEMBER 2014 | VOLUME 15 www.nature.com/reviews/neuro

Our hypothesis is in accord with clinicopathological data after birth157. Nonetheless, proliferative neural stem cells showing: signalling-competent CB1Rs in major axonal can still be identified in the subventricular zone of aged trajectories in human foetal brains111; disrupted endo- patients who have suffered a stroke, with new neurons cannabinoid signalling (particularly CB1R levels) and appearing in the adjacent striatum throughout life158. molecular differentiation of CB1R+ GABA interneurons Interestingly, subventricular zone neuroblasts can be in patients with schizophrenia151,152; and deficient endocan- recruited to sites of brain injury where they might pro- nabinoid signalling in rodent models of autism (neuroligin vide trophic support to help preserve function159, and knockouts40); and deletion of the fragile X gene153. recent evidence suggests that neural stem cells and progenitor cells resident in the subventricular zone might Endocannabinoids in restorative neurobiology also provide ‘local’ trophic support to neurons residing We live in an exciting time for restorative neuroscience, in the proximal striatum160. in which key findings in developmental biology are Epidermal growth factor (EGF) and FGFR signal- being translated to combat devastating human neuro- ling have central roles in driving adult neurogenesis161, logical conditions. In this section, we focus on emerging with their respective ligands operating alongside inhibi- evidence that endocannabinoids regulate the prolif- tory factors such as growth and differentiation factor 11 eration of neural stem cells in the adult brain with the (GDF11)162,163 to maintain the balance between neural hope that persistent neurogenesis might be harnessed stem cell quiescence and proliferation164. This leaves for brain repair154. In the adult rodent brain, there are the cells poised to respond in a positive or a nega- neural stem cell niches in the hippocampus and in the tive manner to other cues, some of which seem to be subventricular zone of the lateral ventricle, and these derived from local microglia165,166. Genetic deletion of generate new neurons that can integrate into mature syn- cannabinoid receptors, or fairly short-term treatment aptic circuits to facilitate specialized forms of learning with CB1R- and CB2R-selective antagonists, suppresses and memory155. In the hippocampus, the generation and neural stem cell proliferation in the hippocampus and the differentiation of new neurons is a fairly local event subventricular zone12,103 (TABLE 2). By contrast, activa- within the dentate gyrus155. In the subventricular zone, tion of cannabinoid receptors with synthetic agonists neural stem cells generally differentiate into migratory and/or by increasing the level of endocannabinoids neuroblasts that travel a considerable distance along the by genetic deletion or pharmacological inhibition of rostral migratory stream (RMS) to populate the olfactory FAAH, stimulates neural stem cell proliferation in bulb with new neurons156. In humans, a robust RMS is a both niches11,12,20,103. Unexpectedly, stimulating endo- transient structure that is only obvious for a few months cannabinoid signalling has more marked effects in

Table 2 | Postnatal neurogenesis following manipulation of endocannabinoid signalling* Phenotype Treatment Response Refs NSC proliferation in dentate gyrus CB1R antagonists Decrease 207 CNR1−/− Decrease 207 DAGL−/− Decrease 15 CB1R agonists Increase 207 CB2R agonists Increase 103 FAAH inhibitors Increase 19 FAAH−/− Increase 19 NSC proliferation in SVZ CB2R antagonists Decrease 11 DAGL inhibitors Decrease 11 CB2R agonists Increase 11 FAAH inhibitors Increase 11 Neuroblast migration CB1R or CB2R antagonists Decrease 24 DAGL inhibitors Decrease 24 New neurons in adult olfactory bulb CB2R antagonists Decrease 11 CB2R agonists Increase 11 FAAH inhibitors Increase 11

CB1R, cannabinoid 1 receptor; CNR1, cannabinoid receptor 1 gene; DAGL, sn-1-diacylglycerol lipase; FAAH, fatty acid amide hydrolase; NSC, neural stem cell; SVZ, subventricular zone. *The impact of endocannabinoids on neurogenesis in the dentate gyrus or olfactory bulb was tested by selectively inhibiting CB1Rs and CB2Rs, by DAGL inhibitors, or by the deletion of genes. Conversely, endocannabinoid signalling can be mimicked or enhanced by CB1R or CB2R agonists, or by inhibition or knockout of FAAH (which results in multifold increases in brain AEA levels206). This wide range of tools11,15,19,20,103,207 has been used to uncover that DAGL-dependent endocannabinoid signalling regulates neural stem cell proliferation in both the dentate gyrus of the hippocampus and the subventricular zone or rostral migratory stream in rodents, with specific effects on neuroblast migration24, and that such signalling regulates the appearance of new neurons in the olfactory bulb.

NATURE REVIEWS | NEUROSCIENCE VOLUME 15 | DECEMBER 2014 | 795

older animals, which suggests that the natural age- Cannabinoid receptors in glioma therapy related decline in neurogenesis might partly reflect a It is widely thought that the optimal strategy for cancer ‘run-down’ in endocannabinoid tone. In this context, treatment would involve targeted therapies capable of whereas treatment of 6 week-old mice with CB2R ago- providing the most efficacious and selective treatment nists increases subventricular neural progenitor cell for each individual tumour patient (‘personalized ther- proliferation by 20–30%, the same treatments results in apy’). Glioblastoma multiforme (grade IV astrocytoma) a 300–400% increase in 6 month-old mice, with an asso- is one of the most malignant forms of human cancer ciated threefold increase in the number of new neurons with very short survival times following diagnosis. The appearing in the olfactory bulb 2 weeks later11. recent finding that a small molecule that selectively 2-AG seems to be the main endocannabinoid that affects membrane turnover in human glioma cells was drives adult neurogenesis as there is a 50% reduction in efficacious in a preclinical glioma model171 reinforces neural stem cell proliferation in the hippocampus and the concept that the maintenance of lipid mass in divid- subventricular zone of young adult mice when DAGLα ing tumour cells is crucial for their continued growth. and/or DAGLβ are knocked out15. Even greater effects Moreover, it is known that cannabinoids inhibit tumour (70–80% loss) are seen in the subventricular zone growth in animal models of glioma through a mecha- following acute pharmacological inhibition of these nism involving inhibition of PI3K–AKT–mTORC1 enzymes11 (TABLE 2). Neural stem cells express both signalling, which induces apoptosis of tumour cells DAGLs as well as CB1R and CB2R11, which suggests without affecting their non-transformed counterparts172 that there is an autocrine signalling pathway. However, (FIG. 5). Additional mechanisms, such as inhibition of it is also possible that 2-AG exerts paracrine effects on angiogenesis173 and invasion174,175, can contribute to neighbouring cells, with this type of ‘short-range’ signal- the CB1R- and CB2R-induced impairment of glioma ling recently shown to be capable of regulating axonal growth in mouse models. Moreover, in mice bearing guidance63. gliomas, cannabinoids potentiate the antitumour effi- In addition to promoting stem cell proliferation, cacy of temozolomide, which is the benchmark agent DAGL-dependent activation of both CB1R and CB2R for the clinical management of gliomas, in a synergistic promotes the migration of neuroblasts along the RMS manner and without overt toxicity176. In a pilot Phase I towards the olfactory bulb, again via an autocrine sig- clinical study, intracranial Δ9-THC administration to nalling mechanism24. As such, endocannabinoid tone nine patients with actively growing recurrent glioblas- stabilizes the leading process on migratory neuroblasts, toma multiforme was safe and could be achieved without perhaps allowing them to better read and/or respond substantial unwanted effects177. Importantly, radiologi- to spatial guidance cues within the RMS. The question cal monitoring and analyses of tumour specimens sup- remains as to how endocannabinoid tone arises and ported the antitumour action of cannabinoids in those how it is maintained in neural stem cells and migratory cancer patients172,177. Notably, endocannabinoid-sensing neuroblasts. There are many candidate mechanisms receptors (especially CB2R and TRPV1) are upregulated as a wide range of growth factors have been implicated in human glioma tissue compared with normal brain in neurogenesis, including FGF2, and have the potential tissue, and their expression positively correlates with to directly and focally stimulate DAGL-dependent endo- tumour grading75,178. The differential receptor expres- cannabinoid signalling95. Interestingly, recent progress in sion patterns thus allow potential endocannabinoid understanding the key downstream effectors that medi- drug targets to induce glioma cell death. These findings ate endocannabinoid signalling has implicated mTORC1 provide ‘proof-of-concept’ that endocannabinoid-based rather than PI3K or ERK102; biochemical and transcrip- medicines can improve the clinical efficacy of classical tional experiments suggest that PI3K activity is mainly cytotoxic drugs in patients with glioma. In line with regulated by the EGF receptor, whereas the EGF and FGF this, a Phase I/II trial for a combination therapy with receptors synergistically regulate the ERK pathway167. cannabinoids and temozolomide is currently ongoing In summary, adult neurogenesis can be considered (ClinicalTrials.gov identifier: NCT01812603). as a form of structural or cellular plasticity operating The mechanism of action seems to involve ER within the developed brain; it is highly dynamic with stress, which is a common theme in cannabinoid- and correlative changes seen following injury, in depression endocannabinoid-triggered glioma cell death (FIG. 5). and in various neurodegenerative and inflammatory ER stress is activated in response to ER-damaging disease states168. A considerable body of evidence now stimuli and aims to lessen the protein load on the ER by supports a key role for endocannabinoid signalling in coordinating a temporal shutdown of protein transla- the regulation of neural stem cell proliferation and the tion and a complex programme of gene transcription lineage commitment and migration of their progeny. to increase the protein-folding capacity of the ER. If Stimulation of neurogenesis in the adult brain is benefi- this transcriptional programme fails to re-establish ER cial, for example, in depression169, but substantial ben- homeostasis, persistent ER stress will induce cell death. efits in the aged, injured and/or diseased brain remain Cannabinoid-induced apoptotic death of glioma cells Ceramide to be shown. Nonetheless, FAAH inhibitors might be was shown to stimulate the CB1R and CB2R-dependent A molecule that consists of useful drugs for this purpose given the marked effects de novo synthesis of ceramide. This sphingolipid accumu- sphingosine and a fatty acyl 11,22 chain; it is therefore one of the they have on neurogenesis in older mice and the fact lates in the ER, thereby inducing ER stress through the structurally simplest forms of that they have been developed for use in humans, albeit phosphorylation and inhibition of eukaryotic initiation sphingolipids. for other conditions170. factor 2α (EIF2α) and the downstream activation of ER

796 | DECEMBER 2014 | VOLUME 15 www.nature.com/reviews/neuro

a Neural progenitor cell b Glioma cell TRKA eCB CB1 or CB2 receptor NGF eCB α γ β Ceramide Cytoplasm GTP Gi P P accumulation α γ PI3K RAS Non Gi? β P P eCB GTP Gi

AKT ER stress Ca2+ depletion? TRPV1 EIF2α P Ca2+ ER GCK-3β mTORC1 ATF4 TRIB3 ATF3

β-catenin p27 PAX6 AKT

mTORC1 Cell growth and/or survival Cell death

Figure 5 | Dysregulation of cannabinoid receptor signalling in glioma cells. a | In Natureneural progenitorReviews | Neuroscience cells, endocannabinoid (eCB) binding to cannabinoid receptors couples to the phosphatidylinositol 3-kinase (PI3K)–AKT–

mammalian target of rapamycin complex 1 (mTORC1) pathway via Gi proteins. Receptor tyrosine kinases that have been transactivated (by phosphorylation) might amplify cannabinoid receptor-mediated signalling by also using PI3K and RAS as molecular effectors. Activated AKT can elicit cell growth and survival effects (that is, it is mitogenic) either by inhibiting glycogen synthase kinase 3β (GSK3β) and activating β-catenin205 or by activating mTORC1, which leads to p27 inhibition102 as well as PAX6 phosphorylation86 and upregulation of PAX6 expression87. b | In glioma cells, eCBs trigger endoplasmic reticulum (ER) stress by engaging (at least) two mechanisms: binding of eCBs to cannabinoid receptors

stimulates de novo synthesis of ceramide in the ER via Gi-dependent and perhaps also via Gi-independent mechanisms179,180; and binding of eCBs to transient receptor potential cation channel subfamily V member 1 (TRPV1) receptors on the ER mediates Ca2+ release from this organelle to the cytoplasm and, conceivably, depletes ER Ca2+ stores75. Ceramide accumulation and Ca2+ depletion in the ER converge at the phosphorylation (that is, inhibition) of eukaryotic initiation factor 2α (EIF2α) and the induction of activating transcription factor 4 (ATF4), which, in turn, triggers cell death by two signalling cascades: upregulation of tribbles homologue 3 (TRIB3) expression, which leads to the inhibition of the AKT–mTORC1 axis180; and upregulation of ATF3 expression75.

stress-related transcription factors such as p8, activating The widely reported induction of glioma cell death transcription factor 4 (ATF4) and C/EBP-homologous by cannabinoid receptor stimulation is in contrast with protein (CHOP; also known as DDIT3)179. In parallel, the well-known proliferative and pro-survival activity of fatty acyl ethanolamides (such as AEA) activate TRPV1 cannabinoid receptors on neural progenitor cells, neu- receptors located on the ER of glioma cells, inducing Ca2+ rons and glial cells. In fact, cannabinoid receptors dis- release into the cytoplasm; this probably occurs together tinctly regulate signal transduction pathways in tumour with ceramide accumulation to deplete ER Ca2+ stores and non-tumour cells (FIG. 5). The molecular basis of and to induce the phosphorylation and inhibition of this ‘yin-yang’ behaviour is incompletely understood. EIF2α and the upregulation of transcription factors such Nevertheless, the possibility that it relies on different pat- as ATF3 and ATF4 (REF. 75) (FIG. 5). These two branches of terns of CB1R and CB2R expression and/or precoupling cannabinoid- and endocannabinoid-mediated ER stress to effectors seems unlikely for several reasons: glioma cell trigger glioma cell death. In the fatty acyl ethanolamide- lines that are resistant to cannabinoid-induced apoptosis induced activation of TRPV1 receptors, cannabinoid express similar amounts of CB1R and/or CB2R compared receptor-mediated ER stress upregulates the expression of with cannabinoid-sensitive lines; pharmacological studies the pseudokinase tribbles homologue 3 (TRIB3), which using cannabinoid receptor subtype-selective antagonists interacts with and inhibits the pro-survival kinase AKT, support that either CB1R or CB2R can induce apoptosis thereby leading to the inhibition of mTORC1 and the in cannabinoid-sensitive glioma cells182,183; and the short- subsequent stimulation of autophagy-mediated apoptotic term (minute-range) coupling of CB1R and CB2R to key cell death180. The molecular targets downstream of ATF3 cell signalling pathways, such as the ERK and AKT path- in the TRPV1-mediated ER stress response remain elu- ways, is similar in glioma cell lines that are sensitive or sive. Incidentally, cannabinoids and endocannabinoids resistant to cannabinoid-induced apoptosis173,184, as well can engage additional ER stress-related mechanisms as in primary astroglia125 or oligodendroglia120. Taken to trigger neuronal death. As such, AEA induces apo- together, this evidence suggests that mechanisms other ptosis of human neuroblastoma cells through a CB1R- than changes in the expression or intrinsic functionality dependent pathway via p38, ERK1, ERK and GRP78 of cannabinoid receptors might account for the differ- (also known as BiP), which is an ER stress sensor that ences in cannabinoid sensitivity of glioma cells. Indeed, triggers activation of p53 and p53 upregulated modulator a series of studies indicates that this ‘yin-yang’ behaviour of apoptosis (PUMA; also known as BBC3)181. could result from the differential capacity of tumour

NATURE REVIEWS | NEUROSCIENCE VOLUME 15 | DECEMBER 2014 | 797

or non-tumour glial cells to induce long-term de novo astrocytes187 and CB1Rs are assumed to be expressed ceramide synthesis in the ER and, in turn, to trigger an on mitochondria8. Although the expression of CB1Rs on ER stress response173,180,184, which determines whether mitochondria is controversial188, future studies aimed at the mitogenic PI3K–AKT–mTORC1 pathway becomes defining sequential endocannabinoid signal recruitment stimulated (in non-tumour cells; FIG. 5a) or inhibited (in along distinct timescales to foci of neuronal and glial tumour cells; FIG. 5b) following cannabinoid receptor differentiation will undoubtedly provide fundamental engagement173,181. insights into the contribution of signalling lipids to shaping the nervous system. These studies will facilitate the Conclusions molecular analysis of neuropsychiatric diseases that have In this Review, we discuss available experimental and presumed developmental origins and in which modifica- translational evidence185 implicating endocannabinoid tions to endocannabinoid signalling have been observed signalling in brain development, from neural stem cell (including autism40, schizophrenia151,189,190, bipolar dis- survival and proliferation, cell fate decisions and the order41,191 and depression151). Attempts to disentangle motility and differentiation of ensuing neuronal and glial complex genetic traits and epigenetic mechanisms must progenies. We highlight multiple niches at which devel- rely, at least partly, on modern molecular and cellular opmental endocannabinoid signalling, encompassing the tools, to reconcile existing controversies in cannabinoid foetal-to-young adult periods, is of importance, including pharmacology. Thus, advances in our understanding of recreational cannabis use during pregnancy and in ado- endocannabinoid actions in subpallial, midbrain and lescence affecting structural and functional brain plas- spinal networks is expected and will facilitate the exploi- ticity185, adult neurogenesis in which endocannabinoids tation of axonal responsiveness and glial scaffolding for might be exploited to promote the colonization of areas therapeutic purposes. Further analysis using combina- of neuronal loss and the molecular pathogenesis of glio- torial platforms is ultimately warranted to define the mas, in which ceramide-induced ER stress is identified importance of endocannabinoid signals among coex- as a promising therapeutic target. Despite great progress isting morphogenetic pathways and to define how they over the past decades, information is limited as to the are influenced by metabolic processes. Thus, we await involvement of endocannabinoids in the maintenance the emergence of consensus views on how targeting the of neuron–glia interplay, particularly the recruitment of cannabinoid system can be used to alleviate the adverse astroglia for metabolic control and oligodendroglia for effects of maternal drug abuse, obesity, diabetes and car- ensheathing of axons. These areas of research are war- diovascular diseases192, which are commonly associated ranted, as functional CB1Rs are probably expressed by with altered circulating endocannabinoid levels193 and (adult) astroglia186, neuron-derived endocannabinoids increased risk of neuropsychiatric and metabolic illnesses can induce Ca2+ signalling and glutamate release from in the offspring194, on future generations.

1. Sang, N., Zhang, J., Marcheselli, V., Bazan, N. G. & 12. Jiang, W. et al. Cannabinoids promote embryonic and mechanisms of cannabinoid receptors and the key Chen, C. Postsynaptically synthesized prostaglandin adult hippocampus neurogenesis and produce metabolic enzymes for endocannabinoids in E2 (PGE2) modulates hippocampal synaptic anxiolytic- and antidepressant-like effects. J. Clin. astroglia. Moreover, it establishes the role of transmission via a presynaptic PGE2 EP2 receptor. Invest. 115, 3104–3116 (2005). endocannabinoids in cell fate decisions and lineage J. Neurosci. 25, 9858–9870 (2005). 13. Pernia-Andrade, A. J. et al. Spinal endocannabinoids commitment. 2. Abad-Rodriguez, J. & Robotti, A. Regulation of axonal and CB1 receptors mediate C-fiber-induced 20. Aguado, T. et al. The endocannabinoid system drives development by plasma membrane gangliosides. heterosynaptic pain sensitization. Science 325, neural progenitor proliferation. FASEB J. 19, J. Neurochem. 103 (Suppl. 1), 47–55 (2007). 760–764 (2009). 1704–1706 (2005). 3. Fyrst, H. & Saba, J. D. An update on sphingosine- 14. Bradshaw, H. B. & Walker, J. M. The expanding field of 21. Berghuis, P. et al. Hardwiring the brain: 1phosphate and other sphingolipid mediators. Nature cannabimimetic and related lipid mediators. Br. endocannabinoids shape neuronal connectivity. Chem. Biol. 6, 489–497 (2010). J. Pharmacol. 144, 459–465 (2005). Science 316, 1212–1216 (2007). 4. Piomelli, D. The molecular logic of endocannabinoid 15. Gao, Y. et al. Loss of retrograde endocannabinoid In this paper, the authors show that axonal signalling. Nature Rev. Neurosci. 4, 873–884 (2003). signaling and reduced adult neurogenesis in CB1Rs can sense extracellular ligand gradients 5. Ahn, K., McKinney, M. K. & Cravatt, B. F. Enzymatic diacylglycerol lipase knock-out mice. J. Neurosci. 30, and can induce growth cone repulsion or pathways that regulate endocannabinoid signaling in the 2017–2024 (2010). collapse. This mechanism is then implicated in nervous system. Chem. Rev. 108, 1687–1707 (2008). This paper reports the generation of DAGL-null disrupted synaptogenesis in the cortex of mice 6. Paria, B. C. et al. Dysregulated cannabinoid signaling mice and provides genetic evidence on the lacking CB1Rs in GABAergic cortical disrupts uterine receptivity for embryo implantation. requirement of DAGL function for continued interneurons. J. Biol. Chem. 276, 20523–20528 (2001). neurogenesis in the mouse forebrain during 22. Vitalis, T. et al. The type 1 cannabinoid receptor is 7. Harkany, T., Mackie, K. & Doherty, P. Wiring and firing postnatal life. highly expressed in embryonic cortical projection neuronal networks: endocannabinoids take center 16. Tanimura, A. et al. The endocannabinoid neurons and negatively regulates neurite growth stage. Curr. Opin. Neurobiol. 18, 338–345 (2008). 2-arachidonoylglycerol produced by diacylglycerol in vitro. Eur. J. Neurosci. 28, 1705–1718 (2008). 8. Benard, G. et al. Mitochondrial CB1 receptors lipase α mediates retrograde suppression of synaptic 23. Leterrier, C. et al. Constitutive activation drives regulate neuronal energy metabolism. Nature transmission. Neuron 65, 320–327 (2010). compartment-selective endocytosis and axonal Neurosci. 15, 558–564 (2012). 17. Devane, W. A. et al. Isolation and structure of a brain targeting of type 1 cannabinoid receptors. J. Neurosci. 9. Kano, M., Ohno-Shosaku, T., Hashimotodani, Y., constituent that binds to the cannabinoid receptor. 26, 3141–3153 (2006). Uchigashima, M. & Watanabe, M. Endocannabinoid- Science 258, 1946–1949 (1992). 24. Oudin, M. J. et al. Endocannabinoids regulate the mediated control of synaptic transmission. Physiol. In this paper, the authors identify anandamide as migration of subventricular zone-derived neuroblasts Rev. 89, 309–380 (2009). the first endogenous ligand of CB1Rs in the brain. in the post-natal brain. J. Neurosci. 31, 4000–4011 10. Regehr, W. G., Carey, M. R. & Best, A. R. Activity- This landmark paper marks the advent of (2011). dependent regulation of synapses by retrograde contemporary endocannabinoid research. 25. Elphick, M. R. & Egertova, M. The neurobiology and messengers. Neuron 63, 154–170 (2009). 18. Pertwee, R. G. et al. International Union of Basic and evolution of cannabinoid signalling. Phil. Trans. R. Soc. 11. Goncalves, M. B. et al. A diacylglycerol lipase-CB2 Clinical Pharmacology. LXXIX. Cannabinoid receptors Lond. B 356, 381–408 (2001). cannabinoid pathway regulates adult subventricular and their ligands: beyond CB1 and CB2. Pharmacol. 26. McPartland, J. M., Norris, R. W. & Kilpatrick, C. W. zone neurogenesis in an age-dependent manner. Mol. Rev. 62, 588–631 (2010). Coevolution between cannabinoid receptors and Cell Neurosci. 38, 526–536 (2008). 19. Aguado, T. et al. The endocannabinoid system endocannabinoid ligands. Gene 397, 126–135 This paper shows that neural progenitor cells in the promotes astroglial differentiation by acting on neural (2007). subventricular zone of the cerebral cortex express progenitor cells. J. Neurosci. 26, 1551–1561 (2006). 27. Leung, H. T. et al. DAG lipase activity is necessary for CB2Rs and use an autocrine DAGL–CB2R signalling This paper provides the molecular identity and TRP channel regulation in Drosophila photoreceptors. arrangement to promote neurogenesis. describes the expression sites and signalling Neuron 58, 884–896 (2008).

798 | DECEMBER 2014 | VOLUME 15 www.nature.com/reviews/neuro

28. Begbie, J., Doherty, P. & Graham, A. Cannabinoid 48. Keimpema, E. et al. Differential subcellular 69. Harkany, T. et al. The emerging functions of receptor, CB1, expression follows neuronal recruitment of monoacylglycerol lipase generates endocannabinoid signaling during CNS development. differentiation in the early chick embryo. J. Anat. 205, spatial specificity of 2-arachidonoyl glycerol signaling Trends Pharmacol. Sci. 28, 83–92 (2007). 213–218 (2004). during axonal pathfinding. J. Neurosci. 30, 70. Morozov, Y. M., Ben Ari, Y. & Freund, T. F. The spatial This paper reports that neuronal commitment 13992–14007 (2010). and temporal pattern of fatty acid amide hydrolase coincides with the upregulation of CB1R in the 49. Yoshida, T. et al. Localization of diacylglycerol lipase-α expression in rat hippocampus during postnatal earliest-born neurons of the chick embryo. This around postsynaptic spine suggests close proximity development. Eur. J. Neurosci. 20, 459–466 (2004). proposal led to subsequent mechanistic studies between production site of an endocannabinoid, 71. Johansson, C. B. et al. Identification of a neural stem implicating CB1R functions in a broad range of 2-arachidonoyl-glycerol, and presynaptic cannabinoid cell in the adult mammalian central nervous system. developmental processes. CB1 receptor. J. Neurosci. 26, 4740–4751 (2006). Cell 96, 25–34 (1999). 29. Watson, S., Chambers, D., Hobbs, C., Doherty, P. & 50. Katona, I. & Freund, T. F. Endocannabinoid signaling 72. Rakic, P. A century of progress in corticoneurogenesis: Graham, A. The endocannabinoid receptor, CB1, is as a synaptic circuit breaker in neurological disease. from silver impregnation to genetic engineering. required for normal axonal growth and fasciculation. Nature Med. 14, 923–930 (2008). Cereb. Cortex 16, i3–i17 (2006). Mol. Cell Neurosci. 38, 89–97 (2008). 51. Bisogno, T. et al. Cloning of the first sn-1-DAG lipases 73. Metin, C., Vallee, R. B., Rakic, P. & Bhide, P. G. Modes 30. Li, L. et al. Endocannabinoid signaling is required for points to the spatial and temporal regulation of and mishaps of neuronal migration in the mammalian development and critical period plasticity of the endocannabinoid signaling in the brain. J. Cell Biol. brain. J. Neurosci. 28, 11746–11752 (2008). whisker map in somatosensory cortex. Neuron 64, 163, 463–468 (2003). 74. Morozov, Y. M. & Freund, T. F. Post-natal development 537–549 (2009). This paper identifies the DAGLs, shows their of type 1 cannabinoid receptor immunoreactivity in 31. Argaw, A. et al. Concerted action of CB1 cannabinoid distribution in both the developing and adult nervous the rat hippocampus. Eur. J. Neurosci. 18, receptor and deleted in colorectal cancer in axon system and suggests a striking switch in DAGL 1213–1222 (2003). guidance. J. Neurosci. 31, 1489–1499 (2011). expression sites during synaptogenesis. The substrate 75. Stock, K. et al. Neural precursor cells induce cell death 32. Diaz-Alonso, J. et al. The CB1 cannabinoid receptor and product specificity of DAGL is also defined. of high-grade astrocytomas through stimulation of drives corticospinal motor neuron differentiation 52. Dinh, T. P. et al. Brain monoglyceride lipase TRPV1. Nature Med. 18, 1232–1238 (2012). through the Ctip2/Satb2 transcriptional regulation participating in endocannabinoid inactivation. Proc. 76. Pineiro, R., Maffucci, T. & Falasca, M. The putative axis. J. Neurosci. 32, 16651–16665 (2012). Natl Acad. Sci. USA 99, 10819–10824 (2002). cannabinoid receptor GPR55 defines a novel autocrine 33. Wu, C. S. et al. Requirement of cannabinoid CB1 53. Marrs, W. R. et al. The serine hydrolase ABHD6 loop in cancer cell proliferation. Oncogene 30, receptors in cortical pyramidal neurons for controls the accumulation and efficacy of 2-AG at 142–152 (2011). appropriate development of corticothalamic and cannabinoid receptors. Nature Neurosci. 13, 77. Andradas, C. et al. The orphan G protein-coupled thalamocortical projections. Eur. J. Neurosci. 32, 951–957 (2010). receptor GPR55 promotes cancer cell proliferation via 693–706 (2010). 54. Huang, G. Z. & Woolley, C. S. Estradiol acutely ERK. Oncogene 30, 245–252 (2011). 34. Dinieri, J. A. et al. Maternal cannabis use alters suppresses inhibition in the hippocampus through a 78. Brown, I. et al. Cannabinoid receptor-dependent and ventral striatal dopamine D2 gene regulation in the sex-specific endocannabinoid and mGluR-dependent -independent anti-proliferative effects of omega-3 offspring. Biol. Psychiatry 70, 763–769 (2011). mechanism. Neuron 74, 801–808 (2012). ethanolamides in androgen receptor-positive and 35. Hurd, Y. L. et al. Marijuana impairs growth in mid- 55. Chavez, A. E., Chiu, C. Q. & Castillo, P. E. TRPV1 -negative prostate cancer cell lines. Carcinogenesis gestation fetuses. Neurotoxicol. Teratol. 27, 221–229 activation by endogenous anandamide triggers 31, 1584–1591 (2010). (2005). postsynaptic long-term depression in dentate gyrus. 79. De Petrocellis, L. et al. The endogenous cannabinoid In this paper, the authors report on foetal growth Nature Neurosci. 13, 1511–1518 (2010). anandamide inhibits human breast cancer cell retardation as a consequence of maternal cannabis 56. Grueter, B. A., Brasnjo, G. & Malenka, R. C. proliferation. Proc. Natl Acad. Sci. USA 95, smoking. The study is particularly important to Postsynaptic TRPV1 triggers cell type-specific long- 8375–8380 (1998). show that many organ systems are sensitive to term depression in the nucleus accumbens. Nature 80. Goswami, C., Schmidt, H. & Hucho, F. TRPV1 at nerve phytocannabinoid disruptors. Neurosci. 13, 1519–1525 (2010). endings regulates growth cone morphology and 36. Wang, X., Dow-Edwards, D., Keller, E. & Hurd, Y. L. 57. Gulyas, A. I. et al. Segregation of two movement through cytoskeleton reorganization. FEBS Preferential limbic expression of the cannabinoid endocannabinoid-hydrolyzing enzymes into pre- and J. 274, 760–772 (2007). receptor mRNA in the human fetal brain. postsynaptic compartments in the rat hippocampus, 81. Goswami, C. & Hucho, T. TRPV1 expression-dependent Neuroscience 118, 681–694 (2003). cerebellum and amygdala. Eur. J. Neurosci. 20, initiation and regulation of filopodia. J. Neurochem. 37. Wang, X., Dow-Edwards, D., Anderson, V., Minkoff, H. 441–458 (2004). 103, 1319–1333 (2007). & Hurd, Y. L. In utero marijuana exposure associated 58. Wilson, R. I. & Nicoll, R. A. Endogenous cannabinoids 82. Antypa, M., Faux, C., Eichele, G., Parnavelas, J. G., & with abnormal amygdala dopamine D2 gene mediate retrograde signalling at hippocampal Andrews, W. D. Differential gene expression in expression in the human fetus. Biol. Psychiatry 56, synapses. Nature 410, 588–592 (2001). migratory streams of cortical interneurons. Eur. J. 909–915 (2004). 59. Kreitzer, A. C., Carter, A. G. & Regehr, W. G. Inhibition Neurosci. 34, 1584–1594 (2011). 38. Tortoriello, G. et al. Miswiring the brain: of interneuron firing extends the spread of 83. Herkenham, M. et al. Cannabinoid receptor Δ9-tetrahydrocannabinol disrupts cortical endocannabinoid signaling in the cerebellum. Neuron localization in brain. Proc. Natl Acad. Sci. USA 87, development by inducing an SCG10/stathmin2 34, 787–796 (2002). 1932–1936 (1990). degradation pathway. EMBO J. 33, 668–685 (2014). 60. Walker, D. J., Suetterlin, P., Reisenberg, M., 84. Howlett, A. C. et al. International Union of This paper is the first molecular analysis of THC Williams, G. & Doherty, P. Down-regulation of Pharmacology. XXVII. Classification of cannabinoid effects on neuronal differentiation, particularly diacylglycerol lipase-α during neural stem cell receptors. Pharmacol. Rev. 54, 161–202 (2002). axon guidance. By unbiased proteomics, SCG10 is differentiation: Identification of elements that 85. Galve-Roperh, I. et al. Cannabinoid receptor signaling identified as the first molecular effector of regulate transcription. J. Neurosci. Res. 88, in progenitor/stem cell proliferation and unwanted THC effects on neuronal morphology. 735–745 (2010). differentiation. Prog. Lipid Res. 52, 633–650 (2013). 39. Marsicano, G. et al. CB1 cannabinoid receptors and 61. Rimmerman, N. et al. Compartmentalization of 86. Bromberg, K. D., Ma’ayan, A., Neves, S. R. & on-demand defense against excitotoxicity. Science endocannabinoids into lipid rafts in a dorsal root Iyengar, R. Design logic of a cannabinoid receptor 302, 84–88 (2003). ganglion cell line. Br. J. Pharmacol. 153, 380–389 signaling network that triggers neurite outgrowth. 40. Foldy, C., Malenka, R. C. & Sudhof, T. C. Autism- (2008). Science 320, 903–909 (2008). associated neuroligin-3 mutations commonly disrupt 62. Bari, M., Battista, N., Fezza, F., Finazzi-Agro, A. & In this paper, the authors define a minimal tonic endocannabinoid signaling. Neuron 78, Maccarrone, M. Lipid rafts control signaling of type-1 transcription factor network downstream from 498–509 (2013). cannabinoid receptors in neuronal cells. Implications CB1Rs that is required for neurite outgrowth. They 41. Minocci, D. et al. Genetic association between bipolar for anandamide-induced apoptosis. J. Biol. Chem. also implicate BRCA1 in cannabinoid signalling, disorder and 524A>C (Leu133Ile) polymorphism of 280, 12212–12220 (2005). which was shown as a candidate to control MAGL CNR2 gene, encoding for CB2 cannabinoid receptor. 63. Keimpema, E. et al. Diacylglycerol lipase α stability by Keimpema in 2013. J. Affect. Disord. 134, 427–430 (2011). manipulation reveals developmental roles for 87. Diaz-Alonso, J. et al. CB1 cannabinoid receptor- 42. Ishiguro, H. et al. Brain cannabinoid CB2 receptor in intercellular endocannabinoid signaling. Sci. Rep. 3, dependent activation of mTORC1/PAX6 signaling schizophrenia. Biol. Psychiatry 67, 974–982 (2010). 2093 (2013). drives TBR2 expression and basal progenitor 43. Ujike, H. et al. CNR1, central cannabinoid receptor 64. Duff, G. et al. Cannabinoid receptor CB2 modulates expansion in the developing mouse cortex. Cereb. gene, associated with susceptibility to hebephrenic axon guidance. PLoS ONE. 8, e70849 (2013). Cortex 7 Mar 2014 [Epub ahead of print]. schizophrenia. Mol. Psychiatry 7, 515–518 (2002). 65. Morozov, Y. M., Torii, M. & Rakic, P. Origin, early 88. Vasquez, C. & Lewis, D. L. The CB1 cannabinoid 44. Sipe, J. C., Chiang, K., Gerber, A. L., Beutler, E. & commitment, migratory routes, and destination of receptor can sequester G-proteins, making them Cravatt, B. F. A missense mutation in human fatty cannabinoid type 1 receptor-containing interneurons. unavailable to couple to other receptors. J. Neurosci. acid amide hydrolase associated with problem drug Cereb. Cortex 19, i78–i89 (2009). 19, 9271–9280 (1999). use. Proc. Natl Acad. Sci. USA 99, 8394–8399 66. Soderstrom, K. & Tian, Q. Developmental pattern of 89. Kearn, C. S., Blake-Palmer, K., Daniel, E., Mackie, K. & (2002). CB1 cannabinoid receptor immunoreactivity in brain Glass, M. Concurrent stimulation of cannabinoid CB1 45. Fiskerstrand, T. et al. Mutations in ABHD12 cause the regions important to zebra finch (Taeniopygia guttata) and dopamine D2 receptors enhances heterodimer neurodegenerative disease PHARC: an inborn error of song learning and control. J. Comp. Neurol. 496, formation: A mechanism for receptor cross-talk? Mol. endocannabinoid metabolism. Am. J. Hum. Genet. 87, 739–758 (2006). Pharmacol. 67, 1697–1704 (2005). 410–417 (2010). 67. Gotz, M. & Huttner, W. B. The cell biology of 90. Hudson, B. D., Hebert, T. E. & Kelly, M. E. Ligand- and 46. Mulder, J. et al. Endocannabinoid signaling controls neurogenesis. Nature Rev. Mol. Cell. Biol. 6, 777–788 heterodimer-directed signaling of the CB1 cannabinoid pyramidal cell specification and long-range axon (2005). receptor. Mol. Pharmacol. 77, 1–9 (2010). patterning. Proc. Natl Acad. Sci. USA 105, 68. Keimpema, E. et al. Nerve growth factor scales 91. Dalton, G. D. & Howlett, A. C. Cannabinoid CB1 8760–8765 (2008). endocannabinoid signaling by regulating receptors transactivate multiple receptor tyrosine 47. Pertwee, R. G. Ligands that target cannabinoid monoacylglycerol lipase turnover in developing kinases and regulate serine/threonine kinases to receptors in the brain: from THC to anandamide and cholinergic neurons. Proc. Natl Acad. Sci. USA 110, activate ERK in neuronal cells. Br. J. Pharmacol. 165, beyond. Addict. Biol. 13, 147–159 (2008). 1935–1940 (2013). 2497–2511 (2012).

NATURE REVIEWS | NEUROSCIENCE VOLUME 15 | DECEMBER 2014 | 799

92. Berghuis, P. et al. Endocannabinoids regulate signal transduction functionality in the human brain. 132. Saez, T. M., Aronne, M. P., Caltana, L. & Brusco, A. H. interneuron migration and morphogenesis by Eur. J. Neurosci. 17, 1747–1754 (2003). Prenatal exposure to the CB and CB cannabinoid transactivating the TRKB receptor. Proc. Natl Acad. 112. Coutts, A. A. et al. Agonist-induced internalization and receptor agonist WIN 55, 212–212 alters migration Sci. USA 102, 19115–19120 (2005). trafficking of cannabinoid CB1 receptors in of early-born glutamatergic neurons and GABAergic 93. Ahn, K. H., Mahmoud, M. M., Shim, J. Y. & hippocampal neurons. J. Neurosci. 21, 2425–2433 interneurons in the rat cerebral cortex. Kendall, D. A. Distinct roles of β-arrestin 1 and (2001). J. Neurochem.129, 637–648 (2013).

β-arrestin 2 in ORG27569-induced biased signaling 113. He, J. C. et al. The G αo/i-coupled cannabinoid receptor- 133. Antonelli, T. et al. Long-term effects on cortical and internalization of the cannabinoid receptor 1 mediated neurite outgrowth involves RAP regulation glutamate release induced by prenatal exposure to the (CB1). J. Biol. Chem. 288, 9790–9800 (2013). of SRC and STAT3. J. Biol. Chem. 280, 33426–33434 cannabinoid receptor agonist (R)-(+)-[2,3dihydro- 94. Maison, P., Walker, D. J., Walsh, F. S., Williams, G. & (2005). 5methyl3-(4morpholinyl-methyl)pyrrolo[1,2,3de]-1, Doherty, P. BDNF regulates neuronal sensitivity to 114. Ade, K. K. & Lovinger, D. M. Anandamide regulates 4benzoxazin-6yl]-1naphthalenylmethanone: an in vivo endocannabinoids. Neurosci. Lett. 467, 90–94 postnatal development of long-term synaptic plasticity microdialysis study in the awake rat. Neuroscience (2009). in the rat dorsolateral striatum. J. Neurosci. 27, 124, 367–375 (2004). 95. Williams, E. J., Walsh, F. S. & Doherty, P. The FGF 2403–2409 (2007). 134. Mereu, G. et al. Prenatal exposure to a cannabinoid receptor uses the endocannabinoid signaling system 115. Zhu, P. J. & Lovinger, D. M. Developmental alteration of agonist produces memory deficits linked to to couple to an axonal growth response. J. Cell Biol. endocannabinoid retrograde signaling in the dysfunction in hippocampal long-term potentiation 160, 481–486 (2003). hippocampus. J. Neurophysiol. 103, 1123–1129 (2010). and glutamate release. Proc. Natl Acad. Sci. USA 100, In this paper, the authors suggest that 116. Yasuda, H., Huang, Y. & Tsumoto, T. Regulation of 4915–4920 (2003). endocannabinoid signalling is regulated by FGFRs excitability and plasticity by endocannabinoids and 135. Antonelli, T. et al. Prenatal exposure to the CB1 and that this coupling underpins FGF-induced PKA in developing hippocampus. Proc. Natl Acad. Sci. receptor agonist WIN 55, 212-2 causes learning neurite outgrowth. USA 105, 3106–3111 (2008). disruption associated with impaired cortical NMDA 96. Brittis, P. A., Silver, J., Walsh, F. S. & Doherty, P. 117. Arevalo-Martin, A. et al. Cannabinoids modulate receptor function and emotional reactivity changes in Fibroblast growth factor receptor function is required OLIG2 and polysialylated neural cell adhesion rat offspring. Cereb. Cortex 15, 2013–2020 (2005). for the orderly projection of ganglion cell axons in the molecule expression in the subventricular zone of 136. Schlosburg, J. E. et al. Chronic monoacylglycerol developing mammalian retina. Mol. Cell Neurosci. 8, post-natal rats through cannabinoid receptor 1 and lipase blockade causes functional antagonism of the 120–128 (1996). cannabinoid receptor 2. Eur. J. Neurosci. 26, endocannabinoid system. Nature Neurosci. 13, 97. Ross, R. A. Anandamide and vanilloid TRPV1 1548–1559 (2007). 1113–1119 (2010). receptors. Br. J. Pharmacol. 140, 790–801 (2003). 118. Gomez, O. et al. The constitutive production of the 137. Wu, C. S. et al. Long-term consequences of perinatal 98. Puente, N. et al. The transient receptor potential endocannabinoid 2-arachidonoylglycerol participates fatty acid amino hydrolase inhibition. Br. vanilloid-1 is localized at excitatory synapses in the in oligodendrocyte differentiation. Glia 58, J. Pharmacol. 171, 1420–1434 (2014). mouse dentate gyrus. Brain Struct. Funct. http:dx.doi. 1913–1927 (2010). 138. Wu, C. S. et al. GPR55, a G-protein coupled receptor org/10.1007/s00429-014-0711-2 (2014). 119. Molina-Holgado, E. et al. Cannabinoids promote for lysophosphatidylinositol, plays a role in motor 99. Maccarrone, M. et al. Anandamide inhibits oligodendrocyte progenitor survival: involvement of coordination. PLoS ONE. 8, e60314 (2013). metabolism and physiological actions of cannabinoid receptors and phosphatidylinositol-3 139. Huizink, A. C. & Mulder, E. J. Maternal smoking, 2-arachidonoylglycerol in the striatum. Nature kinase/AKT signaling. J. Neurosci. 22, 9742–9753 drinking or cannabis use during pregnancy and Neurosci. 11, 152–159 (2008). (2002). neurobehavioral and cognitive functioning in human This paper describes the interplay between AEA 120. Molina-Holgado, F. et al. CB2 cannabinoid receptors offspring. Neurosci. Biobehav. Rev. 30, 24–41 (2006). action at TRPV1 and the metabolic control and promote mouse neural stem cell proliferation. Eur. 140. Goldschmidt, L., Richardson, G. A., Cornelius, M. D. & CB1R engagement of 2-AG, which allow AEA to J. Neurosci. 25, 629–634 (2007). Day, N. L. Prenatal marijuana and alcohol exposure control the ‘endogenous tone’ and physiological 121. Sun, J. et al. WIN55, 212–212 promotes and academic achievement at age 10. Neurotoxicol. action of 2-AG in the striatum. differentiation of oligodendrocyte precursor cells and Teratol. 26, 521–532 (2004). 100. Berrendero, F. et al. Localization of mRNA expression improve remyelination through regulation of the 141. Castaldo, P. et al. Altered regulation of glutamate and activation of signal transduction mechanisms for phosphorylation level of the ERK 1/2 via cannabinoid release and decreased functional activity and cannabinoid receptor in rat brain during fetal receptor 1 after stroke-induced demyelination. Brain expression of GLT1 and GLAST glutamate transporters development. Development 125, 3179–3188 Res. 1491, 225–235 (2013). in the hippocampus of adolescent rats perinatally (1998). 122. Soltys, J., Yushak, M. & Mao-Draayer, Y. Regulation of exposed to Δ9-THC. Pharmacol. Res. 61, 334–341 101. Berrendero, F., Sepe, N., Ramos, J. A., Di Marzo, V. & neural progenitor cell fate by anandamide. Biochem. (2010). Fernandez-Ruiz, J. J. Analysis of cannabinoid receptor Biophys. Res. Commun. 400, 21–26 (2010). 142. Spano, M. S., Ellgren, M., Wang, X. & Hurd, Y. L. binding and mRNA expression and endogenous 123. Shinjyo, N., Piscitelli, F., Verde, R. & Di, M. V. Impact Prenatal cannabis exposure increases heroin seeking cannabinoid contents in the developing rat brain of ω6 polyunsaturated fatty acid supplementation and with allostatic changes in limbic enkephalin systems in during late gestation and early postnatal period. γ-aminobutyric acid on astrogliogenesis through the adulthood. Biol. Psychiatry 61, 554–563 (2007). Synapse 33, 181–191 (1999). endocannabinoid system. J. Neurosci. Res. 91, 143. Szutorisz, H. et al. Parental THC exposure leads to This paper describes the distribution of CB1Rs, 943–953 (2013). compulsive heroin-seeking and altered striatal 2-AG and AEA at successive developmental periods 124. Gomez Del Pulgar, T., de Ceballos, M. L., Guzman, M. synaptic plasticity in the subsequent generation. in foetal rodent brains. The authors suggest a & Velasco, G. Cannabinoids protect astrocytes from Neuropsychopharmacology 39, 1315–1323 (2014). temporal association between receptor expression ceramide-induced apoptosis through the 144. Wang, X., Dow-Edwards, D., Anderson, V., Minkoff, H. and ligand bioavailability. phosphatidylinositol-3 kinase/protein kinase B & Hurd, Y. L. Discrete opioid gene expression 102. Palazuelos, J., Ortega, Z., Diaz-Alonso, J., Guzman, M. pathway. J. Biol. Chem. 277, 36527–36533 (2002). impairment in the human fetal brain associated with & Galve-Roperh, I. CB2 cannabinoid receptors 125. Gomez, O. et al. Cannabinoid receptor agonists maternal marijuana use. Pharmacogenomics J. 6, promote neural progenitor cell proliferation via modulate oligodendrocyte differentiation by activating 255–264 (2006). mTORC1 signaling. J. Biol. Chem. 287, 1198–1209 PI3K/AKT and the mammalian target of rapamycin 145. Jourdain, L., Curmi, P., Sobel, A., Pantaloni, D. & (2012). (mTOR) pathways. Br. J. Pharmacol. 163, Carlier, M. F. Stathmin: a tubulin-sequestering 103. Palazuelos, J. et al. Non-psychoactive CB2 1520–1532 (2011). protein which forms a ternary T2S complex with two cannabinoid agonists stimulate neural progenitor 126. Fernandez-Lopez, D. et al. The cannabinoid tubulin molecules. Biochemistry 36, 10817–10821 proliferation. FASEB J. 20, 2405–2407 (2006). WIN55,212-2 promotes neural repair after neonatal (1997). 104. Fishell, G. & Hanashima, C. Pyramidal neurons grow hypoxia-ischemia. Stroke 41, 2956–2964 (2010). 146. Shin, J. E. et al. SCG10 is a JNK target in the axonal up and change their mind. Neuron 57, 333–338 127. Solbrig, M. V., Fan, Y., Hermanowicz, N., degeneration pathway. Proc. Natl Acad. Sci. USA 109, (2008). Morgese, M. G. & Giuffrida, A. A synthetic E3696–E3705 (2012). 105. Jin, M. et al. Ca2+-dependent regulation of rho cannabinoid agonist promotes oligodendrogliogenesis 147. Fride, E. et al. Inhibition of milk ingestion and growth GTPases triggers turning of nerve growth cones. during viral encephalitis in rats. Exp. Neurol. 226, after administration of a neutral cannabinoid CB1 J. Neurosci. 25, 2338–2347 (2005). 231–241 (2010). receptor antagonist on the first postnatal day in the 106. Ishii, I. & Chun, J. Anandamide-induced 128. Compagnucci, C. et al. Type1 (CB1) cannabinoid mouse. Pediatr. Res. 62, 533–536 (2007). neuroblastoma cell rounding via the CB1 cannabinoid receptor promotes neuronal differentiation and 148. O’Shea, M. & Mallet, P. E. Impaired learning in receptors. Neuroreport 13, 593–596 (2002). maturation of neural stem cells. PLoS ONE. 8, e54271 adulthood following neonatal Δ9-THC exposure. 107. Ford, L. A. et al. A role for Lα-lysophosphatidylinositol (2013). Behav. Pharmacol. 16, 455–461 (2005). and GPR55 in the modulation of migration, 129. Xapelli, S. et al. Activation of type 1 cannabinoid 149. Rubino, T. et al. Changes in hippocampal orientation and polarization of human breast cancer receptor (CB1R) promotes neurogenesis in murine morphology and neuroplasticity induced by cells. Br. J. Pharmacol. 160, 762–771 (2010). subventricular zone cell cultures. PLoS ONE. 8, adolescent THC treatment are associated with 108. Lauckner, J. E. et al. GPR55 is a cannabinoid receptor e63529 (2013). cognitive impairment in adulthood. Hippocampus that increases intracellular calcium and inhibits M 130. Xapelli, S. et al. Modulation of subventricular zone 19, 763–772 (2009). current. Proc. Natl Acad. Sci. USA 105, 2699–2704 oligodendrogenesis: a role for hemopressin? Front. 150. Zamberletti, E. et al. Alterations of prefrontal cortex (2008). Cell Neurosci. 8, 59 (2014). GABAergic transmission in the complex psychotic-like 109. Zhou, D. & Song, Z. H. CB1 cannabinoid receptor- 131. Zimmer, A., Zimmer, A. M., Hohmann, A. G., phenotype induced by adolescent mediated tyrosine phosphorylation of focal adhesion Herkenham, M. & Bonner, T. I. Increased mortality, Δ9-tetrahydrocannabinol exposure in rats. Neurobiol. kinase-related non-kinase. FEBS Lett. 525, 164–168 hypoactivity, and hypoalgesia in cannabinoid CB1 Dis. 63, 35–47 (2014). (2002). receptor knockout mice. Proc. Natl Acad. Sci. USA 96, 151. Eggan, S. M., Stoyak, S. R., Verrico, C. D. & 110. Derkinderen, P. et al. Regulation of a neuronal form of 5780–5785 (1999). Lewis, D. A. Cannabinoid CB1 receptor focal adhesion kinase by anandamide. Science 273, This paper describes the initial characterization of immunoreactivity in the prefrontal cortex: comparison 1719–1722 (1996). CB1R-null mice, showing their premature death of schizophrenia and major depressive disorder. 111. Mato, S., Del Olmo, E. & Pazos, A. Ontogenetic during aging. However, this study failed to identify Neuropsychopharmacology 35, development of cannabinoid receptor expression and developmental abnormalities. 2060–2071 (2010).

800 | DECEMBER 2014 | VOLUME 15 www.nature.com/reviews/neuro

152. Lewis, D. A., Cruz, D., Eggan, S. & Erickson, S. 174. Ramer, R. & Hinz, B. Inhibition of cancer cell invasion 194. Cristino, L. et al. Obesity-driven synaptic remodeling Postnatal development of prefrontal inhibitory circuits by cannabinoids via increased expression of tissue affects endocannabinoid control of orexinergic neurons. and the pathophysiology of cognitive dysfunction in inhibitor of matrix metalloproteinases-1. J. Natl Proc. Natl Acad. Sci. USA 110, E2229–E2238 (2013). schizophrenia. Ann. NY Acad. Sci. 1021, 64–76 Cancer Inst. 100, 59–69 (2008). 195. Reisenberg, M., Singh, P. K., Williams, G. & Doherty, P. (2004). 175. Blazquez, C. et al. Cannabinoids inhibit glioma cell The diacylglycerol lipases: structure, regulation and 153. Jung, K. M. et al. Uncoupling of the endocannabinoid invasion by down-regulating matrix roles in and beyond endocannabinoid signalling. Phil. signalling complex in a mouse model of fragile X metalloproteinase-2 expression. Cancer Res. 68, Trans. R. Soc. B 367, 3264–3275 (2012). syndrome. Nature Commun. 3, 1080 (2012). 1945–1952 (2008). 196. Kaczocha, M., Glaser, S. T. & Deutsch, D. G. 154. Lie, D. C., Song, H., Colamarino, S. A., Ming, G. L. & 176. Torres, S. et al. A combined preclinical therapy of Identification of intracellular carriers for the Gage, F. H. Neurogenesis in the adult brain: new cannabinoids and temozolomide against glioma. Mol. endocannabinoid anandamide. Proc. Natl Acad. Sci. strategies for central nervous system diseases. Annu. Cancer Ther. 10, 90–103 (2011). USA 106, 6375–6380 (2009). Rev. Pharmacol. Toxicol. 44, 399–421 (2004). 177. Guzman, M. et al. A pilot clinical study of 197. Oddi, S. et al. Molecular identification of albumin and 155. Deng, W., Aimone, J. B. & Gage, F. H. New neurons Δ9-tetrahydrocannabinol in patients with recurrent HSP70 as cytosolic anandamide-binding proteins. and new memories: how does adult hippocampal glioblastoma multiforme. Br. J. Cancer 95, 197–203 Chem. Biol. 16, 624–632 (2009). neurogenesis affect learning and memory? Nature (2006). 198. Fu, J. et al. A catalytically silent FAAH1 variant drives Rev. Neurosci. 11, 339–350 (2010). 178. Sanchez, C. et al. Inhibition of glioma growth in vivo by anandamide transport in neurons. Nature Neurosci. 156. Lois, C. & Alvarez-Buylla, A. Long-distance neuronal selective activation of the CB2 cannabinoid receptor. 15, 64–69 (2012). migration in the adult mammalian brain. Science 264, Cancer Res. 61, 5784–5789 (2001). 199. Oddi, S. et al. Evidence for the intracellular 1145–1148 (1994). 179. Carracedo, A. et al. The stress-regulated protein p8 accumulation of anandamide in adiposomes. Cell. Mol. 157. Sanai, N. et al. Corridors of migrating neurons in the mediates cannabinoid-induced apoptosis of tumor Life Sci. 65, 840–850 (2008). human brain and their decline during infancy. Nature cells. Cancer Cell 9, 301–312 (2006). 200. Viscomi, M. T. et al. Selective CB2 receptor agonism 478, 382–386 (2011). This paper defines transcriptional elements by protects central neurons from remote axotomy- 158. Ernst, A. et al. Neurogenesis in the striatum of the which cannabinoids can trigger glioma cell death. It induced apoptosis through the PI3K/AKT pathway. adult human brain. Cell 156, 1072–1083 (2014). also highlights the relevance and the efficacy of J. Neurosci. 29, 4564–4570 (2009). 159. Arvidsson, A., Collin, T., Kirik, D., Kokaia, Z. & phytocannabinoids to cancer therapy. 201. Alpar, A. et al. Endocannabinoids modulate cortical Lindvall, O. Neuronal replacement from endogenous 180. Salazar, M. et al. Cannabinoid action induces development by configuring SLIT2/ROBO1 signalling. precursors in the adult brain after stroke. Nature Med. autophagy-mediated cell death through stimulation of Nature Commun. 5, 4421 (2014). 8, 963–970 (2002). ER stress in human glioma cells. J. Clin. Invest. 119, 202. Katona, I. et al. Molecular composition of the 160. Butti, E. et al. Subventricular zone neural progenitors 1359–1372 (2009). endocannabinoid system at glutamatergic synapses. protect striatal neurons from glutamatergic 181. Pasquariello, N. et al. Characterization of the J. Neurosci. 26, 5628–5637 (2006). excitotoxicity. Brain 135, 3320–3335 (2012). endocannabinoid system in human neuronal cells and 203. Tamamaki, N. et al. Green fluorescent protein 161. Jin, K. et al. Neurogenesis and aging: FGF2 and proteomic analysis of anandamide-induced apoptosis. expression and colocalization with calretinin, HBEGF restore neurogenesis in hippocampus and J. Biol. Chem. 284, 29413–29426 (2009). parvalbumin, and somatostatin in the GAD67–GFP subventricular zone of aged mice. Aging Cell 2, 182. Galve-Roperh, I. et al. Anti-tumoral action of knockin mouse. J. Comp. Neurol. 467, 60–79 (2003). 175–183 (2003). cannabinoids: involvement of sustained ceramide 204. Goebbels, S. et al. Genetic targeting of principal 162. Kim, J. et al. GDF11 controls the timing of progenitor accumulation and extracellular signal-regulated kinase neurons in neocortex and hippocampus of NEX–Cre cell competence in developing retina. Science 308, activation. Nature Med. 6, 313–319 (2000). mice. Genesis. 44, 611–621 (2006). 1927–1930 (2005). 183. Lorente, M. et al. Stimulation of the midkine/ALK axis 205. Trazzi, S., Steger, M., Mitrugno, V. M., Bartesaghi, R. 163. Wu, H. H. et al. Autoregulation of neurogenesis by renders glioma cells resistant to cannabinoid & Ciani, E. CB1 cannabinoid receptors increase GDF11. Neuron 37, 197–207 (2003). antitumoral action. Cell Death. Differ. 18, 959–973 neuronal precursor proliferation through AKT/ 164. Williams, G. et al. Transcriptional basis for the (2011). glycogen synthase kinase3β/β-catenin signaling. inhibition of neural stem cell proliferation and 184. Cudaback, E., Marrs, W., Moeller, T. & Stella, N. The J. Biol. Chem. 285, 10098–10109 (2010). migration by the TGFβ-family member GDF11. PLoS expression level of CB1 and CB2 receptors determines 206. Cravatt, B. F. et al. Supersensitivity to anandamide ONE. 8, e78478 (2013). their efficacy at inducing apoptosis in astrocytomas. and enhanced endogenous cannabinoid signaling in 165. Goncalves, M. B. et al. The COX2 inhibitors, PLoS ONE. 5, e8702 (2010). mice lacking fatty acid amide hydrolase. Proc. Natl meloxicam and nimesulide, suppress neurogenesis in 185. Volkow, N. D., Baler, R. D., Compton, W. M. & Acad. Sci. USA 98, 9371–9376 (2001). the adult mouse brain. Br. J. Pharmacol. 159, Weiss, S. R. Adverse health effects of marijuana use. 207. Jin, K. et al. Defective adult neurogenesis in CB1 1118–1125 (2010). N. Engl. J. Med. 370, 2219–2227 (2014). cannabinoid receptor knockout mice. Mol. Pharmacol. 166. Shigemoto-Mogami, Y., Hoshikawa, K., Goldman, J. E., 186. Han, J. et al. Acute cannabinoids impair working 66, 204–208 (2004). Sekino, Y. & Sato, K. Microglia enhance neurogenesis and memory through astroglial CB1 receptor modulation oligodendrogenesis in the early postnatal subventricular of hippocampal LTD. Cell 148, 1039–1050 (2012). Acknowledgements zone. J. Neurosci. 34, 2231–2243 (2014). 187. Navarrete, M. & Araque, A. Endocannabinoids This work was supported by the Ministero dell’Istruzione, 167. Sutterlin, P. et al. The molecular basis of the potentiate synaptic transmission through stimulation dell’Università e della Ricerca (PRIN 2010–2011 grant to cooperation between EGF, FGF and eCB receptors in of astrocytes. Neuron 68, 113–126 (2010). M.M.), Swedish Medical Research Council (to T.H.), Novo the regulation of neural stem cell function. Mol. Cell 188. Morozov, Y. M. et al. Antibodies to cannabinoid type 1 Nordisk Foundation (to T.H.), Petrus and Augusta Hedlunds’ Neurosci. 52, 20–30 (2013). receptor coreact with stomatin-like protein 2 in mouse Foundation (to T.H.), the Wellcome Trust (to P.D.), the Spanish 168. Zhao, C., Deng, W. & Gage, F. H. Mechanisms and brain mitochondria. Eur. J. Neurosci. 38, 2341–2348 Ministry of Economy and Competitiveness (grant functional implications of adult neurogenesis. Cell (2013). SAF201235759 to M.G.), the European Commission (FP7 132, 645–660 (2008). 189. Eggan, S. M., Hashimoto, T. & Lewis, D. A. Reduced ‘PAINCAGE’ integrated project to T.H.) and the National 169. Santarelli, L. et al. Requirement of hippocampal cortical cannabinoid 1 receptor messenger RNA and Institutes of Health (grant DA023214 to T.H., and grants neurogenesis for the behavioral effects of protein expression in schizophrenia. Arch. Gen. DA011322 and DA021696 to K.M.). The content of this antidepressants. Science 301, 805–809 (2003). Psychiatry 65, 772–784 (2008). report is solely the responsibility of the authors and does not 170. Li, G. L. et al. Assessment of the pharmacology and 190. Veen, N. D. et al. Cannabis use and age at onset of necessarily represent the official views of the US National tolerability of PF04457845, an irreversible inhibitor schizophrenia. Am. J. Psychiatry 161, 501–506 Institutes of Health. M.M. dedicates this Review to his late of fatty acid amide hydrolase-1, in healthy subjects. (2004). father, Giuseppe. Br. J. Clin. Pharmacol. 73, 706–716 (2012). 191. Monteleone, P. et al. Investigation of CNR1 and FAAH 171. Kitambi, S. S. et al. Vulnerability of glioblastoma cells endocannabinoid gene polymorphisms in bipolar Competing interests statement to catastrophic vacuolization and death induced by a disorder and major depression. Pharmacol. Res. 61, The authors declare no competing interests. small molecule. Cell http://dx.doi.org/10.1016/j. 400–404 (2010). cell.2014.02.021 (2014). 192. Krakowiak, P. et al. Maternal metabolic conditions and 172. Velasco, G., Sanchez, C. & Guzman, M. Towards the risk for autism and other neurodevelopmental DATABASES use of cannabinoids as antitumour agents. Nature disorders. Pediatrics 129, e1121–e1128 (2012). Clinicaltrials.gov: http://clinicaltrials.gov Rev. Cancer 12, 436–444 (2012). 193. Di Marzo, V. The endocannabinoid system in obesity NCT01812603 173. Blazquez, C. et al. Inhibition of tumor angiogenesis by and type 2 diabetes. Diabetologia 51, 1356–1367 ALL LINKS ARE ACTIVE IN THE ONLINE PDF cannabinoids. FASEB J. 17, 529–531 (2003). (2008).

NATURE REVIEWS | NEUROSCIENCE VOLUME 15 | DECEMBER 2014 | 801