Effectiveness of biomaterial-based combination strategies for spinal cord repair - a systematic review and meta-analysis of preclinical literature.

STUDY DESIGN: Systematic review and meta-analysis of preclinical literature. OBJECTIVES: To assess the effects of biomaterial-based combination (BMC) strategies for the treatment of Spinal Cord Injury (SCI), the effects of individual biomaterials in the context of BMC strategies, and the factors influencing their efficacy. To assess the effects of different preclinical testing paradigms in BMC strategies. METHODS: We performed a systematic literature search of Embase, Web of Science and PubMed. All controlled preclinical studies describing an in vivo or in vitro model of SCI that tested a biomaterial in combination with at least one other regenerative strategy (cells, drugs, or both) were included. Two review authors conducted the study selection independently, extracted study characteristics independently and assessed study quality using a modified CAMARADES checklist. Effect size measures were combined using random-effects models and heterogeneity was explored using meta-regression with tau2, I2 and R2 statistics. We tested for small-study effects using funnel plot-based methods. RESULTS: 134 publications were included, testing over 100 different BMC strategies. Overall, treatment with BMC therapies improved locomotor recovery by 25.3% (95% CI, 20.3-30.3; n = 102) and in vivo axonal regeneration by 1.6 SD (95% CI 1.2-2 SD; n = 117) in comparison with injury only controls. CONCLUSION: BMC strategies improve locomotor outcomes after experimental SCI. Our comprehensive study highlights gaps in current knowledge and provides a foundation for the design of future experiments.

Electrical Stimulation Directs Migration, Enhances and Orients Cell Division and Upregulates the Chemokine Receptors CXCR4 and CXCR2 in Endothelial Cells.

Natural direct current electric fields (DC EFs) within tissues undergoing angiogenesis have the potential to influence vessel formation, but how they affect endothelial cells is not clear. We therefore quantified behaviours of human umbilical vein endothelial cells (HUVEC) and human microvasculature endothelial cells (HMEC) stimulated by EFsin vitro. Both cell types migrated faster and toward the cathode; HUVECs responded to fields as low as 50mV/mm, but the HMEC threshold was 100 mV/mm. Mitosis was stimulated at 50 mV/mm for HMEC and at 150 mV/mm for HUVECs, but the cleavage plane was oriented orthogonal to the field vector at 200 mV/mm for both cell types. That different field strengths induced different cell responses suggests distinct underlying cellular mechanisms. A physiological electric field also upregulated expression of CXCR4 and CXCR2 chemokine receptors and upregulated phosphorylation of both chemokines in HUVEC and HMEC cells. Evidence that DC EFs direct endothelial cell migration, proliferation and upregulate chemokines involved in wound healing suggests a key role for electrical control of capillary production during healing. Our data contribute to the molecular mechanisms by which DC EFs direct endothelial cell behaviour and present a novel signalling paradigm in wound healing, tissue regeneration and angiogenesis-related diseases.

Contact-mediated control of radial migration of corneal epithelial cells.

PURPOSE: Patients with a heterozygous mutation in the gene encoding the transcription factor, PAX6, have a degenerative corneal opacity associated with failure of normal radial epithelial cell migration across the corneal surface and a reported wound healing defect. This study investigated the guidance mechanisms that drive the directed migration of corneal epithelial cells. METHODS: In vivo corneal epithelial wounding was performed in adult wild-type and Pax6(+/-) mice, and the healing migration rates were compared. To investigate the control of the cell migration direction, primary corneal epithelial cells from wild-type and Pax6(+/-) mice were plated on grooved quartz substrates, and alignment relative to the grooves was assayed. A reconstructed corneal culture system was developed in which dissociated wild-type and genetically mutant corneal epithelial cells could be cultured on a de-epithelialized corneal stroma or basement membrane and their migration assayed with time-lapse microscopy. RESULTS: The Pax6(+/-) cells efficiently re-epithelialized corneal wounds in vivo but had mild slowing of healing migration compared to the wild-type. Cells aligned parallel to quartz grooves in vitro, but the Pax6(+/-) cells were less robustly oriented than the wild-type. In the reconstructed corneal culture system, corneal epithelial cells continued to migrate radially, showing that the cells are guided by contact-mediated cues from the basement membrane. Recombining wild-type and Pax6 mutant corneal epithelial cells with wild-type and Pax6 mutant corneal stroma showed that normal Pax6 dosage was required autonomously in the epithelial cells for directed migration. Integrin-mediated attachment to the substrate, and intracellular PI3Kγ activity, were required for migration. Pharmacological inhibition of cAMP signaling randomized migration tracks in reconstructed corneas. CONCLUSIONS: Striking patterns of centripetal migration of corneal epithelial cells observed in vivo are driven by contact-mediated cues operating through an intracellular cAMP pathway, and failure to read these cues underlies the migration defects that accompany corneal degeneration in patients with mutations in PAX6.

Wireless control of nerve growth using bipolar electrodes: a new paradigm in electrostimulation.

Electrical activity underpins all life, but is most familiar in the nervous system, where long range electrical signalling is essential for function. When this is lost (e.g., traumatic injury) or it becomes inefficient (e.g., demyelination), the use of external fields can compensate for at least some functional deficits. However, its potential to also promote biological repair at the cell level is underplayed despite abundant in vitro evidence for control of neuron growth. This perspective article considers specifically the emerging possibility of achieving cell growth through the interaction of external electric fields using conducting materials as unwired bipolar electrodes, and without intending stimulation of neuron electrical activity to be the primary consequence. The use of a wireless method to create electrical interactions represents a paradigm shift and may allow new applications in vivo where physical wiring is not possible. Within that scheme of thought an evaluation of specific materials and their dynamic responses as bipolar unwired electrodes is summarized and correlated with changes in dynamic nerve growth during stimulation, suggesting possible future schemes to achieve neural growth using bipolar unwired electrodes with specific characteristics. This strategy emphasizes how nerve growth can be encouraged at injury sites wirelessly to induce repair, as opposed to implanting devices that may substitute the neural signals.

Interaction between hedgehog signalling and PAX6 dosage mediates maintenance and regeneration of the corneal epithelium.

PURPOSE: To investigate the roles of intracellular signaling elicited by Hedgehog (Hh) ligands in corneal maintenance and wound healing. METHODS: The expression of Hedgehog pathway components in the cornea was assayed by immunohistochemistry, western blot and reverse-transcription polymerase chain reaction (RT-PCR), in wild-type mice and mice that were heterozygous null for the gene encoding the transcription factor, paired box gene 6 (Pax6).  Corneal epithelial wound healing and cell migration assays were performed after pharmacological upregulation and downregulation of the hedgehog pathway.  Reporter mice, mosaic for expression of the gene encoding ß-galactosidase (LacZ), were crossed to Pax6(+/-) mice, mice heterozygous for the gene encoding GLI-Kruppel family member GLI3, and Pax6(+/-)Gli3(+/-) double heterozygotes, to assay patterns of cell migration and corneal epithelial organization in vivo. RESULTS: Corneal epithelial wound healing rates increased in response to application of Sonic hedgehog (Shh), but only in mice with wild-type Pax6 dosage.  Downregulation of Hedgehog signalling inhibited corneal epithelial cell proliferation.  Pax6(+/-) corneal epithelia showed increased proliferation in response to exogenous Shh, but not increased migration. Desert hedgehog (Dhh) was shown to be the major endogenous ligand, with Shh detectable only by RT-PCR and only after epithelial wounding. The activity of phosphatidylinositol-3-OH kinase-γ (PI3Kγ) was not required for the increased migration response in response to Shh.  Nuclear expression of the activator form of the transcription factor Gli3 (which mediates Hh signalling) was reduced in Pax6(+/-) corneal epithelia. Pax6(+/-)Gli3(+/-) double heterozygotes showed highly disrupted patterns of clonal arrangement of cells in the corneal epithelium. CONCLUSIONS: The data show key roles for endogenous Dhh signalling in maintenance and regeneration of the corneal epithelium, demonstrate an interaction between Pax6 and Hh signalling in the corneal epithelium, and show that failure of Hh signalling pathways is a feature of Pax6(+/-) corneal disease that cannot be remedied pharmacologically by addition of the ligands.

The role of electrical signals in murine corneal wound re-epithelialization.

Ion flow from intact tissue into epithelial wound sites results in lateral electric currents that may represent a major driver of wound healing cell migration. Use of applied electric fields (EF) to promote wound healing is the basis of Medicare-approved electric stimulation therapy. This study investigated the roles for EFs in wound re-epithelialization, using the Pax6(+/-) mouse model of the human ocular surface abnormality aniridic keratopathy (in which wound healing and corneal epithelial cell migration are disrupted). Both wild-type (WT) and Pax6(+/-) corneal epithelial cells showed increased migration speeds in response to applied EFs in vitro. However, only Pax6(+/+) cells demonstrated consistent directional galvanotaxis towards the cathode, with activation of pSrc signaling, polarized to the leading edges of cells. In vivo, the epithelial wound site normally represents a cathode, but 43% of Pax6(+/-) corneas exhibited reversed endogenous wound-induced currents (the wound was an anode). These corneas healed at the same rate as WT. Surprisingly, epithelial migration did not correlate with direction or magnitude of endogenous currents for WT or mutant corneas. Furthermore, during healing in vivo, no polarization of pSrc was observed. We found little evidence that Src-dependent mechanisms of cell migration, observed in response to applied EFs in vitro, normally exist in vivo. It is concluded that endogenous EFs do not drive long-term directionality of sustained healing migration in this mouse corneal epithelial model. Ion flow from wounds may nevertheless represent an important component of wound signaling initiation.

Electrical dimensions in cell science.

Cells undergo a variety of physiological processes, including division, migration and differentiation, under the influence of endogenous electrical cues, which are generated physiologically and pathologically in the extracellular and sometimes intracellular spaces. These signals are transduced to regulate cell behaviours profoundly, both in vitro and in vivo. Bioelectricity influences cellular processes as fundamental as control of the cell cycle, cell proliferation, cancer-cell migration, electrical signalling in the adult brain, embryonic neuronal cell migration, axon outgrowth, spinal-cord repair, epithelial wound repair, tissue regeneration and establishment of left-right body asymmetry. In addition to direct effects on cells, electrical gradients interact with coexisting extracellular chemical gradients. Indeed, cells can integrate and respond to electrical and chemical cues in combination. This Commentary details how electrical signals control multiple cell behaviours and argues that study of the interplay between combined electrical and chemical gradients is underdeveloped yet necessary.

Physiological strength electric fields modulate human T cell activation and polarisation.

The factors and signals driving T cell activation and polarisation during immune responses have been studied mainly at the level of cells and chemical mediators. Here we describe a physical driver of these processes in the form of physiological-strength electric fields (EFs). EFs are generated at sites where epithelium is disrupted (e.g. wounded skin/bronchial epithelia) and where T cells frequently are present. Using live-cell imaging, we show human primary T cells migrate directionally to the cathode in low strength (50/150 mV/mm) EFs. Strikingly, we show for the first time that EFs significantly downregulate T cell activation following stimulation with antigen-activated APCs or anti-CD3/CD28 antibodies, as demonstrated by decreased IL-2 secretion and proliferation. These EF-induced functional changes were accompanied by a significant dampening of CD4+ T cell polarisation. Expression of critical markers of the Th17 lineage, RORγt and IL-17, and the Th17 polarisation mediator phospho-STAT3 were reduced significantly, while STAT1, ERK and c-Jun phosphorylation were comparatively unaffected suggesting STAT3 modulation by EFs as one mechanism driving effects. Overall, we identify electrical signals as important contributors to the co-ordination and regulation of human T cell functions, paving the way for a new research area into effects of naturally occurring and clinically-applied EFs in conditions where control of T cell activity is paramount.

Roles for IFT172 and Primary Cilia in Cell Migration, Cell Division, and Neocortex Development.

The cilium of a cell translates varied extracellular cues into intracellular signals that control embryonic development and organ function. The dynamic maintenance of ciliary structure and function requires balanced bidirectional cargo transport involving intraflagellar transport (IFT) complexes. IFT172 is a member of the IFT complex B, and IFT172 mutation is associated with pathologies including short rib thoracic dysplasia, retinitis pigmentosa and Bardet-Biedl syndrome, but how it underpins these conditions is not clear. We used the WIM cell line, derived from embryonic fibroblasts of Wimple mice (carrying homozygous Leu1564Pro mutation in Ift172), to probe roles of Ift172 and primary cilia in cell behavior. WIM cells had ablated cilia and deficiencies in directed migration (electrotaxis), cell proliferation and intracellular signaling. Additionally, WIM cells displayed altered cell cycle progression, with increased numbers of chromatids, highlighting dysfunctional centrosome status. Exposure to a physiological electric field promoted a higher percentage of primary cilia in wild-type cells. Interestingly, in situ hybridization revealed an extensive and dynamic expression profile of Ift172 in both developing and adult mouse cortex. In vivo manipulation of Ift172 expression in germinal regions of embryonic mouse brains perturbed neural progenitor proliferation and radial migration of post-mitotic neurons, revealing a regulatory role of Ift172 in cerebral morphogenesis. Our data suggest that Ift172 regulates a range of fundamental biological processes, highlighting the pivotal roles of the primary cilium in cell physiology and brain development.

Prioritising guidance cues: directional migration induced by substratum contours and electrical gradients is controlled by a rho/cdc42 switch.

Coordinated cell migration is a fundamental feature of embryogenesis but the intracellular mechanism by which cells integrate co-existing extracellular cues to yield appropriate vectoral migration is unknown. Cells in the cornea are guided by a naturally occurring DC electric field (EF) (electrotaxis) as they navigate non-planar substrata but the relative potencies of electrotaxis and guidance by substratum shape (contact guidance) have never been determined. We tested the hypothesis that vectoral migration was controlled by selective activation of rac, cdc42 or rho in response to a 150 mV/mm EF or to a series of parallel substratum nanogrooves (NGs) 130 nm deep. EFs and NGs were presented singly or in combination. Electrotaxis of dissociated bovine corneal epithelial cells (CECs) on planar quartz required signalling by cdc42 and rho but not rac. Contact guidance by substratum NGs required rho but not cdc42 or rac activities. When an EF and NGs were superimposed in parallel, cathodal electrotaxis along NGs was enhanced compared to that on planar quartz but when they were superimposed orthogonally (vertical NGs with horizontal EF) cells were recruited from contact guidance to electrotaxis, suggesting that the EF was more potent. However, increasing the EF to 250 mV/mm was insufficient to recruit the majority to electrotaxis. Consistent for the cues in isolation, when an EF (150 mV/mm) and NGs were superimposed orthogonally, rac activity was not essential for either contact guidance or electrotaxis. However, attenuation of cdc42 signalling abolished electrotaxis and enhanced contact guidance relative to controls (no drug), whereas inhibiting rho signalling enhanced electrotaxis and rho stimulation enhanced contact guidance. Our data are consistent with the idea that migrating CECs use a cdc42/rho "switch" to sort vectoral cues, with cdc42 controlling electrotaxis and rho controlling contact guidance.

Alignment of corneal and lens epithelial cells by co-operative effects of substratum topography and DC electric fields.

Corneal and lens epithelial cells (CECs and LECs) in the eye encounter precisely ordered fibre arrays on the nanoscale in tandem with an endogenous electric field (EF). Prosthetic biomaterials often incorporate topographical features intended to mimic those in situ. However, the cellular basis for control of cell morphology by nanotopography or by an EF is not clear. We examined cell axis alignment in response to substratum nanotopography and a physiological EF separately and in combination. Bovine CECs aligned parallel to substratum nanogrooves (NGs) as shallow as 14 nm but LECs were less sensitive. Actin filaments of both cell types concentrated at substratum ridges so we tested the mechanistic roles of rho, rac and cdc42, molecules that control cytoskeletal organization. CEC alignment to 130 nm deep NGs was prevented by the inhibition of rho, but not by the inhibition of cdc42, rac, or the rho effectors myosin light chain kinase or rho kinase. Conversely, CEC alignment was enhanced by the activation of rho. CECs on planar quartz substrata aligned orthogonal to an EF of 150 mV/mm. Alignment required signalling by cdc42 and rho but not rac, and was accompanied by lamellipodial reorganisation and cell migration toward the cathode. When CECs on vertically oriented NGs were exposed simultaneously to a horizontal EF, they aligned more robustly than to either cue alone and the enhanced alignment required rho signalling. Therefore, nanoscale substratum features and EFs co-operate to control cell axis alignment via rho, and cdc42-mediated intracellular signals, which can be exploited in tissue engineering.

Controlling Nerve Growth with an Electric Field Induced Indirectly in Transparent Conductive Substrate Materials.

Innovative neurostimulation therapies require improved electrode materials, such as poly(3,4-ethylenedioxythiophene) (PEDOT) polymers or IrOx mixed ionic-electronic conductors and better understanding of how their electrochemistry influences nerve growth. Amphibian neurons growing on transparent films of electronic (metal) conductors and electronic-ionic conductors (polymers and semiconducting oxides) are monitored. Materials are not connected directly to the power supply, but a dipole is created wirelessly within them by electrodes connected to the culture medium in which they are immersed. Without electrical stimulation neurons grow on gold, platinum, PEDOT-polystyrene sulfonate (PEDOT-PSS), IrOx , and mixed oxide (Ir-Ti)Ox , but growth is not related to surface texture or hydrophilicity. Stimulation induces a dipole in all conductive materials, but neurons grow differently on electronic conductors and mixed-valence mixed-ionic conductors. Stimulation slows, but steers neurite extension on gold but not on platinum. The rate and direction of neurite growth on PEDOT-PSS resemble that on glass, but on IrOx and (Ir-Ti)Ox neurites grow faster and in random directions. This suggests electrochemical changes induced in these materials control growth speed and direction selectively. Evidence that the electric dipole induced in conductive material controls nerve growth will impact electrotherapies exploiting wireless stimulation of implanted material arrays, even where transparency is required.

Requirement of Pax6 for the integration of guidance cues in cell migration.

The intricate patterns of cell migration that are found throughout development are generated through a vast array of guidance cues. Responding integratively to distinct, often conflicting, migratory signals is probably crucial for cells to reach their correct destination. Pax6 is a master transcription factor with key roles in neural development that include the control of cell migration. In this study, we have investigated the ability of cells derived from cortical neurospheres from wild-type (WT) and Pax6-/- mouse embryos to integrate diverging guidance cues. We used two different cues, either separately or in combination: substratum nanogrooves to induce contact guidance, and electric fields (EFs) to induce electrotaxis. In the absence of an EF, both WT and Pax6-/- cells aligned and migrated parallel to grooves, and on a flat substrate both showed marked electrotaxis towards the cathode. When an EF was applied in a perpendicular orientation to grooves, WT cells responded significantly to both cues, migrating in highly oblique trajectories in the general direction of the cathode. However, Pax6-/- cells had an impaired response to both cues simultaneously. Our results demonstrate that these neurosphere derived cells have the capacity to integrate diverging guidance cues, which requires Pax6 function.

Has electrical growth cone guidance found its potential?

Many neurobiologists spurn the existence and use of direct-current (DC) electric fields (EFs) in nervous system development and regeneration. This is despite direct measurement of EFs in embryos and adults, and evidence that EFs are required for normal development, dramatically influence the rate and direction of nerve growth in vitro, and promote nerve regeneration in vivo. The notion that growth cones use EFs as guidance cues was dismissed partly because there was no convincing evidence that naturally occurring EFs influence nerve growth at the single-cell level in vivo. Recent work indicates that growth cones can be guided by EFs in vivo and, intriguingly, that in vitro guidance by chemotropic gradients and EFs might invoke similar mechanisms. Ongoing clinical trials to assess the effectiveness of DC EFs in promoting the regeneration of human spinal cord could allow EFs to achieve their potential.

Electric fields are novel determinants of human macrophage functions.

Macrophages are key cells in inflammation and repair, and their activity requires close regulation. The characterization of cues coordinating macrophage function has focused on biologic and soluble mediators, with little known about their responses to physical stimuli, such as the electrical fields that are generated naturally in injured tissue and which accelerate wound healing. To address this gap in understanding, we tested how properties of human monocyte-derived macrophages are regulated by applied electrical fields, similar in strengths to those established naturally. With the use of live-cell video microscopy, we show that macrophage migration is directed anodally by electrical fields as low as 5 mV/mm and is electrical field strength dependent, with effects peaking ∼300 mV/mm. Monocytes, as macrophage precursors, migrate in the opposite, cathodal direction. Strikingly, we show for the first time that electrical fields significantly enhance macrophage phagocytic uptake of a variety of targets, including carboxylate beads, apoptotic neutrophils, and the nominal opportunist pathogen Candida albicans, which engage different classes of surface receptors. These electrical field-induced functional changes are accompanied by clustering of phagocytic receptors, enhanced PI3K and ERK activation, mobilization of intracellular calcium, and actin polarization. Electrical fields also modulate cytokine production selectively and can augment some effects of conventional polarizing stimuli on cytokine secretion. Taken together, electrical signals have been identified as major contributors to the coordination and regulation of important human macrophage functions, including those essential for microbial clearance and healing. Our results open up a new area of research into effects of naturally occurring and clinically applied electrical fields in conditions where macrophage activity is critical.

The core planar cell polarity gene, Vangl2, directs adult corneal epithelial cell alignment and migration.

This study shows that the core planar cell polarity (PCP) genes direct the aligned cell migration in the adult corneal epithelium, a stratified squamous epithelium on the outer surface of the vertebrate eye. Expression of multiple core PCP genes was demonstrated in the adult corneal epithelium. PCP components were manipulated genetically and pharmacologically in human and mouse corneal epithelial cells in vivo and in vitro. Knockdown of VANGL2 reduced the directional component of migration of human corneal epithelial (HCE) cells without affecting speed. It was shown that signalling through PCP mediators, dishevelled, dishevelled-associated activator of morphogenesis and Rho-associated protein kinase directs the alignment of HCE cells by affecting cytoskeletal reorganization. Cells in which VANGL2 was disrupted tended to misalign on grooved surfaces and migrate across, rather than parallel to the grooves. Adult corneal epithelial cells in which Vangl2 had been conditionally deleted showed a reduced rate of wound-healing migration. Conditional deletion of Vangl2 in the mouse corneal epithelium ablated the normal highly stereotyped patterns of centripetal cell migration in vivo from the periphery (limbus) to the centre of the cornea. Corneal opacity owing to chronic wounding is a major cause of degenerative blindness across the world, and this study shows that Vangl2 activity is required for directional corneal epithelial migration.

A role for L-alpha-lysophosphatidylinositol and GPR55 in the modulation of migration, orientation and polarization of human breast cancer cells.

BACKGROUND AND PURPOSE: Increased circulating levels of L-alpha-lysophosphatidylinositol (LPI) are associated with cancer and LPI is a potent, ligand for the G-protein-coupled receptor GPR55. Here we have assessed the modulation of breast cancer cell migration, orientation and polarization by LPI and GPR55. EXPERIMENTAL APPROACH: Quantitative RT-PCR was used to measure GPR55 expression in breast cancer cell lines. Cell migration and invasion were measured using a Boyden chamber chemotaxis assay and Cultrex invasion assay, respectively. Cell polarization and orientation in response to the microenvironment were measured using slides containing nanometric grooves. KEY RESULTS: GPR55 expression was detected in the highly metastatic MDA-MB-231 breast cancer cell line. In these cells, LPI stimulated binding of [(35)S]GTPgammaS to cell membranes (pEC(50) 6.47 +/- 0.45) and significantly enhanced cell chemotaxis towards serum. MCF-7 cells expressed low levels of GPR55 and did not migrate or invade towards serum factors. When GPR55 was over-expressed in MCF-7 cells, serum induced a robust migratory and invasive response, which was further enhanced by LPI and prevented by siRNA to GPR55. The physical microenvironment has been identified as a key factor in determining breast tumour cell metastatic fate. LPI endowed MDA-MB-231 cells with the capacity to detect shallow (40 nm deep) grooved slides and induced marked cancer cell polarization on both flat and grooved surfaces. CONCLUSIONS AND IMPLICATIONS: LPI and GPR55 play a role in the modulation of migration, orientation and polarization of breast cancer cells in response to the tumour microenvironment.

Hardwiring the brain: endocannabinoids shape neuronal connectivity.

The roles of endocannabinoid signaling during central nervous system development are unknown. We report that CB(1) cannabinoid receptors (CB(1)Rs) are enriched in the axonal growth cones of gamma-aminobutyric acid-containing (GABAergic) interneurons in the rodent cortex during late gestation. Endocannabinoids trigger CB(1)R internalization and elimination from filopodia and induce chemorepulsion and collapse of axonal growth cones of these GABAergic interneurons by activating RhoA. Similarly, endocannabinoids diminish the galvanotropism of Xenopus laevis spinal neurons. These findings, together with the impaired target selection of cortical GABAergic interneurons lacking CB(1)Rs, identify endocannabinoids as axon guidance cues and demonstrate that endocannabinoid signaling regulates synaptogenesis and target selection in vivo.