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• W2989218046 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Endocannabinoids are recently recognized regulators of brain development, but molecular effectors downstream of type-1 cannabinoid receptor (CB1R)-activation remain incompletely understood. We report atypical coupling of neuronal CB1Rs, after activation by endo- or exocannabinoids such as the marijuana component ∆9-tetrahydrocannabinol, to heterotrimeric G12/G13 proteins that triggers rapid and reversible non-muscle myosin II (NM II) dependent contraction of the actomyosin cytoskeleton, through a Rho-GTPase and Rho-associated kinase (ROCK). This induces rapid neuronal remodeling, such as retraction of neurites and axonal growth cones, elevated neuronal rigidity, and reshaping of somatodendritic morphology. Chronic pharmacological inhibition of NM II prevents cannabinoid-induced reduction of dendritic development in vitro and leads, similarly to blockade of endocannabinoid action, to excessive growth of corticofugal axons into the sub-ventricular zone in vivo. Our results suggest that CB1R can rapidly transform the neuronal cytoskeleton through actomyosin contractility, resulting in cellular remodeling events ultimately able to affect the brain architecture and wiring. https://doi.org/10.7554/eLife.03159.001 eLife digest Our brains are full of cells called neurons, which are connected to each other in complex networks that send messages around the brain. The way the neurons connect to each other, known as brain wiring, differs widely between individuals. Moreover, our brain wiring changes in response to our environment and experiences throughout our lives, from developing embryo to old age. One way this happens is through the action of chemicals called cannabinoids. Produced naturally in the body, cannabinoids are also found in the popular recreational drug cannabis that is increasingly being used in medicine to treat chronic pain and other conditions. However, cannabis misuse can have negative side effects on the brain leading to memory loss and mental illness, especially in young people. Cannabinoids can be detected by a group of proteins called cannabinoid receptors, but it is not clear how this leads to changes in brain wiring. Roland et al. now show that detection of cannabinoids by a type-1 cannabinoid receptor triggers a series of events that change how neurons grow and connect with each other. Detection of the cannabinoid by the receptor leads to the activation of an enzyme called ROCK. This, in turn, activates a motor protein called non-muscle myosin II that inhibits the growth of neurons. Roland et al. suggest that this prevents the neurons from reaching their neighbors and forming new connections. Investigating how this works in individuals with medical conditions that alter brain function could help inform us how cannabis could be used more safely. https://doi.org/10.7554/eLife.03159.002 Introduction The endocannabinoid (eCB) system is emerging as an important regulator of brain wiring during development with a variety of functions, ranging from lineage segregation of stem cells to refinement of synaptic functions in complex neuronal networks (Williams et al., 2003; Berghuis et al., 2007; Harkany et al., 2008; Mulder et al., 2008; Vitalis et al., 2008; Watson et al., 2008; Wu et al., 2010). In both the embryonic and adult brains, eCB action is predominantly mediated by CB1 cannabinoid receptors (CB1Rs), which is one of the most highly expressed neuronal G-protein-coupled receptors (GPCRs), known to couple to Gi/o heterotrimeric proteins (Howlett, 2005), but the molecular mechanisms by which CB1R shapes developing neurons remain mostly unknown. The exact role of eCBs in shaping the neuronal architecture is also under debate, since several reports indicate neurite retraction, while others found the induction of neurite outgrowth following CB1R activation (review in Gaffuri et al., 2012). Likewise, currently it is difficult to reconcile the locally repulsive effects of eCBs, reported at axonal growth cones (Berghuis et al., 2007; Argaw et al., 2011), and their role of mediating efficient directional axonal growth and shaping well-fasciculated axonal tracts (Mulder et al., 2008; Vitalis et al., 2008; Watson et al., 2008). During neuronal development, an elaborate balance of positive and negative regulators is necessary to establish precise neuronal structure. This structure is stabilized by the cytoskeleton, which, similar to non-neuronal cells, is composed of two major polymers, the highly plastic filamentous-actin (F-actin) and the more stable microtubule (MT) networks. Actin filaments are often cross-linked to a molecular motor protein, the non-muscle myosin II (NM II), whose contractile properties further endow the actomyosin network with highly dynamic control of cell behavior and architecture (Vicente-Manzanares et al., 2009). The cytoskeleton is mainly regulated by Rho-like GTPases that control a wide variety of effector mechanisms such as actin polymerization and branching, actomyosin contractility, focal adhesions, microtubule dynamics, and membrane transport (Kaibuchi et al., 1999; Etienne-Manneville and Hall, 2002; Hall and Lalli, 2010). Downstream protein kinases such as the Rho-associated, coiled coil-containing kinase (ROCK) are the key activator proteins of these convergent-signaling pathways. Interestingly, ROCK is associated with particular CB1R-induced phenotypes. In CB1R-over-expressing B103 cells, the endocannabinoid anandamide induces cell rounding via ROCK (Ishii and Chun, 2002), and CB1R activation results in RhoA- and ROCK-dependent repulsion of growth cones of cultured hippocampal neurons (Berghuis et al., 2007), but neither the coupling mechanism of CB1R to ROCK nor the cytoskeletal targets downstream of CB1R-activated ROCK are identified yet. Since Rho-activated effectors operate over a large range of spatial and temporal scales, understanding of eCB-mediated structural plasticity requires the identification of the precise spatial and temporal dynamics of CB1R-mediated cytoskeletal modifications. In this study, by using highly resolved live imaging approaches, we report that CB1R-activation rapidly and reversibly contracts the neuronal actomyosin cytoskeleton through an unusual coupling to G12/G13 proteins that produce Rho- and ROCK-mediated NM II activation. In addition, we show that chronic CB1R-mediated activation of actomyosin contractility may mediate lasting changes in neuronal and cerebral morphology. Results CB1R-activation results in rapid retraction of actin-rich growth cones In order to investigate the spatio-temporal dynamics of cannabinoid-induced cytoskeletal modifications, we have established a sensitive, specific, and highly accessible experimental assay system to study neuronal remodeling downstream of CB1R activation. We have visualized highly dynamic neuronal growth cones in cultured hippocampal neurons, where the activation of endogenous CB1Rs results in repulsion (Berghuis et al., 2007), by labeling endogenous F-actin with fluorescent LifeAct. This actin-binding peptide allows observation of the dynamic actin network without perturbing natural reorganization kinetics (Riedl et al., 2008). Time-lapse microscopy of live neurons, expressing Flag-CB1R-eGFP and LifeAct-mCherry, showed numerous F-actin-rich dynamic growth cones (Figure 1A) advancing at individually variable velocities (Figure 1A,B), but yielding a fairly constant mean growth rate of 20–30 µm/hr (Figure 1D). In addition to growth cones, axonal F-actin was also present in filopodia and in isolated patches on the shaft of the distal axonal region (Figure 1—figure supplement 1). Strikingly, bath application of 100 nM WIN 55,212-2 (WIN), a synthetic cannabinoid agonist, led to a rapid retraction of the F-actin-rich domain (Figure 1A), with mean retraction amplitude of 62.2 µm ± 5.2 (Figure 1C–E). Retraction was already detectable at 2 min after agonist exposure and typically reached a plateau between 10 and 20 min (Figure 1C,D). The morphology of retracted axons was characterized by an F-actin-rich retraction bulb (arrowheads on Figure 1A and Figure 1—figure supplement 1) and a thin membranous trailing remnant (open arrowheads on Figure 1A and Figure 1—figure supplement 1), the latter of which was not included in the length measurement. Pre-treatment with the CB1R selective antagonist/inverse agonist AM281 (AM) (1 µM) inhibited retraction (Figure 1D,E). Figure 1 with 1 supplement see all Download asset Open asset CB1R activation induces retraction of actin-rich growth cones. Cultured DIV8 hippocampal neurons co-expressing Flag-CB1R-eGFP and LifeAct-mCherry on (A–G) and LifeAct-mCherry only on (H and I). (A) Treatment with CB1R agonist WIN55,212-2 (WIN, 100 nM, added at 0 min) induces rapid retraction of the F-actin-rich domain (arrowheads). Open arrowheads: growth cone position at 0 min. (B) Progression of individual growth cones in control conditions. (C) WIN-induced retraction of individual growth cones. (D) Mean values of growth cone progression in control condition or after treatment with WIN with or without pre-treatment with the CB1R-specific antagonist AM281 (AM, 1 µM). WIN-induced growth cone retraction is effectively abolished by AM. (E) Amplitudes of growth cone retraction induced by different exo- and endocannabinoids, calculated as the net difference of mean growth cone position in the pre-treatment (PRE on D) and post-treatment (POST on D) time intervals from at least three independent experiments. (F) Concentration-response curve of WIN-induced retraction, 9 to 27 neurons per concentration from two independent experiments expressed as percentage of maximal retraction, Emax = 52.2 µm. (G) WIN-induced retraction (25 nM at 40 min) is fully reversible after WIN-washout (at 70 min), n = 9. (H) Mean values of growth cone retraction downstream of endogenous CB1R activation, from four pooled independent experiments, outliers were removed in accordance with the Grubb's test. (I) Amplitudes of growth cone retraction downstream of endogenous CB1R activation after treatment with WIN (100 nM), 2-AG (1 µM), or with WIN (100 nM) after pre-treatment with the CB1R-specific antagonist AM281 (AM, 1 µM). WIN-induced growth cone retraction is effectively abolished by AM. Values in D, F, G, and H are mean ± SEM; values in E and I are presented as boxplots; n.s = p > 0.05, ***p < 0.001, calculated using Kruskal–Wallis one-way ANOVA followed by Dunn's post-tests on (E and I) and paired t-test on (H). Scale bar: 20 µm. https://doi.org/10.7554/eLife.03159.003 Further pharmacological characterization showed that several other chemically distinct CB1R agonists, the endocannabinoid 2-arachidonoylglycerol (2-AG) (1 µM), the principal psychoactive marijuana constituent Δ9-tetrahydrocannabinol (Δ9-THC) (1 µM) and the synthetic agonists CP55,940 (100 nM) and HU-210 (100 nM) also produced significant retraction (Figure 1E). The retraction was saturable and concentration-dependent with a half-maximal effective concentration (EC50) value of around 20 nM for WIN (Figure 1F). When treatment with 25 nM WIN was followed by ligand-free wash-out, growth cone progression resumed normally showing the reversibility of cannabinoid-induced growth cone retraction (Figure 1G). Finally, this retraction was not a result of CB1R over-expression since treatment with 100 nM WIN or 1 µM 2-AG induced significant retraction with similar kinetics in neurons transfected only with LifeAct-mCherry (Figure 1H–I). However, the mean amplitude of retraction was lower and responses were more variable than in Flag-CB1R-eGFP-expressing neurons (compare Figure 1C,D with Figure 1H,I), as expected in a heterogeneous neuronal population expressing endogenous CB1Rs at highly variable levels (Leterrier et al., 2006). In addition, growth cone advance rapidly resumed even in the continued presence of 100 nM WIN (Figure 1H). In conclusion, our results show that cannabinoids trigger a rapid, saturable, and reversible retraction of actin-rich growth cones downstream of both endogenous and overexpressed CB1Rs. G12/G13 heterotrimeric proteins, Rho GTPase, ROCK, myosin II, and F-actin microfilaments mediate CB1R-induced rapid growth cone retraction First, we investigated which cytoskeletal elements act downstream of CB1Rs to induce rapid growth cone retraction. We expressed, in addition to LifeAct-mCherry, a GFP-tagged version of End-binding protein 3 (EB3-eGFP), which binds to endogenous microtubule (MT) plus ends without changing MT growth parameters and thus allows the visualization of MT structure and dynamics (Stepanova et al., 2003). Indeed, MTs in the entire neuron were labeled in green, with many bright comet-like fluorescent dashes in all the neuronal compartments, moving randomly in the cell body and directionally in axons and distal dendrites, representing dynamic MT plus ends (Stepanova et al., 2003). During 100 nM WIN-induced retraction the dynamics of the two main cytoskeletal polymers, F-actin and MTs, was remarkably different (Figure 2A). A significant portion of F-actin redistributed in the first 2–4 min after stimulation from its original location in growth cones into a more homogenous cable-like pattern on the distal axonal shaft (Figure 2B and Figure 2—figure supplement 1). In contrast, MTs bent during the same time frame forming periodic local loops (Figure 2A,A’,B and Video 1) before finally consolidating into a homogenously labeled retraction bulb. The F-actin cables (bundles of F-actin filaments, which are not separately resolved here by diffraction-limited microscopy), often co-localized with regions displaying periodic bends in MTs (Figure 2B'), suggesting that an F-actin-related force pulls strongly enough to bend MTs. This effect was not the result of the over-expression of the cytoskeletal markers EB3-eGFP or LifeAct-mCherry, since we could observe similar periodic MT bends, detected by post hoc immunohistochemistry, in neurons not expressing these markers (Figure 2—figure supplement 2). Figure 2 with 3 supplements see all Download asset Open asset CB1R-induced retraction is mediated by non-muscle myosin II dependent actomyosin contraction. Cultured hippocampal neurons co-expressing Flag-CB1R-eCFP, LifeAct-mCherry, and EB3-eGFP at DIV6 were treated by WIN (100 nM) at 0 min. (A) Microtubules (MT) bend and form small loops (arrowhead on A′) in the first 4 min (B) F-actin is reorganized from the growth cone tips and isolated patches to homogenous cable-like distribution in distal axonal shaft. (C–H) Pre-treatment with: (C and D) MT polymerization inhibitor nocodazole (10 µM), (E and F) actin polymerization inhibitor cytochalasin D (1 µM), (G and H) Non-muscle myosin II-inhibitor blebbistatin (25 µM). Scale bars: 5 µm on (A′) and (B′), 20 µm elsewhere. https://doi.org/10.7554/eLife.03159.005 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg CB1R activation induces retraction of actin-rich growth cones. Dynamic, F-actin-rich growth cone of a cultured hippocampal neuron co-expressing CB1R-eCFP, LifeAct-mCherry, and EB3-eGFP at DIV6 treated with 100 nM WIN at 10 min. Scale bar: 20 μm. https://doi.org/10.7554/eLife.03159.009 To investigate the requirement for polymerized actin microfilaments and MTs in these retractions, we depolymerized MTs with nocodazole (10 µM) and F-actin with cytochalasin D (1 µM) (Forscher and Smith, 1988). Nocodazole pre-treatment stopped growth cone advance but WIN still induced significant retraction (Figure 2C,D, Figure 3E and Video 2). In contrast, cytochalasin D inhibited both growth cone advance and WIN-induced retraction (Figure 2E,F, Figure 3E and Video 3) showing that while the presence of both F-actin and MTs is necessary for growth cone advance, as reported previously (Dent et al., 2003), only F-actin is necessary for CB1R-induced retraction. Figure 3 with 2 supplements see all Download asset Open asset CB1Rs activate non-muscle myosin II through heterotrimeric G12/G13 proteins, Rho GTPase, and ROCK. Cultured hippocampal neurons at DIV6 co-expressing a combination of LifeAct-mCherry, Flag-CB1R-eCFP, and EB3-eGFP as indicated and treated by WIN (100 nM) at 0 min. (A–B) Representative LifeAct-mCherry expressing growth cones (delimited with a dotted line) at 2 min after treatment with vehicle (A) or WIN (100 nM, B), labeled with a phospho-Myosin Light Chain (phosphoMLC) antibody. Arrowheads show the distal axon adjacent to the F-actin-rich growth cone where WIN induces rapid and strong upregulation of myosin light chain phosphorylation. (C) pMLC labeling intensity at the distal 50–60 µm of the axon, adjacent to the actin-rich growth cone, from neurons expressing LifeAct-mCherry (A) or co-expressing LifeAct-mCherry and Flag-CB1R-eCFP (B). The region-of-interest used to measure pMLC labeling intensity is delimited with a dotted line on a representative growth cone on Figure 3—figure supplement 1. (D) Amplitude of 100 nM WIN-induced growth cone retraction in neurons co-expressing LifeAct-mCherry and EB3-eGFP pre-treated with 25 µM blebbistatin or 10 µM Y-27632. (E) Amplitude of 100 nM WIN-induced growth cone retraction in neurons co-expressing LifeAct-mCherry, EB3-eGFP, and Flag-CB1R-eCFP pre-treated with: 1 µM cytochalasin D; 25 µM blebbistatin; 25 µM blebbistatin + 10 µM Y-27632; 10 µM Y-27632; 30 µM ML-7 + 10 µM Y-27632; 30 µM ML-7; 10 µM nocodazole; 100 ng/µl PTX. (F) Effect of siRNA-mediated knock-down of endogenous myosin IIA, IIB or of endogenous G12/G13 proteins on growth cone-retraction induced by 100 nM WIN in neurons co-expressing the three constructs, as compared to control (luciferase) siRNA. Results are pooled from at least two independent experiments, and outliers were removed in accordance with Grubb's test. Results in are expressed as boxplots. n.s p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001 calculated using Student's t-test on (C), Kruskal–Wallis one-way ANOVA followed by Dunn's post-tests on (D) and (E), and using one-way ANOVA followed by Newman–Keuls post-tests on (F). Scale bar: 10 µm. https://doi.org/10.7554/eLife.03159.010 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Effect of microtubule depolymerization on CB1R-induced growth cone retraction. Dynamic, F-actin-rich growth cone of a cultured hippocampal neuron co-expressing Flag-CB1R-eGFP and LifeAct-mCherry at DIV6, pre-treated with 10 µM Nocodazole at 20 min before treatment with 100 nM WIN at 40 min. Scale bar: 20 μm. https://doi.org/10.7554/eLife.03159.013 Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Effect of actin depolymerization on CB1R-induced growth cone retraction. Dynamic, F-actin-rich growth cone of a cultured hippocampal neuron co-expressing CB1R-eCFP, LifeAct-mCherry, and EB3-eGFP at DIV6 pre-treated with 1 µM cytochalasin D at 20 min before treatment with 100 nM WIN at 40 min. Scale bar: 20 μm. https://doi.org/10.7554/eLife.03159.014 A likely candidate for the generation of such rapid F-actin-related force, which is capable of bending microtubules, is non-muscle myosin II (NM II), an ATPase protein with actin cross-linking and contractile properties, which is activated by the phosphorylation of its regulatory light chain. The two main activators of NM II are myosin light chain kinase (MLCK) and ROCK, the latter being already known to participate in CB1R-induced cytoskeletal modifications (Ishii and Chun, 2002; Berghuis et al., 2007). This raises the possibility that ROCK- and/or MLCK-induced NM II contractility is responsible for the force-generation reported above. In order to directly investigate the implication of NM II, we pre-incubated neurons, for 20 min before WIN stimulation, with the highly selective NM II ATPase inhibitor blebbistatin (25 µM) that blocks NM II in an actin-detached state without perturbing F-actin polymerization (Kovacs et al., 2004). Blebbistatin pre-treatment induced substantial morphological changes of the growth cone, which continued to move forward in a rather disorganized fashion (Figure 2G and Video 4), typically transforming the growth cone lamellipodia into several dynamically advancing filopodia, as reported previously (Rosner et al., 2007). Remarkably, blebbistatin completely abolished WIN-mediated retraction of these dynamically advancing F-actin-rich structures (Figure 2G,H, Figure 3E and Video 4), suggesting that the main force-generating factor downstream of CB1R activation is actomyosin contractility. This inhibitory effect of blebbistatin was concentration dependent with half-maximal value of inhibition (EC50) of 116 nM (Figure 2—figure supplement 3). Immunocytochemical analysis of WIN-treated F-actin-rich growth cones at 2 min after the addition of WIN strikingly showed rapid and strong up-regulation of myosin light chain phosphorylation in the distal axon, adjacent to the F-actin-rich growth cone (Figure 3A–C and Figure 3—figure supplement 1), at the right place for the subsequent NMII-dependent contraction, both in neurons transfected only with LifeAct-mCherry (Figure 3A–C) and with Flag-CB1R-eCFP and LifeAct-mCherry (Figure 3C). Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Effect of NM II inhibition on CB1R-induced growth cone retraction. Dynamic, F-actin-rich growth cone of a cultured hippocampal neuron co-expressing Flag-CB1R-eGFP and LifeAct-mCherry at DIV6 pre-treated with 25 µM blebbistatin at 20 min before treatment with 100 nM WIN at 40 min. Only LifeAct-mCherry emission is visualized here. Scale bar: 20 μm. https://doi.org/10.7554/eLife.03159.015 Next, we investigated the mechanism coupling CB1R to the ROCK/NM II pathway. First, we showed that NMII-dependent growth cone contraction is not a result of CB1R over-expression, since treatment with blebbistatin (25 µM) or the ROCK inhibitor Y-27632 (10 µM) (Figure 3D) significantly inhibited endogenous CB1R-induced retraction of growth cones, previously presented on Figure 1I, in neurons transfected only with LifeAct-mCherry and EB3-eGFP. Then we used neurons expressing Flag-CB1R-eCFP, LifeAct-mCherry, and EB3-eGFP, our high-throughput experimental read-out, to characterize in detail the molecular mechanism of CB1R-induced actomyosin contractility. The amplitude of WIN-mediated retraction was significantly reduced by pre-treatment with the Rho inhibitor C3 transferase (1 µg/ml, Figure 3—figure supplement 2), the ROCK inhibitor Y-27632 (10 µM) (Figure 3E), but not by the MLCK-specific inhibitor ML-7 (30 µM) (Figure 3E). Treatment with the inactive (R)-(+)-blebbistatin (25 µM) stereoisomer was ineffective (data not shown). The implication of neuronal NM II was further confirmed by siRNA knock-down of endogenous NM IIA and NM IIB (Miserey-Lenkei et al., 2010), which resulted in significant reduction of WIN-mediated contractility as compared to control (anti-luciferase) siRNA (Figure 3F). Next, we investigated which heterotrimeric G-protein family couples CB1Rs to Rho activation. Notably, treatment with pertussis toxin (100 ng/µl), a specific inhibitor of Gi/o heterotrimeric proteins, which are generally considered as the main signaling pathway of CB1Rs (Howlett, 2005), did not decrease significantly cannabinoid-induced growth cone retraction (Figure 3E), similarly to a previously reported finding for ROCK-mediated induced cell rounding after anandamide treatment (Ishii and Chun, 2002). Another family of heterotrimeric G-proteins, G12/G13, may mediate rapid growth cone collapse, neurite retraction, and cell rounding in neuronal cell lines in response to certain GPCR agonists such as lysophosphatidic acid (LPA) (Katoh et al., 1998; Kranenburg et al., 1999). Therefore, we inactivated endogenous G12/G13 proteins in our hippocampal neuronal cultures by using two pools of 4 different siRNAs directed against rat G12- or G13-alpha proteins, respectively. Used separately, neither pool decreased WIN-induced growth cone retraction as compared to control (anti-luciferase) siRNA (Figure 3F). However, when we combined together 2 siRNAs of each pool, each resulting mixed pools efficiently inhibited WIN-mediated contractility (Figure 3F). These results show that the presence of either G12 or G13 is necessary and sufficient for CB1R-induced actomyosin contraction. Finally, to verify that CB1R-induced retraction is not an artifact of altered adhesion properties of growth cones in vitro, we co-transfected Flag-CB1R-eCFP, EB3-eGFP, and LifeAct-mCherry into embryonic rat brains using in utero electroporation at embryonic day 16 (E16). In organotypic slices prepared from the offspring between postnatal day 4 and 6 (P4–P6), numerous corticofugal F-actin-rich growth cones from layer II–III pyramidal neurons could be visualized by video microscopy at 48 hr after slice preparation (Figure 4A). Application of 1 µM WIN resulted in significant retraction of growth cones (Figure 4B,D and Video 5) through activation of CB1R since this effect could be prevented by pre-treatment with 5 µM AM281 (Figure 4D). This retraction displayed slower kinetics ex vivo than in vitro (compare to Figure 1) probably due to limited diffusion of the highly hydrophobic WIN into the slice and/or into differences in adhesive and mechanistic properties within the organotypic brain slice. Previously, we have shown that at around P5, cortical projection neurons still express CB1R, albeit at lower levels than at birth (Vitalis et al., 2008), thus we have replicated these experiments by expressing only the cytoskeletal markers EB3-eGFP and LifeAct-mCherry. WIN-mediated activation of endogenous CB1Rs typically led to arrest or retraction of numerous growth cones (Figure 4C,E). The relatively mild averaged effect is probably due to the variable level of endogenous CB1R expression in these neurons. Importantly, pre-treatment with blebbistatin (25 µM) efficiently blocked this effect (Figure 4E). Figure 4 Download asset Open asset Activation of exogenous or endogenous CB1Rs modifies growth cone dynamics ex vivo. Progression of dynamic, F-actin-rich corticofugal growth cones from organotypic slices cultured for 24 to 48 hr, prepared from P4-6 rat brains, previously electroporated in utero at E16 to express EB3-eGFP, LifeAct-mCherry, with or without Flag-CB1R-eCFP, was followed by time-lapse imaging. (A) Experimental design and illustration of a typical transfected cortical area (A) and of a typical labeled growing axon (B). For the illustration, the organotypic section was fixed and EB3-eGFP signal was enhanced by incubation with an anti-GFP antibody. (B–E) Response to CB1R agonist WIN (1 µM, added at 0 min). The F-actin-rich growth cone is indicated by arrowheads. Open arrowheads indicate growth cone position at 0 min (B, D) WIN-induced retraction in growth cones expressing EB3-eGFP, LifeAct-mCherry, and Flag-CB1R-eCFP is abolished by pre-treatment with 5 µM CB1R-specific antagonist AM281. (C, E) WIN-induced retraction in growth cones expressing EB3-eGFP and LifeAct-mCherry is abolished by pre-treatment with blebbistatin (25 µM). Results are pooled from at least two independent experiments and are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001, calculated using Student's t-test. Scale bar: 100 µm on A, 20 µm elsewhere. https://doi.org/10.7554/eLife.03159.016 Video 5 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg CB1R activation induces retraction of actin-rich growth cones in organotypic slices. Dynamic, F-actin-rich corticofugal growth cones from organotypic slices were cultured for 24 to 48 hr, prepared from P4-6 rat brains, previously electroporated with EB3-eGFP, LifeAct-mCherry, and Flag-CB1R-eCFP in utero (See Figure 3). Treatment with 1 µM WIN at 30 min induces retraction of the growth cone. Scale bar: 20 μm. https://doi.org/10.7554/eLife.03159.017 In conclusion, we show that CB1R activation significantly reorganizes growth cones through MLCK/ROCK-mediated NM II activation. This large-scale actomyosin contractility ultimately leads to the remodeling of MT structure in the distal axonal segments. In the developing brain, both activation of endogenous CB1Rs and actomyosin contractility are required for path-finding of CB1R expressing corticofugal axons In the embryonic brain, developing corticofugal axons express high levels of CB1Rs (Figure 5B’,B’’" @default.
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• W2989218046 title "Author response: Cannabinoid-induced actomyosin contractility shapes neuronal morphology and growth" @default.
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