Concerted action of CB1 cannabinoid receptor and deleted in colorectal cancer in axon guidance

Abstract

Endocannabinoids (eCBs) are retrograde neurotransmitters that modulate the function of many types of synapses. The presence of eCBs, their CB1 receptor (CB1R), and metabolizing enzymes at embryonic and early postnatal periods have been linked to developmental processes such as neuronal proliferation, differentiation, and migration, axon guidance, and synaptogenesis. Here, we demonstrate the presence of a functional eCB system in the developing visual system and the role of CB1R during axon growth and retinothalamic development. Pharmacological treatment of retinal explants and primary cortical neuron cultures with ACEA, a selective CB1R agonist, induced a collapse of the growth cone (GC). Furthermore the application of AM251, a CB1R inverse agonist, to the neuronal cultures increased the surface area of GC. In vivo, intraocular injection of ACEA diminished retinal projection growth, while AM251 promoted growth and caused aberrant projections. In addition, compared with their wild-type littermates, CB1R-deficient adult mice revealed a lower level of eye-specific segregation of retinal projections in the dorsal lateral geniculate nucleus. Finally, we found that pharmacological modulation of CB1R affected the trafficking of Deleted in colorectal cancer (DCC) receptor to the plasma membrane in a PKA-dependent manner. Moreover, pharmacological inhibition or genetic inactivation of DCC abolished the CB1R-induced reorganization of the GC. Overall, these findings establish a mechanism by which the CB1R influences GC behavior and nervous system development in concerted action with DCC.

Figure 1.

Expression of the eCB system in retinal projections. A, Retinal ganglion cell projections of E14–E15 mice retinal explants cultured for 1 DIV and immunolabeled for CB1R, NAPE-PLD, DAGLα, FAAH, MGL, and NFL. B, C, A retinal ganglion cell and a primary cortical neuron immunostained for CB1R. D, Western analysis of CB1R expression in primary cortical neuron cultures at different DIVs. Molecular weight markers are indicated on the right side of the panel.

Figure 2.

Pharmacological activation and blockade of the CB1R reorganizes the morphology of the GC and modulates axonal growth and navigation. A, D, Photomicrographs of retinal projection GCs cultured for 1 DIV (A) and of primary neurons cultured for 2 DIV (D) treated with a CB1R agonist (ACEA), an inverse agonist (AM251), an antagonist (O2050), or FSK. B, C, E, F, GC surface area and filopodia number analysis following a 60 min pharmacological treatment of retinal explant (B, C) and cortical neuron cultures (E, F). G, Photomicrographs of retinal explant cultures treated with ACEA or AM251. H, Quantification of retinal projection growth subsequent to treatments with CB1R agonist or inverse agonist. I, Photomicrographs of RGC projection GCs taken at t = 0 min and 60 min during GC turning assay experiments. Arrows indicate the direction of the microgradient, while arrowheads show the neurite stimulated. I′, Representative photomicrograph of the microgradient created during drug stimulation. J, Scatter plot analysis of GC behavior as a result of CB1R agonist, db-cAMP, or vehicle application. K, L, Quantifications of neurite elongation and GC turning responses following drug stimulation. Values are presented as means ± SEM. * indicates a significant change compared with the control group, p < 0.01 (B, C, E, F, H).

Figure 3.

The CB1R modulates growth cone morphology through the cAMP/PKA pathway. A, Western blot analysis of downstream kinases previously suggested to modulate eCB action. Molecular weight markers are indicated on the right side of the panel. B, Quantification of the optical density for P-AKT, P-ERK1/2, and P-S6 following 10 min stimulation with ACEA or AM251. C, Photomicrographs of cortical neuron cultures immunolabeled for cAMP following 15 min stimulation with ACEA, AM251, O2050, or FSK. D, Quantification of cAMP fluorescence intensity at the GC. # and * indicate significant changes compared with the control group, p < 0.05 and p < 0.0001, respectively. E, Western blot analysis of PKA phosphorylation following 10 min stimulation with ACEA or AM251. Molecular weight markers are indicated on the right side of the panel. F, Quantification of the optical density for P-PKA. G, Photomicrographs of GCs treated with CB1R inverse agonist and agonist, FSK and PKA inhibitors. H, I, GC surface area and filopodia analysis subsequent to the addition of a CB1R inverse agonist and simultaneous inhibition of PKA or the activation of PKA and the addition of a CB1R agonist. Values are presented as mean ± SEM. * indicates a significant change compared with the AM251 group and # with the FSK group (p < 0.0001).

Figure 4.

DCC is necessary for CB1R-induced reorganization of the GC. A, E14–E15 mice RGC projections immunolabeled for DCC, CB1R, and L1. B, C, Immunocytochemical and Western blot analysis of DCC expression in primary retinal neuron cultures obtained from CB1R^−/− mice. D, E, Immunocytochemical and Western blot analysis of CB1R expression in primary neuron cultures obtained from dcc^−/− mice. F, Total and biotinylated proteins analyzed by Western blot with antibodies directed against either DCC, β-actin, CB1R, or NCAM. For Western blots, molecular weight markers are indicated on the right side of the panel. G, H, GC surface area and filopodia number analysis of cortical neuron cultures treated with αDCCfb followed by the addition of either a CB1R inverse agonist or antagonist (AM251 or O2050, respectively) or FSK. I–L, GC surface area and filopodia number analysis of dcc^−/− (I, K) and dcc^+/+ (J, L) primary neuron cultures treated with either ACEA, AM251, O2050, or FSK. Values are presented as mean ± SEM. * indicates a significant change compared with the AM251, O2050, or FSK in G and H, and the control group in J and L (p < 0.05).

Figure 5.

Pharmacological modulation of CB1R activity modulates retinal projection development in vivo. A, Photomicrographs of P5 retinal projections in the DTN in the control, ACEA, and AM251 groups. Lower panel shows higher magnification of retinal projections. B, C, Quantification of retinal projection development in the DTN; axon collateral growth (B) and collateral branch number (C) of treated groups compared with the control group. D, Photomicrographs of retinal projections in the SC in the control and AM251-treated groups. E, Retinogeniculate projection patterns visualized following CTb conjugated to Alexa-546 (CTb-546; red) and CTb-488 (green) injections into left and right eyes of CB1R^−/− and CB1R^+/+ adult mice. F, Quantification of the percentage of the dLGN receiving overlapping inputs in CB1R^−/− and CB1R^+/+ adult mice. Data are presented as means ± SEM. * indicates a significant change compared with the control group in C (p < 0.05) and the CB1R^+/+ group in F (p < 0.005).