ABSTRACT
Navigation of retinal projections towards their targets is regulated by guidance molecules and growth cone transduction mechanisms. Here, we present in vitro and in vivo evidences that the cannabinoid receptor 2 (CB2R) is expressed along the retino-thalamic pathway and exerts a modulatory action on axon guidance. These effects are specific to CB2R since no changes were observed in mice where the gene coding for this receptor was altered (cnr2 (-/-)). The CB2R induced morphological changes observed at the growth cone are PKA dependent and require the presence of the netrin-1 receptor, Deleted in Colorectal Cancer. Interfering with endogenous CB2R signalling using pharmacological agents increased retinal axon length and induced aberrant projections. Additionally, cnr2 (-/-) mice showed abnormal eye-specific segregation of retinal projections in the dorsal lateral geniculate nucleus (dLGN) indicating CB2R's implication in retinothalamic development. Overall, this study demonstrates that the contribution of endocannabinoids to brain development is not solely mediated by CB1R, but also involves CB2R.
Subject(s)
Axons/metabolism , Endocannabinoids/metabolism , Geniculate Bodies/metabolism , Receptor, Cannabinoid, CB2/genetics , Retinal Ganglion Cells/metabolism , Animals , Axons/ultrastructure , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Embryo, Mammalian , Gene Expression Regulation, Developmental , Geniculate Bodies/cytology , Geniculate Bodies/growth & development , Mice , Mice, Knockout , Netrin Receptors , Neurogenesis/physiology , Primary Cell Culture , Receptor, Cannabinoid, CB1/genetics , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/deficiency , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Retinal Ganglion Cells/cytology , Visual Pathways/physiologyABSTRACT
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.
Subject(s)
Axons/physiology , Neurons/metabolism , Receptor, Cannabinoid, CB1/physiology , Receptors, Cell Surface/physiology , Retina/metabolism , Tumor Suppressor Proteins/physiology , Animals , Cannabinoid Receptor Modulators/metabolism , Cell Membrane/metabolism , Cells, Cultured , Cyclic AMP/physiology , Cyclic AMP-Dependent Protein Kinases/physiology , DCC Receptor , Drug Inverse Agonism , Growth Cones/physiology , In Vitro Techniques , Mice , Mice, Knockout , Neurotransmitter Agents/metabolism , Protein Transport , Receptor, Cannabinoid, CB1/agonists , Receptor, Cannabinoid, CB1/genetics , Retina/embryology , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/ultrastructure , Thalamus/embryology , Thalamus/metabolismABSTRACT
During development, retinal ganglion cells (RGCs) extend their axons toward their thalamic and mesencephalic targets. Their navigation is largely directed by guidance cues present in their environment. Since cAMP is an important second messenger that mediates the neural response to guidance molecules and its intracellular levels seem to decrease significantly following birth, we tested whether modulation of the cAMP/protein kinase A (PKA) pathway would affect the normal development of RGC axons. At postnatal day 1, hamsters received a unilateral intraocular injection of either 0.9% saline solution, 12 mM of the membrane-permeable cAMP analogue (dibutyryl cAMP; db-cAMP), or 10 microM of the PKA inhibitor KT5720. Intraocular elevation of cAMP significantly accelerated RGC axonal growth while inhibition of PKA activity decreased it. Moreover, when highly purified RGC cultures were treated with forskolin (an activator of adenylate cyclase) or cAMP analogues (db-cAMP and Sp-cAMP), neurite length, growth cone (GC) surface area and GC filopodia number were significantly increased. This indicates that intraocular elevation of cAMP acts directly on RGCs. Since these effects were prevented by PKA inhibitors, it demonstrates that cAMP also exerts its action via the PKA pathway. Taken together, these results suggest that the cAMP/PKA cascade is essential for the normal development of retinothalamic projections.
