ENDOCANNABINOIDS INHIBIT AXOTOMY-INDUCED EXCITOTOXICITY IN A MODEL OF SYNAPTIC PLASTICITY

Endocannabinoids are lipid signaling mediators that exert an important neuromodulatory role and confer neuroprotection in several types of brain injury. Neuroinflammation, microglial activation, oxidative stress, and excitotoxicity are variably combined in neurodegenerative diseases following trauma and can be modulated by endocannabinoids (for review, Rossi et al., Exp. Neurol. 2010). Unlike mammalian, the nervous system of low vertebrates (fish, amphibians and reptilia) is promptly able to regenerate neurons and spinal nerves after unjury. In the lizards, tail loss transects spinal nerves and the cut axons elongate in the regrowing tail, providing a natural paradigm of robust regenerative response of injured spinal motoneurons (Terni, Arch Ital Anat Embriol., 1920). On this basis we have investigated the possible involvement of the endocannabinoid system in the survival of motoneurons committed to axonal elongation in the context of the neuroregenerative response that follows caudotomy. By our previous investigations, a typical chromatolytic reaction to axotomy (cellular hypertrophy and nuclear eccentricity) was mostly showed in the first two weeks after caudotomy (reactive phase) (Cristino et al., J. Comp. Neurol. 2000). On this basis, in the present work, we compared the endocannabinoid system of regenerating motoneurons, at 10 days (reactive phase) and at 4 months after caudotomy (regenerative phase), to those of intact motoneurons. Single and multiple immunohystochemistry were performed for CB1 and CB2 receptors, 2-AG endocannabinoid synthesizing (DAGL alpha) and degrading (MAGL) enzymes, for glutamate and GABA vescicular transmitter transportes (VGluT1 and VGAT, respectively) and for the glial marker GFAP and for the activated microglia Iba-1. Intact motoneurons exhibited DAGL- immunoreactivity (-ir) in the somatodendritic compartment and were immersed in a meshwork of MAGL/CB1-ir puncta on VGluT1-ir fibers. Very low CB2-ir expression was found in the gray matter of intact spinal cord which showed an intense and diffuse GFAP-ir and the lowest Iba-1 reactivity. During the reactive fase to axotomy, a general decrease of markers of the endocannabinoid system was observed, except for CB2-ir which, diffusely, colocalize with Iba-1-ir microglial cells. In the regenerative phase a strong DAGL-and CB1-ir, on both glutamatergic and GABAergic axon terminals surrounding motoneurons, was observed. CB-2-ir and Iba-1-ir came back to normal levels. Our data suggest a model of retrograde control by CB1 over glutamate release. In the reactive phase the wide-ranging temporary loss of contact between some presynaptic axons and the motoneurons could explain the endocannabinoid system down regulation. Cannabinoids exert anti-glutamatergic and anti-inflammatory actions through activation of the CB1 and CB2 receptors, respectively. Activation of CB1 receptors may therefore inhibit glutamate release from presynaptic nerve terminals and reduce the postsynaptic calcium influx in response to glutamate receptor stimulation. Meanwhile, CB2 receptors may influence inflammation, whereby receptor activation reduces microglial activation, resulting in a decrease in microglial secretion of neurotoxic mediators. These results collectively lead to a model of the motoneuron survival and axonal regeneration whereby 2-AG mediates the CB1-induced inhibition of glutammate release.