| The cannabinoid 1 receptor (CB1R) is a G-protein coupled receptor that binds the active constituents of marijuana (cannabis). CB1R is activated by the endocannabinoids anandamide and 2-arachidonylglycerol to modulate neurotransmission in the central nervous system. CB1R has also been implicated as a modulator of developmental processes in the mammalian brain, including progenitor cell proliferation and fate determination. Controversy exists on how CB1R regulates neuronal differentiation in the adult brain. Moreover, no evidence exists for the modulation of neuronal differentiation by CB1R in the adult spinal cord. The purpose of this thesis was to determine whether inhibition of CB1R could promote neuronal differentiation of adult spinal cord- derived progenitor cells in vitro. To test this hypothesis, adult spinal cord cultures were treated with the CB1R antagonist AM251, and the extent of neuronal differentiation was assessed by quantification of cells positive(+) for the neural progenitor cell marker Nestin, the immature neuronal markers betaIII tubulin and Doublecortin (DCX), as well as their capacity to incorporate ethynyldeoxyuridine (EdU). AM251 treatment decreased the number of Nestin+ cells, and increased the number of betaIII tubulin+ and DCX+ cells. AM251's effect was blocked by concomitant administration of CB1R agonists, WIN55,212-2 or ACEA. These results suggest that CB1R antagonism induces neuronal differentiation of adult spinal cord progenitors in vitro. To determine if the chronic absence of CB1R also promoted neuronal differentiation, spinal cord cultures from adult CB1R knock-out (KO) mice were characterized. Consistent with our hypothesis, spinal cord cultures from CB1R KO contained significantly higher levels of DCX+ cells compared to wild type (WT) cultures. Moreover, AM251 promoted neuronal differentiation in cultures derived from WT, but not from CB1R KO mice. Since CB1R modulates synaptic transmission, and synaptic transmission has been shown to influence progenitor cell fate, we evaluated whether the AM251-induced neuronal differentiation was affected by chronic inactivity. Interestingly, either the presence of the voltage-dependent sodium channel blocker tetrodotoxin (TTX), or the removal of mature neurons, inhibited the AM251-induced increase in DCX+ cells. In summary, antagonism or absence of CB1R promotes neuronal differentiation in adult spinal cord cultures, which may be mediated, in part, by TTX-sensitive neuronal activity. |
