No abstract

Endocannabinoids (eCBs) are retrograde lipid neuromodulators involved in many physiologically important processes. However, their release and dynamics in the brain remain largely unknown, in part due to lack of probes capable of reporting real-time eCBs with sufficient spatiotemporal resolution. Here, we developed a new G protein-coupled receptor Activation Based eCB sensor GRABeCB2.0 using the human CB1 cannabinoid receptor and a circular-permutated EGFP. GRABeCB2.0 exhibited proper cell membrane trafficking, ~seconds kinetics, high specificity and robust fluorescence response to eCBs at physiological concentrations. Using GRABeCB2.0, we detected evoked eCB dynamics in both cultured neurons and acute brain slices. Interestingly, we also observed spontaneous compartmental eCB transients that spread ~11 μm in cultured neurons, suggesting locally-restricted eCB signaling. By expressing GRABeCB2.0in vivo, we readily observed foot-shock elicited and running triggered eCB transients in mouse amygdala and hippocampus, respectively. Lastly, using GRABeCB2.0 in an epilepsy model, we observed a spreading eCB wave following a calcium wave in mouse hippocampus. In summary, GRABeCB2.0 is a powerful new probe to resolve eCB release and dynamics under both physiological and pathological conditions.

Programmed cell death (PCD) has an important role in sculpting organisms during development. However, much remains to be learned about the molecular mechanism of PCD. We found that ectopic expression of tousled-like kinase (tlk) in Drosophila initiated a new type of cell death. Furthermore, the TLK-induced cell death is likely to be independent of the canonical caspase pathway and other known caspase-independent pathways. Genetically, atg2 RNAi could rescue the TLK-induced cell death, and this function of atg2 was likely distinct from its role in autophagy. In the developing retina, loss of tlk resulted in reduced PCD in the interommatidial cells (IOCs). Similarly, an increased number of IOCs was present in the atg2 deletion mutant clones. However, double knockdown of tlk and atg2 by RNAi did not have a synergistic effect. These results suggested that ATG2 may function downstream of TLK. In addition to a role in development, tlk and atg2 RNAi could rescue calcium overload-induced cell death. Together, our results suggest that TLK mediates a new type of cell death pathway that occurs in both development and calcium cytotoxicity.