A fluorescent sensor for spatiotemporally resolved endocannabinoid dynamicsin vitroandin vivo

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.

Cannabis use, abuse, and withdrawal: Cannabinergic mechanisms, clinical, and preclinical findings

“…This depolarization-induced synaptic depression has been described at GABAergic synapses (labeled DSI) and glutamatergic synapses (labeled DSE) (Diana & Marty, 2004). The duration of this depression appears to coincide with the duration of extracellular increases in eCBs (Kim & Alger, 2004;Liput et al, 2020;Pan et al, 2009). Pharmacological and genetic knockout experiments indicate that depolarization and activation of postsynaptic voltage-gated calcium channels (VGCCs) produces increased 2-AG production and release and this eCB traverses the synaptic cleft in a retrograde manner to act on presynaptic CB1 and induce depression (Castillo et al, 2012).…”

“…Indeed there appears to be an ever expanding palette of sensors specific to different neurotransmitters and neuromodulators (O'Banion & Yasuda, 2020), including eCBs (Dong et al, 2020;Liput et al, 2020), which will allow for unprecedented analysis of how different neural circuits respond to various cannabis treatment procedures.…”

A solution to temporal credit assignment using cell-type-specific modulatory signals

“…Indeed, our computational experiments suggest that the level of modulatory input decreases over training iterations and sharply rises in response to changes in task condition (Figure S7). Rapidly maturing technologies on in-vivo monitoring of neuromodulators [72, 73, 41] suggest that such predictions of our model can be tested experimentally.…”

Postsynaptic synucleins mediate vesicular exocytosis of endocannabinoids

“…A 920-nm wavelength was used to excite the GRABeCB2.0 sensor, and fluorescence was collected using a 495-540-nm filter. Electrical stimulation consisted of 10 pulses (0.2 ms duration) delivered at 20 Hz (Dong et al, 2020). Pharmacological experiments included addition of 10 µM AEA and/or 10 µM AM251 to the ACSF perfusion at 2-3ml/min.…”