In vivo endocannabinoid dynamics at the timescale of physiological and pathological neural activity

Abstract

The brain's endocannabinoid system is a powerful controller of neurotransmitter release, shaping synaptic communication under physiological and pathological conditions. However, our understanding of endocannabinoid signaling in vivo is limited by the inability to measure their changes at timescales commensurate with the high lability of lipid signals, leaving fundamental questions of whether, how, and which endocannabinoids fluctuate with neural activity unresolved. Using novel imaging approaches in awake behaving mice, we now demonstrate that the endocannabinoid 2-arachidonoylglycerol, not anandamide, is dynamically coupled to hippocampal neural activity with high spatiotemporal specificity. Furthermore, we show that seizures amplify the physiological endocannabinoid increase by orders of magnitude and drive the downstream synthesis of vasoactive prostaglandins that culminate in a prolonged stroke-like event. These results shed new light on normal and pathological endocannabinoid signaling in vivo.

Keywords: 2-AG; AEA; COX-2; EP(1); MAGL; PGE(2); endocannabinoid; postictal hypoxia; prostaglandin; seizure.

Figure 1.. Locomotion- and seizure-driven endocannabinoid increase depends on 2-AG, not AEA.

(A) Experimental set-up. 2-photon imaging of calcium and eCB signals from awake, head-fixed mice. (B) Enzymes involved in eCB synthesis and hydrolysis and corresponding inhibitors (abbreviated) used in the study. (C) eCB (green) and calcium (magenta) traces, averaged across cells, from a representative mouse during locomotion showing altered eCB signal with 2-AG perturbation. (D) Representative traces during seizure activity showing a similar pharmacological sensitivity to that of eCB activity during locomotion C. (E) Summary data for C reported as a percent change from vehicle. One-way ANOVAs were performed on the maximum ΔF/F for each session’s run-triggered average (see Figure S1B). n=12 for DO34, JZL184, and URB597 (eCB: F_3,11=26.43, Dunnett post-test **p<0.01; calcium F_3,11=3.18). n=6 for LEI401 (eCB: F_4,5=8.61, calcium; F_4,5=3.57, LEI non-significant for both). (F) DO34 significantly prolonged electrographic seizure duration (n=12 group: Friedman test F_3,11=9.65, Dunnett post-test *p<0.05; n=6 group: Friedman test F_4,5=8.42, LEI non-significant). (G) Summary data for D reported as a percent change in the area under curve for eCB (n=12 group: F_3,11=156.0, Dunnett post-test ****p<0.0001, n=6 group: F_4,5=49.73, LEI non-significant) and calcium (n=12 group: F_3,11=2.00, n=6 group: F_4,5=4.54). See also Figure S1A.

Figure 2.. Spatiotemporal characteristics of activity-dependent 2-AG signaling.

(A) eCB and calcium traces were pulled from active neurons identified in the baseline period (no seizure). Inset shows neighboring cells with traces displayed in E. (B) eCB traces from each cell were moved forward and backward in time to determine the optimal temporal offset (yielding maximum Pearson r value). Histogram shows an example from a vehicle locomotor session from one mouse with a mean lag of 1s across cells. (C) The temporal offset, or eCB lag, was prolonged by JZL pre-treatment for locomotor sessions (F_4,5=14.98, Dunnett post-test **p<0.01). (D) Seizure traces similarly show a prolonged temporal offset by JZL (F_4,5=26.13, Dunnett post-test ****p<0.0001) (E) eCB traces from each cell were correlated to the calcium activity within its region of interest and to all other cells to determine spatial specificity (Pearson r values). Example traces from a vehicle session show stronger within cell coupling. (F) Reduced correlation values were observed between cell relative to within for locomotor sessions (one-sample t-tests). No significant differences were found on the amount of reduction in correlation value across drug treatments (F_4,5=1.67). Note the lower correlation values with DO34 (see Figure S1C). (G) Reduced correlation values were observed between cell vs. within for seizure recordings (one-sample t-tests). No significant differences were found on the amount of reduction in correlation value across treatments (F_4,5=0.39). Note the lower correlation values with JZL184 (see Figure S1D).

Figure 3.. The endocannabinoid-prostaglandin pathway leads to severe postictal hypoxia.

(A) Experimental set-up (left) and molecular pathway with associated pharmacological inhibitors (right). (B) Changes in oxygen profiles following drug pre-treatment. X and Y scales apply to all four graphs. (C) All four drugs significantly reduced the area below 10mmHg (One sample t-test, DO34 – t₄=7.51; JZL184 – t₄=5.75; celecoxib – t₄=22.73; ONO8130 – t₄=8.03). **p<0.01, ****p<0.0001. See also Figure S2 and Figure S3.