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. 2011 Nov;164(6):1672-83.
doi: 10.1111/j.1476-5381.2011.01486.x.

COX-2 and fatty acid amide hydrolase can regulate the time course of depolarization-induced suppression of excitation

A Straiker  1 , J Wager-Miller, S S Hu, J L Blankman, B F Cravatt, K Mackie
Affiliations

COX-2 and fatty acid amide hydrolase can regulate the time course of depolarization-induced suppression of excitation

A Straiker et al. Br J Pharmacol. 2011 Nov.

Abstract

Background and purpose: Depolarization-induced suppression of inhibition (DSI) and excitation (DSE) are two forms of cannabinoid CB(1) receptor-mediated inhibition of synaptic transmission, whose durations are regulated by endocannabinoid (eCB) degradation. We have recently shown that in cultured hippocampal neurons monoacylglycerol lipase (MGL) controls the duration of DSE, while DSI duration is determined by both MGL and COX-2. This latter result suggests that DSE might be attenuated, and excitatory transmission enhanced, during inflammation and in other settings where COX-2 expression is up-regulated.

Experimental approach: To investigate whether it is possible to control the duration of eCB-mediated synaptic plasticity by varied expression of eCB-degrading enzymes, we transfected excitatory autaptic hippocampal neurons with putative 2-AG metabolizing enzymes: COX-2, fatty acid amide hydrolase (FAAH), α/β hydrolase domain 6 (ABHD6), α/β hydrolase domain 12 (ABHD12) or MGL.

Key results: We found that overexpression of either COX-2 or FAAH shortens the duration of DSE while ABHD6 or ABHD12 do not. In contrast, genetic deletion (MGL(-/-)) and overexpression of MGL both radically altered eCB-mediated synaptic plasticity.

Conclusions and implications: We conclude that both FAAH and COX-2 can be trafficked to neuronal sites where they are able to degrade eCBs to modulate DSE duration and, by extension, net endocannabinoid signalling at a given synapse. The results for COX-2, which is often up-regulated under pathological conditions, are of particular note in that they offer a mechanism by which up-regulated COX-2 may promote neuronal excitation by suppressing DSE while enhancing conversion of 2-AG to PGE(2) -glycerol ester under pathological conditions.

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Figures

Figure 1
Figure 1
Overexpression of COX-2 with endogenous MGL shortens the duration of DSE. (A) ‘Dose’–response for DSE using a range of depolarizations from 50 ms to 10 s. The wild-type DSE dose–response is shown for comparison. (B) Averaged DSE time courses for neurons transfected with COX-2. Arrow indicates the point at which the cell is depolarized for 3 s. (C) Recovery time courses for COX-2-transfected and control cells. t1/2-values with 95% CIs are listed below the curves.
Figure 2
Figure 2
Overexpression of FAAH with endogenous MGL also shortens DSE duration. (A) ‘Dose’–response for DSE of FAAH-transfected neurons (FAAH T/F) using a range of depolarizations from 50 ms to 10 s. The wild-type DSE dose–response is shown for comparison. (B) Averaged DSE time courses for neurons transfected with FAAH and FAAH transfected neurons subsequently treated with URB597 (250 nM). (C) DSE recovery time courses for FAAH-transfected and control cells. t1/2-values with 95% CIs are listed below the curves. (D) Micrograph shows FAAH staining in autaptic hippocampal cultures. Scale bar = 25 µm. (E) Micrograph shows FAAH staining does not colocalize with the astrocyte cell marker GFAP. Scale bar = 25 µm. (F) Micrograph shows that FAAH is expressed in neurons transfected with FAAH, and that it is expressed in processes that are labelled and unlabelled with dendritic marker MAP2. Left panel shows HA11 staining against the HA-tagged FAAH protein. Centre panel shows dendritic marker MAP2. Right panel shows the composite of the left and centre panels, with overlap in yellow. Scale bar = 15 µm.
Figure 3
Figure 3
ABHD6 and ABHD12 do not speed up DSE recovery. (A) ‘Dose’–response for DSE in ABHD6-transfected neurons (ABHD6 T/F) using a range of depolarizations from 50 ms to 10 s. The wild-type DSE dose–response is shown for comparison. (B) Averaged DSE time courses for neurons transfected with ABHD6. Arrow indicates the time point at which the neuron is depolarized for 3 s. (C) Recovery time courses for ABHD6-transfected and control cells. t1/2-values with 95% CIs are listed as inset. (D) ‘Dose’–response for DSE in ABHD12-transfected neurons (ABHD12 T/F) using a range of depolarizations from 50 ms to 10 s. The wild-type DSE dose–response is shown for comparison. ABHD12 decreases the maximum inhibition for 10 s depolarization. (E) Averaged DSE time courses for neurons transfected with ABHD12. Arrow indicates the point at which the neuron is depolarized for 3 s. (F) Recovery time courses for ABHD12-transfected and control neurons. t1/2-values with 95% CIs are listed as insets. (G) Micrograph shows that ABHD6 is expressed in neurons transfected with ABHD6 in processes that are labelled and unlabelled with the dendritic marker MAP2. Left panel shows HA11 staining of the HA-tagged ABHD6 protein (red). Centre panel shows the dendritic marker MAP2. Right panel shows the composite. Scale bar = 15 µm. (H) Transfected ABHD12 is also widely expressed in neurons. Left panel shows ABHD12-HA11 staining. Centre panel shows MAP2 staining. Right panel is the composite. Scale bar = 25 µm.
Figure 4
Figure 4
Summary of changes to DSE t1/2 after overexpression of four endocannabinoid-metabolizing enzymes. Bar graph shows t1/2-values of DSE (s) for untransfected neurons (control) versus neurons transfected with COX-2, FAAH, ABHD6 or ABHD12. Error bars represent 95% CIs with a Bonferroni correction for multiple comparisons (see Methods).
Figure 5
Figure 5
Robust DSE requires an optimal level of MGL. (A) Sample time course shows epscs in MGL−/− neuron after DSE stimulation (arrow), 2-AG treatment (5 µM) and reversal by CB1 antagonist SR141716 (200 nM). Example of WT DSE time course is included for comparison. (B) Averaged DSE time courses from MGL−/− and MGL+/+ cultures. (C) Scatter plot of inhibition values after the DSE stimulus in MGL−/− neurons shows that DSE is diminished in most neurons. (D) Bar graph shows epsc inhibition in response to DSE or 2-AG (5 µM) in MGL−/− neurons. (E) WIN55212-2 responses are desensitized in MGL KO neurons. Concentration–response curves for WIN55212-2 in MGL KO and wild-type (C57) neurons. The CD1 wild-type WIN55212-2 concentration–response is shown for comparison (adapted from Straiker and Mackie, 2005). (F) Averaged DSE time course for wild-type neurons transfected with MGL under control conditions or after treatment overnight with MGL blocker JZL184 (200 nM). Arrow indicates the point at which the cell was depolarized for 3 s. (G) Sample time courses in response to 2-AG (5 µM) in MGL-transfected neuron (the incomplete recovery is probably due to 2-AG-induced LTD; Kellogg et al., 2009) and in MGL-transfected neurons treated overnight with JZL 184 (200 nM). (H) Bar graph shows average 5 µM 2-AG responses for untransfected neurons, MGL-transfected neurons and MGL-transfected neurons treated overnight with JZL184 (200nM). (I) Micrograph shows MGL-transfected neuron stained for HA11 and MAP2, a dendritic marker. Bottom panel shows overlay of the two channels (yellow represents overlap, scale bar = 25 µm). (J) Micrographs show MGL antibody (top panel) staining for the axonal marker, 2H3 (centre panel) and overlap (bottom panel, overlap in yellow) in a WT untransfected neuron. (K) Same layout as (J), but in MGL−/− neuronal cultures. Panels (J–K) show that the MGL antibody stains axons. Scale bar = 20 µm.
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