Article Figures & Data
Figures
- Figure 1.
DAGL-α−/− mice and C57BL/6 mice treated with the DAGL inhibitor DO34 show disrupted TBS-induced LTP in CA1 of the hippocampus. The magnitude of LTP was significantly decreased in both (A) DAGL-α−/− mice (n = 8) compared with DAGL-α+/+ (n = 9) and DAGL-α+/− mice (n = 11), as well as in slices from (B) DO34-treated C57BL/6J mice (n = 7) compared with VEH-treated (n = 9) or DO53-treated mice (n = 8). The magnitude of PTP during the initial induction phase was also significantly decreased in the DO34 group compared with VEH or DO53. Data are mean ± SEM.
- Figure 2.
DAGL-α−/− mice are profoundly impaired in MWM Fixed Platform task performance. A, Fixed Platform task experimental timeline (days). B, During MWM Fixed Platform acquisition, DAGL-α−/− mice (n = 13) exhibited longer distances to the platform than both DAGL-α+/+ mice (n = 15) and DAGL-α+/− mice (n = 13), as well as showed probe trial performance deficits of (C) longer distances to the prior platform position, (D) fewer platform entries, and (E) a lower spatial preference for the target quadrant. No significant difference in (F) cued task performance or (G) swim speed suggests that the DAGL-α−/− mice performance deficits were unaffected by sensorimotor or motivational impairments. No significant differences were evident in body weight at either (H) baseline or (I) during Fixed Platform acquisition. Data are mean ± SEM. **p < 0.01 versus DAGL-α+/+ mice. ***p < 0.001 versus DAGL-α+/+ mice. $p < 0.05 versus DAGL-α+/− mice. $$p < 0.01 versus DAGL-α+/− mice. $$$p < 0.001 versus DAGL-α+/− mice.
- Figure 3.
DAGL-α−/− mice exhibit altered MWM search strategy. DAGL-α−/− mice (n = 13) (A) used more nonspatial and thigmotaxic swim paths than DAGL-α+/+ mice (n = 15) on Fixed Platform day 10 and (B) spent more time in the MWM outer ring than both DAGL-α+/+ and DAGL-α+/− mice (n = 13) during Fixed Platform acquisition. C, Fixed Platform day 10 representative swim traces show spatial (direct and self-orienting) swim paths in DAGL-α+/+ mice, spatial (direct) and nonspatial (circling) swim paths in DAGL-α+/− mice, and nonspatial (circling and thigmotaxic) swim paths in DAGL-α−/− mice. Data are mean ± SEM. **p < 0.01 versus DAGL-α+/+ mice. ***p < 0.001 versus DAGL-α+/+ mice. $p < 0.05 versus DAGL-α+/− mice.
- Figure 4.
DO34 delays MWM Fixed Platform acquisition but does not affect probe trial performance. A, Fixed Platform task acquisition experimental timeline (days). B, During MWM Fixed Platform acquisition, mice treated with 30 mg/kg DO34 (n = 12) exhibited longer distances to the platform than VEH (n = 15), 0.3 mg/kg DO34 (n = 12), and 30 mg/kg DO53 (n = 12). C, Fixed Platform task probe trial (expression) experimental timeline (days). Following drug-free MWM Fixed Platform acquisition training, no change in performance was seen at probe trial between drug treatments (VEH n = 9; or 30 mg/kg DO34 n = 8) for any measure, (D) distances to the prior platform position, (E) platform entries, or (F) spatial preference for the target quadrant. No difference in (G) cued task performance suggests that the high-dose DO34 did not affect sensorimotor or motivational components of MWM performance. H, Mice administered 30 mg/kg DO34 showed increased swim speeds during Fixed Platform acquisition compared with VEH-treated mice on days 1, 2, 4, 5, and 8, but 0.3 mg/kg DO34 reduced swim speeds on days 7, 9, and 10. No significant differences were evident between groups on Fixed Platform training day 1 across trials (data not shown), or in body weight at (I) baseline, yet a significant interaction between drug and day on body weight throughout (J) acquisition training showed reductions compared with VEH at 30 mg/kg DO34 (days 2–15), 3 mg/kg DO34 (days 4, 8, 13), 0.3 mg/kg DO34 (day 4), and 30 mg/kg DO53 (days 2, 3, 4, 5, 7, 13). Data are mean ± SEM. *p < 0.05 versus VEH. **p < 0.01 versus VEH. ***p < 0.001 versus VEH. $$p < 0.01 versus 0.3 mg/kg DO34. ###p < 0.01 versus 30 mg/kg DO53.
- Figure 5.
DO34 produces modest and selective changes to MWM search strategy. DO34-treated mice (0.3 mg/kg, n = 12; 3 mg/kg, n = 11; 30 mg/kg, n = 12) (A) showed no significant change in swim path strategy than VEH-treated mice (n = 15) on Fixed Platform day 10, but (B) mice administered 30 mg/kg DO34 showed a modest increase in the time spent in the MWM outer ring during Fixed Platform acquisition than VEH-treated mice. C, Fixed Platform day 10 representative swim traces show spatial (self-orienting and direct) and nonspatial (circling and scanning) swim paths in VEH, and DO34-treated (0.3, 3, 30 mg/kg) mice. Data are mean ± SEM. p < 0.05 DO34 (30 mg/kg) versus VEH-treated mice.
- Figure 6.
DO34 does not affect extinction or forgetting tasks. A, Experimental timeline for the extinction (days/weeks) of mice trained drug-free in Fixed Platform task (same cohort as per probe trial [expression]; Fig. 4). B, Experimental timeline for additional mice trained drug-free in Fixed Platform task before a forgetting task (days/weeks). C, No change in either the extinction of the Fixed Platform task, or forgetting performance, was seen between VEH (n = 9 and n = 6, respectively) and 30 mg/kg DO34-treated mice (n = 8 and n = 6, respectively) (MWM Fixed Platform acquisition day 1 and 10 of VEH and 30 mg/kg DO34 for both extinction and forgetting tasks included for comparison). Data are mean ± SEM.
- Figure 7.
DO34 delays MWM reversal learning but does not produce perseverative behavior. A, Experimental timeline for mice trained in a reversal task (days) following drug-free Fixed Platform training. B, During MWM reversal, mice treated with 30 mg/kg DO34 (n = 15) exhibited longer distances to the platform than VEH (n = 15) on reversal days 1, 2, and 5 (MWM Fixed Platform acquisition day 1 and 10 of VEH and 30 mg/kg DO34 included for comparison). C, During the same MWM reversal trials, mice treated with 30 mg/kg DO34 also exhibited a delayed inhibition of spatial preference for the quadrant containing the previous platform location compared with VEH. D, During the same MWM reversal trials, mice treated with 30 mg/kg DO34 also exhibited a delayed acquisition of spatial preference for the quadrant containing the new platform location compared with VEH. Data are mean ± SEM. *p < 0.05, **p < 0.01.
- Figure 8.
DAGL-α−/− mice, but not DO34 treated mice, show OL spatial memory deficits. A, Experimental protocol for OL testing of DAGL-α+/+, DAGL-α+/−, and DAGL-α−/− mice. During sample phase, no difference in (B) total exploration time (C) and no preference for either object (preassigned stationary or moved) were seen between DAGL-α+/+ (n = 13), DAGL-α+/− (n = 15), and DAGL-α−/− (n = 7) mice. During choice phase (D), a significant decrease in discrimination ratio was evident in DAGL-α−/− and DAGL-α+/− mice compared with DAGL-α+/+ mice. E, In the evaluation of DO34 on OL spatial memory, during sample phase (F), DO34 treatment (n = 15) lowered the total exploration time compared with VEH (n = 14), yet (G) no preference for either object (preassigned stationary or moved) was seen between DO34 and VEH-treated mice. During choice phase (H), DO34 produced no significant change in discrimination ratio compared with VEH-treated mice. Data are mean ± SEM. *p < 0.05 versus either DAGL-α+/+ or VEH-treated control mice.
- Figure 9.
DAGL-α−/− mice show profound alterations in brain endocannabinoids. A–D, The DAGL-α substrate SAG was elevated in all four brain regions in DAGL-α−/− mice (n = 8) compared with both DAGL-α+/+ (n = 8) and DAGL-α+/− mice (n = 8). DAGL-α−/− mice possessed decreased levels of 2-AG and AA, respectively, in (E,M) hippocampus, (F,N) PFC, (G,O) striatum, and (H,P) cerebellum. DAGL-α+/− mice showed modest 2-AG reductions in striatum, hippocampus, and cerebellum compared with DAGL-α+/+ mice. AEA levels of DAGL-α−/− mice were significantly reduced in (I) hippocampus and (L) cerebellum, but not in (J) PFC or (K) striatum. Data are mean ± SEM. *p < 0.05 versus DAGL-α+/+ mice. **p < 0.01 versus DAGL-α+/+ mice. ***p < 0.001 versus DAGL-α+/+ mice. $$p < 0.01 versus DAGL-α+/− mice. $$$p < 0.001 versus DAGL-α+/− mice.
- Figure 10.
DO34-treated mice show brain region-selective alterations in endocannabinoids and related lipids. A–D, The DAGL-α substrate SAG was elevated in all four brain regions at 2 h after DO34 injection (n = 5) compared with VEH (n = 5), and compared with 24 h (n = 8) in PFC and striatum. At 2 h after DO34 injection, 2-AG and AA were reduced, respectively, in (E,M) hippocampus, (F,N) PFC, (G,O) striatum, and (H,P) cerebellum. At 24 h after DO34 injection, 2-AG was reduced only in (E) hippocampus and (H) cerebellum, whereas AA showed reductions across (M–P) all four brain areas. No changes in (I,J,K,L) AEA were evident compared with VEH. Data are mean ± SEM. *p < 0.05 versus VEH. **p < 0.01 versus VEH. ***p < 0.001 versus VEH. #p < 0.05 versus 24 h. ##p < 0.01 versus 24h. ###p < 0.001 versus 24h.
