Elevation of endogenous anandamide impairs LTP, learning, and memory through CB1 receptor signaling in mice

Abstract

In rodents, many exogenous and endogenous cannabinoids, such as anandamide (AEA) and 2-arachidonyl glycerol (2-AG), have been shown to play an important role in certain hippocampal memory processes. However, the mechanisms by which endogenous AEA regulate this processes are not well understood. Here the effects of AEA on long-term potentiation (LTP), hippocampal-dependent learning and memory tasks, pERK1/2, pCaMKIV, and pCREB signaling events in both cannabinoid receptor type 1 (CB1R) wild-type (WT) and knockout (KO) mice were assessed following administration of URB597, an inhibitor of the fatty acid amide hydrolase (FAAH). Acute administration of URB597 enhanced AEA levels without affecting the levels of 2-AG or CB1R in the hippocampus and neocortex as compared to vehicle. In hippocampal slices, URB597 impaired LTP in CB1R WT but not in KO littermates. URB597 impaired object recognition, spontaneous alternation and spatial memory in the Y-maze test in CB1R WT mice but not in KO mice. Furthermore, URB597 enhanced ERK phosphorylation in WT without affecting total ERK levels in WT or KO mice. URB597 impaired CaMKIV and CREB phosphorylation in WT but not in KO mice. CB1R KO mice have a lower pCaMKIV/CaMKIV ratio and higher pCREB/CREB ratio as compared to WT littermates. Our results indicate that pharmacologically elevated AEA impair LTP, learning and memory and inhibit CaMKIV and CREB phosphorylation, via the activation of CB1Rs. Collectively, these findings also suggest that pharmacological elevation of AEA beyond normal concentrations is also detrimental for the underlying physiological responses.

Keywords: CB1 null mice; CREB; CaMKIV phosphorylation; URB597; anandamide; fatty acid amide hydrolase; long-term potentiation; mitogen-activated protein kinase.

FIGURE 1

Pharmacological inhibition of FAAH enhances AEA but not 2-AG levels in the hippocampus and cortex of CB1R WT and KO mice. (A-C) The hippocampal or cortex total extracts were subjected to LC-MS analysis for the AEA (A-C) and 2-AG (D) contents (30 min) (n = 10 mice/group) (**p < 0.05, ***p < 0.001vs. compared to respective vehicle or genotype). (E) The hippocampal PM fractions were subjected to Western blot and probed with CB1R specific Ab. Representative blot is shown for the hippocampal PM extracts (n = 6 mice/group). The error bars represent the SEM (two-way ANOVA with Bonferroni’s post hoc tests). HP, hippocampus; NC, neocortex.

FIGURE 2

URB597 inhibits LTP in hippocampal slices from adult CB1R WT mice but not KO mice. (A) A schematic diagram showing the positions of the stimulating and recording electrodes in the CA1 region of the hippocampus. (B) A summary graph showing the field input/output relationships in WT slices. (C) Time course of the averaged fEPSP slopes in WT slices with and without URB597 (solid line 1 μM for 30 min). (D) A summary graph showing the field input/output relationships for KO slices with and without URB597. (E) Time course of the averaged fEPSP slopes in KO slices. Arrows denote the time of tetanic stimulation. (F) A combined plot of the averages of the fEPSP slopes for WT and KO slices with and without URB597. Each point is presented as the mean ± SEM (n= 5 mice/group; 10 slices/group) (Two-way ANOVA with Bonferroni’s post hoc tests), ***p < 0.001 vs. CB1WT + Vehicle; # p < 0.05 vs. CB1WT + Vehicle; $ p < 0.05 vs. CB1WT + URB597.

FIGURE 3

Acute administration of URB597 impairs novel object recognition in adult mice. (A) The level of exploration at el and e2 (1 or 4 or 24 hrs), and the time spent exploring the two objects at T1 and T2 (at 1, 4 and 24 hrs) in WT or KO mice treated with vehicle or URB597. (B-D) Discrimination indices (d2) obtained from the WT or KO mice treated with vehicle or URB597 at 1 hr (B), 4 hrs (C) and 24 hrs (D) retention intervals. One-way ANOVA with Bonferroni’s post hoc test; ***p < 0.001 vs. CB1WT + Vehicle; # p < 0.05 vs. CB1WT + Vehicle; $ p < 0.05 vs. CB1WT + URB597; @ p < 0.001 vs. CB1WT + Vehicle.

FIGURE 4

Acute administration of URB597 impairs spontaneous alternation performance in adult mice. (A) Spatial working memory of CB1R WT and KO mice treated with or without URB597. *** p < 0.001, # p < 0.05 versus CB1WT + V; @ p < 0.05 versus CB1WT + V; $ p < 0.05 versus CB1WT + URB597). (B) The number of arm entries for WT and KO mice (** p < 0.001 versus CB1WT + V) (** p < 0.001 versus CB1KO + V). (C) The time spent in each arm for WT and KO mice with or without URB597 treatment. Each point is the mean ± SEM (n= 8 mice/group). One-way ANOVA with Bonferroni’s post hoc test.

FIGURE 5

URB597 administration impairs spatial memory performance as measured by Y maze. (A-B) Discrimination ratio for arm entries (A) and dwell time (time in the arm) (B) of WT and KO mice treated with or without URB597, 1 h after the first encounter with the partially opened maze. The dashed line indicates chance performance (0.5). (C) The percentage of animals selecting the novel arm as the first choice is shown for WT and KO mice treated with or without URB597, 1 h after the first encounter with the partially opened maze. Each point is the mean ± SEM (n= 8 mice/group). One-way ANOVA with Bonferroni’s post hoc test; ***p < 0.001 vs. CB1WT + Vehicle; # p < 0.05 vs. CB1WT + Vehicle; $ p < 0.05 vs. CB1WT + URB597; @ p < 0.001 vs. CB1WT + Vehicle.

FIGURE 6

Effect of acute administration of URB597 on pERK, pCaMKIV and pCREB levels in the hippocampus. (A) The levels of pERK1/2 and ERK1/2 in hippocampal total extracts. Representative blots are shown for the hippocampal total extracts (n = 6 mice/group). **p < 0.01 vs. CB1WT + Vehicle (0 min). (B) The levels of pCaMKIV, pCREB, CaMKIV and CREB. Representative blots are shown for the hippocampal nuclear extracts (n = 6 mice/group). Student’s t-test was used for statistical analysis. The error bars represent SEM. ***p < 0.001 vs. CB1WT + Vehicle (0 min); **p < 0.01 vs. CB1WT + Vehicle (0 min); *p < 0.05 vs. CB1WT + Vehicle (0 min).