CB2 Cannabinoid Receptor Knockout in Mice Impairs Contextual Long-Term Memory and Enhances Spatial Working Memory

Abstract

Neurocognitive effects of cannabinoids have been extensively studied with a focus on CB1 cannabinoid receptors because CB1 receptors have been considered the major cannabinoid receptor in the nervous system. However, recent discoveries of CB2 cannabinoid receptors in the brain demand accurate determination of whether and how CB2 receptors are involved in the cognitive effects of cannabinoids. CB2 cannabinoid receptors are primarily involved in immune functions, but also implicated in psychiatric disorders such as schizophrenia and depression. Here, we examined the effects of CB2 receptor knockout in mice on memory to determine the roles of CB2 receptors in modulating cognitive function. Behavioral assays revealed that hippocampus-dependent, long-term contextual fear memory was impaired whereas hippocampus-independent, cued fear memory was normal in CB2 receptor knockout mice. These mice also displayed enhanced spatial working memory when tested in a Y-maze. Motor activity and anxiety of CB2 receptor knockout mice were intact when assessed in an open field arena and an elevated zero maze. In contrast to the knockout of CB2 receptors, acute blockade of CB2 receptors by AM603 in C57BL/6J mice had no effect on memory, motor activity, or anxiety. Our results suggest that CB2 cannabinoid receptors play diverse roles in regulating memory depending on memory types and/or brain areas.

Figure 1

Contextual, but not cued, fear memory of CB2R KO mice is impaired. (a) Baseline freezing behavior of mice during the conditioning period. A mouse in a fear conditioning chamber was presented with a tone for 30 s, 3 times every 2 min. During the last 2 s of the tone, an electric foot shock was delivered. The freezing time of KO mice was similar to that of WT mice in each of the 2 min periods. (b) Contextual fear memory was tested 24 h after the fear conditioning. Mice were placed in the same conditioning chamber for 5 min and the freezing time was measured. The freezing behavior was also analyzed in the early (0–2 min) and late (4-5 min) phases of the 5 min period. ^∗∗ P < 0.01; ^∗ P = 0.02; t-test. (c) Cued fear memory was assayed after the contextual memory test in a modified conditioning chamber. A tone was presented for 3 min and the freezing behavior was monitored before (for 2 min) and during the tone. Error bars represent SEM.

Figure 2

Spatial working memory of CB2R KO mice is enhanced when assessed with a spontaneous alternation test in a Y-maze. (a) Representative tracking of mouse movement for 3 min in a Y-maze. Consecutive entries into three different arms were counted as an alternation. (b) KO mice displayed a higher rate of spontaneous alternation compared with WT mice. ^∗ P = 0.012, t-test. (c) The total number of arm entries was not significantly different between the two strains. Error bars represent SEM.

Figure 3

The levels of motor activity and anxiety assessed in an open field arena are normal in CB2R KO mice. (a) Representative tracking of mouse movement for 5 min in an open field arena (40 × 40 cm²). (b–e) During the 5 min exploration period, KO and WT mice showed no significant difference from each other in the time spent in the center area (20 × 20 cm²) of the arena (b), the travel distance in the center area (c), the total travel distance in the arena (d), or the mean speed of movement (e). Error bars represent SEM.

Figure 4

The anxiety level of CB2R KO mice is similar to that of WT mice when assayed in an elevated zero maze. (a) Representative tracking of mice in an elevated zero maze for 5 min. Dashed lines indicate the walls in two closed quadrants. (b-c) KO and WT mice spent similar time (b) and traveled similar distances (c) in the closed quadrants of the zero maze. (d) The total travel distance of KO mice in the zero maze for 5 min was not significantly different from that of WT mice. Error bars represent SEM.

Figure 5

Acute blockade of CB2Rs had little effect on fear memory. (a) Adult male C57BL/6J mice were fear-conditioned with a tone and a foot shock, as illustrated in Figure 1(a). The animals were injected with AM630 (3 mg/kg, i.p.), a CB2R antagonist, 3 min after the conditioning. (b) Baseline freezing behavior of mice during the conditioning session. The mice were randomly divided into two groups for injection with AM630 or vehicle. (c) Contextual fear memory of AM630-treated mice was not significantly different from that of control mice. The freezing time was counted during a 5 min session and also analyzed for the first 2 and the last 1 min of the session. (d) Cued fear memory was not affected by acute treatment with AM630. A tone was presented for 3 min. There was no significant difference in freezing behavior between the two groups either before or during the tone. Error bars represent SEM.

Figure 6

Acute blockade of CB2Rs had no effect on working memory, locomotion, or anxiety. (a) Adult male C57BL/6J mice were injected with AM630 (3 mg/kg, i.p.) and, 1 h after the injection, used for behavioral tests in a Y-maze, an open field arena, and an elevated zero maze. (b) The rate of spontaneous alternation or the number of arm entries of AM630-treated mice in a Y-maze was not different from that of control mice. (c) In an open field arena, AM630- and vehicle-injected mice displayed similar behavioral patterns in the time spent in the center area, distance traveled in the center area, total travel distance, and travel speed. (d) In an elevated zero maze, the time and distance in the closed quadrants and the total travel distance of AM630-administered mice were similar to those of control mice. Error bars represent SEM.