CB1 Activity Drives the Selection of Navigational Strategies: A Behavioral and c-Fos Immunoreactivity Study

Abstract

To promote efficient explorative behaviors, subjects adaptively select spatial navigational strategies based on landmarks or a cognitive map. The hippocampus works alone or in conjunction with the dorsal striatum, both representing the neuronal underpinnings of the navigational strategies organized on the basis of different systems of spatial coordinate integration. The high expression of cannabinoid type 1 (CB₁) receptors in structures related to spatial learning-such as the hippocampus, dorsal striatum and amygdala-renders the endocannabinoid system a critical target to study the balance between landmark- and cognitive map-based navigational strategies. In the present study, mice treated with the CB₁-inverse agonist/antagonist AM251 or vehicle were trained on a Circular Hole Board, a task that could be solved through either navigational strategy. At the end of the behavioral testing, c-Fos immunoreactivity was evaluated in specific nuclei of the hippocampus, dorsal striatum and amygdala. AM251 treatment impaired spatial learning and modified the pattern of the performed navigational strategies as well as the c-Fos immunoreactivity in the hippocampus, dorsal striatum and amygdala. The present findings shed light on the involvement of CB₁ receptors as part of the selection system of the navigational strategies implemented to efficiently solve the spatial problem.

Keywords: AM251; Circular Hole Board; amygdala; dorsal striatum; endocannabinoid system; hippocampus; mice; spatial learning.

Figure 1

Circular Hole Board (CHB). (A) apparatus. (B) experimental procedures. (C) experimental groups: On the left, animals injected with AM251 (AM251 group, n = 20) and tested (n = 17) or not (n = 3) in the CHB. On the right, animals injected with vehicle (VHL group, n = 17) and tested (n = 3) or not (n = 3) in the CHB. To analyze c-Fos expression, we selected six animals (of which three belonging to the AM251 group and three to the VHL group) that used the Landmark-related Navigational Strategy (L-NS) in the CHB, and six animals (of which three belonging to the AM251 group and three to the VHL group) that used the Cognitive Map-related Navigational Strategy (CM-NS) in the CHB.

Figure 2

Behavior in the free exploration trial. Total distance (A), velocity (B), visited holes (the mouse put at least its nose in the hole) (C) and rim stretched attend postures (the mouse looked over the edge of the board) (D) exhibited by animals injected with AM251 or vehicle (VHL). The data presented as mean and standard errors were analyzed by one-way ANOVAs.

Figure 3

Behavior in the training trials. Total distances (A), velocity (B), visited holes (C), first hole exploration latency (D), exit hole exploration latency (E), perseverations (the mouse visited the same hole or at least two adjacent holes twice in a row) (F), rim stretched attend postures (G), grooming (H) and defecations (I) exhibited by animals injected with AM251 or vehicle (VHL). The data are presented as mean and standard errors. For each parameter, the values of trials 1-2 (T1-2), 3-4 (T3-4) and 5-6 (T5-6) were mediated and analyzed by two-way ANOVAs (group x trials). Significant group effect: @: p ≤ 0.05; @@@: p < 0.0005; significant Interaction: * p ≤ 0.05.

Figure 4

Behavior in the test trial. Total distances (A), velocity (B), visited holes (C), first hole exploration latency (D), exit hole exploration latency (E) and navigational strategy (F) exhibited by animals injected with AM251 or vehicle (VHL). For parameters A–E, the data were analyzed by one-way ANOVAs, while the frequencies of the parameter Navigational Strategy were compared by χ² test. * p ≤ 0.05.

Figure 5

c-Fos activation in hippocampal CA1 and CA3 and dentate gyrus (DG), basolateral amygdala (BLA), dorsolateral (DL) and dorsomedial (DM) striatum exhibited by animals injected with vehicle (VHL) according to the landmark- or cognitive map-related navigational strategy carried out in CHB and in No test condition. Data were analyzed by Kruskal-Wallis test followed by Mann-Whitney U when appropriate. VHL animals which carried out the cognitive map-related navigational strategy showed a number of c-Fos⁺ cells significantly (@, p = 0.05) higher than the VHL animals which carried out landmark-related navigational strategy in all regions of interest but in CA1. Furthermore, VHL animals that carried out the cognitive map-related navigational strategy showed a number of c-Fos⁺ cells significantly (*, p = 0.05) higher than the not tested VHL animals in all regions analyzed. In comparison to not tested VHL animals, the VHL animals that carried out the landmark-related navigational strategy showed a number of c-Fos⁺ cells significantly (+, p = 0.05) higher in CA1 and DM Striatum, lower in CA3, BLA and DL Striatum and not statistically different in DG.

Figure 6

c-Fos activation in hippocampal CA1, CA3 and dentate gyrus (DG), basolateral amygdala (BLA) and dorsolateral (DL) and dorsomedial (DM) striatum exhibited by animals injected with AM251 according to the landmark- or cognitive map-related navigational strategy carried out in CHB and in No test condition. Data were analyzed by Kruskal-Wallis test followed by Mann-Whitney U when appropriate. In DM Striatum, AM251 animals that carried out the cognitive map-related navigational strategy expressed a number of c-Fos⁺ cells significantly (*, p = 0.05) higher than the not tested AM251 animals, while the AM251 animals that carried out the landmark-related navigational strategy expressed a number of c-Fos⁺ cells significantly (+, p = 0.05) higher than the not tested AM251 animals.

Figure 7

Comparison of normalized c-Fos activations in hippocampal CA1 and CA3 and dentate gyrus (DG), basolateral amygdala (BLA) and dorsolateral (DLS) and dorsomedial (DMS) striatum exhibited by animals injected with vehicle (VHL) or AM251 according to the landmark- or cognitive map-related navigational strategy carried out in CHB. Data were analyzed by Kruskal-Wallis test followed by Mann-Whitney U when appropriate. In CA3 and DLS, the VHL or AM251 animals that carried out the cognitive map-related navigational strategy expressed a normalized number of c-Fos⁺ cells significantly (*, p = 0.05) higher than the VHL or AM251 animals that carried out the landmark-related navigational strategy. In DMS, VHL (but not AM251) animals that carried out the cognitive map-related navigational strategy expressed a number of c-Fos⁺ cells significantly (*, p = 0.05) higher than the VHL animals that carried out the landmark-related navigational strategy, and higher (#, p = 0.05) than the AM251 animals using the cognitive map-related navigational strategy.

Figure 8

Representative photomicrographs of c-Fos+ cells (highlighted in the black boxes) in hippocampal CA1, CA3 and dentate gyrus (DG) of an animal treated with vehicle at lower (4×) (on the left, Scale bar: 1000 μm) and higher (20×) (on the right, Scale Bar: 200 μm) magnification.