*3.1. Open Field*

The open field exploration test represents a unique opportunity to systematically assess novel environment exploration and general locomotor activity and provides initial screening for anxiety-related behaviors in rodents [43]. Two factors influence anxietylike behaviors in the open field. The first is social isolation resulting from the physical separation from cage mates when performing the test. The second is the stress created by the brightly lit, unprotected, novel test environment [44,45]. To assess the behavioral effects of adolescent exposure to THC, we treated female and male mice of two different genotypes (GHSR +/+ WT) and GHSR −/− (KO) during adolescence. We assessed anxietylike behaviors in young adulthood (Figure 1A).

In our experimental setting, parameters that evaluate general locomotion showed no differences amongs<sup>t</sup> the groups, suggesting locomotion was unaffected by THC exposure (Figure 2A–E). Further, we did not observe significant differences in the in the open field parameters indicative of anxiety-like behaviors in all groups independently of genotype and/or treatment (Figure 3A–E). For all main effects and interaction, *p* ≥ 0.05.

**Figure 2.** Open field, general locomotion. WT-vehicle *n* = 19, WT-THC *n* = 18, KO-vehicle *n* = 19, KO-THC *n* = 18; Male and female mice were included. (**A**) Time mobile. (**B**) Total distance travelled. (**C**) Average speed. (**D**) Freezing time. (**E**) Number of freezing episodes. Data are expressed as mean ± SEM Two-way ANOVA plus post hoc comparison test.

#### *3.2. Elevated Zero Maze*

Elevated zero maze is the master test for assessing anxiety-like behaviors in mice. The test exploits the natural tendencies of mice to explore novel environments [46].

We did not observe significant differences in the willingness of mice to explore open environments in all groups independently of genotype and/or treatment. For all main effects and interaction, *p* ≥ 0.05 Figure 4A–E.

**Figure 3.** Open field, anxiety-related parameters. WT-vehicle *n* = 19, WT-THC *n* = 18, KO-vehicle *n* = 19, KO-THC *n* = 18; Male and female mice were included. (**A**,**B**) Number of entries in the central or peripheral zone. (**C**,**D**) Time spent in central or peripheral zone. (**E**,**F**) Total distance traveled in central or peripheral zone. Data are expressed as mean ± SEM. Two-way ANOVA plus post hoc comparison tests.

**Figure 4.** Elevated Zero Maze. WT-vehicle *n* = 19, WT-THC *n* = 18, KO-vehicle *n* = 19, KO-THC *n* = 18. Male and female mice were included. (**A**) Entries in open arms. (**B**) Time spent in open arms. (**C**) Distance travelled in open arms. (**D**) Average speed in open arms. (**E**) Time spent freezing in open arms. Data are expressed as mean ± SEM. Two-way ANOVA plus post hoc comparison test.
